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The Toxicity Effect of Monocrotophos 36% E.C on the Biochemical Change in Catla Catla (Hamilton, 1882)
Authored by Pugazhendy K
Abstract
This study was undertaken to find out the Catla catla fresh water fish biochemical changes in the fish muscles, liver and kidney. Toxicity were calculated probit analysis, The total carbohydrate contented was estimated by the technique of Roe , Protein was estimated by Lowry method and total lipids were estimated by the method of floch methods. The results shown on total carbohydrate level in muscle 6.43mg/g after treatment 2.70mg/g, liver carbohydrate control 17.55mg/g after treatment 16.10mg/g changes accrued 7.41% and kidney carbohydrate control 1.44mg/g after treatment 0.53mg/g was gradually decreased. Then total protein in muscles 23.20mg/g after sub lethal concentration at 96hours treated 17.80mg/g decreased and liver protein 23.12mg/g treated fish 19.40mg/g changes accrued 13.58% kidney protein level 9.60mg/g after treatment in sub lethal concentration in 96hours 6.24mg/g were decreased and Total lipid level are in muscles 1.78mg/g after 96hours 0.89mg/g liver lipid 7.10mg/g after treatment 4.35mg/g then kidney 2.09mg/g, 0.92mg/g simultaneous gradually decreased muscles, liver and kidney. The Monocrotophos affects not only fishes but also organisms in the food chain through the procedure of expenditure of one by the other those human begins affected various genetic disorders absolutely insecticides.
Keywords: Monocrotophos; Catla catla; Carbohydrates; Protein; Lipids
Introduction
Pesticides are generally used in contemporary agriculture to aid in the manufacture of high quality food. However, some pesticides have the probable to cause serious environment and health directly or ultimately [1]. Long-term expose function of toxic chemical as well as pesticides in different ecosystems, which due to their high efficacy and easy use have eliminate some biological methods of pest control, was caused by ecological pollution [2]. Developed and developing countries which are succeeding quickly in the field of agriculture, technology and industry are incessantly release a variety of kinds of harmful substance into the biosphere and in this manner cause a brutal hazard to the environment [3,4]. The most important source of irrigate pollution are household, agricultural and industrialized waste which are discharge into ordinary water body [5].
Domestic sewages are sprint decomposed commencement agriculture field weighed down with pesticides and fertilizers contaminate the water body. Frequently worn pesticides can be injurious livelihood organisms, pets, and their surroundings. These dropout frequently enclose an assortment of type of pollutant such as heavy metals, radioactive elements, pesticides, herbicides and sarcastic substance like acids and basis [6] reuse bring into play of pesticides causes element pollution consequences in the near future health hazard to be alive stock, above all to fish, birds, frogs, and mammals all live animals [7].
Rhythmic experience to sub-lethal dose of some pesticides can cause physiological and behavioral, biochemical and histological changes in fish getting directly [8] that reduce populations, such as leaving behind of nests and broods, decreased imperviousness to disease and greater than earlier than failure to let alone predators [9]. Nowadays modern globalization assortments of factor poignant formers are using authenticity of pesticide, insecticide, herbicide using agricultural countryside. The pesticide mainly two types Organochlorine, organophosphate and various types of mechanism like pesticide insecticide in topical year monocrotophos is organophosphate using their field of scheming the creepy-crawly pest. Aquatic water bodies are frequently polluted with a numerous of potentially dangerous substance [10].
Fishes play a very important accountability in human nourishment fish proteins are well balanced with obligatory amino acids and are analogous to other proteins of animals' origin [11]. A supplementary fish contains lipids particularly omega fatty acids from the human nutrious position of inspection, eminent and excellence in attendance in maritime and cultivable fishes. Various toxicity data symbolize for assortment of pesticide such as organophosphate, organochlorine, carbamide and pyrethroid pesticides have been reported for number of fish species noted various approaches and momentous researchers find out like Visvanathan et al. [12], [13-18]. The enzyme activity slowly but surely level of decreases mercury chloride (PbCl3) using pesticide martin [19]. Metabolic activity and physiological activity studies alone do not satisfy the accomplished various toxic pathological circumstances of tissue under toxic stress. The natural physiological accomplishment of an organisms get dispersed on reporting to toxicants, stress, it induces its effect first at cellular or even at molecular level, but ultimately cause physiological, pathological and biochemical modification [20].
The biochemical changes occurrence in the body of the organisms give first indication of stress several investigators have description a quantity of change in biochemical parameter of aquatic organisms due to pesticides reporting [21-25]. Carbohydrates form one of the major source of energy forerunner under any stress circumstance. Total carbohydrate satisfied decreased during the revelation to monocrotophos in the air breathing fish Anabas scandens maximum decrease in the brain tissues observed on 21st day [26]. The total decrease in carbohydrate level has been noted in the liver and muscle of Heteropneustes fossilis showing to Butachlor [27]. Chlorpyrifos, an organophosphate compound diminish hepatic glycogen level due to inactivation of enzymes concerned in the carbohydrate metabolism in the fresh water fishes, such as Catla catla, Labeo rohita and Cirrhinus mrigala [28]. Momentous fall in the entire biochemical constituent in all the tissues except glucose prompt to propose that the fish cultured in the aquatic systems earlier to the industrial locations would not have the predictable nutritive value [29]. Because of their low insistence, frequent application of these pesticides are life form skillful for the have power over of pests in agricultural fields and thereby large quantities find their way into water bodies.
Protein being the essential substance is desirable for growth and enlargement and also serves as energy source during the stress condition. The total protein point of muscle and liver are decreased in freshwater telelost fish, Channa punctatus showing to Nuvacron [30]. Decreasing trends have been reported in gill, liver, muscle and brain tissues of O. mossambicus exposed to Quinalphos [31]. Lipids are in general triglycerides that can dish up as metabolic reserves. Phospholipids show a quick diminish given that it is aggressively degraded due to the pesticide stress [32,33] reported decrease in liver lipid content of Barbus chonchonius exposed to Aldiocarb for 15 and 30days for sublethal concentration.
In general, the end point worn in toxicity studies is mortality, survival and growth with acute toxicity tests, the consideration are quite apposite, but for long-term sub lethal concentration's this applicable parameter are easier said than done to ascertain [34]. Organophosphate (OP) pesticides are pronouncement increasing use in recent years since they are biodegradable therefore keep it up in the environment only for a short time. Therefore, in the present study enhance and accommodating to such as good finding an the assessment of biochemical components like a total carbohydrates, protein and total lipids, in the muscle liver kidney and studies of the fresh water fish in Catla catla. Now day’s farmers are using an assortment of pesticide and insecticide monocrotophos in their grassland of cultivation devious the insect pest. Residual of this pesticide alters in to the ecosystem and trouble the healthy environment and aquatic forms. Aquatic farm contains fish and other organism. But the fish is mostly affected by pesticide residuals.
Experimental
Acclimatization
Healthy freshwater fish Catla catla finger links of the weight (10±1g) and length (8.0±0.5cm) were selected for the experiment and were collected from the local commercially culture farm Kumbakonam near Swamimalai HAQ fish culture pond. Fish were screen for any pathogenic infections. Glass contamination aquaria were washed with 1% KMnO4 to keep away from fungal contamination and then dried in the sun light. In good physical shape fishes were then transfer to glass aquaria (35:20:20cm) containing dechlorinated tap water. Fish were acclimatized to laboratory conditions for 10 to 15days prior to carrying out tests. The rate of humanity during becoming accustomed was less than 10%. They were habitually fed with marketable food. Chlorinated tap water was misrepresented every day to take not here face and foodstuff bits and pieces.
Toxicity test
Toxicity tests were conduct in harmony with customary methods. Accumulation solution of monocrotophos 36% EC with an attentiveness of 0.1ml per liter (equivalent to 1ppm) was prepared in distilled water. Based on the progressive bisection of intermission on a logarithmic scale, log concentrations were fixed after conducting the assortment pronouncement test. The fish were starved for 24hours aforementioned to their use in the experiments as not compulsory by luggage compartment to avoid any intrusion in the toxicity of pesticides by excretory products. After the adding together of the toxicant into the test tank with 10liters of water have twenty fish, mortality was record after 24, 48, 72 and 96hours. Five replicates are maintained concurrently. Percent mortality was premeditated and the morals were transfer into probit scale.
Probit analysis was carried out as recommended by Finney [34]. Deterioration appearances of probit alongside logarithmic transformations of concentrations were made. Off the record limits (upper and lower) of the regression line with chi-square test were calculated by an automated curriculum SPSS Version 14 for Probit analysis.
Total carbohydrate estimation
The total carbohydrate at ease was humdrum by the practice of a 10% homogenate of tissue muscles, liver, kidney was equipped using 5% TCA and this was centrifuged at 3000rpm for 10minutes. Samples were cooled in the dark at space temperature for 30minutes. The supernatant was collect and the optical density was deliberate in a spectrophotometer (Hitachi 2205) at a wavelength of 620nm a blank explanation. Blank was geared up by integration 1ml of distilled water with 4ml of Biuret reagent. The total carbohydrate content was intended in mg/g of tissue.
Total protein estimation
Protein was estimated by the method of 1% tissue homogenate were outfitted in 10% TCA and centrifuged at 3000rpm for 15minutes [35]. The gal set was liquefy in 1ml of 1N NaOH to the higher than 5ml of alkaline copper reagent was added and after 10minutes, 0.5ml of folin phenol reagent was considered after was accompanying and quickly The mugginess content was estimated by subtracting the dry heaviness (dried in hot air oven) recognized wet of the tissue muscles, liver, kidney [36].
Lipid estimation
The total lipids were extract by the method of to find out total lipid, known volume of conduct experiment samples were homogenized with 1ml of methanol and 2ml of chloroform to which again 2ml of chloroform : methanol (2:1v/v) was added and mixed systematically [37]. To this, 0.2ml-0.09% sodium chloride explanation was added. The above mixture was poured into independently funnel, mixed and permissible to situate for few hours.
The lower phase was alienated and 0.5ml of extract was deliberate and poured into a clean test tube. It was allowed to try in vacuum dedicators over silica gel, dissolve in 0.5ml concentrated sulphuric acid and mixed well. The tube was plug with nonabsorbent cotton wool and placed in a sweltering water bath for 10minutes and the tubes were cooled at room temperature. 0.3ml of this acid absorbs was taken for experimental analysis.0.5mg of cholesterol for stand and, 0.5ml of distilled water for blank unconnectedly. To each tube, 5ml of vanillin reagent was added. Mixed well and allowable to stand for half an hour and the developed color were deliberate at 250nm.
Results
After sub lethal concentration all the way through the path of experiment surveillance were made to approximation how the animal reacts to the toxicity consequence of the pesticide monocrotophos. From the Table 1 it is manifest that the control commonplace carbohydrate in muscle 6.43mg/g after the exposure period of 24hrs. The carbohydrate was reduced to 5.83 mg/g liver carbohydrate 7.55mg/g and 15.40mg/g 1.42mg/g respectively for 48, 72, 96hrs of exposure period. Liver tissues shows control 17.55mg/g after 96 hours changes 16.10mg/g kidney have significant changes be in indict of tissues 1.44mg/g treatment 0.53mg/g 37% changes carbohydrate level ( Figure 1). Homogenate cell were notice protein have recurrently changes occurs 23.20mg/g after treatment sub lethal concentration 17.80mg/g from muscles cells. The results shown inTable 2 the liver cells 23.12mg/g and changes accuracy 19.40mg/g protein level were slowly but surely decrease kidney control protein shows were 9.60mg/g after 96hours total protein level was decrease 32.39% protein amalgamation have been accrued total protein level decreased in highest level because the metabolic activity and urinary waste was remove therefore total protein level decreases in kidney tissues ( Figure 2).
Value are Mean ± SD of six observation _ or + indicate percentage decrease or increase over control. (n=6)
The body alteration and synthesis problem regulation in present of lipids are total values gradually decreased lipids level control Catla catla fish 1.78mg/g muscles and changes 0.89mg/g liver tissues gradually decreased on 4.34mg/g then kidney tissues control 2.09mg/g after 96 hours sub lethal concentration 0.92 lipid level was decreased after 48hour highest level in kidney 28% changes (Figure 3) (Table 3). The total carbohydrate, protein, lipids and various tissues like Muscles, liver and kidney gradually decreased shows on carbohydrate, protein and lipids.
Discussion
The ever-increasing use of pesticides causes chemical pollution results potential health hazards to live stock, in particular to fish, frogs, birds and mammals [38]. The physiological movements in animal were effected with the aid of muscle. The following are the principal constituents of muscle: water 75%, protein 20%, minerals and organic compounds 5%. Proteins are perhaps the most essential and typical of all the constitutions of livelihood cells. Proteins constitute the fabric material of protoplasm. Apart from formation of protoplasm, protein is an important constituent of the various cellular membranes in conjugation with lipids. Most of the biological active compounds are proteins including enzymes. The use of protein fuels is inadequate for the reason that they cannot be store like lipids and carbohydrates.
Proteins are complex substance with high molecular compound weight form not only the structural framework, but also gears and levers of the operating mechanism in the living wage body. Proteins are useful for the polypeptide chains of amino acid molecules. The proteins are useful for the transport and storage. Specific proteins transport many small molecule and ions. A protein was complex that guides the formation of neural networks in higher organisms. The primary function of protein food is to supply the amino acids needed for the growth, repair and general maintenance of the structural and catalytic machineries of living.
Carbohydrate and protein are the chief nutrients of the animals. They have a variety of function. The carbohydrate supplies energy in the form of ATP molecules, which are formed during TCA cycle. The proteins in different tissues differ in composition and properties [19]. In the present study the protein content in the muscle and liver kidney of Catla catla is decreased with the low concentration of pesticide Monocrotophos. Even with the same concentration longer exposure resulted in decreased amount of protein content (Table 1) which indicate that the tissue protein endure proteolysis. The result in the production of free amino acids, which are used in TCA cycle for energy production under, stresses. There are similar reports of effects of toxicants on total protein in other fishes by Mhadhbi et al. [6], Nagaraju et al. [7] and Pugazhendy et al. [39].
In the present study the result obtains clearly indicate that there was a decreased amount of protein and glycogen content to resist the effect of pesticides. That is to provide immediately energy to the combating elements of the body and protect all systems of the body from the harmful effect of the pesticide. With regard to carbohydrate in the fish exposed to different hours of exposure of pesticide Monocrotophos there was no much change within 24hours of treatment with low concentration 0.040ppm fish kept prolonged exposure up to 96 hours the carbohydrate content was observed in decreased amount (Table 1) the carbohydrate content was found more and more in decreased. Such reduction in store carbohydrate content has been reported in Labeo rohita exposed to monocrotophos effect of monocrotophos was reported by Patil et al. [32]. A fall in carbohydrate levels clearly indicates its rapid utilization to meet the enhanced energy demands in pesticides treated individuals through glycolysis or hexose monophosphate pathway
The term lipid was used by the biochemist to describe that group of substances of animal origin, which is inexplicable in water, but soluble in fat solvents. All cells contain lipid in the form of globules scattered in the cytoplasm. The concentration is much higher in cells forming adipose tissue. In the present study hours of exposure periods has decreased amount of lipid. Totally concluded pesticide posses through high health impact entire food chains various human disorders attains various aspects in human body then avoid pesticide using biopesticides.
Conclusion
The results in the present study showed that the various hours of exposure in Catla catla fresh water fish against monocrotophos caused by various physiological changes and pathology in their organs muscles liver and kidney they were associated with various cell signals and metabolic activity in the exposure. It can be used as a sensitive model to monitor the aquatic pollution, aquatic animals and using organophosphate various via human genetic disorders in young one generation so agriculture filed avoid Organo (OP) pesticide controlled only bio pesticides.
A Review on the Safety of Inhalation of Propylene Glycol in E-cigarettes
Authored by Cotta KI
Abstract
Electronic cigarette (e-cigarette) are being used as a safe alternative to tobacco smoking, or as a smoking cessation device. However, the FDA presumes that e-cigarettes are as hazardous as conventional cigarettes. The Tobacco Control Task Force of the American Association of Public Health Physicians instead has indicated that e-cigarettes closely resemble Nicotine Replacement Therapy products. Since Propylene glycol (PG) constitutes around 89-90% of the e-cigarettes formulation, the objective here is to review the safety of inhalation of PG. All animal and human studies that analyzed the effect of the inhalation of PG have indicated that, PG does not appear to pose a significant hazard via the inhalation route. In fact, in several of these animal studies the concentrations of PG used were higher compared to the concentration used in e-cigarettes and did not give rise to any toxic effects. However, there are no human studies at the level of e-cigarette concentrations.
This review throws some light in terms of the safety aspects of inhalation of PG particularly seen in animal studies as it relates to e-cigarette concentration. Since most of the results came from animal studies. The information gained can be used as a platform to conduct human studies to check the short term and long term effect of propylene glycol in e-cigarettes. Therefore, further human studies using PG concentrations similar to that in e-cigarettes need to be conducted to confirm the safety of inhalation of PG from e-cigarettes.
Keywords: Electronic cigarette; Propylene glycol; Inhalation; E-cigarettes; PG
Introduction
An electronic cigarette, or e-cigarette, is an electrical device that simulates the act of cigarette smoking by producing an inhaled mist bearing the physical sensation, appearance, and often the flavor and nicotine content of inhaled tobacco smoke. The primary stated use of the e-cigarette is a safe alternative to tobacco smoking, or as a smoking cessation device, while it attempts to deliver the experience of smoking without, or with greatly reduced, adverse health effects. However, the FDA in a July 22, 2009 press conference adopted the position that it will presume that e-cigarettes are as hazardous as conventional cigarettes. An opposing view is held by the Tobacco Control Task Force of the American Association of Public Health Physicians who has indicated that e-cigarettes closely resemble Nicotine Replacement Therapy (NRT) products approved by the FDA. Tests performed by the FDA have shown that e-cigarettes have similar nicotine levels and trace contaminants as NRT products. The Ruyan e-cigarettes use micro-electronics to vaporize, very small quantities of nicotine dissolved in propylene glycol into a fine aerosol with each puff.
Nicotine and Propylene Glycol are two small molecules with known safety profiles. Propylene glycol (PG) is generally recognized as safe by oral, dermal or inhalation routes and has been a common ingredient in all American made tobacco cigarettes for seven decades (AAPHP website). The cartridge liquid is tobacco-free and no combustion occurs (Ruyan cartridge report). There is a lot of literature emerging related to e-cigarettes. Most of the literature available so far related to e-cigarettes indicates.
Consumer based survey regarding personal view about vaping.
Chemical analysis of the e-cigarette cartridges, solutions and mist.
Nicotine content, delivery and Pharmacokinetics. Since Propylene glycol (PG) constitutes around 65-90% of the e- cigarettes formulation, the objective here is to review the safety of inhalation of PG. This was done by reviewing all the available animal and human studies involving the inhalation of PG at concentration levels near those used in e-cigarettes.
Pharmacokinetics of propylene glycol
Metabolism: Propylene glycol is metabolized into pyruvic acid (a normal part of the glucose metabolism process, readily converted to energy), acetic acid (handled by ethanol metabolism), lactic acid- a normal acid generally abundant during digestion and propionaldehyde [1].
Half life: According to the World Health Organization (WHO) PG has a relatively short plasma half-life (4-8 hours).
Elimination: The route of elimination of PG depends on the dose administered and not on the route of exposure. It is mainly excreted in the urine as the glucuronide conjugate, but 12-45% is excreted unchanged [2]. Renal clearance decreases with dose (390mL/min/1.73m2 at a dose of 5g/day, but only 144 mL/ min/1.73m2 at a dose of 21g/day) [3].
Safe levels of propylene glycol: The inhaled dose of PG from normal e-cigarette use is 0.3 to 0.45g/day and if used more intensively, could result in 0.9g/day (Ruyan e-cigarette cartridge report). In terms of systemic levels, serious toxicity generally occurs only at plasma concentrations over 1g/L, which requires extremely high in takeover a relatively short period of time. Considering that the volume of blood in an average adult is 3L. Serious toxicity in an average adult can only occur at plasma amount over 3g. Thus an inhaled dose of 0.3 to 0.45g/day of PG is significantly lower compared to the concentration that can cause serious systemic toxicity.
Animal studies indicating the inhalation safety of propylene glycol
Suber et al. [4] exposed rats by nose-only inhalation to a mean target aerosol concentrations of 0.054, 0.54 or 1.2g propylene glycol/day. These exposure levels are higher compared to the inhaled dose of PG from e-cigarettes (0.3 to 0.45g/day) (Ruyan e-cigarette cartridge report). The rats in this study did not show any significant differences in terms of respiratory rates, minute volumes or tidal volumes. The mean terminal body weights for male rats did not change. However the mean body weights for female animals exposed to 1.2g/day were significantly lower than those of the female controls from day 50 onwards. This decrease in weight was consistent with a decrease in feed consumption in the female rats exposed to high concentration of propylene glycol. The nasal passages of the animals exposed to medium (0.54g propylene glycol/day) and those exposed to high (1.2g propylene glycol/day) showed a significant increase in the number of goblet cells or an increase in the mucin content of the existing goblet cells.
Most of the rats had nasal hemorrhage and ocular discharge, and this may have been caused due to the dehydration of the nares and eyes caused due to the physical irritation of propylene glycol upon the nasal epithelium in the rat. This study has shown that the rats exposed to propylene glycol by the inhalation route at the concentrations administered resulted in changes in clinical, gross pathological, histopathological or organ-weight variables that were not life-threatening. The changes thus observed in organ weight and clinical pathology parameters did not indicate a toxic effect on any single organ system or blood component. The PG exposure levels in this rat study (0.054, 0.54 or 1.2g/day) are higher when compared to the inhaled dose of PG from e-cigarettes (0.3 to 0.45g/day) and Suber et al. [4] have indicated that PG does not appear to pose a significant hazard via inhalation of either the vapor or a vapor/aerosol mixture. In addition, this study also supports previous studies indicating that propylene glycol is not a systemic toxin when administered by inhalation or by other routes [5-8]. Thus the PG concentration used in e-cigarettes appears not to have any toxic effects as shown by this in-vivo study but further human studies need to be conducted to confirm this.
