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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Polyethylene Glycol-Based Solid Dispersions to Enhance Eprosartan Mesylate Dissolution and Bioavailability

Authored by  Ismaiel A Tekko*,

Abstract

Purpose: The aim of this study was to improve the dissolution of eprosartan mesylate (ESM) using solid dispersions (SDs). Various compositions of polyethylene glycol polymers (PEG) were tested in attempt to improve ESM bioavailability after oral administration.

Methods: ESM solid dispersions (SDs) were prepared (by solvent method) using mixtures of PEG 3350 or PEG 6000 at various drug-polymer ratios (1:1), (1:2), (1:3), (1:4), (1:5) w/w with or without Tween 80 added at 0.1% of drug weight. Physical mixtures (PMs) of drug and carriers were also prepared at same ratios and used. Drug solid dispersions and physical mixtures were characterized in terms of drug content and drug dissolution using USP II dissolution apparatus. Drug dissolution enhancement ratio (ER) from SD in comparison to the crude drug was calculated. Drug- polymer interactions were also evaluated using Differential Scanning Calorimetry (DSC) and Fourier transform infrared (FT-IR).

Results: The in vitro dissolution studies showed that SDs prepared using both polymers produced a remarkable improvement in drug dissolution which reached around 32% in comparison to the crude drug (p<0.05). The dissolution enhancement ratio was polymer-concentration dependent but up to a certain concentration and this varied according to the used polymer. Adding Tween 80 to the SD did not show further dissolution enhancement but reduced the required amount of the polymer to get the same dissolution enhancement. The DSC and FT-IR studies indicated that using SD resulted in transformation of drug from crystalline to amorphous form.

Conclusion: This study indicated that SD prepared by using both polymers i.e. PEG 3350 and PEG 6000 improved the in vitro dissolution of ESM remarkably which which could result in improving in improving the drug bioavailability, but further in vivo studies are required (Graphical Abstract).

Keywords: Eprosartan mesylate; Solid dispersions; Solvent method; PEG 3350; PEG 6000; Dissolution test

Introduction

Cardiovascular diseases are the cause of most mortalities worldwide with a staggering 17 million deaths per year [1]. One of these lifelong cardiovascular diseases is hypertension. Hypertension has a silent mode of progression which results in only one out of four hypertensive people to seek medical care [1]. Hypertension can be controlled by different therapeutic strategies using one or combination of drugs that acts like beta blocker, angiotensin-converting enzyme inhibitors or Angiotensin II receptors antagonists [2]. Eprosartan mesylate (ESM) is a potent antihypertension drug marketed as oral tablets (Teventen®). It acts by antagonising the Angiotensin II receptors, resulting in peripheral blood vessels wideness and blood pressure reduction [3]. The drug decreases sympathetic norepinephrine production which also results in blood pressure reduction as well [4]. The drug is not only well-tolerated but it also presents effective benefit in the secondary prevention of cerebrovascular events. ESM has a low potential for serious adverse events and has not been associated with any clinically significant drug interactions. All of these demonstrate ESM as a promising agent for solo or combination antihypertensive strategies [5]. The recommended daily starting and maintenance dose of ESM when used as monotherapy is 735.8 mg, equivalent to 600 mg Eprosartan, available as the commercial Teveten® 600 mg tablets. In parallel, ESM has low water solubility (< 0.1 mg/ml) and is classified as a Class II drug according to Biopharmaceutical Classification Systems (BCS). Considering the drug water-solubility value, the drug requires over 5 L of water to dissolve the total dose which consequently lead to low and variable oral bioavailability (approximately 13%) [6]. Furthermore, this is a relatively high dose and requires to be incorporated into large tablets which are less preferred in terms of patient compliance [7]. ESM successful formulation strategy needs to address drug solubility which is the rate-limiting step in drug bioavailability