Montharu et al. [9] in their study subjected each rat to a dose equivalent to 25mg/kg/day of propylene glycol for 4 days. The concentrations of propylene glycol used in this study are significantly higher compared to the amount of propylene glycol inhaled from-cigarettes which is in the range of 4.29mg/kg/ day to 6.4mg/kg/day. In this study by Montharu et al. [9] the biochemistry and histopathology test results demonstrated that, 25mg/kg propylene glycol presented limited cellular reaction and was extremely well tolerated in a similar way to deionized water. However it was observed that during the administration of propylene glycol the respiratory arrests were the most frequent side effect and occurred on the third day of administration. As suggested by Montharu et al. [9] one explanation for this occurrence could be a rapid broncho constriction caused due to the irritating and/or inflammatory potential of the molecule.
These results indicate that with respect to inhalation of aerosols, propylene glycol may be considered as a solvent with acceptable toxicity. In fact, Propylene glycol is one of the solvents included in the FDA inactive ingredients guide for various preparations including inhalation. The concentrations of propylene glycol used in this study are significantly higher compared to the amount of propylene glycol inhaled from- cigarettes which is in the range of 4.29mg/kg/day to 6.43mg/ kg/day. Secondly this study only analyzed the short term effect of propylene glycol only for 4days. Thus indicating that, the inhalation of propylene glycol in e-cigarettes at the low concentrations used appears to be safe. However further human studies to check the long term effect of inhalation of propylene glycol have to be conducted.
Werley et al. [10] in their study exposed rats and dogs to high concentrations of PG aerosol followed by comprehensive systemic evaluations, especially involving the respiratory system. In case of the acute and 7-day exposure studies the rats were exposed to a concentration of up to 214mg/kg and 174mg/ kg of PG respectively, which is relatively high compared to the inhaled dose of PG from e-cigarettes of 4.29mg/kg to 6.43mg/ kg. When the rats were exposed repeatedly by the inhalation route for the 7 days exposure it did not yield remarkable inlife findings. In acute inhalation studies in the rat, bleeding was observed around the eyes and from the nose when dosed at 214mg/kg. This finding was also reported by Suber et al. [4] This bleeding can be associated with one of the chemical properties of PG. PG is known to be hygroscopic, it absorbs moisture from its surroundings, and is used as humectants. When PG is deposited on the skin and mucus membranes it absorbs moisture thus causing the tissues to dry, resulting in small tissue breaks with minor bleeding.
In a 28 days inhalation toxicity study of PG in rats, rats were exposed up to 216mg/kg/day PG aerosol, which again is significantly higher, compared to the inhaled dose of 4.29mg/ kg/day to 6.43mg/kg/day of PG from e-cigarettes. This high exposure gave rise to "minimal" laryngeal squamous metaplasia; this lesion is commonly observed in many different inhalation exposure studies and is probably related to the unique sensitivity of the larynx, and its capacity for efficient deposition of particles. The no-observed-effect-level (NOEL) for the 28-day rat study was determined to be approximately 20mg/kg/day. Dogs were exposed to attain up to 60mg/kg/day of PG in lungs which again is significantly higher compared to the inhaled dose of 4.29mg/kg/day to 6.43mg/kg/day of PG from e-cigarettes. It is important to note that in the dog study where the concentration administered was lower and, the tissue surfaces were much larger as compared to the rats the clinical observation of bleeding around the eyes and nose were not observed.
Safety pharmacology studies in the dogs were unremarkable. The NOEL for dogs in the 28-day study was determined to be 6.05mg/kg/day. This study indicated that when aerosol PG is administered by various dose routes including the inhalation route it has a relatively low toxic potential in dogs. In addition, no histopathological effects were observed on tissues like the larynx, trachea and lung. However, dogs treated for 28 days showed a decrease in some assessments of red blood cells. A treatment related decrease in hemoglobin level, red blood cell concentration and hematocrit were observed which were possibly attributable to red blood cell breakdown. Although the mechanism for this process is not clear, some veterinary texts suggest PG as an "oxidant" capable of producing intravascular hemolysis in animals. But these effects had no impact upon the health of the dog and were not clinically significant. The red blood cell count was still within the normal ranges for dogs for this age, strain and sex.
Overall, PG inhalation exposure in both rats and dogs produced limited toxicological findings thus indicating that CAG- PG exposures could be safely conducted in man by the inhalation route. Additionally, all the side effects were observed at PG exposure levels hundreds of times higher than the exposure levels of 4.29mg/kg/day to 6.43mg/kg/day which correspond to e-cigarette levels of PG. Although this study only analyzed the impact of short-term exposure to PG vapor and the effects of long-term (months or years) exposure are unknown. It does provide valuable information for e-cigarette users and policy makers. This study shows that no measurable harmful effects were observed with high concentrations of PG vapor and this is fully consistent with its designation by the FDA as "generally recognized as safe". Indicating that, the inhalation of PG at such low concentrations from e-cigarettes appears to be safe, but have to be confirmed with further human studies.
Heck et al. [11] performed a study to determine if subchronic exposure to smoke from cigarettes containing nonvolatile humectant ingredients like propylene glycol would influence the incidence, severity of toxicological endpoints, or persistence of respiratory-tract histopathology that were otherwise observed following exposure to similar cigarettes made without humectant ingredients. American- style tobacco blend containing propylene glycol at 2.2% w/w tobacco, were used to prepare the filtered test cigarettes used in this study. This amount to exposure levels of 95mg/kg of PG. Smoke exposures were conducted for 1h/ day, 5days/wk. for 13 wk. The exposure levels in this study were higher compared to the inhaled dose of PG from e-cigarettes which are 4.29mg/kg/day to 6.43mg/kg/day.
This study shows the addition of propylene glycol to cigarettes, has no meaningful effect on the site, occurrence, or severity of respiratory-tract changes or on the measured indices of pulmonary function. It indicates that the addition of propylene glycol to cigarettes does not significantly affect the biological activity of inhaled cigarette smoke in this rat model. Thus indicating that the Inhalation of PG in rats did not have any toxic effects, however this was short term animal study conducted only for 13weeks. The effects of long-term (months or years) exposure in humans are unknown. Therefore further human studies need to be conducted to check the long-term effects of inhalation of PG.
Robertson et al. [12] performed a study to determine the safety of employing the vapors of propylene glycol in atmospheres inhabited by human beings. In this study, monkeys and rats were exposed to an atmosphere supersaturated with high concentrations of vapors of propylene glycol for periods of 12 to 18months. The concentrations of propylene glycol used were 0.23-0.35mg/liter (0.04-0.07mg/kg) and 0.17-0.35mg/ liter (2-4.12mg/kg) for monkeys and rats, respectively. Thus, the dose of propylene glycol used in this study for monkeys were lower and for rats were approximately similar compared to the inhaled dose of PG from e-cigarettes which is 4.29mg/kg to 6.43mg/kg. Propylene glycol did not have any impact on growth rates, blood counts, urine examinations, kidney function tests, fertility and general condition of the animals, with the exception that the rats in the glycol atmospheres exhibited consistently higher weight gains.
Examination at autopsy as well failed to reveal any differences between the animals kept in glycolized air and those living in the ordinary room atmosphere. Extensive histological study of the lungs done to determine whether the glycol had produced any generalized or local irritation indicated absence of irritation. The liver, spleen, kidneys and bone marrow were also normal. Thus the results of these experiments along with the absence of any observed ill effects when patients were exposed to propyleneglycol vapors for months at a time provide assurance that air containing these vapors in amounts up to the saturation point is completely harmless. Propylene has been known to be essentially non-toxic [13,14].However since this was an animal study, further human studies need to be conducted to check the effects of inhalation of PG in e-cigarettes.
Wang et al. [15] performed studies to evaluate the potential toxicity of aerosolized cyclosporine formulated in propylene glycol when given to rats and dogs for 28days by the inhalation route. In case of rats, much higher doses of propylene glycol were evaluated (total inhaled doses of PG here were 106.2, 348.8, and 777.0mg/kg/day). The highest total inhaled dose (777.0mg/ kg/day) is approximately 7times the clinical exposure based on a pulmonary deposited dose. In case of dogs, total inhaled doses of PG were 61.7, 106.9, and 133.9mg/kg/day. Thus the exposure levels in this study were significantly higher compared to the inhaled dose of PG from e-cigarettes which are 4.29mg/ kg/day to 6.43mg/kg/day. The results from the propylene glycol vehicle arm of this 28 day inhalation toxicity studies in both rats and dogs demonstrated that there were no respiratory or systemic effects from high doses of propylene glycol relative to air controls.
Similarly Venitz et al. [16] in their study delivered inhaled nominal doses ranging from 3-60mg/kg/day, over a 6- to 60-minute exposure interval to dogs for 28days. The PG aerosol exposure in this study as well did not have any apparent toxicity upon lung, kidney or liver. Thus the results from these two studies indicate that the concentration of PG used in e-cigarettes will not have any respiratory or systemic and will not have any apparent toxicity upon lung, kidney or liver. However these were animal studies and were conducted only for 28days. Hence further studies to check the long term effects of PG inhalation in humans need to be conducted.
Gaworski et al. [17] performed a study to examine the biological activity and analytical smoke chemistry of mainstream smoke from cigarettes containing three different amounts (low, medium, high) (4, 7 and 10% respectively) (i.e. 28,900, 54,300 and 77,900mg/kg) of added PG. All these above mentioned concentrations are significantly higher than the inhaled dose of PG from e-cigarettes which is 4.29mg/kg/day to 6.43mg/kg/day.
In this study they compared the biological activities with that of otherwise similar cigarettes, containing no added PG. It was observed that the addition of PG to the tobacco material reduced the presence and severity of some histopathological lesions (e.g., goblet cell hyperplasia in the nose, epithelial keratinization in the larynx, focal accumulation of alveolar macrophages, in females only) which are typically seen in smoke inhalation studies with rats.
As suggested by Gaworski et al. [17] one reason for this decrease in severity of some histopathological lesions could be that when PG is added to experimental cigarette tobacco this consequently reduced the concentrations of several of the major smoke constituents, including nicotine. These reductions in smoke constituents are probably the result of a dilution effect by PG displacing nicotine, as well as an increase in water content of the particulate phase from the burned tobacco, rather than an effect of PG. However, the responses shown from the cigarette with no added PG and the three cigarettes with added PG were very similar, and more importantly did not indicate any substantial increase in toxicity related to the levels of PG. Since the exposure levels in this study were higher than the inhaled dose of PG for e-cigarettes and the results did not indicate any toxic effects, it indirectly indicates that the concentration of PG used in e-cigarettes is safe for inhalation.
Human studies indicating the inhalation safety of propylene glycol
\Wieslander et al. [18] performed a study to evaluate the effects of exposure to a PG mist, at exposure levels occurring during normal aviation emergency training. The physiological effects studied included tear film stability, nasal patency and lung function, as well as subjective symptoms. The design was experimental and showed acute effects on ocular and throat symptoms, and decreased tear film stability in non asthmatic subjects after 1 minute exposure to PG mist from an artificial smoke generator. The exposure concentration of PG (geometric mean 309mg/m3) (99.29mg/kg) was quite high, compared with other exposure measurements of this compound in work environments.
Exposure measurements in house painters who used water based paints showed exposure concentrations of PG ranging from <0.1 to 12.7mg/m3 (mean 2.6mg/m3) (0.84mg/kg), but no measurable exposure to PG was found in motor servicing work [19]. These studies showed that exposure to PG can be high from smoke generators, compared to other occupational applications. It was concluded from the results that short exposures to PG mist from smoke generators may cause acute irritative ocular and upper airway effects in non-asthmatic subjects, and some symptoms were more common in women and subjects with atopy. Some subjects may present with minor lower airway obstruction, cough, and mild dyspnoea.
Thus, sensory hyper reactivity could be one mechanism behind the development of a combination of cough, slight airway obstruction, and mild dyspnoea in a few of those exposed to PG mist. However, when children were exposed to, airborne PG (mean concentration 22.17mg/kg, maximum 30.21mg/kg) for air sterilization, continuously during several weeks. The mucous membranes in the upper respiratory tract did not show any negative effects. It was observed that the concentrations of PG used in these studies were higher than the inhaled dose of PG for e-cigarettes which is 4.29mg/kg/day to 6.43mg/kg/day. Indicating that the concentration of PG used in e-cigarettes may be at safe levels [20].
Greenbaum et al. [21] in their study observed that the use of the new formulation of 0.025% Rhinalar containing a reduced amount of propylene glycol (5%) (0.04g/day) considerably reduced nasal burning and stinging and throat irritation compared with the original formulation which contained 20% (0.16g/ day) propylene glycol. The dose (gram/day) used in this study are lower than the inhaled dose of PG from e-cigarettes which is 0.3 to 0.45g/day.
Environmental protection agency (EPA) report
General toxicity observations: The EPA upon reviewing the available toxicity information has concluded that there are no endpoints of concern for oral, dermal, or inhalation exposure to propylene glycol. In these studies, dose levels near or above testing limits (as established in the OPPTS 870 series harmonized test guidelines) were employed in experimental animal studies and no significant toxicity was observed. However the report did not mention the concentration of the dose administered. The acute toxicology profile for propylene glycol in case of oral exposure in rats resulted in LD50 range of 8000-46000mg/kg [22,23].
Based on the EPA report as a result of the Phase IV review of propylene glycol for reregistration under Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), ecological effects data requirements were waived due to its high volatility, known low toxicity, and available data. Data obtained from published studies related to propylene glycol provide an additional confirmation of the low toxicity of the compound to fish and aquatic invertebrates (Table 1).
The EPA report has clearly mentioned that there is a reasonable certainty no harm will result to the general population or any subgroup from the use of propylene glycol. The agency arrived to this conclusion based on all available information on the toxicity, use practices and exposure scenarios, and the environmental behavior of propylene glycol and dipropylene glycol. Because no toxicological endpoints were identified for propylene glycol, by oral, dermal, or inhalation exposure, the agency determined that exposure to it does not result in human health effects of concern [24,25].
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Conclusion
Suber et al. [4], Werley et al. [10] Venitz et al. [10] and Wang et al. [15] through their studies indicated that rats and dogs did not exhibit any respiratory or systemic toxicological effects due to inhalation of high doses of propylene glycol relative to air controls. In addition, these studies also support previous studies indicating that propylene glycol is not a systemic toxin when administered by inhalation Robertson et al. [12] or by other routes [5-8]. Heck et al. [11] demonstrated that the addition of glycerin and propylene glycol to cigarettes, has no meaningful effect on the site, occurrence, or severity of respiratory-tract changes or on the measured indices of pulmonary function. It indicates that the addition of glycerin and propylene glycol to cigarettes does not significantly affect the biological activity of inhaled cigarette smoke in the rat model. Similarly several cigarette analysis conducted by Gaworski et al. [17] indicated that cigarettes with no added PG and cigarettes with added PG were very similar, and more importantly did not indicate any substantial increase in toxicity related to the high levels of PG inclusion.
Gaworski et al. [17] observed that the addition of PG to the tobacco material reduced the presence and severity of some histopathological lesions (e.g., goblet cell hyperplasia in the nose, epithelial keratinization in the larynx, focal accumulation of alveolar macrophages, in females only) these are typically seen in smoke inhalation studies with rats. As suggested by Gaworski et al. [17] one reason for this decrease in severity of some histopathological lesions could be that when PG is added to experimental cigarette tobacco this consequently reduced the concentrations of several of the major smoke constituents, including nicotine. In addition, the EPA report has also clearly mentioned that there is a reasonable certainty no harm will result to the general population or any subgroup from the use of propylene glycol.
The agency arrived at this conclusion based on all available information on the toxicity, use practices and exposure scenarios, and the environmental behavior of propylene glycol. Because no toxicological endpoints were identified for propylene glycol by oral, dermal, or inhalation exposure, the EPA determined that exposure to it does not result in human health effects of concern. It is important to note that this EPA report was published prior to the advent of the e-cigarettes. All the animal studies show that no measurable harmful effects were observed with high concentrations of PG vapor and this is fully consistent with its designation by the FDA as "generally recognized as safe". In fact, propylene glycol is one of the solvents included in the FDA inactive ingredients guide for various preparations including inhalations. Several of the animal studies employed concentrations of PG higher than the concentrations used in e-cigarettes and did not give rise to any toxic effects. Based on animal studies that are testing at higher levels it is safe to use PG in humans. However, there are no human studies at the level of e-cigarette concentrations. Therefore, further human studies using PG concentrations similar to that in e-cigarettes need to be conducted to confirm the safety of inhalation of PG from e-cigarettes.
This review throws some light in terms of the safety aspects of inhalation of PG particularly seen in animal studies as it relates to e-cigarette concentration. There are not many human studies checking the inhalation safety of PG. Since most of the results came from animal studies. The information gained can be used as a platform to conduct human studies to check the short term and long term effect of propylene glycol in e-cigarettes.
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Where We Stand In the Global Eradication of Polio
Authored by Ternullo S
Abstract
Despite almost Three decades of progress and the availability of effective vaccines, global WPV eradication has remained elusive. The end game eradication strategy was developed in 2013 and implementation has begun. One dose of IPV was incorporated into routine immunization schedules in fall of 2015 followed by withdrawal of tOPV and its replacement by bOPV for routine use globally in April 2016. Continued challenges exist as the 2018 target date for eradication approaches. Challenges include technical challenges for maintenance of cold chains for surveillance and assurance of OPV potency, warfare and security challenges, hostility toward vaccinators, low literacy rates, politically motivated misinformation, and religious misinformation resulting in vaccine refusal. The lack of accurate health information via government and media channels at a grass roots level and a lack of reliable governmental commitment to immunization and building network infrastructure have also presented challenges. Successful eradication will require addressing these challenges.
The post-eradication plan includes the ultimate withdrawal of all live-attenuated polio vaccines (OPV) for routine use at the specified trigger points, routine reliance on IPV, maintenance of adequate global stockpiles of appropriate vaccines in case of outbreaks, development of pharmaceutical antiviral for polio, and planning for an appropriate response in the event of a WPV or VDPV escape from a storage or production facility. We are on the cusp of a polio-free world but continued progress requires cooperation of parents, community and religious leaders, governments and political leaders at all levels, international technical and healthcare agencies, and major aid donors.
Keywords: Polio; Immunization; Eradication; Immunization; Epidemiology; WHO; Vaccination
Introduction
Eradicable infectious agents are those where the chain of transmission can be broken by an intervention and where there is no reservoir such as a chronic or persistent carrier state, chronic infection, or a non-human host. The only disease eradicated globally is small pox, with the last case recorded in Somalia in 1977. Despite two highly effective vaccines and a disease restricted to humans with very rare cases of chronic persistent infections, the eradication of polio has been elusive. The less favorable geopolitical climate during the polio eradication effort, characterized by multiple global conflicts, as compared to the Cold War smallpox eradication era has contributed to the difficulties encountered [1]. The Global Polio Eradication Initiative (GPEI) was launched at the World Health Assembly (WHA) in 1988 and signaled an international push for a polio-free world by the year 2000.
Epidemiology of polio
Polio has affected populations for millennia and polio- induced paralysis has been found depicted in Egyptian hieroglyphs [2]. Outbreaks were sporadic during the 1880s in the U.S. and Scandinavia until the turn of the century when epidemics began in Scandinavia followed by other European countries and the United States. Unlike other communicable diseases, polio flourished in areas with advanced sanitation and has been referred to as "the middle-class plague". Ever worsening epidemics were characterized by their unpredictability and in temperate climates were strongly seasonal and typically occurred between August and October. There was a striking latitudinal gradient in the timing of epidemics across the temperate climates, but in tropical areas, no such gradient or seasonality existed. The magnitude and regularity of the outbreaks increased in many countries from the pre-WWII era to the post-WWII baby boom area, hitting a peak in the developed world during the 40s and 50s. Polio was largely eradicated in these developed countries through mass immunizations by the 1980s [3,4].
Since before 1955 there has been evidence that some pediatric injections and procedures could induce polio infection and paralysis. Tonsillectomies were documented to increase the risk of bulbar polio. Injection-induced polio was well documented in Italy, Germany, and France in children being vaccinated or injected with antibiotics. In the United States the increase in the incidence of polio roughly correlated with an increase in the number and frequency of immunization programs and paralysis developed in the limb injected with DPT vaccine [5]. Booster shots were sometimes suspended in the U.S. during epidemics. The concern resurfaced with the expanded vaccinations against polio during the 1980s and health care workers began reporting this phenomenon again after vaccination against common childhood diseases. A case- controlled study in Romania demonstrated that the elevated risk of vaccine-associated paralytic polio (VAPP) between 1970 and 1994 was not a result of vaccine components but resulted from the administration of multiple intramuscular injections of antibiotics within 30 days of receipt of live-attenuated oral polio vaccine (OPV).
The chronologic proximity of injections increased the risk of paralysis by a factor of 2-10 times [6]. Several epidemics erupted in India during the 1990s providing new clinical evidence. Tissue injury by an injection assisted the poliovirus' (PV) systemic spread and entry into the spinal cord. Therefore, until wild poliovirus (WPV) was eradicated in endemic regions, polio vaccination needed to be undertaken before other pediatric immunizations to reduce the risk of provoking polio.
Clinical aspects of polio
Polio presents difficulties in recognizing infections since between 95% and 99% of patients are asymptomatic; surveillance and monitoring. About 24% of patients have minor non-specific symptoms such as fever, sore throat, upset stomach, or flu-like illness. Between 1% and 5%, of patients, develop an aseptic meningitis with stiffness of the legs or paresthesias once the minor symptoms resolve. Only about 2% of people experience viral replication in the CNS. Typically, there is complete recovery within 2-10 days. Fever than 1% of polio cases result in limb paralysis. Respiratory paralysis resulting in death occurs in 5%-10% of those patients. The rates of paralysis increase substantially with age. Paralysis occurs in 1/1000 cases for infants but 1/75 cases for adults.
Historically, in areas with poor sanitation, enteroviruses, including polio, were prevalent and infected infants between 6 and 12months of age, These infants were still partially protected through passively acquired immunity resulting in a lower incidence of paralysis. Speculation on the epidemics that occurred in well-developed countries during the 1950s has surrounded the observation that with improved hygiene, the age of first WPV infection was postponed in children past 12 months. At that age, the child had neither passive immunity from mother nor active immunity against the enterovirus, resulting in a higher incidence of paralysis than in younger children [3].
The World Health Organization (WHO) estimates that there are 10-20 million survivors' worldwide living with various disabilities after acute poliomyelitis. In the developed world, most of these survivors are aged 50 or older [2]. The socioeconomic consequences of polio can be profound. When compared with controls that had never had polio, a higher percentage of paralytic polio survivors remained childless, were unemployed despite a higher educational level, were more likely to receive a disability pension, and had higher health costs whether or not continued paralysis was present [7]. Post-polio syndrome (PPS) affects between 25% and 40% of all polio survivors, both those immediately disabled during the acute stage of the disease and those who recover with few or no residual symptoms but who begin to experience new or worsening disabling symptoms after years of stability. The risk of PPS is greatest for females, those with respiratory disturbances during the acute phase of the disease, and those who required the use of orthoses and aids during the recovery period [8].
Progress toward global eradication of polio
More than 60 years after the development of the first vaccine, the disease has still not been eradicated globally, though progress has been made and polio is on the verge of eradication (see Appendix 1 for timeline). Polio was generally eliminated within developed countries within 25-30 years of the first polio vaccine [9]. Target dates for global eradication were missed in 2000, 2012 and 2015 and the current target for eradication is 2018. Despite our failure to eradicate polio completely, GPEI achievements have been impressive. Polio rates have decreased from an estimated 350,000 cases of WPV yearly at the launch of the campaign in 1988 to only 21 cases through August of 2016. The global polio burden is less than 0.02% of the burden reported in 1988 when the GPEI was launched [10]. Of the WPV cases reported in 2016, 19 were reported in the only remaining endemic countries: Afghanistan and Pakistan. The remaining 2 cases were reported in Nigeria and based on mutation viral typing, had been circulating undetected for several years, indicating gaps in the country's surveillance system.
Available vaccines
Salk vaccine (IPV)
The Salk vaccine is considered the safer of the two available vaccines since its trivalent strains are killed and it is effective in inducing humoral immunity without risk of live viral shedding or mutations that could produce VAPP. It is considered the vaccine of choice in areas where polio has been eradicated for long periods of time. Most developed countries utilize only IPV for routine immunization and the United States converted to routine use of IPV in 1997, approximately 18 years after the last reported case of polio in the United States. IPV is not effective in inducing mucosal immunity and therefore has a limited role during polio outbreaks. It is also limited in its ability to produce secondary immunity in contacts of the vaccinated individual since no live virus shedding occurs in stools. Its role in the rapid control of outbreaks and eradicating WPV in endemic countries is minimal when compared to OPV (Table 1).
Switching to IPV alone in a region where there is continued circulation of WPV is a risky strategy and its exclusive use is limited to countries where polio has been eradicated and reliable environmental surveillance programs are available [11]. The requirement for IM administration, which necessitates health care provider administration, can result in decreased parental acceptance due to discomfort and perceptions of a greater potential for harm. Since it is estimated that IPV will remain part of the routine immunization schedule for at least 10 years during the transition away from all OPV [12], a sufficient supply of affordable IPV must be available to meet global needs, which is anticipated to present a serious challenge.
Sabin vaccine (OPV)
Vaccination with live PV leads to its replication in the gut with subsequent induction of both systemic immunity and local antibody production in the gastrointestinal mucosa. Mucosal immunity typically has a secondary beneficial effect in reducing the probability, duration, and concentration of poliovirus shedding in feces upon subsequent challenges with live PV [13]. The effectiveness of trivalent OPV (tOPV) has been documented serologically and clinically in controlling outbreaks. One dose of tOPV produces immunity in more than 95% of recipients in many situations. All countries need rapid access to a supply of OPV for emergent responses when a rapid block of transmission is required. Vaccination with tOPV results in better overall titers than vaccination with IPV. Other advantages of OPV include its ease of administration by non-health care personnel, its low cost, and its acceptance by parents due to the absence of perceptible pain. Interference can occur between the live strains of poliovirus contained in the OPV. Therefore, the mOPV would be expected to have the most robust response, the bOPV exhibits some diminution of response to the separate strains and the tOPV has the most attenuated response to the individual strains. Currently available bOPV has been documented to produce a better immune response against WPV1 and 3, than tOPV [14] with potential to hasten the eradication of the remaining types 1 and 3.
Sabin-like viruses are normally cleared from the stool by one month from the receipt of the vaccine, but concerns exist as to the potential for chronic poliovirus excretion in children with primary immunodeficiency disease (PID). The risk appears to be low for chronic excretion of iVDPV but several studies have documented continued positive fecal tests for immunization strains of PV for periods of months. Of 531 PID patients in Asia and Africa, only 3% tested positive for VDPV excretion for a prolonged period. Although no chronic shedding state has been identified, difficulty still exists in determining whether shedding for as long as 6 months occurred secondary to reduced mucosal clearance in these individuals or from re-acquisition of strains from the environment or through contact with recent OPV recipients.
Although the immunogenicity of OPV in well-nourished, relatively healthy children is usually robust, some concerns have been raised about humoral response in children in underdeveloped countries. In some areas, children have required 10 or more doses of OPV because other circulating viruses and bacteria that infect their gut interfere with the production of an effective immune response [10]. OPV response in infants with malnutrition, diarrhea, and shorter durations of breastfeeding has been shown to be less robust than in infants not experiencing these conditions. Studies have estimated the immunogenicity of a single dose of tOPV in developing countries to be less than half that of developed countries, while IPV immunogenicity appears to remain intact [15]. Zimbabwean infants perinatally infected with HIV also exhibited impaired mucosal and humoral immune responses to OPV and prolonged shedding after their third dose of OPV when compared to children without HIV [16]. However, in contrast, Schoub reported that cell-mediated immunodeficiency such as that produced with HIV did not seem to result in persistent excretion in his population. Mortality in patients with immunodeficiency-related vaccine-derived polio virus (iVDPV) has been reported to be up to 62% [17].
By 2008 WHO had conceded that ultimately, the global eradication of polio was incompatible with the continued use of live-attenuated OPV [18]. Opportunities for mutational change may occur when the attenuated Sabin viral strains circulate between the GI tracts of susceptible individuals in populations where WPV has been eliminated but vaccination rates have fallen resulting in populations highly susceptible to infection. These strains can recombine with other type C enteroviruses within the GI tract and ultimately acquire phenotypical characteristics of neuro virulence and transmissibility comparable to those of WPV. Vaccine viruses that have mutated but still have <1% nucleotide sequence changes from the original vaccine strain in the VP1 gene are termed vaccine-derived poliovirus (VDPV). Rarely these neurovirulent and transmissible mutational changes can result in VAPP. The longer the period between vaccine administration and viral clearing, the more likely mutation is to occur. The polio outbreak in 2015 in Ukraine originated as a VDPV that spread in an environment of low immunization coverage [19].
VAPP is extremely rare and occurs in 1 in 2.7million children receiving their first OPV dose and 1 in 6.9 million subsequent doses. This is approximately 1 case per 2.5 million doses of OPV [20]. Data collected in Latin America between 1992 and 2011 estimated the risk of VAPP to be 1 case in 2.56-4.10 million newborns [21]. The type 3 strain is the most common isolate in VAPP in vaccine recipients but type 2 is the most common isolated in contacts who develop VAPP [6]. By 2011, the type-2 component of OPV was responsible for 90% of the cVDPV cases and 38% of VAPP cases [22]. Transverse myelitis has been estimated to occur once for every 300 million to % billion doses distributed. Because of these rare, but significant, complications, upper and middle-income countries have changed their routine vaccination product to IPV, which induces humoral immunity but does not carry the risk of cVDPV or VAPP. In these countries, the risks of tOPV administration were greater than the risk of contracting polio in their well-immunized populations.
Another concern associated with the use of OPV is the cold-chain required for its use in the field and the difficulties associated with maintaining that chain to ensure stability in tropical and underdeveloped areas. Some studies suggest that vaccine vial monitors that are meant to monitor the cold-chain and therefore protect the potency of OPV by means of a color change are not sufficiently accurate to predict the continued potency of the products [23].
Optimal use of OPV and IPV together
In the decades after the availability of both the OPV and IPV, it became clear to public health officials that he vaccines could be used together to boost population immunity utilizing their respective strengths. Polio could be eradicated rapidly using the greater herd immunity and mucosal titers created by the OPV and once eradicated, the IPV could be administered to maintain the required 90%-95% immunity of a population and minimize the development of VDPV and VAPP during routine immunization in endemic countries. Prior to the switch to bOPV in April 2016, IPV was used to manage the risk of population susceptibility to cVDPV and during the immediate post-cessation period to prevent gaps in protection against polio type 2. IPV can boost immunity to types1 and 3 poliovirus and mitigate the risks of paralysis by priming the population against type 2 and ensuring better immune responses to OPV. Simultaneous administration of IPV and OPV substantially boosts intestinal and systemic immunity beyond that of the OPV alone [24].
IPV boosts mucosal immunity to PV in recipients previously given OPV and helps close the population humoral immunity gap in those who had not seroconverted after OPV. Overall a combined bOPV and IPV schedule is expected to provide higher levels of protection against poliomyelitis than tOPV alone [12]. Data from Oman, Ivory Coast, India, and The Gambia indicates that a single dose of IPV is sufficient to induce seroconversion in 90% of children who remained seronegative after OPV doses. A single dose of IPV can be rapidly boosted by a second dose in cases of threatened or actual outbreak scenarios. Approximately 98% of Cuban infants 4 months of age who did not convert after a single priming IPV converted after receiving a second dose of IPV.
GPEI endgame strategy (2013-2018)
In May of 2015, all WHO Member States endorsed the WHA resolution to complete implementation of the "Polio Eradication and Endgame Strategic Plan 2013-2018". All WHO Member States committed to implementing containment of WPV type 2 in essential lab and vaccine production facilities by 2015 and of type 2 OPV within 3months of global withdrawal in April 2016 [25]. The goals of the plan are the following:
Certify eradication and containment of all WPV transmission in all WHO regions by 2018.
Strengthen routine immunization systems by strong OPV vaccination and gradually increased reliance on IPV after interruption of PV transmission has occurred.
Maintain high levels of population immunity.
Strengthen networks for accurate and complete detection of circulating polioviruses and strong AFP surveillance.
Sequential cessation of the use of type-specific OPV to eliminate the risks for VAPP, chronic VDDP infections of immune deficient persons, and outbreaks of cVDPV.
Develop a plan for mainstreaming essential polio network functions into ongoing public health programs.
Surveillance is a major component of the eradication campaign. Quick case-based AFP surveillance requires a capability for reverse cold-chain from the field to laboratory that is challenging in tropical and impoverished areas. Continued environmental surveillance for poliovirus in sewage or other samples from the local environments is a necessity in current and post- eradication surveillance since AFP surveillance alone is insufficient due to the low percentage of polio-infected patients that go on to develop symptoms. Continued environmental surveillance allows detection of circulating WPV or cVDPV in areas where AFP is not detected as in Israel's silent epidemic in 2013. Israel’s immunization rate using IPV was at least 95% and no cases of paralytic disease were reported, but virologic studies from 25 sewage sites were positive for poliovirus with virologic types indicating introduction from Pakistan and divergence to Egypt, Israel, and Syria [26]. In April of 2016, in agreement with the Endgame goals, tOPV was taken out of use and replaced by the more immunogenic bOPV.
The Sabin strain type 2 was removed from the tOPV since WPV type 2 has not been detected globally since 1999 and is considered eradicated. Significantly, the only type 2 polio cases that have been reported since 1999 have been VDPV. Trigger points have already been set for the continued phasing out of OPV live-attenuated strains as the corresponding WPV is eradicated. The GPEI plan is to complete withdrawal of OPV 3 years after the last WPV is detected globally with a goal to end OPV use entirely by 2020 [27]. The Strategic Advisory Group of Experts on Immunization (SAGE) had also recommended that at least 1 dose of IPV be incorporated into routine infant immunization programs at or after 14 weeks of age by fall of 2015.
Strategies for increasing polio vaccine coverage to obtain the required 80%-90% coverage at district and national levels include national immunization days (NIDS) in scheduled campaigns and subnational days as well (SNIDS) with mop-up activities in response to identified cases of WPV or cVDPV. This strategy allows regions to boost population immunity quickly to multiple targeted age groups including older children or adults. Afghanistan, Nigeria, and Pakistan, the last remaining countries with endemic WPV are using the recommended combination of bOPV during their national and subnational immunization days and IPV within routine immunization schedules.
Challenges and Opportunities
During the last 6 decades, it has become apparent that sustained collaborative support is critical to achieve the ultimate goal of certification of a polio-free world. A lesson learned from smallpox eradication efforts has been that assets from a global health initiative can disappear very quickly once the initial goal has been attained. The need for the Expanded Program on Immunization (EPI) emerged as one of the legacies of the smallpox eradication initiative that has proven its value in providing ongoing assistance to maintain global immunity through immunization over the last 4 decades [28]. Challenges to the complete eradication in the remaining endemic countries of Pakistan and Afghanistan have included continued threats to security in these countriesand the targeted killing of polio vaccinators . These incidents have occurred as recently as January 2016 in Quetta . In Pakistan, bans on vaccination in Taliban-controlled areas have also hindered progress since 2012, Other challenges include cultural issues that limit accessibility to women and their children, refugee migrations, decreased government commitment at various levels, and surveillance gaps. Because of these challenges, full immunization rates for Pakistan in 2013 were at 54% compared with 95% in neighboring Bangladesh [29].
The result has been that in 2013 WHO declared polio an international public-health risk and called for mandatory polio vaccination for everyone travelling to Afghanistan, Nigeria, Pakistan, and several other countries in the region due to the threat of spread from endemic areas. Herd immunity requires high routine coverage of at least 90% for all three doses. Even a relatively brief, 2 year, interruption of tOPV in Romania in the early 1980s was sufficient to result in a contingent of children highly susceptible to infections and resulted in re-implantation of WPV and the emergence of a polio epidemic [6]. In Northern Nigeria politically motivated, rumors resulted in a 10-month ban on tOPV administration. During that period WPV spread to 12 countries from Nigeria and by 2006, 20 previously, WPV- free countries had been reinfected as far away as Indonesia and Yemen. The result was 1475 reported cases of paralytic polio. Effects continued through 2012 when Nigeria was the only country with endemic circulation of WPV in the WHO African Region [30]. In Somalia in 2013 an immunization ban by armed militants led to half a million children going unimmunized for 3 years and the reintroduction of WPV led to a major outbreak 6. In 2015, inadequate vaccine coverage again fueled a polio outbreak in the Ukraine. In 2014, only 50% of Ukrainian children had full immunization coverage against polio. Vaccine coverage through routine immunizations should approach 95% to prevent outbreaks, however, the immunization coverage in Ukrainian children younger than 1-year of age was only 14% in 2015. These rates reflected a public distrust of immunization and an insufficient vaccine supply [19]. The outbreak originated from environmental shedding of VDPV and was fueled by the low vaccination rates.
Timing of IPV doses, now in routine use became more important with the switch to bOPV and was adversely impacted aggravated by the narrow windows of opportunity in some regions. To maximize immunization with potentially infrequent immunizer contacts, the timing of the IPV was determined to be at the same time as the DTP3 immunization, which typically occurs between 3.5 and 6 months of age. SAGE has recommended that the IPV be administered at or after 14 weeks of age. The optimal age decision was based on maximizing IPV immunogenicity when passive immunity was no longer present, the need to protect typ-2 naïve infants against VDPV and VAPP at an early age, and the benefit of vaccination at an age when there is maximal coverage and minimal dropout. The current recommendation was a result of the relative gain in immunogenicity achieve by the administration of IPV together with DTP 3, which outweighed the risk of VDPV type 2 in the first 3 months of life and the potential lowering of vaccination coverage due to dropout between DTP1 and DTP3 immunization encounters [12]. Avoidance of injection-induced polio is also a consideration in countries where the disease is still endemic and OPV is in continued use.
Security threats have highlighted the importance of routine immunization strategies rather than relying on campaigns that may not reach insecure or inaccessible areas [27]. Vaccination campaigns need to be opportunistically planned to take advantage of narrow windows of opportunity such as ceasefires or mass migrations. Clinical trials in Pakistan and Bangladesh showed that 2-3 doses of mOPV with a 1-2week interval were non-inferior at inducing serum-neutralizing antibodies to the same vaccines given with the standard 4-week intervals and that giving IPV simultaneously with OPV substantially boosted intestinal and systemic immunity [24]. Although studies have been done in military recruits [31], there is little information on the immunogenicity of giving more than 3-4 concurrent vaccinations to children living in underdeveloped nations. Faridi reported simultaneous administration of live BCG vaccine and tOPV to term infants prolonged scar formation to BCG, which is regarded as evidence of successful immunization [32].
Refusal of vaccination has been linked with multiple variables including a lack of access to functional radio and TV [33], lower literacy rates, frequency of OPV campaigns and vaccination fatigue [34], the parenteral route of administration [35], lower maternal utilization of antenatal care services, birth order, younger maternal age, and maternal education at less than a primary level [36]. Despite Fatwas supporting vaccination by Grand Imam of Al Azhar and other leading Islamic clerics, support from the Islamic Advisory Group for Polio Eradication of Al-Azhar University, and the International Islamic Authority's support of immunization as a parental responsibility under Islamic law, one third of refusals have been based on religious grounds in Nigeria, India, and Pakistan.
Misinformation has fueled rumors of contamination of vaccines with estrogens, HIV, or pork. Rumors deserve special mention since they have resulted in vaccination boycotts and bans within the decade in Somalia, Nigeria, Pakistan, and Ukraine. The significance of rumors has been underestimated, especially when fueled by local conditions such as the 2011 CIA (US) attempt to obtain DNA samples from the children in Abbottabad, KPP as part of the search for Osama bin Laden. The scheme involved vaccinating children against hepatitis B, and further eroded public trust in immunization. Historical experience with the pharmaceutical industry in the Kano region of Nigeria undermined trust after a bacterial meningitis outbreak 7 years earlier when a pharmaceutical manufacturer allegedly conducted a clinical trial without license or informed consent and several children died [37].
Eradication of polio not only requires the development of high levels of population immunity, but also a network for laboratory and epidemiological surveillance. Almost all countries are susceptible to importation of polio by mobile populations. Because of the low ratio of symptomatic patients to infected patients, the absence of clinical disease is not a reliable indicator of polio transmission. Paralytic polio as a syndrome is indistinguishable from other paralytic syndromes such as Guillain-Barre and therefore case detection and ascertainment requires high surveillance with rapid laboratory follow-up and remains a challenge in underdeveloped countries.
Surveillance networks must have performance capabilities to follow-up on all acute flaccid paralysis (AFP) with two stool samples, 24 hours apart within 14 days of paralysis. Public health networks must have a rapid response plan to institute appropriate interventions to interrupt PV transmission in outbreaks and surveillance for absence of WPV in countries where WPV is considered eradicated. Documentation of this is required for a country or region to be certified as polio-free [38].Polio laboratory networks require a global specialized laboratory, regional reference laboratories and national and provincial laboratories all capable of providing surveillance of AFP cases and viral analysis of ambient environmental cultures. Capabilities must include poliovirus isolation, identification, and intratypic differentiation and sequencing to determine whether WPV or VDPV strains are involved and a general area of origin [39].Since protocols require substantial resources to build, accurate global case surveillance was not achieved until the middle to late 1990s when WHO provided substantial technical assistance to polio endemic countries. As the current situation in Nigeria indicates, there remain areas where surveillance is limited and these "black spots" allow polioviruses to circulate undetected.
Post eradication risks and mitigation
The WHO has planned for stockpiles of over 2 billion doses of all three types of OPV which could be available globally should polio reappear in the post-eradication era. Outbreak response capacity must be maintained, including the management of stockpiles of appropriate type 2-containing vaccines initially and subsequently types 1 and 3 as they are eliminated from the OPV. Countries could potentially need emergent access to all three WPV types as a combination of monovalent, bivalent, and trivalent OPV in addition to the routine use of IPV. Stockpiling of doses and assuring that sufficient manufacturing capabilities are maintained carries with it the risk of inadvertent exposure to live polio strains from manufacturing facilities and storage sites. Planned surveillance and the capability for an emergent response are required in this situation. An escape incident occurred in a production plant in the Netherlands which used WPV for IPV manufacture and caused the asymptomatic infection of a factory worker and his child in 2004. The WHO plan also outlines principles of containment once strain-specific eradication occurs.
In 2006, the Polio Antiviral Initiative (PAI) began as a result of the National Research Council report that concluded that it would be desirable to develop at least two antiviral drugs to supplement vaccination for the control of poliomyelitis outbreaks in a post eradication era. The partnership between the CDC, WHO, FDA, and two other United States-based groups coordinates efforts of drug sponsors in developing polio antivirals. There are currently several drugs in the pipeline, including capsid and protease inhibitors. The situations for which a polio antiviral would be anticipated to be helpful would be for immunodeficient individuals chronically shedding poliovirus, for persons exposed to poliovirus secondary to unintentional occupational exposure, and in conjunction with IPV for communities exposed to a cVDPV in the post-eradication era. It would be anticipated that compassionate use IND applications would be available for use in the first two situations.
Conclusion
We are on the cusp of a polio-free world but continued progress requires cooperation of parents, vaccinators, community and religious leaders, state, provincial, and national government, political leaders, international technical agencies, and major aid donors. Eradicating polio globally requires a high level of political commitment and intense coordination to achieve and then to maintain high levels of population immunity to prevent outbreaks from "black pockets" created by insufficient surveillance. The endgame strategy includes the ultimate withdrawal of attenuated oral vaccine, development of antiviral drugs directed at the poliovirus, and improved routine surveillance in local and regional networks.
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Extractive Spectrophotometric Methods for the Determination of Dothiepin in Pure and Pharmaceutical Formulations
Authored by Rambabu C
Abstract
Two sensitive and accurate extractive spectrophometric methods have been developed for the estimation of dothiepin in pure and pharmaceutical dosage forms. The developed methods are based on the formation of colored solvent extractible ion-association complexes of the drug with bromocresol blue [BCB] and eriochrome black-T [EBT]. The extracted complexes showed absorbance maxima at 418 and 508 nm respectively. Beer's law is obeyed in the concentration ranges between 16 - 56 and 5-17.5μ/mL for the two methods respectively. The effective concentration of dye, pH and optimum conditions are established for these methods. The methods are applied for the determination of the drug in commercial tablets and results of analysis are validated statistically through recovery studies.
Keywords: Dothiepin; Spectrophotometric methods; BCB; EBT; Dichloromethane; Chloroform
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Introduction
Dothiepin also known as dosulopin, is a thio analogue of amitriptyline which has been used extensively. It is a safe and effective agent for the treatment of major depressive disorders [1]. Although the onset of action is comparable to that of other tricyclic antidepressants, dothiepin may cause fewer intolerable side effects and have less cardiotoxicity than the other compounds. In addition, dothiepin reduces the anxiety associated with some major depressive episodes. These features suggest that dothiepin may be particularly helpful for treating anxious depressed patients and patients who have underlying cardiac disease or who are elderly [2] (Figure 1).
Few validated methods for quantification of dothiepin like HPLC [3-5], LCMS [6], spectrophotometry [7,8] are so far reported. The determination of dothiepin along with other anti depressants [9-11] and capillary electrophoresis method of analysis [12] is also reported. In this study, we report two simple extractive spectrophotometric methods for the determination of the dothiepin.
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Materials and Methods
Apparatus
Spectral and absorbance measurements were carried out by using ELICO UV-Visible double beam spectrophotometer model SL-159 equipped with 1.0cm thickness matched quartz cells. Systronics digital pH meter was used to adjust and determine the hydrogen ion concentration of the buffer solution.
Preparation of Standard drug solution
Pharmaceutical grade dothiepin (99.8% pure), was used in method development. A stock standard solution containing1.0mg. mL-1 was prepared by dissolving accurately weighed (100mg) of pure drug with double distilled water in 100mL calibrated flask. This stock solution is further diluted appropriately with double distilled water to get a working standard concentration of 200μg.mL-1 for the proposed methods.
Reagents and solutions
All chemicals and reagents used were of analytical grade or pharmaceutical grade. All solutions were prepared in doubly distilled water.
Hydrochloric acid (0.1M, SD Fine Chemicals, India)
Prepared by diluting 8.5mL of concentrated acid to 1 liter of double distilled water.
Buffer solution, pH 4.0
Prepared by mixing 50mL of 1.0 M sodium acetate solution with 39.5mL of 1.0M HCl solution and diluted to 250mL with doubly distilled water. The pH of the solution was adjusted to an appropriate value with the aid of a pH meter.
BCB method (M1)
Aliquots of the drug (0.8-2.8mL) were taken in a series of 25mL separating funnels. 0.5mL of 0.1M HCl solution and 2.0mL of 0.2% BCB solution were added, shaken well and made the solution to 15mL with distilled water. Then the color of the aqueous layer was extracted with 10mL of dichloromethane. The absorbance of the organic layer was measured at 418nm against a reagent blank.
EBT method (M2)
Into a series of separating funnels, appropriate amount of the working standard drug solutions were pippetted out. To each funnel 2.0 mL of pH 4.0 buffer solution and 1.0 ml of 0.1% w/v eriochrome black-T were added. Shaken well for 2min and the volume of the aqueous phase was made up to 15mL with distilled water. A 10 mL of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance at 508nm.
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Results and Discussion
Method development
In order to ascertain the optimum wavelength of maximum absorption (λmax) of the colored species formed in the above methods, specified amounts of dothiepin were taken and colors were developed separately by following the above proposed procedures.The absorption spectra were scanned on a spectrophotometer in the wave length region from 400 to 800nm against similar reagent blank or distilled water and are shown in Figure 2 & 3. These spectra show a single well-defined peak with characteristic absorption maxima where as the blank in each method has low or no absorption in this region. The wavelength of absorption maxima of each proposed method was used for the visible spectrophotometric analysis of dothiepin in bulk samples.
Optimization studies of experimental variables for the proposed procedures
The optimization studies for the color development by the proposed methods for the assay of dothiepin were established by varying the parameters one at a time, keeping the others fixed and observing the effect produced on the absorbance of the colored species and were found to be same as described in Table 1.
Method validation
Optical Characteristics: In order to test whether the colored species formed in the above methods, adhere to Beer's law, the absorbances at appropriate wavelengths of a set of solutions containing varying amounts of dothiepin and specified amounts of reagents (as given in the recommended procedures for each method) were recorded against the corresponding reagent blanks. The Beer's law plots of these systems are recorded graphically (Figure 3 & 4).
Linearity range and analytical data: Linearity ranges for each proposed spectrophotometric method for quantitative analysis of dotheipin, were made by plotting calibration curves over the concentration ranges cited. The statistical parameters (optical characteristics) such as Beer's law limits, correlation coefficient, Sandell’s sensitivity, molar absorptivity, percent relative standard deviation (calculated from six replicate samples containing 3/4th of the amount of the upper Beer’s law limits) were calculated for all the proposed methods and the results are summarized in Table 2.
Precision: The precision of each proposed method was ascertained from the absorbance values obtained by actual determination of six replicates of a fixed amount of dothiepin (32.0μg/mL) for BCB and (10.0μg/mL) for EBT methods in Intraday and Inter day and the results are summarized in Table 3. The percent relative standard deviation (0.43 and 0.36 for Intraday precision & 0.51 and 0.42 for Inter day precision) and percent range of error (at 0.05 and 0.01 confidence limits) were calculated for the proposed methods (Table 3).
Recovery studies: To ensure the accuracy and reproducibility of the results obtained, known amounts of pure drug was added to the previously analyzed formulated samples and the samples were reanalyzed by the proposed methods. The percentage recoveries thus obtained were given in Table 4.
Application to the analysis of commercial sample: In order to check the validity of the proposed methods, the drug Dosulepin was determined in commercial formulations. From the results of the determination it is clear that there is a close agreement between the results obtained by the proposed methods and the label claim. These results indicate that there was no significant difference between the proposed methods and the reference methods with respect to accuracy and precision (Table 5).
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Conclusion
Two sensitive spectrophotometric methods have been developed, optimized and validated for the determination of dothiepin in pure drug and in tablet dosage forms. The simplicity, sensitivity and selectivity make these methods a suitable alternative to the HPLC methods. Other characteristics such as short performance time, ease of handling, no requirement of either expensive equipments or specialized technicians also suggest that the procedures developed by the authors can be adopted as routine laboratory methods in quality control laboratories where modern instruments are not available.
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Immunomodulatory Activity of Methanolic Extract of Drypetes Roxburghii Leaves
Authored by Kala C
Abstract
The immunomodulatory potential of methanolic extract of leave extract of Drypetes roxburghii leaves was evaluated. For this models like estimation of serum Immunoglobulin level and carbon clearance test were used All the Data was analyzed by one way ANOVA followed by Dunnet's test at *p<0.05, **p<0.01, ***p<0.001. Serum immunoglobulin level was raised from normal 61.4±1.47 NTU up to 69.083±1.549** and 101.416±1.367*** NTU in Drypetes roxbhrghii low dose (DHLD) (100mg/kg) and Drypetes roxbhrghii high dose (DRHD) (200mg/kg) respectively. Phagocytic index of animals in control group was found to be 1.8±0.186 and was raised up to 2.98±0.069ns and 3.97±0.690** Drypetes roxbhrghii low dose (DHLD) (100mg/kg) and Drypetes roxbhrghii high dose (DRHD) (200mg/kg) respectively. This study showed that methanolic extract of Drypetes roxburghii leaves possessed immunostimulatory activity.
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Introduction
An immunomodulator may be defined as biological or synthetic substance can modulate any of the components of innate and adaptive immune system. Immunomodulators may be classified as immunoadjuvant, immunostimulants and immunosuppressant [1]. The immune system is continuously balancing between quiescence and preparedness for activation and attack [2]. The active agents of immunotherapy are collectively called immunomodulator. Immunotherapy is defined as the "disease treatment by inducing, enhancing, or suppressing an immune response". Immunomodulatory regimens offer an attractive approach as they often have fewer side effects than existing drugs, including less potential for creating resistance in microbial diseases [3]. A range of synthetic, natural and recombinant immunomodulatory compounds are available. levamisole, isoprinosine, pentoxifilline, and thalidomide are some of the most significant used synthetic immunomodulators. These synthetic immunomodulating drugs possess numerous benefits but their general deliberate use is limited by its adverse side effect profile and demands the search for a more safer and effective agents exerting immunomodulatory activity [4]. Several herbal preparations are used to enhance the body's immune status. Many plants constituent like saponins, glycosides, polysaccharides alkaloid, flavonoids, sterols and sterolins have unique ability to modulate immune system [5].
Putranjiva roxburghii Wall (syn. Drypetes roxburghii Wall.) is an evergreen tree of tropical region belonging to family Euphorbiaceae family [6]. The leaves are reported to contain β-amyrin and its esters, putrone, putrol, putranjivic acid, methyl putrajivate, stigmasterol and hydrocarbons, triterpene roxburghonic acid and biflavones [7].
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Material and Methods
Chemicals
Ethanol, CMC, zinc sulphate, barium chloride and sulphuric acid (7664-93-9) were purchased from CDH, pvt ltd, New Delhi.
Plant material
Leaves of Drypetes roxburghii was procured and authenticated from NBRI, Lucknow
Extraction and fractionation
The leaves were dried in shade at room temperature. The dried leaves were powdered by using grinder to coarse powder, packed into Soxhlet column and the extracted 70% ethanol for 48hrs. The excess of solvent was removed using rotatory flash evaporator. The obtained crude extract was stored in airtight container in refrigerator below 10 °C for further studies [8].
Acute oral toxicity Study (LD50)
Animals were fasted prior to test drug administration. Following the period of fasting animals was weighed and then the test substance administered in a single dose of 2000mg/kg to animals by oral gavage. After the test drug administration, food was withheld for next 3-4hours. Following administration, animals were closely observed for next 4hours to see any clinical symptom, any change in behavior or mortality. After 6 hours of test administration the animals weighed again. A careful clinical examination was made once in each day for next 14days (OECD 4/26, 2006) [9].
In-vivo Carbon clearance test
Animals (Wistar albino rats) were divided into three group. Each group contained six animals. Group I served as control and received 1% CMC (p.o), group II and group III received DHLD (100mg/kg, p.o) and DRHD (200mg/kg, p.o). On day 7, all the animal of entire group was treated with intravenous injection of Indian ink dispersion (0.3ml per 30g). 50μl of Blood samples were withdrawn in EDTA solution (125mM, 5μl) by retro orbital puncture at interval of 0 and 15minutes. Blood samples were added to 2ml of 0.1% sodium carbonate to lyse the erythrocytes. Absorbances of samples were taken at 660nm. After 15min of blood collection animals were sacrificed and livers and spleen were collected and weighed.
Rate of carbon clearance (K) and Phagocytic index (a) were calculated by using following formula.
Rate of carbon clearance
Phagocytic index
Where OD0 is the log absorbance of blood at 0min; OD15 is log absorbance of blood at 15min; T2 is the last time point of blood collection; T1 is the first time point of blood collection. Rate of carbon clearance and phagocytic index of treated group animals were compared with the control group animals [10,11].
Effect on serum immunoglobulins
Animals (Wistar albino rats) were taken for the study. They were divided into three group, each group contained six rats. Group I was taken as control group and animals in this group were treated with 1% CMC (p.o). Animals in group II and group III received DHLD (100mg/kg, p.o) and DRHD (200mg/kg, p.o). All the treatments were done up to 21 days. Blood samples were collected after 6hours of the last dose and serum was separated. Serum was than analyzed for immunoglobulin level.
For each serum sample to be analyzed, a control tube containing 6ml of distilled water and a test tube containing 6ml of zinc sulphate solution were prepared. To each, 0.1ml of serum was added from a pipette. They were inverted to enable complete mixing of the reagents and left to stand for 1 hr at room temperature. The first tube served as blank and the second tube was taken as sample. The turbidity developed was measured using a digital nepheloturbidity meter. The turbidity obtained (sample-blank) was compared with that obtained with standard barium sulphate (BaSO4) solution. The standard BaSO4 solution was prepared by adding 3ml of barium chloride solution (1.15% w/v) to 97ml of 0.2 N sulphuric acid. The turbidity was expressed in NTU [12].
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Result
Acute oral toxicity
Acute oral toxicity No sign of mortality was found at the dose of 2000mg/kg. Hence two doses were selected 100mg/ kg as lower dose and 200mg/kg as higher dose to check the effectiveness of test drug (Table 1 & 2).
Results are expressed as mean±sem, (n=6), analysed by one way ANOVA followed by Dunnet's test. *P<0.05, **p<0.01, ***p<0.001 when Arthritic control group compared with other treated groups
Results are expressed as mean±sem, (n=6), analysed by one way ANOVA followed by Dunnet's test. * P<0.05, **p<0.01, ***p<0.001 when Arthritic control group compared with other treated groups.
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Discussion
Two models were used to evaluate the immunomodulatory activity of leaves of Putranjiva roxburghii. Zinc sulphate turbidity test was used for rough estimation of Immunoglobulins present in the serum. . When a zinc sulphate is added to serum containing immunoglobulin it causes precipitation of the same imparting cloudy appearance of the solution. The carbon clearance assay was used to evaluate the effect of extract on reticuloendothelial cell mediated phagocytosis. When colloidal carbon (in ink) is injected intravenously, the macrophages from reticulo endothelial call engulf the carbon particles of the ink. Rate of clearance of carbon particles from blood is known as phagocytic index.
The immunostimulatory activity of Putranjiva roxburghii may be contributed to presence of flavanoids. Also, previously leaves of Putranjiva roxburghii were reported to have antioxidant potential and anti-oxidants act as immunomodulator [13].
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Conclusion
Estimation of serum Ig level, phagocytic index. The findings suggested that extract of leaves of Putranjiva roxburghii possess the capacity to modulate immune system.
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Prescription Notes Auditing in a Medical College and Hospital of Himachal Pradesh
Authored by Kansal D
Abstract
Introduction: Prescription audit is necessary to know the prescription practices and to improve rational pharmacotherapy. In India few publications are available on prescription audit.
Materials and method: This was an observational study, undertaken between August 2016 to October 2016 for which data was collected from our OPDs. Results were compiled, tabulated and analyzed using Microsoft excel. Charts were drawn to present all the findings.
Results: In total 317 medications were prescribed in 113 prescription notes. Therefore average number of drugs/prescription was 2.80. Demographic profile shows more females (58.40%) as compared to males (41.60%). Maximum patients (73.45%) were from adult age group, followed by children (14.15%). Most common dosage form was oral in 83.59%, topical were prescribed in 9.14% and injectable were recommended in 7.25%. Drugs were prescribed by generic name in 31.23% and as fixed dose combinations in 21.45%. Complete diagnosis was written in 63.71%. Findings were written in 60.17%. Signatures were done in 92.92%. Only 78.76% prescriptions were complete in terms of dose, route, frequency and duration. Advice was written only in 41.59% of prescriptions. On evaluation 54% prescriptions were semi rational (Scoring between14-18), 41.59% were rational (scoring between 20-24) and 4.42% were unacceptable (scoring less than 12).
Conclusion: Our study showed that there is scope for improvement in prescribing patterns in areas of writing of complete prescriptions in respect of diagnosis, signature, advise, dose, frequency and duration.
Keywords: Prescription auditing; Drug utilization pattern; Rational pharmacotherapy
Introduction
With prescription audit the main aim is to improve rational use of drugs. Irrational prescriptions can lead to adverse drugs reactions or sub therapeutic treatment and antimicrobial resistance. Irrational prescribing is a universal problem. Examples include: poly pharmacy, inadequate dosage, use of antimicrobials even for non-bacterial infections and excessive use of injections when oral forms are available [1].
The World Health Organization (WHO) has formulated a set of "core prescribing indicators" for improvement in rational drug use in outpatient practice. It includes the prescribing indicators, the patient care indicators and the facility indicators [2]. Based on these indicators, studies have been carried out all over the world and even in India [3].
Aim and objectives
o We will analyze the prescriptions under these subheadings.
o Department wise distribution of the patients.
o Demographic profile of population i.e. male female ratio and age-distribution.
o Number of drugs per prescription.
o Completeness of diagnosis, findings, signatures, dose, frequency and duration of treatment.
o Drug dosage forms; oral/injectable/topical.
o Evaluation of prescription as Rational (score 20-24), Semi Rational (score 14-18) and Unacceptable (score less than 12).
Material and Methodology
This was an observational, prospective study carried out over a period of 3 month between August to October 2016. The data was collected from OPDs of our hospital. These prescriptions were analyzed based on the objective of the study. Complete information i.e. specialty wise distribution, age, sex, demographic profile of patients, prescription profile and number of drugs per prescription. These were uploaded on M.S. Excel sheet. These results were analyzed and deficiencies were observed.
Evaluation of prescriptions was done by twelve questions. Each carries 2 marks so total marks were 24.
These twelve questions were:
a) Completeness of Prescription:
I. Diagnosis
II. Findings
III. Signature
b) Whether prescription corroborates with symptoms/ Diagnosis
IV. Selection of core drugs
V. Selection of symptomatic drug
VI. Relevant advices for patients
c) Prescribing behavior:
VII. Generic prescription
VIII. Essential drugs prescription
IX. Judicious investigations
d) Dosage schedule:
X. Dose
XI. Frequency
XII. Duration of therapy
Evaluation scheme
Total marks 24 (100%)
Marks obtained 18 to 24 = Rational.
Marks obtained 12 to 18= Semi rational.
Marks obtained <12 = unacceptable.
Results
In total 317 drugs were prescribed in 113 prescriptions. So average number of drugs/prescription were 2.80 drugs.Specialty wise distribution shows that 27.4% patients were from Medicine, 16% were from Orthopedics, 11.5% each were from Pediatrics and skin and rest 33.6% patients were from ENT, OBG, Dental, Surgery, Chest and TB, Eye, Emergency medicine and Psychiatry (Figure 1). Demographic data (Figure 2) shows female 58% and male 42%. 73.5% of patients were in the 20-60 years age group (Figure 3). Findings were written in 68(60%) prescriptions. Diagnosis was complete in 72(64%) patients. Advices were written in 47(41.59%) prescriptions. And 89(79%) of prescriptions notes were complete in dose/ frequency and duration. Signatures were done in 105(93%) prescriptions (Figure 4).
Evaluation of these prescription was done as rational (score 20-24), semi rational (14-18) and unacceptable (score less than 12). Result of these prescriptions were maximum of semi rational 61(54%), rational 47(42%) and unacceptable 5(4%) (Figure 5). 75 prescriptions contained 2-3 drugs. In only 27 prescriptions more than 3 drugs were prescribed (Figure 6). Drugs prescribed by generic names were 99(31.23%) and drugs prescribed as FDC were 68(21.45%) (Figure 7). Most commonly oral drugs were prescribed (84%) followed by topical in (9%) and least were injectable (7%) (Figure 8). Deficiencies observed in prescriptions were diagnosis not written in 38 prescription notes (33.62%), incomplete either in dose, frequency or duration 24(21.23%), Duration of treatment was not written in 13 (11.50%) and signature was not done in 8 prescription notes (7.07%) (Figure 9).
Discussion
Prescription auditing is an important tool to improve rational pharmacotherapy. It is very important document for health administrators and clinicians groups for both decision making and drafting policies. In our study total drugs prescribed were 317 in 113 prescription notes. Therefore average numbers of drugs/prescriptions were 2.80. So result was quite satisfactory to the recommended limit of 2.0 [2]. This limit is to prevent drug- drug interactions and unwanted side effects. FDCs were used in 21 % of prescriptions. This figure is also quite low compared to two indian studies which reported 75% and 60 % usage of FDCs respectively [4-10]. FDCs sometimes may be irrational. Our study also show Poly pharmacy. 52% of prescriptions shows either 3 or more than 3 drugs. Even this poly pharmacy is a big concern and it is reported in various studies [5,9].
In 84% prescriptions oral formulation, in 9% topical and in 7% prescriptions injectable were prescribed. Although use of injectable was high from other studies but it was at par with Indian study which reported 7% [3]. We need further decrease in number of injectable, to reduced blood born infections [6]. In our study antibiotics were prescribed in 18.3% of cases. More than one antibiotics was prescribed in 7% of cases. Results were satisfactory and are much lower than other study [7] in which around 50% of patients received more than one antibiotic. Overuse of antibiotics leads to drug resistance.
In 31% of prescriptions generic drugs were prescribed. This is very low as compared to other studies, few study reported very high (73.4%) use of generic names [8]. Generic prescribing decreasing the chances of dispensing errors. Lot of deficiencies were observed in prescription writing like diagnosis not written in 38(34%), signature not done in 8(7%), and duration of treatment not written in 13(11.50%) of prescription notes. Around 24(21.23%) of prescriptions were incomplete either in dose/ frequency or duration. So there was a scope for improvements in the prescription writing.
Conclusion
From our study it is concluded that there is scope for improvement in prescribing patterns in areas of writing diagnosis, findings, signature, advise, dose, frequency and duration. Educating the clinicians about prescription writing is very important aspect. Regular updates of clinicians are also required for improving rational pharmacotherapeutics.
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C-Peptide-Boon or Bane in Diabetes
Authored by Eerike M
Abstract
Chronic hyperglycemia in diabetes will produce microvascualr & macrovascular complications. Strict glycemic control can reduce but not completely prevent the microvascular complications. C-peptide, a connecting peptide is one of the drugs under clinical trials for treatment of diabetic complications. Previously C-peptide was considered as a byproduct of insulin synthesis, but now it has been proven, as an active hormone with various actions. Replacement with C-peptide has been proven to reduce the type-1 diabetic complications but not in type 2 diabetic complications. This review is focusing on mechanism of action and actions of C-peptide, its role in type1 & type 2 diabetic complications, clinical trials related to C-peptide has been discussed.
Keywords: Hyperglycemia; Microvascular complications; C-peptide; Diabetes; Clinical trial
Abbreviations: DM: Diabetes Mellitus; WHO: World Health Organization; cGMP: cyclic Guanosine Monophosphate; eNOS: Endothelial Nitric Oxide Synthase; GLUT: Glucose Transporter; NO: Nitric Oxide: TNF: Tumor Necrosis Factor; NGF: Nerve Growth Factor; IGF-1: Insulin like Growth Factor-1
Introduction
Diabetes mellitus (DM) is the one of common metabolic disorder characterized by hyperglycemia. Prevalence of diabetes is steadily increasing. It has been estimated that 422million adults were suffering with diabetes in 2014 (8.5% of adult population). WHO projected that diabetes will be the 7th leading cause of death in 2030 [1]. Diabetic complications are classified into acute and chronic or long term complications. Acute complications include diabetic ketoacidosis & hyperglycaemic hyperosmolar state. Chronic complications are classified into vascular & non vascular. Depending on size of the blood vessel involved, vascular complications are divided into microvascular & macrovacular. Microvascualr complications include diabetic retinopathy, nephropathy & neuropathy. The exact etiology by which chronic hyperglycemia will produce these complications is not known.
Studies showed that hyperglycemia is directly linked with development of diabetic complications [2]. Strict glycemic control can reduce, but not completely prevent the morbidity & mortality due to chronic complications [3]. For the past 2 decades a lot of research is going on new drugs for diabetes and its complications. New drugs under trial are Ranirestat, is an aldose reductase inhibitor for diabetic neuropathy and protein kinase beta inhibitor, Ruboxistaurin & Fenofibrate [4] for diabetic retinopathy. Recently Fenofibrate, a hypolipidemic drug was approved in Australia for diabetic retinopathy. C-peptide, a peptide is also under trial for treatment of diabetic complications. The role of C-peptide in diabetic complications with mechanism and clinical trials related to c-peptide has been discussed.
Discussion
C-peptide, discovered in 1967 by Steiner [5], is a connecting peptide which connects insulin's A-chain to its B-chain in the proinsulin molecule. C-peptide and insulin are co-secreted in equal amounts into circulation. It consists of 31 amino acids with molecular weight of 3600kDa. The half-life of C-peptide is about 2-5times longer than that of insulin. Hence, serum C-peptide is used as a substitute marker for monitoring of endogenous insulin production. C-peptide test has been used as a substitute marker for monitoring the course of type 1 and type 2 diabetes and determining the effects of interventions designed to preserve and improve residual pancreatic beta cell function. C-peptide is removed from the peripheral circulation at a constant rate. Metabolised in the proximal renal tubules, and about 5-10% is excreted unchanged in the urine. This is in contrast to insulin where most of the insulin will undergo metabolism in portal circulation.
In patients with type 1 & some patients with type 2 diabetes, destruction of the beta cells results in deficiency of both insulin and C-peptide. These patients routinely receive insulin injections to compensate for the lack of endogenous insulin production, but no replacement of C-peptide is given. Till 1980 C-peptide was considered as inert and by product during insulin synthesis. Now it is considered a bioactive peptide with diverse tissue and cell-specific actions in various physiologic states and diseases [6].
J Wahren [7] first started looking at C-peptide effects in diabetes in the 1980s. In1990, he had published part of research on the beneficial effects of C-peptide in animal studies. The Diabetes Control and Complications Trial also showed that this C-peptide had relation with diabetic complications. Results of a cross sectional study conducted from 1994 to 2004 on 471 type 1 diabetic patients was showed that subjects with the lowest fasting C-peptide levels were found to have the highest rate of microvascular complications [7]. All these studies are showing that C-peptide has definitive role in diabetes especially chronic complications.
Mechanism of action
It has been reported that C-peptide interacts with G-protein coupled receptor present in endothelial, fibroblast, neuronal, and renal tubular at very low concentration [8] and produce its actions through 3 post receptor mechanisms.
Interaction with Na+ K+ ATPase: It has been reported that Na+ K+ ATPase activity is reduced due to hyperglycemia in diabetes [9]. C-peptide has regulatory influence on this enzyme.
Activation of endothelial nitric oxide synthase (eNOS): In physiological range, C-peptide has been found to activate eNOS and increase in the synthesis of nitric oxide (NO) in endothelial cells. It is well known that NO causes vascular smooth muscle relaxation by increasing cGMP levels and improves blood flow through NO induced vasodilatation.
Insulin receptor mediated signalling pathway: C-peptide causes Insulin receptor substrate 1 tyrosine phosphorylation, and with downstream effects leading to glucose transporter (GLUT) mobilization, promotion of amino acid uptake & glycogen synthesis.
Pharmacological actions Anti inflammatory, Cytoprotective & antiapoptotic: Patients with type 1 diabetes and microvascular complications show increased levels of several inflammatory markers as compared with patients without complications [10]. C-peptide produce the anti-inflammatory & cytoprotective action by antagonizing adhesion molecule expression, decreases inflammatory cytokine secretion, decreases reactive oxygen species (ROS) formation in endothelial cells and leukocytes and also decreases apoptosis by decreasing caspase3 & increased Bcl2.
Circulatory effects: In type I diabetes microvascular complications are due to endothelial dysfunction and decrease in microvascular blood flow. C- Peptide increases eNos expression in vascular endothelial cells thereby increases microvascular blood flow by relaxing vascular smooth muscles. It also increases blood flow by improving the erythrocyte deformability through enhancing Na+ K+ ATPase activity.
Role of C-Peptide in Type I diabetic complications
Diabetic neuropathy: Neuropathy in type 1 diabetes progresses more rapidly & shows a more marked decline of nerve conduction velocity than neuropathy in type 2 diabetes. Supplementation of C-peptide in these patients restores the activity of Na+K+ ATPase and also improves nitric oxide (NO) availability in dose dependent manner. It also increases endoneurial blood flow and conduction velocity even in the presence of highly elevated glucose levels. In addition, it improves nerve structural abnormalities by increasing neurotropic factors such as nerve growth factor (NGF), neurotrophin3 & insulin like growth factor (IGF-1).
Diabetic Nephropathy: C-peptide supplementation in diabetic nephropathic patient causes reduction in intraglomerular pressure by constriction of the afferent and relaxation of efferent glomerular arteriole. It also reduces diabetes-induced structural changes of the glomeruli by decreasing apoptosis & mesangial expansion through decrease in transcription factors TNF-alpha & beta and increase in IGF-1.
Diabetic retinopathy: C-peptide replacement reduces the expression of protein fibronectin, prevents vascular permeability & regulates extracellular matrix expression.
Role in Type 2 DM complication
Effect of C-peptide in type 2 DM is controversial: Type 2 DM is associated with hyper insulinemia state, so when there is an increased secretion of insulin, simultaneously C-peptide levels also will be increased. In many studies it has been found that micro vascular complications are at higher level with stimulated C-peptide levels than with basal levels. The C-peptide in type 2 diabetes showed pro inflammatory and pro atherogenic effects [11] in spite of its anti inflammatory and cytoprotective action. Kim ST et al reported that in type 2 diabetes, basal C-peptide levels can be correlated to intima media thickness and it can be used as a substitute marker of subclinical atherosclerosis [12].
It has been hypothesized that these negative effects of C- peptide in type 2 diabetes are due to
a. In type 1 and type 2 diabetes there may be difference in the levels of C-peptide and insulin in the circulation
b. Difference in the level of inflammation
c. C-peptide may have tissue and cell specific actions.
Clinical trials
C-peptide has been tested in 300 type 1 diabetic patients in nineteen clinical trials to study its effects in diabetic complications [13]. The results were positive with early phases of clinical trials. In 2012 to 2015, phase II b clinical trials were conducted with long acting c-peptide in 250 type1 diabetic neuropathy patients. Long acting C-peptide was given as subcutaneous injection once a week dose in these trials but the results were not satisfactory. Long acting C-peptide showed marked improvement in vibration perception threshold with no effect on nerve conduction velocity compared to placebo [14]. Further research is required to find the reason for negative effects of C-peptide in these trials.
Conclusion
To conclude C-peptide has been reported to have beneficial role in type 1 diabetic microvascular complications but not in type 2 diabetic complications. It showed positive results in early phases of clinical trials but not in phase II b clinical trials in diabetic neuropathy. Further studies are required to find the reasons for the negative results of C-peptide in these trials and hoping modified form of C-peptide with positive results will come in future.
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An Overview of Neptunia prostrata: A Source of Herbal Medicine of Ethnopharmacological Importance
Authored by Acharya PC
Introduction
Plants are used in the treatment of various diseases from the ancient time. The use of plants as a source of medicine lies deep in the root of antiquity. The traditional Indian system of medicine, Ayurveda and Unani are based on the use of plants, animals and other natural substances. The history of medicinal plants dates back to Rigveda, perhaps the oldest repository of human knowledge, which was written in about 4500-1600 B.C and Ayurveda (about 2500 B.C) which provide the detail accounts of many herbal drugs [1]. Most of the medicinally active substances investigated in the 19th and 20th centuries used in the form of crude extract [2,3]. Medicinal plants and natural product derived medicines are widely used in traditional cultures all over the world and they are becoming increasingly popular in modern society as natural alternatives to synthetic medicines [4]. Several herbal cosmetics, nutraceuticals and remedies are flooded in the market nowadays.
In the last few decades there has been an exponential growth in the field of herbal medicine and it is gaining popularity in developing and developed countries owing to its natural origin and lesser adverse effects [5,6]. The World Health Organization (WHO) estimated that approximately 80% of world population rely mainly on traditional medicines, mostly plant drugs in their health care [7]. A plant is biosynthetic laboratory for myriads of chemical compounds like carbohydrates, proteins and lipids that are consumed as food also compounds like glycosides, alkaloids, steroids, volatile oils, tannins etc. that produces biological effects as well [8]. Natural products from medicinal plants provide limitless opportunities for new drug leads due to the diverse and abundant availability. Due to the rise in the adverse effects and resistance to the chemically synthesized drugs, world has turned to ethnopharmacognosy exploring several plant based drugs with biological activities like anticancer, antimicrobial, antioxidant, antidiarrheal, analgesic and wound healing activity etc [9-11]. In this context, this article focuses on the ethonomedicainal uses of the plant Neptunia prostrata used in the north eastern region of India.
Ethno-medicinal uses of Neptunia prostrata
Neptunia prostrata (Synonym: Neptunia oleracea) belongs to the family Mimosaceae and is an annual floating aquatic herb usually distributed in lakes and marshy places all over India [12,13]. It is found in 25 different states of India including the north-eastern state Tripura [14,15]. As an edible medicinal plant, Neptunia prostrata (Figure 1) is well-known to the herbal practitioners of Tripura as well as other states of North-eastern region of India. The tribals of Tripura cultivate this plant both as vegetable as well as medicinal plant and prepare various tasty delicacy dishes with this vegetable [15,16]. The plant has been used for different types of remedies like gastritis, acidity, constipation, dysentery, and also reported to possess hepatoprotective, analgesic and antimicrobial activity [14-18]. The ethnopharmacological approaches used for the treatment of several aliments has been compiled in Table 1 [19-22].
Future prospects
According to the folklore, Neptunia prostrata is a useful herb to treat several ailments including hepatitis or jaundice. However, identification of bio-active molecules behind the treatment of these diseases has not been explored or documented till date. Therefore, it can be a great opportunity to explore the plant for isolation and identification of the bioactive components from this astounding medicinal herb.
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Spermidine for a Long, Dementia-Free Life?
Authored by Tsikas D
Abstract
Spermidine, N-(3-aminopropyl) butane-1,4-diamine, is an endogenous basic compound known for more than 90 years. The biosynthesis of spermidine, its pharmacological properties including distribution, metabolism and excretion have been thoroughly investigated. Spermidine and other polyamines have been early associated with growth and aging, and were found to stabilize various cellular and subcellular components including nucleic acids due to their polyvalent cationic structures. Polyamines including spermidine have been found at elevated circulating and urinary concentrations in a variety of diseases including cancer and psoriasis. Thus, decreasing the concentration of polyamines mainly by inhibiting their biosynthesis is regarded as a realistic therapeutical strategy. Yet, more recent studies suggest that exogenous spermidine exerts beneficial effects on the brain where it protects from age-induced memory impairment, protects the heart and extents lifespan.
Supplementation of the natural compound spermidine has emerged as an autophagy and mitophagy inducer in a mice model of aging and hypertension and lowered high blood pressure. In humans, high dietary spermidine intake was found to correlate with reduced blood pressure and decreased risk of cardiovascular disease and related death. Scientists in this area of research suggest that integration of spermidine in common diets may represent a cardio- and vascular-protective autophagy inducer in humans. The present article provides a brief review of the recent literature reporting on spermidine and takes the chance to make a first evaluation of the pharmacological potential of spermidine to reverse age-induced memory impairment, to protect the heart and to extent lifespan.
Keywords: Aging; Autophagy; Cancer; Dementia; Helicobacter pylori; Memory, to Mitophagy; Polyamines; Supplementation
Abbreviations: DMFO: α-difluoromethylornithine; GABR: Global Arginine Bioavailability Ratio; NOS: Nitric Oxide Synthase
Introduction
Spermidine, N-(3-aminopropyl) butane-1,4-diamine, is a low-molecular-mass organic base (C7H19N3, CAS 124-20-9) and belongs to the polyamine family which includes spermine and putrescine, the direct precursor of spermidine (Figure 1).At ambient temperature (22-25 °C) spermidine is a colorless liquid or a white powder. In biological samples, the two primary amine groups and the secondary amine group of spermidine are protonated so that the spermidine molecule is a polyvalent cation. Spermidine is a natural compound and widely distributed in plants, animals and humans. Scientific reports on spermidine and putrescine exist for more than 90 years, whereas articles on spermine appeared 25 years earlier (Table 1).
Spermidine is biosynthesized by microorganisms, plants and animals including humans [1-3]. Two possible biosynthetic pathways are illustrated in a simplified form in Figure 1. Hydrolysis of L-arginine by arginase yields L-ornithine which is decarboxylated by ornithine decarboxylase to form putrescine (butane-1,4-diamine or 1,4-diaminobutane). Putrescine can also be formed by hydrolysis of agmatine which is produced from L-arginine by the action of arginine decarboxylase. Putrescine is alkylated by the decarboxylated S-adenosyl-methionine which regularly serves as the major methyl (CH3) group donor for methyl transferases. Spermidine and other polyamines have been and are still subject of continuous scientific research from many different disciplines (Table 1). Spermidine's pharmacological properties including distribution, metabolism and excretion have been thoroughly investigated. A major fraction of injected spermidine is excreted in the urine [4]. Spermidine and other polyamines have been early and widely associated with growth (Table 1) and aging. They were found more than 50 years ago to stabilize various cellular and subcellular components including nucleic acids due to their polyvalent cationic structures.
Several analytical methods have been reported for the simultaneous measurement of spermidine, spermine and putrescine in human biological samples, including HPLC and GC-MS [5,6] and LC-MS/MS [7]. Polyamines including spermidine have been found at elevated circulating and urinary concentrations in a variety of diseases including cancer and psoriasis (Table 1). In plasma spermidine concentration was found to be 220 nM in healthy humans and 200-800 nM in cancer patients. Mean spermidine concentrations in urine were found to be also in the nM-range, e.g., 130 nM in healthy subjects, 186 nM in diabetic patients and 680 nM in patients with severe complications [7].
Measurement of elevated spermidine concentrations in subjects suffering from various diseases has early prompted research on developing drugs to inhibit polyamine biosynthesis. One of these drugs is α-difluoromethylornithine (DMFO) [2]. More recent studies suggest that spermidine supplementation may exert beneficial effects on the brain and the heart, and can even extent lifespan. A selection of these studies is provided in Table 2 [8-31] and is briefly reviewed and discussed in the section that follows. Escherichia coli (E. coli) has been very often associated with polyamines (Table 1). The pathogenic Helicobacter pylori (H. pylori) from subjects infected with the bacterium produces large amounts of spermidine and histamine in culture [32]. The potential implication of H. pylori-derived spermidine in diseases remains to be investigated. Spermidine is considered an inducer of autophagy. For recent reviews and commentaries to autophagy and longevity and underlying mechanisms see References [33-42]. For the polyamine content of food see the recent review by Kalač [43].
Discussion
Spermidine and other polyamines are biologically active compounds and have numerous biological activities in humans. More recent non-human studies suggest polyamines as potential candidates for diseases associated with autophagy or impairment of memory due to advanced age. Due to their pharmacological potential, their toxicity and health risks have been thoroughly investigated twenty years ago. Oral acute toxicity of spermidine was determined to be 600mg (4.1mmol)/kg body weight in Wistar rats, with the no-observed-adverse-effect being 83mg (0.57mmol)/kg body weight [43]. Pharmacokinetic data for spermidine and other polyamines in humans are very scarce and need to be generated in carefully performed studies. The toxicity and health risks data observed in animals and the polyamine concentrations measured in healthy and diseased subjects provide an approximate idea of the dose regimen for spermidine supplementation in humans.
Despite the long interest in polyamines, reference values and intervals have not been established thus far. Moreover, reported circulating and urinary polyamine concentrations vary considerably. As an example, plasma spermidine concentration lies between 0.2μM (healthy subjects) and 0.8μM (cancer patients) [6]. Spermidine concentration seems to be ten times higher in packed red blood cells (5-8μM) [43-45] compared to plasma, suggesting accumulation of spermidine in human erythrocytes. To our knowledge, there is only a single paper reporting on circulating spermidine concentration in healthy humans of both genders aging between 31 and 106 years. In the Group 1 (age range, 31-56 years) the spermidine median [95%CI interval] concentration was approximately 63 [48-100] pmol/mg whole blood proteins. In the Group 2 (age range, 60-80 years) the spermidine concentration was approximately 23 [20-30] pmol/mg protein, i.e., about three times lower than in Group 1. In the Group 3 (age range, 90-106 years) the spermidine concentration was approximately 70 [48-81] pmol/mg protein, i.e., even higher than in Group 1 [18]. This is a very important but also surprising observation and needs evaluation in forthcoming studies.
Eisenberg et al. [31] reported that "In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease."
As the authors acknowledged in their paper, estimation of dietary spermidine intake on the basis of food-frequency questionnaires is an indirect method. In our opinion, this estimation is not eligible to draw far reaching conclusions that spermidine is the elixir for cardioprotection and lifespan extension. This must yet be demonstrated in further studies. In this context, we should remember that the mean daily dietary intake of spermidine in Western countries ranges between 7.9mg (54μmol) and 12.6 mg (87μmol) [43]. Even if these dietary spermidine amounts would be completely absorbed, the spermidine concentrations that would occur in blood and tissue would be not high enough to exert the reported effects in the majority of the studies in which spermidine was used mostly in the lower mM-range (Table 2). Eisenberg et al. [31] assumed that the blood pressure lowering effect of polyamines is presumably due to an increase of the bioavailability of L-arginine, but not data were reported about the nitric oxide (NO) synthesis from L-arginine. Based on the same thoughts and estimations stated above, dietary spermidine, and most likely other dietary polyamines, cannot considerably contribute to the global arginine bioavailability ratio (GABR), especially in regard to the about 1000 times higher dietary intake of L-arginine. Thus, food is likely to contribute to polyamines via bactericidal metabolism of exogenous L-arginine.
Therefore, if spermidine and other polyamines can indeed lower the blood pressure in humans, the underlying mechanisms are unlikely to be enhancement of the GABR. In mice, spermidine supplementation (3mM spermidine in drinking water for 4 weeks; 3mM in vitro in cultured aortic rings) has been reported to improve arterial aging in terms of a normalization aortic pulse waive velocity, endothelium-dependent dilation, autophagy, and even to reduce oxidative stress (based on nitrotyrosine measurement) in the older animals [46]. Unfortunately, in this paper no circulating concentrations were reported for spermidine and for the NO metabolites nitrite and nitrate. Very recently, the L-arginine metabolism has been investigated in rat vestibular nucleus and cerebellum. Measured biomarkers included arginine, citrulline, ornithine, glutamine, glutamate, GABA, spermidine, spermine, putrescine and agmatine and NOS activity by measuring the 3H-L-arginine to 3H-L-citrulline conversion rate [47].
This assay is associated with remarkable pitfalls, most notably with unspecificity, in tissue homogenates because of their capacity to produce and utilize L-citrulline by alternative pathways [48]. The study revealed that brain L-arginine metabolism is influenced by age. With regard to spermidine, age-related differences were found in the cerebellum but not in the vestibular nucleus complex of the rats (age, 4 v. 24 months), and not between middle-aged and aged rats (12 vs. 24 months). With minor exceptions [11,15], reported effects of exogenous spermidine have been observed at very high concentrations, mostly at 3mM (435mg/L) to 5mM (725mg/L). These concentrations are three and four orders of magnitude higher than spermidine concentrations found in plasma and red blood cells of humans, respectively. Although supplementation of high amounts of spermidine, putrescine or spermine is feasible from a pharmaceutical point of view, it has first to be demonstrated that supplementation of polyamines to reach concentrations that were turned out to be effective in flies, worms and small animals (mice and rats), is safe in humans.
Unfortunately, in the majority of the studies reported in the last decade, drug safety has not been implemented. Also, the potential formation of catabolic and metabolic reaction products of polyamines has not been considered at all. However, catabolism of polyamines produces many toxic compounds including H2O2 and aldehydes [43,49]. And last but not least important, spermidine can be nitrosated to N-nitrosospermidine which is further converted by cyclisation to N-nitrosopyrrolidine [50] which is classified as a possible carcinogen. Formation of nitrosopolyamines from polyamines and inorganic nitrite, that is abundantly present in human saliva, is favored under acidic conditions as they prevail in the stomach. In this context, a possible involvement of H. pylori which produces spermidine in large amounts [8] is worthy of investigation
A first research paper on spermidine and spermine in tissues of rats of increasing age revealed that the spermidine content decreased in all tissues with increasing age, with the fall being most marked during the first month of life [51]. The spermidine/ spermine molar ratio in the tissues was highest immediately after birth. In contrast to other tissues, in rat brain the spermidine/ spermine increased from 1.3 to 2.1 during the first 9 months. Similar results were found in liver and kidney of rats aged 1, 3, 6, 13 and 22 months [52].
In human brain, polyamines are heterogeneously distributed [53,54]. The highest spermidine levels were measured in white matter (20nmol/mg protein) and thalamus (9.3nmol/ mg protein) [29]. In contrast to rodents, spermidine levels in occipital cortex of neurologically normal human brain were found to increase markedly from birth, to reach maximal tissue content at the age of about 40 years and not to decrease up to senescence [53]. Spermidine tissue content was found to correlate positively with age from birth to adulthood (p=0.71, P<0.01, for day 1 to 50 years; p=0.40, P<0.01, for day 1 to 103 years). In contrast to spermidine, putrescine and spermine tissue concentrations were found to be independent of age. In older adults (mean age, 58-85 years), spermidine content in several brain regions was found to decrease with age [54]. Among the degenerative movement disorders investigated (Parkinson's disease, Huntington's disease to Progressive supra nuclear palsy), only spermidine putamen content was lower compared to healthy controls.
H. pylori has been declared as a definite gastric carcinogen [55]. The prevalence for H. pylori is about 50% around the world. This bacterium is considered to be responsible for about 90% of noncardia gastric cancer. The precise molecular and cellular mechanisms of gastric cancer development associated with H. pylori infection are still elusive. One possible mechanism could involve misregulation of p27 expression by H. pylori [55]. In cultures of this bacterium isolated from subjects infected with the H. pylori we measured large amounts of spermidine and histamine [8]. High circulating and urinary concentrations of polyamines are found in cancer patients [6]. One may therefore ask the question whether spermidine produced by H. pylori might be involved in gastric cancer. The potential implication of H. pylori-derived spermidine in diseases remains to be investigated. A very recent study revealed that spermidine, spermine and putrescine at very low concentrations (range, 8nM to 10|iM) induced proliferation and migration of certain cancer cells by up regulating among others ornithine decarboxylase and by down regulating p27 [56].
Conclusion
Since the fall of mankind, human beings strive for longevity in good physical and mental health. Can Science, the modern Prometheus, disclose us the secret of the Gods? Are natural polyamines the elixir for long life and against dementia? Might spermidine be the core-polyamine for a long and dementiafree life? Many sophisticated studies published in the last decade suggest that spermidine exerts beneficial effects in the cardiovascular system and in the brain, at least in yeast, flies, worms and small animals. Yet, in many respects human biology differs from that of rodents behave. With exception of isolated cells, where spermidine exerted remarkable effects at concentrations of the order of 20nM, too high spermidine concentrations have been supplied to achieve measurable biological effects.
Within a few years, there is already a general belief that spermidine is an inducer of autophagy and that this prolongs life. Spermidine, being an L-arginine metabolite, seems to be associated with NO which plays multiple roles in the renal, cardiovascular and central nervous systems. Yet, can the results observed in microorganisms and small animals be translated to man? Is high dietary spermidine intake a risk-free option for good physical and mental health? Data from human studies on spermidine are very rare, contradictory and not convincing for example with respect to the inverse correlation between spermidine and blood pressure or risk of cardiovascular disease and related death. In the past, observations from the use of many antioxidants at high concentrations in vitro and in vivo animal experiments raised the expectations for health-promotion in humans, but large clinical studies were very disappointing [57]. For the same reasons, most notably use of irrelevantly high concentrations/doses and disregard of health-risk effects, this may also happen to the use of spermidine and/or other polyamines as supplements. It’s a long way to Longevity!
Happy Thanksgiving Day
Happy Thanksgiving Day
Genotoxicity: Mechanisms, Testing Guidelines and Methods
Authored by Rajendra SV
Abstract
Genotoxicity is one of the major causes for cancer. Genotoxins are agents that can cause the damage of DNA or chromosomal structure thereby causing mutations. It can be chemical or radiation. This damage in the somatic cells will lead to various diseases ranging to cancer whereas the damage to the germ cell will lead to heritable diseases. Better identification and understanding of genotoxins would enable us to prevent the potential damage that can be caused by these genotoxic agents. In this article we discuss about the basic of genotoxicity and the importance of genotoxic studies.
Keywords: Genotoxins, Mutagens, DNA Damage, Chromosomal mutation, Testing guidelines.
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Introduction
Genotoxicity is a word used in genetics that describes the possession of substance that has destructive effect on the genetic material of the cell (DNA, RNA) thus affecting the integrity of the cell. Genotoxins are mutagens that can cause genotoxicity leading to the damage of DNA or chromosomal material thus causing mutation. Genotoxins can include chemical substance as well as radiation. Genetic toxicology is the branch of science that deals with the study of agents or substances that can damage the cell’s DNA and chromosomes. It is noted that often genotoxicity is confused with mutagenicity. All mutagens are genotoxic however all genotoxic substances are not mutagenic [1].
Genotoxins can be of the following category depending on its effects [2]:
o Carcinogens or cancer causing agents
o Mutagens or mutation causing agents
o Teratogens or birth defect causing agents
The damage of genetic material of somatic cells may lead to malignancy (cancer) in eukaryotic organisms. Whereas the genetic damage of the germ cells may lead to heritable mutations causing birth defects (Figure 1). Mutations can be of any form; which may include duplication, insertion or deletion of genetic information. These mutations can cause varying range of problems in the host, from a wide variety of diseases to cancer [3,4] One of the best ways to control the damage due to mutagens and carcinogens is to identify the substance or chemical, i.e. antimutagens /anticlastogens (which suppress or inhibit the mutagenesis process by directly acting on the cell mechanism) and demutagens (which destroy or inactivate the mutagens partially or fully thereby affecting less population of cell) from the medicinal plants so that it can be used as antimutagenic and anticarcinogenic food or drug additives [5-7].
Importance of genotoxicity studies
Genotoxicity studies can be defined as various in-vitro and in-vivo tests designed to identify any substance or compounds which may induce damage to genetic material either directly or indirectly by various mechanisms. These tests should enable the identification of hazard with respect to DNA damage and fixation [8]. Genetic change play only a part in the complex process of heritable effects and malignancy which include the fixation of the damage to the DNA by gene mutation or large scale chromosomal damage or recombination or numerical chromosomal changes. These tests play an important role in predicting if the compound have the potential to cause genotoxicity and carcinogenicity by testing them positive [9]. As a part of safety evaluation process, regulatory authorities all over the globe require information on the genotoxic potential of the new drugs. Genotoxicity is usually evaluated along with other toxicological end points during the safety assessment [10] (Figure 2).
During the early testing stages; the same testing assays are carried out for predicting both the potential heritable germ cell damage as well as the carcinogenicity because these endpoints have common precursors. The relationship between exposure to particular chemical and carcinogenesis has been established whereas such relationship has been difficult to establish for heritable diseases, genotoxic studies have been mainly associated and used for the prediction of carcinogenicity of a compound [11,12]
Classifiaction of carcinogens
EU classification of carcinogens [
13
]
o Carcinogen category 1-shown to cause cancer in humans
o Carcinogen category 2-causes cancer in animal tests, and most probably also in humans
o Carcinogen category 3-possibly carcinogenic, but evidence supporting carcinogenicity is inadequate for the classification to category 2.
Iarc (International Agency For Research On Cancer) classification of carcinogens [
4
]
o IARC class 1-The substance is carcinogenic to humans.
o IARC class 2A-The substance is probably carcinogenic to humans.
o IARC class 2B-The substance is possibly carcinogenic to humans.
o IARC class 3-The substance is not classifiable to as to its carcinogenicity to humans.
o IARC class 4-The substance is probably not carcinogenic to humans.
Agents that can cause direct or indirect damage to the DNA
Reactive oxygen species are known to be genotoxic in nature, thus any chemical or substance that may increase the reactive oxygen species (ROS) production might evidently add to the endogenously produced ROS and may lead to non-linear relationships of dose-effect. The following agents are capable of damaging the DNA directly or indirectly; [14]
o Electrophilic species that form covalent adducts to the DNA
o Reactive oxygen species
o Ultra violet and ionizing radiations.
o Nucleoside analogues
o Topoisomerase inhibitors
o Protein synthesis inhibitors
o Some herbal plants like Aconite, Alfa-alfa, Calamus,Aloe vera, Isabghol etc.
Anti-mutagen is any agent that decreases the effect of spontaneous and induced mutations. There are mainly two mechanisms of anti-mutagenesis:
Desmutagenesis in which the factors on the mutagens are somehow inactivated,
Bio-antimutagenesis, in which the factors act on the process of mutagenesis or by repairing the damaged DNA that result in the decreased frequency of DNA mutation [15]. Our cells have several DNA repair system by which they try to control the DNA mutations naturally.
The five major pathways through which the cell repair the damaged DNA are: [16-19]
o Direct repair
o Base excision repair( BER)
o Nucleotide excision repair (NER)?
o Mismatch repair
Single/ double strand break repair
Mechanism of genotoxicity
The damage to the genetic material is caused by the interactions of the genotoxic substance with the DNA structure and sequence. These genotoxic substance interact at a specific location or base sequence of the DNA structure causing lesions, breakage, fusion, deletion, mis-segregation or nondisjunction leading to damage and mutation [20]. For example, in its high-valent oxidation state the transition metal chromium interacts with the DNA so that DNA lesions occur leading to carcinogenesis. Researchers have found that the mechanism of damage and base oxidation products for the interaction between DNA and high-valent chromium are relevant to in-vivo formation of DNA damage leading to cancer in chromate- exposed human population, thus making high valent chromium a carcinogen (Figure 3).
Reactive oxygen species causes one of the most abundant oxidative lesions in DNA and is 8- hydroxydeoxyguanosine (8- OHdG), which is a potent mutagenic lesion. Oxidants as well as free radical when present in the cellular system can adversely affect and alter the structure of lipids, proteins as well as DNA. Reactive aldehydes like 4-hydroxynonenal (4-HNE) are generated by the decomposition of lipid peroxyl radicals or primary free radical intermediate of lipid peroxidation. 4-Hydroxynonenal is involved in many of the oxidative stress related diseases such as atherosclerosis, fibrosis, neurodegenerative diseases it. Many studies have indicated that 4-Hydroxynonenal can stimulate the cell proliferation, differentiation as well as cytoprotective response through its effects on various signalling pathway [21].
Standard test battery for genotoxicity
The standard test battery for genotoxicity recommends the following for genotoxicity
Evaluation [22-24] (Table 1).
o Testing for gene mutation in bacteria
o In-vitro: cytogenetic evaluation of chromosomal damage with mammalian cells or mouse lymphoma assay.
o In-vivo: test for chromosomal damage using rodent hematopoietic cells (Figure 4).
Purpose of genotoxicity assays
Assays even though inexpensive, have high statistical power and can be reproduced and have the ability to detect a wide variety of genotoxic end-points. It also allows the detection of a drug's potential to cause genotoxicity even in the early stage of drug development. They are designed in such a way that it can be more sensitive to damage so as to enhance the identification of hazard [25,26].
In-Vitro testing methods
There are many in-vitro genotoxicity testing methods available. Some of the commonly used tests which are also a part of the standard battery are [27]:
• Bacterial reverse mutation test which is otherwise called as Ames test whose endpoint is the gene mutations in the bacterial cell [28-29].
• Mammalian chromosome aberration test with the end point of chromosome aberration [30-31].
• Mammalian cell gene mutation test or the mouse lymphoma test whose end point is the gene mutations [32,33].
Bacterial reverse mutation test: The Bacterial reverse mutation test was developed by Ames. B thus the name Ames test. The amino acid requiring strains of Salmonella typhimurium and Escherichia coli are used in the bacterial reverse mutation test to detect the mutation points which may involve substitution, deletion or addition of one or few of base pairs of DNA [34]. The main principle of the test is that after identifying the mutation it reverts it back and restore the functional capability of the mutant cell to synthesize Histidine. In this test the reverent bacteria cells are identified by the ability of the parent test strain to grow in the absence of amino acids. The bacterial reverse mutation test being rapid, inexpensive and easy to perform is commonly used as an initial screening test for genotoxicity or mutagenicity [35,36].
Mammalian chromosome aberration test: The main purpose of the mammalian chromosome aberration test is to identify the agents which can cause structural mutations in chromosomes or chromatids, chromatid mutation being the common [37,38]. Other type of chromosomal changes like polyploidy and duplication can also be found using this test. A positive test result shows a potential mutagenic or carcinogenic of the agent but there is not a perfect correlation [39].
Mammalian cell gene mutation test: This test is used to find the gene mutations caused by the chemical substances. The commonly used cell lines include L5178Y mouse lymphoma cells, the CHO, CHO-AS52 and V79 lines of Chinese hamster cells, and TK6 human lymphoblastic cells [40-41]. It detects the end points like thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyltransferase (HPRT), and a transgene of xanthine- guanine phosphoribosyltransferase (XPRT) mutation [42,43].
In-vivo genotoxicity testing methods
The in-vivo genotoxicity test or assays are done supplemental to in-vitro assay if an in-vitro positive result is obtained. Some of the in-vivo tests done are as follows [44].
In-vivo comet assay: It is one of the commonly used in-vivo test used for hazard assessment of agents which have potential for genotoxicity or mutagenicity [45]. It helps in detecting the DNA damage and detects a broad variety of primary DNA lesions which cannot be identified by any other tests. This test can be applied to a wide variety of tissues or any special cell types. Being sensitivity to even low level of DNA damage it requires only small amount of cells per sample and it can be completed in a short period of time [46].
In-vivo micronuclei test/In-vivo chromosome aberration test: It is a test done to identify the damage done chromosome or spindles. On exposure to the mutagen the cell may undergo damage and on division it will form smaller micronucleus additional to the main nucleus [47].
Go to
Conclusion
Genotoxins are agents that can interact with the DNA thus causing mutations and damaging its structure and may lead to cancer. They act by changing the chromosomal structure by addition, deletion, duplication, forming rings etc. The mutations may lead to a wide variety of diseases to cancer. It is very important to do genotoxicity studies so as to avoid the potential damage that can be caused by it. These genotoxicity tests are done to identify if a drug or other substance have the potential to cause mutation and genotoxicity. By doing so they help us identifying the hazards in the early stage of drug development itself. Identification of the genotoxic agents helps us understand the mechanism of the mutation and genotoxicity thereby paving us way to better prevent the frequency of such mutation and genotoxicity.
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Etilefrine Could Improve Response to Standard Medical Therapy in Chronic Hepatitis C Egyptian Patients with Cirrhotic Refractory Ascites: A Randomized Pilot Study-Juniper Publishers
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Ascites is a frequent complication of cirrhosis that accounts for over 75% of episodes of ascites. Patients with cirrhotic ascites have marked splanchnic vasodilation and arterial hypotension with subsequent activation of vasoconstrictive and anti-natriuretic mechanisms. One of the most serious complications in cirrhotic patients with ascites is the occurrence of refractoriness that is the inability to resolve ascites by the standard medical treatment. The aim of this study is to evaluate the effects of etilefrine on systemic hemodynamics, renal function and control of ascites in chronic hepatitis C (CHC) patients with cirrhotic refractory ascites receiving standard medical treatment (SMT) with low sodium diet and maximal diuretic doses of 160mg/day of furosemide and 400mg/day of spironolactone.
Methods: A total of 50 CHC patients with cirrhotic refractory ascites were prospectively studied after 1 month administration of SMT (n=25) or SMT plus etilefrine (n=25), in a randomized controlled study.
Results: A significant increase in 24-h urinary output, urinary sodium excretion, mean arterial pressure (MAP), and decrease in body weight, plasma renin activity and plasma aldosterone concentration (P<0.05) was noted after 1 month in the SMT/etilefrine group. Furthermore, the effective diuretic doses and the need for large-volume paracentesis were significantly reduced in the SMT/etilefrine group compared to the SMT group after 1month of therapy. No significant changes in the aforementioned parameters were noted in the SMT group, except that MAP was significantly decreased. There was no significant change in the score of the model for end-stage liver disease (MELD) in the SMT/etilefrine group; however, there was significant deterioration in the MELD score in the SMT group.
Conclusion: These results suggest that the addition of etilefrine to SMT improves the systemic hemodynamics and enhances water and sodium excretion, providing better control in patients with refractory cirrhotic ascites treated with SMT alone.
Keywords: Refractory ascites; Etilefrine; Systemic hemodynamics; Plasma renin activity; Renal function; Plasma aldosterone concentration
Abbreviations: CHC: Chronic Hepatitis C; SMT: Standard Medical Therapy; MAP: Mean Arterial blood Pressure; MELD: Model for End-stage Liver Disease
Introduction
Ascites is the most common of the three major complications of cirrhosis; the other complications are hepatic encephalopathy and variceal hemorrhage [1]. Approximately 50% of patients with "compensated" cirrhosis develop ascites during 10 years of observation [1]. Ascites is the most common complication of cirrhosis that leads to hospital admission [2]. Development of fluid retention in the setting of cirrhosis is an important landmark in the natural history of chronic liver disease: approximately 15% of patients with ascites succumb in 1 year and 44% succumb in 5 years [3]. Many patients are referred for liver transplantation after development of ascites. Refractory ascites develops in approximately 5-10% of all cases of cirrhosis- related ascites and carries a high mortality rate [4]. The available therapies for patients with refractory ascites are repeated large volume paracentesis, trans jugular intra hepatic portosystemic shunts, peritoneovenous shunts, and liver transplantation [5,6].
The mechanism by which refractory ascites develops in cirrhosis is related to splanchnic vasodilatation followed by maximal activation of the sympathetic nervous system (SNS) and the renin - angiotensin- aldosterone system (RAAS) [7-10]. Splanchnic and peripheral vasoconstrictors (octreotide, midodrine, and terlipressin) increase effective arterial volume and decrease activation of the renin-angiotensin system with resultant increase in renal sodium excretion [6]. Vasopressors causing splanchnic vasoconstriction have been used in hepatorenal syndrome [11-13] for the prevention of post-paracentesis circulatory dysfunction [14-16], improving circulatory and renal function in patients with cirrhotic ascites [17,18] and for control of ascites in patients with refractory ascites [19]. Combined use of midodrine with standard medical therapy (SMT) was found to improve the systemic hemodynamics without any renal or hepatic dysfunction and is superior to SMT alone for the control of refractory cirrhotic ascites [20]. Clonidine, a centrally-acting presynaptic ɑ2- adrenergic receptor agonist, when given with spironolactone has been shown to cause rapid mobilization of ascites by significantly decreasing the sympathetic activity and renin- aldosterone levels [21,22]. Synthetic vasopressin-V2 receptor antagonists are being evaluated for mobilization of ascites by increasing the excretion of solute-free water [23-25]. Etilefrine is a sympathomimetic agent with a potent stimulating effect on peripheral ɑ-adrenoceptors and a mild agonist effects on β1- and β2-adrenoceptors. It has a potent vasoconstrictor effect. It has a stimulant effect on the cardiovascular system where it raises blood pressure to normal, improves cardiac performance and tissue perfusion. Etilefrine is indicated in hypotension and, hypotensive circulatory disorders [26-28]. There are no published reports on the combined use of etilefrine and SMT in patients with refractory cirrhotic ascites. Therefore, this study is designed to investigate whether the long-term use of etilefrine would improve systemic hemodynamics and control of ascites in patients with cirrhosis and refractory ascites.
Methods
The study protocol was approved by the local ethics committee and was conducted in accordance with the principles of Helsinki declaration. Written informed consent was obtained from all patients before enrollment in the study. A total of 58 chronic hepatitis C patients with refractory cirrhotic ascites were evaluated for inclusion in the study between November 2015 and May 2016. Eight of the enrolled patients were lost during the study period for different personal unidentified reasons. Accordingly, 50 CHC patients with cirrhosis and refractory ascites with stable renal function (creatinine level <1.5 for at least 7days), attending Gastroenterology and Hepatology Department of a specialized hospital were prospectively included in the study. Diagnosis of cirrhosis was based on clinical, biochemical and ultrasonography findings with or without liver biopsy [29,30]. Inclusion criteria were as follows: presence of refractory ascites; patients less than 60 years of age and no treatment with drugs known to affect systemic or renal hemodynamics within one week before initiation of the study.
Exclusion criteria were as follows: presence of marked hepatic encephalopathy, GIT bleeding, hepatorenal syndrome, hepatocellular carcinoma, bacterial peritonitis, portal vein thrombosis, arterial hypertension, diabetes, intrinsic renal or cardiovascular disease. Refractory ascites is defined as
A. fluid overload that is unresponsive to sodium- restricted diet and high-dose diuretic treatment (400mg/d of spironolactone and 160mg/d furosemide),
B. buildup of fluid that recurs rapidly after therapeutic paracentesis or
C. development of diuretic-related complications that exclude the use of an effective diuretic dosage. Patients were randomized to either SMT alone (n=25) or SMT plus oral etilefrine (n=25). Etilefrine is active ingredient of EffortilR produced by Chemical Industries Development, Egypt under license of Boehringer Ingelheim International, Germany. Etilefrine was blindly given orally at a dose of 5 mg/8h. SMT was defined by dietary restriction of sodium (≤2g/day, starting at least 7 days before the start of the study), treatment with a combination of a loop diuretic (furosemide 160mg/d) and a distally-acting aldosterone antagonist (spironolactone 400mg/d) and repeated large volume paracentesis (LVP) along with intravenous albumin (8g/L of ascitic fluid removed).
During the study period, diuretic doses were reduced by a 40mg for furosemide and100 mg for spironolactone for a mean weight loss >0.8kg over 4 days from the previous weight. Large- volume paracentesis was performed when ascites is tense and symptomatic. Frequency of paracentesis sessions over the 4 weeks preceding the study was obtained from patient files. All patients were subjected to baseline clinical and biochemical workup including, body weight, mean arterial blood pressure, 24-h urinary output, 24-h urinary sodium excretion, liver function tests and renal function tests. These parameters were assessed at baseline and at weekly intervals for 1 month. Measurement of mean arterial blood pressure (MAP) was calculated as diastolic blood pressure+((systolic blood pressure-diastolic blood pressure)/3). Three measurements were taken each 1-hour apart and the mean was calculated. Plasma renin activity and plasma aldosterone concentration were evaluated at baseline and after 1 month (endpoint). Plasma renin activity (PRA) was measured by radioimmunoassay using RIA plasma renin activity kit (Diasorin, Stillwater, MN, USA). Plasma aldosterone concentration (PAC) was measured by radioimmunoassay using ALDO-RIACT aldosterone kit (Cisbio, Parc Marcel Boiteux, France). Diuretic requirements were assessed at baseline, at weekly intervals and at endpoint. Patients were instructed to undergo tapping when become symptomatic.
Outcome measures
The primary endpoints of the study were partial or complete control of ascites. Complete response was defined as the elimination of ascites (as assessed by clinical examination and abdominal ultrasonography); a partial response was defined as the presence of ascites not requiring paracentesis; and absence of a response was defined as the persistence of ascites requiring paracentesis [31]. Secondary endpoints include alteration of diuretic requirements, changes in the scores of end-stage liver disease, liver and renal function, and frequency of other complications of cirrhosis (e.g., encephalopathy, upper GIT hemorrhage or development of hepatorenal syndrome) after 1 month of therapy.
Statistical analysis
Data were analyzed using SPSS for MS-Windows (version 17.0, SPSS, Chicago, IL, USA). The baseline patient characteristics (clinical as well as biochemical) were compared between two groups (SMT or SMT plus etilefrine) by using Kruskal-Wallis ANOVA, Chi-square test or Fisher's exact test as appropriate. Intra-group comparisons were done using multiple repeated- measures analysis of variance. The paired t-test was performed to detect mean and standard deviation of prevalues (baseline) and post values (endpoint at 1 month) of the same variable of the same patients. The results were reported as mean values ±SD. A p-value of ≤ 0.05 was taken as significant.
Results
SMT, standard medical therapy, MAP, mean arterial pressure; MELD, model for end-stage liver disease; INR, international normalized ratio. *Baseline values between groups 1, and 2 are not significantly different (P> 0.05). Data are expressed as mean ± SD.
The demographic characteristics and baseline clinical and biochemical parameters were similar between SMT and SMT/ etilefrine groups (Table 1). Baseline body weight did not differ significantly between the two groups (p>0.05). There was a significant decrease in mean body weight in SMT/etilefrine group at 1-month as compared to baseline (p<0.05) however; it did not change in the SMT group (Table 2) (Figure 1).
SMT, standard medical therapy, MAP, mean arterial pressure; MELD, model for end-stage liver disease.
*1-month values are significantly different from baseline (P<0.05) in the etilefrine group but not in the SMT group. Data are expressed as mean ±SD.
Baseline MAP did not differ between SMT and SMT/etilefrine groups (p>0.05). There was a significant increase in mean arterial pressure in SMT/etilefrine group at 1-month as compared to baseline (p<0.05) and a significant decrease (p<0.05) in the SMT group (Table 2) (Figure 2). Baseline urine output did not differ between SMT and etilefrine groups (p>0.05). The urine output was significantly higher in the SMT/etilefrine group (p<0.05) but not the SMT after 1 month of treatment as compared to baseline (Table 2) (Figure 3). Baseline urinary sodium excretion was comparable in the SMT and SMT/etilefrine groups (p>0.05). Urinary sodium excretion significantly increased in the SMT/ etilefrine group after treatment at 1-month as compared to baseline (p<0.05); however, it did not change in the SMT group (Figure 4).
Baseline values for plasma renin activity (Table 1) were similar in both treatment groups (p>0.05). Plasma renin activity significantly decreased at 1-month (p<0.05) only in the SMT/etilefrine group with no change in the SMT group compared to baseline (Figure 5). Baseline plasma aldosterone concentrations did not differ between the two groups (p>0.05). Plasma aldosterone concentrations decreased significantly in the SMT/etilefrine group at 1-month as compared to baseline (p<0.05); however, it did not change in the SMT group (Figure 6). Baseline values for serum creatinine in both SMT and SMT/ etilefrine groups were similar (p>0.05, (Table 1). There was no significant change in serum creatinine in both groups after 1-month treatment as compared to baseline (p>0.05, (Table 2).
Baseline serum bilirubin and INR were similar in both groups (p>0.05, (Table 1) but there were significant increase in their values at 1 month only in the SMT group (p<0.05, (Table 2). Baseline MELD score was similar in both treatment groups (p>0.05; (Table 1). There was a significant deterioration in MELD score in SMT group at 1 month (p<0.05) with no change in the SMT/etilefrine group as compared to baseline (p>0.05. The need for LVP (≥5L) was significantly reduced in the SMT/etilefrine group (p<0.05; (Figure 7) at lmonth compared to baseline value. No significant change was noted in the SMT group. Fifteen patients from the SMT group required LVP compared to only six in the SMT/etilefrine group over the 1-month period of therapy.
SMT, standard medical therapy.
As depicted in Table 2, diuretic requirements were significantly declined from baseline in the SMT/etilefrine group (p<0.05) with no change in the SMT group. There was higher rate of partial response to treatment and better control of ascites in the SMT/etilefrine group (p<0.05; (Table 3) (Figure 8) compared to SMT group at 1 month of treatment. Mild abdominal pain that subsided on its own was noted in three patients in the SMT group. In the SMT/etilefrine group, mild headache was developed in two patients, which disappeared with time without discontinuation of therapy.
Follow-up
The 1-month morbidity and mortality of the study is depicted in Table 4. In SMT group, encephalopathy developed in two patients, upper gastrointestinal bleeding, spontaneous bacterial peritonitis (SBP) and renal failure developed in one patient each. One case of SBP was recorded in the SMT/etilefrine group. The 1-month mortality was two in the SMT group and was related to sepsis during the follow-up period. No mortalities were recorded in the SMT/etilefrine group.
Discussion
Splanchnic arterial vasodilatation induced by nitric oxide [32] and glucagons [33] leads to disturbance of systemic hemodynamics reflected as reduced arterial blood pressure, reduced vascular resistance, and decreased effective blood volume with activation of potent vasoconstricting systems such as the sympathetic nervous system, the renin-angiotensin- aldosterone system, in addition to nonosmotic release of vasopressin [34-36]. This results in renal vasoconstriction, avid sodium and fluid retention with development of ascites [37]. The administration of arterial vasoconstrictors has been associated with improvement in systemic hemodynamics and renal function in cirrhotic patients with ascites [38]. The efficacy of vasoconstrictors in advanced cirrhotic ascites might be related to failure of the activated endogenous vasopressor systems to counteract the arterial vasodilatation [39], probably due to reduced arterial reactivity to vasopressors [40]. Administration of intravenous arterial vasoconstrictors such as metaraminol [41], norepinephrine [42,43], angiotensin II [44] and terlipressin [45] in cirrhotic ascitic patients has been associated with improvement of systemic hemodynamics without harmful effects on renal function. Midodrine, a potent peripherally acting oral a-adrenergic receptor agonist, either alone [19,20,46] or in combination with octreotide and albumin [47] has been used to improve renal hemodynamics in cirrhotic patients with ascites. In these studies, midodrine-induced splanchnic vasoconstriction improved systemic hemodynamics with better control of ascites without any renal or hepatic dysfunction.
Combined use of midodrine with tolvaptan, an aquaretic vasopressin V2 receptor antagonist, has been found to control ascites and improve response to diuretic therapy. Rai et al. [48], reported that midodrine (by causing splanchnic vasoconstriction, increasing effective arterial blood volume and improving renal perfusion) and tolvaptan (by increasing free water clearance) acting at different sites in combination were more effective in combating increased renal sodium retention and refractoriness to diuretic therapy and better controlled ascites. There are no studies in literature on the short- or longterm use of combination of etilefrine and standard medical therapy in patients with cirrhotic refractory ascites, therefore the results of the present study will be compared to previous studies in which different vasopressor agents were used for the control of refractory ascites.
In our study, we compared the changes in systemic hemodynamics (MAP), renal excretory function (24-hour urinary sodium excretion, 24-hour urinary output), plasma renin activity and plasma aldosterone concentration in patients with cirrhotic refractory ascites after 1-month treatment with SMT alone or in combination with etilefrine. Changes in models for end-stage liver disease scores, the need for paracentesis and diuretic requirements were also compared in both groups.In the current study, there was a significant increase in the mean arterial pressure (MAP) with etilefrine while there was no change in the SMT group. Etilefrine-induced increase in MAP may be related to reducing venous pooling and counteracting reflex arteriolar vasodilatation [28].
In agreement with our results, Kalambokis et al. [17,38] reported an improvement in circulatory function manifested as a significant increase in MAP after short-term use of midodrine [17] and chronic combined use of midodrine with octreotide [38] in patients with nonazotemic cirrhotic ascites. Similar results were reported after long-term use of midodrine with SMT [19,20,48], and combined use of midodrine with SMT and clonidine [20]. On contrary, Oda et al. [49] reported that a 3-month course of midodrine produced no change in MAP in cirrhotic patients with refractory ascites. In comparison to baseline values, our results showed a meaningful improvement in renal hemodynamics and function reflected as a significant increase in twenty-four-hour urine output and urinary sodium excretion in the etilefrine/SMT group but not in the SMT group. These findings agree with those observed with previous studies employing midodrine plus SMT [19,20,48] , combined use of midodrine with SMT and clonidine [20], and combined use of midodrine with SMT and tolvaptan [48]. In another study, a single dose of terlipressin (vasopressin V1 receptor agonist) showed marked increase in urinary sodium excretion in patients with and without refractory ascites [18]. The results of our study disagreed with a previous study [50] that found no change in 24-h urine volume after two-week midodrine therapy.
In the present study a significant decrease in plasma renin activity (PRA) and plasma aldosterone concentration was noted only in the etilefrine/SMT group after one-month therapy compared to baseline. This effect is possibly related to etilefrine-induced suppression of the renin-angiotensin- aldosterone system. Similar results with other vasoconstrictors were reported by Singh et al. [19, 20] and Rai et al. [48] after long-term use of SMT/midodrine regimen [19,20,48], combined SMT/midodrine/clonidine therapy [20] or combined SMT/ midodrine/tolvaptan therapy [48]. In an earlier study, no change in PRA was noted in patients with refractory ascites maintained on a 3-month course of midodrine therapy [49]. In the current study, significant reduction in mean body weight was observed in patients receiving etilefrine plus SMT compared to those treated with SMT alone. This comes in concordance with previous findings of decreased body weight by midodrine [38,50]. The reduction in body weight may be related to a drop in fluid accumulation by etilefrine-induced vasoconstriction with reflex inactivation of the renin-angiotensin-aldosterone system (RAAS). Similar explanation were reported in previous studies in which the authors observed a sig-nificant reduction in plasma renin and aldosterone con-centration and a trend toward a reduction in the volume of ascitic fluid removed by paracentesis following the administration of midodrine [38,50]. No change in body weight was reported in a number of previous studies utilizing different vasoconstrictors including midodrine [19,20,48].
Rate of response to treatment, measured as need for large- volume paracentesis (≥5L) and reduction of ascites with SMT alone or combined SMT/etilefrine therapy was measured. There was higher rate of response to treatment, reflected as a significant decrease in the number of times of paracentesis in the combined SMT/ etilefrine therapy at 1 month. There was no significant change in rate of response to treatment in the SMT group. These results come in harmony with some previous studies in which the vasoconstrictor midodrine was used with various daily doses [20,48,50]. In another study, midodrine along with octreotide and albumin given for 1 month showed lesser requirement of paracentesis in eight patients with refractory ascites [47]. In agreement with previous studies evaluating midodrine [17,19,20,48], midodrine and clonidine [20], midodrine plus octreotide [38] or midodrine plus tolvaptan [48] in cirrhotic patients with ascites, our results did not show significant change in hepatic function or MELD score in the combined SMT/etilefrine group. A significant deterioration in MELD score was noted in the SMT group at 1 month. In one previous study [38], combined use of SMT with midodrine and tolvaptan showed significant improvement in MELD score at 1 and 3 month of therapy. In another pilot study, significant deterioration in the MELD score was observed during treatment with midodrine at 1 month of therapy [47]. Diuretic needs for furosemide and spironolactone were significantly reduced in the SMT/etilefrine group at 1 month compared to baseline. Diuretic doses were reduced by increments of 40 mg (furosemide) /100mg (spironolactone) for each ≥0.8kg mean decrease in body weight from the previous weight over 4 days of therapy according to the criteria of International Ascites Club [51].
The reduction of diuretic requirements and subsequent enhancement in diuretic response may be related to etilefrine- induced improvement in renal perfusion and/or its inhibitory effect on RAAS. No significant change in diuretic needs was noted in patients receiving SMT alone. Reduction in diuretic needs with better control of ascites was reported in one earlier pilot study [20]. There was higher rate of partial response to treatment (defined as ascites requiring no paracentesis) and better control of ascites in the SMT/etilefrine group compared to SMT group at 1 month of treatment (76 versus 40%). Etilefrine use was well tolerated by the majority of patients. Only two patients developed mild headache that resolved spontaneously within few days without discontinuation of treatment.
Conclusion
To our knowledge, this is the first study of long-term use of combined SMT/etilefrine therapy in patients with cirrhotic refractory ascites. In patients receiving combined SMT/ etilefrine therapy, we observed a significant increase in MAP, 24-h urinary output, 24-h urinary sodium excretion and a significant reduction in body weight, plasma renin activity and aldosterone concentration. There was a considerable reduction in the need for large volume paracentesis and diuretic therapy. Large multicentre, randomized-controlled trials are required before combined SMT/etilefrine therapy can be routinely recommended.
Conflict of interest
The study was done using 50 blood smears of patients with different age groups.
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Comparison of Morphological Analysis of RBC through Peripheral Smear and Automated Method-Juniper Publishers
Aim: To compare the morphology of red blood cells by peripheral smear and automated method.
Objective: Comparison of morphology of red blood cells by using peripheral smear and automated method.
Background: Counting of red blood cells (RBC) in blood cell images is very important to detect as well as to follow the process of treatment of many diseases like anaemia, leukaemia etc. However, locating, identifying and counting of -red blood cells manually are tedious and timeconsuming that could be simplified by means of automatic analysis.
Reason: To know which is the most effective method of analysing morphology of RBC.
Keywords: Red blood cells; Observers; Automated method
Introduction
Red blood cells (RBCs), also known as erythrocytes, are the most common type of blood cells in our body and it is the principal means of delivering oxygen to the body tissues.The red blood cells are typically biconcave disks: flat and depressed in the centre, with a dumbbell-shaped cross section, and a rim on the edge with the torus shapeddisk [1]. Red blood cells in mammals are unique amongst vertebrates as they do not have nuclei when mature.Anaemia is the lack of red blood cells and/ or haemoglobin. This results in a reduced ability of blood to transfer oxygen to the tissues [2]. Normal, mature RBCs are biconcave, disc-shaped, a nuclear cells measuring approximately 7-8 microns in diameter on a peripheral blood smear with an internal volume (MCV) of 80-100 femto liters (fL). The term used to describe RBCs of normal size is "normocytic". Anaemia is classified by the size of the red blood cells which is either done automatically or on microscopic examination of a peripheral blood smear. The size is reflected in the mean corpuscular volume (MCV). If the cells are smaller than normal (under 80fl), the anemia is said to be microcytic; if they are normal size (80- 100fl), normocytic; and if they are larger than normal (over 100fl), the anaemia is classified as microcytic [3]. MCV measures only average cell volume. The MCV can be normal while the individual red cells of the population vary wildly in volume from one to the next. Such an abnormal variation in cell volume is called anisocytosis [1]. The degree of anisocytosis in a sample of blood is known as the red cell distribution width (RDW).
Materials and Methods
The study was done using 50 blood smears of patients with different age groups.
Blood samples
All venous blood specimens were collected in tubes containing ethylenediaminetetraacetic acid (K3EDTA) and then were analysed
Automated method
After thorough mixing of each blood sample on an automated mixer for 3-5min, a mean corpuscular value was obtained in which value between 80-100 were considered normal.
Manual method
Thin air-dried blood smears made after thorough mixing of each sample were stained manually with leishman’s stain and examined under light microscopy with a X100 oil-immersion lens.
Results and Discussion
Manual method was done with two different observers (pathologists) and the results obtained were compared with the results obtained from automated method. The similarities among the results are shown in the graph shown in Figure 1. Out of 50, 38% of the results were same with both the observes and the automated method. 24% of the results were same between 2 observers 17% of the results were common between one observer and the automated value. Only 4% of the results were different among all the observers and the automated method. Among all 50 slides when examined by the observerl, 24 slides were normocytic, in case of observer 2, 39 were normocytic and in automated method 26 were normocytic. Most cases of normocytic anemia are caused by blood loss, suppressed production of RBCs, or hemolysis [2]. Macrocytic cells were seen in 9 slides by observer 1, only one slide had macrocytic cells by observer 2 and 3 slides in automated method. Macrocytic is usually seen to differentiate between megaloblastic and nonmegaloblastic causes megaloblastosis is seen with and folate deficiency, MDS and CDA, HIV infection, and rare inborn errors of metabolism, while nonmegaloblastic causes include liver and thyroid disease, alcohol, Down syndrome, aplastic anemia, and reticulocytosis. Medications can be responsible for both megaloblastic and non-megaloblastic anemia, while RBC agglutination may lead to spurious macrocytosis [2]. Microcytic cells are seen in 17 slides by observer 1,10 slides had microcytic cells by observer 2 and 20 slides were microcytic in automated method.In classic cases, the morphological differentiation of the three common microcytic anemias is straightforward [4].
Conclusion
The review of red blood cell morphology is the most important step in the evaluation of a patient with anemia. It can be very useful in evaluating microcytic, normocytic, and macrocytic anemias and is especially helpful in the patients with hemolysis [5]. It can be concluded that for diagnostic purposes results obtained from two different observers or results from both the observers and the automated value can be considered.
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Safety Based Limits for the Control of Impurities in Drug Substances and Drug Products: A Review-Juniper publishers
Safety based impurity limits are key to the effective reduction and control of impurities in medicinal products. Impurity limits now have a greater focus on daily exposure limits, with the introduction of various approaches, such as permitted daily exposure (PDEs), acceptable intake (AIs), threshold of toxicological control (TTCs) and staged TTCs all aimed at defining a virtually safe dose (VSD). This in turn led to the introduction of less than lifetime (LTL) limits for mutagenic impurities, which is based on an application of Haber's law which states that concentration and exposure times are both critical for assessing likely safety risk to patients. Surprisingly, LTLs have not been applied to the other specific classes of impurities or indeed general impurities.
A recent publication has suggested that a LTL limit for general impurities of 5 mg or 0.7% (whichever is lower), for clinical studies with durations of less than 6 months is warranted. ICH S9 indicated that there was no additional impact on patient safety for impurities where the parent drug substance is extremely toxic and impurity levels (even mutagenic impurities) for oncology products could be controlled at higher levels. However, there has been little regulatory appetite for broadening this entirely pragmatic approach to other therapeutic areas, where life expectancy is equally short, i.e. rare diseases.
Keywords: Haber's law; Duration of dosing; Safety-based limits; Acceptable daily intakes; Acceptable intakes; Permitted daily exposure; Virtually safe dose; Less than lifetime limits
Introduction
Paracelsus, the medieval physician, who is often viewed as the father of modern toxicology, was the first person to appreciate that "the dose makes the poison". This essentially means that very toxic materials can be used therapeutically at very low concentrations and conversely even safe materials can be toxic if overdosed. This in turn led to Haber's law, which basically states that, the incidence and/or severity of any toxic effect is dependent on the total exposure to the toxic agent; that is, the exposure concentration (c) rate(or dose) multiplied by the duration time (t) of exposure (i.e. cxt). This law is often utilised in setting exposure limits for toxic components. The major caveat, is that establishing acceptable daily intakes (ADIs) for long-term exposures to a toxic substance when only data from short-term studies are available, does require the use of an uncertainty or safety factor.
For example, cancer risk estimates are typically based on the average lifetime daily dose (LDD), which in turn is derived from the total cumulative exposure, using Haber's law, i.e. cxt. Gaylor [1] proposed a modified Haber’s law to better extrapolate safe levels based on shorter term exposure intervals and this takes the form of:
c3x t=c’3x t'
where c and t are the known safe exposure levels(c) based on the longer exposure duration (t) and c’ and t' are the projected safe concentration (c’) based on the pre-defined shorter exposure duration (t’). Haber’s law is equally germane to impurities as it is to medicinal products.
Impurities in New Drug Substances and New Drug Products (Ich Q3a/ Ich Q3b)
One of the first international guidance that used safety based limits for impurities was the international conference on harmonization (ICH) Q3A [2]. This provided an overview of the typical impurities that were found in new drug substances and their controls. Impurities were evaluated based on both chemistry considerations, including "classification and identification of impurities, report generation, listing of impurities in specifications, and a brief discussion of analytical procedures"; and safety considerations, including "specific guidance for qualifying those impurities that were not present, or were present at substantially lower levels, in batches of a new drug substance used in safety and clinical studies"
Impurities were further delineated into identified and unidentified classes, both of which were included as specification tests [3]. This includes unidentified impurities that were known to be present at levels greater than pre-defined reporting, identification and qualification threshold (Table 1). Those unidentified impurities are often defined on the drug substance specification "by an appropriate qualitative analytical descriptive label (e.g., "unidentified A", "unidentified with relative retention of 0.9")"
1. The amount of drug substance administered per day;
2. Reporting Threshold: A limit above (>) which an impurity should be reported. Reporting threshold is the same as reporting level in Q2B (4);
3. Higher reporting thresholds should be scientifically justified;
4. Lower thresholds can be appropriate if the impurity is unusually toxic;
5 Identification Thresholds: A limit above (>) which an impurity should be identified;
6 Qualification Thresholds: A limit above (>) which an impurity should be qualified
The reporting threshold was linked to the capability of the supporting analytical methodology [4]. Identification threshold was the limit where the unknown impurity was required to be identified by appropriate analytical methodology. Whereas, the qualification threshold necessitated acquiring and evaluating pre-clinical safety data that "establishes the biological safety of an individual impurity or a given impurity profile at the level(s) specified".
Interestingly, although the derivation of the reporting threshold was linked to method capability, the derivation of the identification and qualification threshold limits were never fully delineated, apart from linkage with the maximum daily dose of the product. In addition, for those impurities "known to be unusually potent or to produce toxic or unexpected pharmacological effects, the quantitation/detection limit of the analytical procedures should be commensurate with the level at which the impurities should be controlled". Again, the implicit meaning of this statement was never fully articulated, but it was the genesis for the subsequent guidance on mutagenic impurities, initially termed genotoxic impurities [5].
Similar guidance was provided for impurities typically found in new drug products. These impurities are usually termed degradation products [6]. There was greater delineation of the thresholds in terms of dose (Table 2). However, it was never fully explained why the various thresholds, in terms of maximum daily dose, could not be aligned. Thus there is the confusing scenario that the reporting thresholds are above or below 1g; whereas, the identification thresholds are divided into four (>2g, >10mg- 2g, 1mg-10mg and <1mg); whilst the qualification thresholds were also divided into four, but were not aligned with the classes defined in the identification thresholds (>2g, >100mg-2g, 10mg- 100mg and <10mg).
1 The amount of drug substance administered per day;
2 Thresholds for degradation products are expressed either as a percentage of the drug substance or as total daily intake (TDI) of the degradation product. Lower thresholds can be appropriate if the degradation product is unusually toxic;
3 Higher thresholds should be scientifically justified.
The other confusing aspect was that the maximum daily dose (mg/day) and the maximum strength of a product (mg) are often not the same value. Thus for instance, the anti-malarial drug quinine sulfate [7] has a maximum therapeutic dose from the product label of 648mg every 8hours, i.e. 1944mg/day; whereas, the highest dose strength are 324mg/capsule. The analysts testing and releasing quinine sulfate capsules will do so on the commercial product (324mg/capsule), not the maximum dose taken by the patient (1944mg/day). In addition, although reporting thresholds are always measured as percentage values and are easily aligned with the data output from the method used by the analyst; identification and qualification thresholds are measured in either percentage values or mg/day values (Table 3).
The other aspect of having safety based limits for impurities is that it does not reflect the duration of treatment use for that drug and results in the same limits being proposed irrespective of whether the drug is proscribed pro ne rata (PRN) or as required, e.g. for constipation, mild pain, etc., or through life time treatments, e.g. for treatment of high blood pressure, etc.
ICH Q3A [2] and Q3B [6] were always intended to be only applicable to marketed products, but the regulatory expectations during clinical development often exceed what is actually required. For example, it isn't unusual to see the following expectations [8]:
"For phase I expect structure (or identifier) and origin For phase II expect Limit of Detection and Quantification and actual impurity levels to be established (aligned with ICH Q3A, Q3B, etc)".
However, by phase II the final synthetic route and process of the drug substance are rarely identified or optimised, and the attrition rate of phase II drugs is still very high [9]. What the regulatory guidance enshrined in ICH M3 [10], actually states with respect to impurities is, "If specific studies are warranted to qualify an impurity or degradant, generally these studies are not warranted before phase 3 unless there are changes that result in a significant new impurity profile (e.g., a new synthetic pathway, a new degradant formed by interactions between the components of the formulation). In these latter cases, appropriate qualification studies can be warranted to support phase 2 or later stages of development"
Residual Solvent Impurities (ICH Q3C)
Although residual solvents are mentioned in ICH Q3A [2], a separate guideline, ICH Q3C [11], was developed to provide safety based guidance on the allowable limits of common residual solvents within pharmaceuticals. As there are "no therapeutic benefits from residual solvents, all residual solvents should be removed to the extent possible to meet product specifications, good manufacturing practices, or other quality based requirements". Additionally, ICH Q3C recommends the use of less toxic solvents. Thus, solvents that are known to be highly toxic (Class 1) should be avoided during the production of drug substances, excipients, and especially drug products, unless their usage can be justified using a risk-benefit assessment [12]. In addition, some solvents with intermediate toxicity (Class 2) should also be limited from a patient safety perspective. Ideally, the least toxic solvents (Class 3) should always be used where practical. Recommended limits for all solvents may change as additional safety data become available. In addition, supporting safety data for new solvents may be added to the guidance.
Although tolerable daily intake (TDI) and acceptable daily intake (ADI) were both in common usage, ICH Q3C (12) introduced a new term, permitted daily exposure (PDE) to avoid confusion of differing values for ADI's for the same substance. In addition to avoidance of class 1 solvents, the concept of "as low as reasonably practicable" (ALARP) was introduced and is applied to class 2 solvents and often to class 3 solvents. Indeed, regulatory agencies will often use process capability arguments to drive down residual solvent levels below the safety based limits [13] derived from ICH Q3C.
Residual Elemental Impurities (ICH Q3D)
Residual elemental impurities were also mentioned in ICH Q3A [2], but again a separate guideline, ICH Q3D (14) was developed to provide safety based guidance on the allowable limits of residual elements within pharmaceuticals. As elemental impurities provide no therapeutic benefit to the patient, "their levels in the drug product should be controlled within acceptable limits" [14].
The ICH Q3D guideline is sub-divided into three parts: the derivation and assessment of toxicity data; the establishment of a PDE for each elemental impurity derived for three different routes of administration (oral, inhaled and parenteral); and application of a risk based approach to control elemental impurities (as per ICH Q9 (12)). One difference from ICH Q3C [11] is that applicants are not expected to tighten the safety based limits based on process capability considerations, as long as the residual elemental impurities do not exceed the PDE values. However, in certain cases, levels below the PDE may be warranted when lower levels have been shown to positively impact on other critical quality attributes (CQAs) of the drug product; for example, element catalyzed drug degradation (this is particularly common with oxidative degradation mechanisms [15]. In addition, for those elements with higher PDEs, lower limits may have to be assessed from a pharmaceutical quality perspective. Residual elements are classified into 5 different categories: class 1, 2a, 2b, 3 and others (Table 4).
ICH Q3D [14] provides a platform for developing an ICH Q9 (12) aligned risk-based control strategy to limit elemental impurities within the drug product. Although, the guidance had highlighted the risk inherent from both drug substance and excipients, the reality based on a multi-product assessment is that the risk is low. Li et al. [16] tested 190 samples from 31 different excipients and 15 samples from eight different drug substances for residual elemental impurities. The results show relatively low levels of elemental impurities are present in the samples tested.
Residual mutagenic impurities (ICH M7)
ICH M7 [5] is focused on DNA reactive impurities that can potentially cause DNA damage, when present at low levels, and thus can potentially cause cancer in man. Importantly, other types of toxic impurities that are non-mutagenic will typically have a threshold mechanism and as such usually do not pose carcinogenic risk in man, at the levels typically seen for impurities.
A Threshold of Toxicological Concern (TTC) approach was introduced to describe an "acceptable intake for any unstudied chemical that poses a negligible risk of carcinogenicity or other toxic effects", this equates to a virtually safe dose (VSD). The methodologies that underpin the TTC are universally considered to be very conservative, as they use a simple linear extrapolation from the TD50 dose (i.e. dose giving a 50% tumor incidence) to a 1 in 106 likelihood of cancer occurrence.
A default TTC value of 1.5μg/day corresponding to a theoretical 10-5 excess lifetime risk of cancer can therefore be justified for mutagenic impurities. Some high potency groups referred to as the "cohort of concern", e.g. aflatoxin-like-, N-nitroso-, and alkyl-azoxy compounds; were identified where the default TTC would still pose a significant carcinogenic risk. These high potency compounds were excluded from the TTC approach.
ICH M7 [5] bases acceptable intakes for mutagenic impurities on established risk assessment approaches (see ICH Q9 [12]. As such, acceptable risk during the early development phase is established at a higher theoretically calculated risk level of approximately one additional cancer incidence per million, i.e. 1 in 106 risk levels. For later stages in development (Phase III) and for commercial products, the risk level is reduced to one in one hundred thousand, i.e. 1 in 105 risk levels. It is worth highlighting, that these risk levels represent a small theoretical increase in risk when compared to the overall lifetime incidence of developing cancer in man, which is greater than 1 in 3.
The initial risk assessment is undertaken on the drug substance synthetic pathway to identify real or potential impurities that may be reactive and thereby mutagenic in nature. In parallel, the formulation and manufacturing process are also assessed for the formation of any reactive degradants (both real and potential), that could be realistically expected to form during long term, real-time storage conditions. In silico structure- based assessments, i.e. Derek Nexus, Sarah Nexus, etc., are used for predicting mutagenicity based upon QSAR (quantitative structure activity relationships) approaches. These findings are then reviewed by toxicology experts to provide any additional understanding as to the relevance of these predictions (both positive and negative), and in the case of contradictory outcomes to understand those differences. Based on this assessment, impurities are categorised into five different classes in order of decreasing regulatory concern (Table 5).
1. For class 1 compounds, i.e. those which are known mutagenic carcinogens, an AI (acceptable intake) or a PDE (permitted daily exposure)approach has been introduced (ICH M7(R1) (17)). These limits are based on either (i) linear extrapolations from TD50 (AI) or (ii) threshold-based PDEs. There are 10 compounds covered by the AI approach and a further 3 covered by the PDE approach.
2. LTL (less than lifetime limits)
3. TTC (Threshold of Toxicological Concern)
It is anticipated that monitoring and control strategies (including analytical methods) will be less developed during earlier clinical phases, where overall development experience is of necessity limited, compared to later clinical phases and commercial manufacture. ICH M7 [17] proposesa control strategy using four control options for mutagenic impurities, of these only one includes control of the mutagenic impurity on the API specification (option 1). Options 2 and 3 define some levels of in-process control; whereas, option 4 is centred on process understanding alone, typically termed "Purge Arguments" [18].
It is should be emphasised that these established cancer risk assessments are based on lifetime exposures, i.e. 75 years. Thus, Less-Than-Lifetime (LTL) exposure based limits can be derived both during development and commercial use. LTLs can have higher acceptable intakes of mutagenic impurities and still maintain comparable risk levels, which is obviously an application of Haber's law. Therefore, the carcinogenic effect is predicated on both duration of exposure and dose. Thus for example, "if the compound specific acceptable intake is 15 |ig/day for lifetime exposure, the less than lifetime limits (Table 6) can be increased to a daily intake of 100 |ig (>1-10 years treatment duration), 200 |ig (>1-12 months) or 1200|ig (<1month)" [5]. This LTL approach may also be appropriate "in diseases with reduced life expectancy, limited therapeutic alternatives or chronic diseases with late onset" [19].
It is worth emphasising that exceeding the default TTC or LTL limits is not necessarily linked with an increased cancer risk in man, given the extremely conservative suppositions employed in the evolution and derivation of the TTC or LTL values. For instance, higher exposure to a potential mutagenic impurity, e.g. formaldehyde, may be reasonable when exposure can be significantly greater from other sources, e.g. endogenous metabolism, food, etc. The most likely increase in cancer incidence is actually much less than 1 in 100,000. In addition, in cases where a mutagenic compound is a non-carcinogen in a rodent bioassay, there would be no predicted increase in cancer risk. Based on all the above considerations, any exposure to an impurity that is later identified as a mutagen is not necessarily associated with an increased cancer risk for patients already exposed to the impurity. A risk assessment would determine whether any further actions would be taken
In principle, ICH M7 does not apply to advanced cancer therapeutic indications (covered by ICH S9 (20)), where the drug is itself genotoxic. ICH M7 does not apply to established excipients, flavouring agents and certain biological products, including herbal medicines. Existing commercial products are also exempted, apart from where there are new safety data (including new mutagenic data) for existing impurities; significantly, structural alerts alone do not trigger regulatory concern. However, ICH M7 does cover changes to marketed products, including new marketing applications and postapproval submissions.
Impurities in oncology products (ICH S9)
ICH S9 [20] was developed to provide guidance for nonclinical studies for the development of anticancer pharmaceuticals used in clinical trials for the treatment of patients with advanced disease and limited therapeutic options. During the development of oncology products, supporting clinical studies often involve cancer patients whose prognosis is poor and projected lifetime is short (<2 years).
As such, the guideline objectives are to facilitate and accelerate the development of these anticancer pharmaceuticals whilst protecting patients from unnecessary adverse effects. In addition, ethical use of animals, in accordance with the 3R principles (reduce/refine/replace) are paramount. Importantly, the principles described in other ICH guidelines need to be considered in the development of oncology products; whereas, those specific situations where requirements for nonclinical testing may diverge from other guidance are described in ICH S9.
Additionally, the dose levels used in these clinical oncology studies are often at the top end of the tolerable range [21] and often result in adverse effect dose levels. Hence, "the type, timing and flexibility called for in the design of nonclinical studies of anticancer pharmaceuticals can differ" significantly from nononcology pharmaceuticals. Historically, limits for impurities (see ICH Q3A [2] and Q3B [6] have been based on a negligible risk to the patient. In oncology products this consideration, whilst important, is not as important as patient wellbeing and exceeding the ICH Q3A [2]/Q3B [6] limits for impurities may be applicable and an appropriate justification should be included in the marketing application.
This explanation should include an overview of the disease being treated, including patient prognosis, the nature of the drug itself (pharmacology, genotoxicity and carcinogenicity, etc.), the total duration of treatment, and the impact of any reduction in impurity levels on manufacturability. Furthermore, the qualification of these impurities may include reflections on the concentration tested in supporting nonclinical studies compared to the levels seen in clinical batches. In addition, TTC, LTL and AI/PDE limits for mutagenic impurities (see ICH M7 [5]) are inappropriate for oncology products and justifications can be used to set higher limits. Interestingly, the guidance does not specifically say that the applicant can default to ICH Q3A [2]/ Q3B [6] limits, although this is often inferred. Impurities that are also metabolites can be considered to be suitably qualified.
Interestingly, regulators have been very unwilling to extend the philosophy of ICH S9 [20] beyond oncology products, for instance into rare diseases [22], where lifetime expectancy can be similarly short, i.e. <2 years and where patient expectations are equally high.
New Reflections on Impurities
Harvey et al. [23] used a variety of chemical databases to demonstrate that the 1mg/day impurity level for an unqualified impurity of unknown toxicity, proposed by ICH Q3A [2] (Table 1) is indeed a robust prediction of a virtually safe dose (VSD) for non-mutagenic impurities. Then using the modified Haber's law, where C=1 mg and t=75 years (i.e., 27375 days) and t is 6 months or 182 days they determined a VSD for this shorter exposure interval of 5 mg/day (i.e. 5 times higher than existing ICH Q3A limit). However, for very potent drugs with effective doses of <1mg, a 5mg/day limit for a related impurity isn't realistic from either a safety or quality perspective. Therefore, the authors also introduced a percentage cut off based on 5x the ICH Q3A qualification threshold of 0.15%; i.e. 0.7%. Thus the proposed limits for drug substances are 5mg or 0.7%, whichever is lower.
This allows applicants to adopt the existing ICH Q3A guideline which were developed for commercial products and apply them to development products, in much the same way that the ICH M7 guidelines allows LTL limits for mutagenic impurities, for early clinical development. For drug products, similar LTL limits for non-mutagenic impurities can be derived based on a modification of Haber’s law. The additional constraint of a percentage limit of 0.7% need not be applied to drug products as the more potent the drug substance becomes, the lower the dose required. The authors therefore suggested a limit of 5 mg or 2%, whichever is lower, for exposure intervals of <6 months, for general drug substance impurities, i.e. non-mutagenic.
In addition to absolute amounts of unknown impurities, the other key focus is those impurities with unusually high and/ or specific toxicities. Whilst it is recognised that mutagenic impurities constitute the greatest threat to patient safety and they have been addressed via ICH M7; there are other classes of non-mutagenic impurities that will still give cause for concern. The three principal classes of toxic impurities are
(i) polyhalogenated, dibenzodioxins, dibenzofurans and biphenyls that are non-mutagenic carcinogens, which have specific regulatory framework with respect to acceptable exposure levels [24],
(ii) organophosphates or carbamates that are neurotoxins and have their own threshold of concern [25] and (iii) β- lactam like impurities that have the potential to cause anaphylaxis and which currently do not have any threshold of concern [23].
It is worth highlighting that
(a) These impurities are extremely rare and do not reflect the typical structure of impurities generated by medicinal research [26,27] and
(b) That these structural motifs (if present) would be highlighted and addressed as part of the ICH M7 risk assessment [5] , as "the findings from any mutagenic risk assessment are also reviewed by toxicology experts"
Conclusion
Safety based impurity limits are a core consideration of all of the existing ICH Q3 guidance documents. However, there has been an evolution in the approach toward impurities since the publication of the initial guidance [2,6]. Whereas, ICH Q3A [2] and Q3B [6] provide general guidance on impurities in drug substances and drug products, respectively and mainly focus on absolute levels of impurities, i.e. percentage based limits; later guidance focused on individual impurity classes; i.e. residual solvents [11], residual elemental impurities [14] and mutagenic impurities [5] and had a greater focus on daily exposure limits. In the latter cases, this led to the introduction of various impurity specific limits, such as PDEs, Als, TTCs and staged TTCs, all aimed at defining a virtually safe dose (VSD). This in turn led to the introduction of LTL limits for mutagenic impurities.
LTLs are based on an application of Haber's law which states that concentration and exposure times are both critical for assessing likely safety risk to patients. Surprisingly, LTLs have not been applied to the other specific classes of impurities or indeed general impurities. In order to address this deficiency, Harvey et al. [23] have assessed the underpinning data behind the current "1mg or 1%, whichever is lower" limit in ICH Q3A [2] /Q3B [6], and they found this to be based on robust science and they proposed an ancillary LTL for general impurities of 5 mg or 0.7%, whichever is lower, for clinical studies with durations of less than 6 months.
Logically, the toxicity of the parent drug substance also affects how we deal with impurities, even very toxic impurities. Thus, there is limited, if any, additional impact on patient safety for impurities where the parent drug substance is mutagenic, carcinogenic or cytotoxic. Accordingly it was recognised that for oncology products, impurity levels (even mutagenic impurities) could be controlled at higher levels. This "higher level" wasn’t defined but is based on an overview of the disease being treated, including patient prognosis, and the nature of the drug itself. Additionally, from a risk based perspective and an understanding of Haber's law, an overt focus on impurity control makes little sense if the life expectancy of the affected patient is short, i.e. less than 2 years [20]. Interestingly, there has been little regulatory appetite for widening this entirely pragmatic approach to impurity control to other therapeutic areas, where life expectancy is equally short, i.e. rare diseases [22].
Conflict of Interest
The author is an individual CMC consultant and declares that there are no conflicts of interest.
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Nasal Delivery of Proteins and Peptides-Juniper publishers
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Nasal delivery of protein/peptide drugs has gained considerable amount of attention over the past decade, since it has several advantages over the oral delivery as it avoids harsh gastrointestinal environment, has relatively less enzymatic degradation and relatively highly permeable epithelial mucosa. Nasal products of several small molecular weight (MW < 4000Da) protein/peptide drugs have been approved by the US FDA. This review is focused on the challenges associated with nasal delivery of protein/peptide drugs and possible solutions through various drug delivery technologies.
Keywords : Nasal absorption; Intranasal delivery; Proteins; Peptides; Nasal mucosa
Introduction
Recent years have shown that nasal route can be a potential route for the systemic delivery of protein/peptide drugs as it has a considerably large absorption area (150cm2) which is highly vascularized and has permeability similar to or higher than the small intestinal mucosa. Nasal delivery of protein/peptide drugs also offers other benefits such as ease of administration, noninvasive administration, rapid onset of action, and the avoidance of gastrointestinal degradation and hepatic first-pass effects [1]. Nasal route can be utilized to enhance the delivery of drugs to the brain as nose to brain delivery bypasses the blood-brain barrier [2]. However, nasal delivery also suffers from several limitations such as low membrane permeability to hydrophilic molecules (especially when the molecular weight is over several thousand daltons), small applicable volume per dose, enzymatic degradation, and mucociliary clearance [3].
Thus far, several protein/peptide drugs with largest molecular weight being 3432 Da, have been successfully formulated into nasal delivery products and approved by the US FDA. These commercially available drugs include Desmopressin (1183Da), Nafarelin Acetate (1321Da), Oxytocin (1007Da), and Salmon Calcitonin (3432Da) [4]. Nasal delivery of larger molecular weight protein/peptide drugs still remain a challenge, mainly due to the low permeability caused by the drugs being hydrophilic and large in size. Several drug delivery technologies have been explored for these macromolecules but have gained limited success.
hallenges & Delivery Technologies
Main limitation to nasal delivery of large molecular weight protein/peptide drugs is the low membrane permeability. The rate of permeation is highly sensitive to molecular size for compounds with molecular weight (MW) ≥300 Da [5]. A large number of therapeutic agents, peptides and proteins in particular have shown that for compounds >1k Da, bioavailability can be directly predicted from the knowledge of MW. In general, the bioavailability of these large molecules ranges from 0.5% to 5% [6]. Use of permeation enhancers can help in increasing the transport of proteins and peptides across the nasal membrane [7].
Absorption enhancers such as bile salts, surfactants, fluidic acid derivatives, phosphatidylcholines, cyclodextrins and cell- penetrating peptide (CPP) have been studied to enhance the intranasal absorption of protein/peptide drugs [8]. Absorption enhancers are effective in improving the nasal absorption by either increasing the fluidity of the bilayer of the epithelial cell membrane and there by opening aqueous pores as a result of calcium ion chelation or by increasing the intracellular delivery using functional moieties [7,9]. Mucolytic agent along with nonionic surfactant has been shown to enhance the nasal absorption of calcitonin in rats and achieved 3.5 times higher bioavailability as compared to the commercial calcitonin nasal spray Miacalcin [10].
Delivery of insulin from nose to the distal regions of the brain was significantly enhanced by L- or D-penetratin as CPP [9]. However, absorption enhancers tend to cause severe nasal irritation and damage the nasal membrane at the concentrations required to effectively promote the nasal absorption [7]. Drug carrier systems such as liposomes, emulsions, nanoemulsions, nano/micro particles and niosomes with permeation-enhancing function, have been evaluated to deliver protein and peptide drugs through nasal cavity. Intranasal delivery of human growth hormone (hGH) - 22kDa protein, using glutathione (permeation enhancer) added microparticles as delivery system increased the relative bioavailability approximately by three folds as compared to the microparticles without glutathione [4]. H102, a novel β-sheet breaker peptide, was encapsulated into liposomes to reduce its degradation and increase its brain penetration through intranasal administration for the treatment of Alzheimer's disease [11].
The study showed 2.92 fold larger AUC in hippocampus with liposomes than a solution of H102 peptide. Mitra et al. [12] reported enhanced absorption of insulin through rat's mucosa using emulsion system. The AUC was observed 4 times higher when insulin was loaded in o/w emulsion as compared to pure buffer solution of insulin. Sintov et al. [13] demonstrated that intranasal delivery of o/w micro emulsion system with 20% water content (insulin in aqueous phase) achieved an absolute bioavailability of 7.5% using rabbit as an animal model. Mucociliary clearance (MCC) is a normal defense mechanism of the nasal cavity that clears mucus as well as substances adhering to the nasal mucosa (bacteria, allergens etc.) and drains them into the nasopharynx for eventual discharge into the gastrointestinal tract. Whenever a substance is nasally administered, it is cleared from the nasal cavity in about 21 min [14]. Thus, MCC reduces the retention time of the drug at the absorption mucosa, resulting in a low bioavailability [3].
Use of mucoadhesive agents has been explored to prolong the intimate contact time of the formulation on the nasal mucosa by adhering to the surface of the mucus layer and thereby enhancing the bioavailability. Nazar et al. [15] studied the effect of insulin loaded hydrogels and observed significant decrease in glucose levels in diabetic rats over the period of 24h. However, it is generally difficult to achieve a satisfactory nasal absorption of macromolecular drugs by increasing the retention time alone because it has to simultaneously overcome the physical barrier of the epithelium for a drug to permeate into the systemic circulation. Also intranasal formulations with mucoadhesive agents have not gained much of a commercial success due to the patient discomfort.
Proteolytic enzymes (amino peptidases and proteases) present in nasal mucosa can be another barrier for intranasal delivery of protein/peptide drugs although the enzymatic activity in nasal cavity is relatively lower than the gastrointestinal tract [16]. Zhou et al. [17] found that the amino peptidase activity in nasal tissue was about half of that in the intestinal tissue. Enzyme inhibitors have been investigated to protect the protein/peptide drugs from the enzyme activity present in nasal mucosa [18]. Inhibitors with a trypsin-inhibiting activity have been proved to be useful in enhancing the nasal absorption of salmon calcitonin [19].
However, proteolytic enzyme inhibitors themselves cannot facilitate the penetration of drugs across the epithelial membrane and therefore, are generally unable to considerably improve bioavailability in the absence of absorption enhancers. Furthermore, the enzyme inhibitors will affect the normal metabolism of the body, resulting in serious side effects. Therefore, enzyme inhibitors do not seem to be an effective and safe method of improving the nasal absorption of proteins and peptides [20]. Another limitation to nasal delivery is the applicable volume of the formulation which is restricted to 25- 250μL per dose. Several approaches have been explored to use this volume effectively which includes the use of solubilizers and gelling agents [21].
Conclusion
Low molecular weight protein/peptide drugs have been effectively delivered via nasal mucosa. But there is still a need to develop drug delivery systems that can deliver the large molecular weight protein/peptide drugs via nasal route.
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