To overcome the issue problem of low oral bioavailability of ESM, efforts to increase its dissolution rate are needed. Several techniques have been proposed to increase the dissolution rate of poorly watersoluble drugs like salt formation, micronization [8], surfactants addition [9] and solid dispersions (SDs) [10]. SD method involves a dispersion of one or more active ingredients in an inner carrier or matrix in the solid state [11]. This method has many advantages like particle size reduction, improving wettability properties of the compound surface, and drug transformation into its amorphous form [12]. Various carriers have been employed including Polyethylene glycols (PEGs) [13,14], Poly(vinylpyrrolidone) (PVP) [15], Polyacrylate [16] etc. A further increase in drug solubility and dissolution can be achieved by the addition of inclusion surfactants such as Tween 80 in the formulation containing a polymeric carrier. This is attributed to the surfactant ability to prevent precipitation and/or protect a fine crystalline precipitate from agglomeration into much larger hydrophobic particles, thus enhancing the wettability of particles [11,17]. For these reasons, SD technique was selected as formulation strategy for ESM. Different methods have been proposed to prepare SD including fusion method, spray drying, lyophilization, supercritical fluid, and solvent method [18]. Solvent method was selected in this work for ESM solid dispersion formulation. SD prepared using PEG polymers mixtures (PEG 3350 and PEG 6000) with Tween 80 at certain ratios have been used successfully to enhance the solubility and dissolution rate of several molecules like Daidzein [17], Piroxicam [11], Silybin [13] and Norfloxacin [14]. However, these well studied polymeric combination have not been reported to enhance the dissolution rate of ESM. The objective of this study was to evaluate the potential of enhancing the dissolution rate of EMS with SD technique using two grades of PEG polymers; PEG 3350, PEG 6000. The PEGs were tested individually at various (drug: carrier) ratios or in combination with 0.1 %w/w Tween 80 of drug weight. Physical mixtures of the same carriers at the same ratios were also prepared and evaluated as controls. The chemical structure of ESM and carriers are presented in Figure 1. The prepared SDs were characterized in terms of their drug content, in vitro drug dissolution, thermal properties using differential scanning calorimetric (DSC) and drug-carrier molecular interactions using Fourier Transform Infrared Spectroscopy (FTIR). Also, formulations morphology was evaluated using a highresolution microscope and compared with the crude drug and its physical mixtures (Figure 1).

Materials and Methods

Eprosartan mesylate of pharmaceutical grade (batch no. EM0061010, purity 99%) was purchased from Andhra Pradesh, India. PEG 3350 and Tween 80 were purchased from Sigma-Aldrich, Germany. PEG 6000 from Sasol, Germany. Nylon filters (0.45 μm, 13 mm) obtained from Millipore, USA. All other used chemicals and solvents were of analytical grade and were provided from Sigma- Aldrich, Germany.

Preparation of solid dispersions (SDs)

Solid dispersions of ESM (SD-ESM) were prepared by solvent method [19]. ESM, PEG 3350, PEG 6000 and Tween 80 were accurately weighed using Sartorius R180D analytical lab scale balance (Sartorius GmbH, Germany) and mixed according to Table 1. The mixtures were dissolved in an appropriate volume of methanol and acetone (25:75 v/v). Solvents were, then, evaporated at 45°C under reduced pressure using a Rotary Evaporator (Buchi 215 Rotavapor, Germany) operated at the speed of 100 rpm. The final obtained mass was cooled immediately using an ice bath and then pulverized and passed through a sieve with a pore size of 112 μm to produce the SDs. The SDs were left to dry for 48 hours at room temperature to remove residual solvents and then stored in airtight containers for further tests (Table 1).

Table 1: Composition of the various formulations of eprosartan mesylate solid dispersion (ESM-SD). PEG stands for polyethylene glycol.

Preparation of physical mixtures (PMs)

Physical mixtures having the same composition of solid dispersions were prepared by simply triturating and homogenizing the ESM drug with the polymers and Tween 80 in a porcelain mortar. The mixtures were then sieved with a sieve of pore size 112 μm and stored in airtight containers for further tests.

Eprosartan mesylate assay

A quantitative drug assay was carried out employing a developed and validated HPLC method from Patel and co-worker [20], using an apparatus of (HPLC LC 20 – AT, Shimadzu, Japan) equipped with DAD (SPDM20-A/D2 ) and auto-sampler, ODS column (RP C18, 5μ,ID: 250×4.5mm) housed at 25ºC. The mobile phase consisted of formic acid 0.5%: methanol: and acetonitrile (80:25:20 v/v/v) pumped at 1 ml/minute. ESM drug was detected at wavelength of 232 nm. ESM peak. The developed assay was checked for linearity, selectivity, precision, intermediate precision and accuracy.

Eprosartan mesylate formulation yield

The SD`s drug content was determined in triplicates by accurate weighing of amount equivalent to 50 mg of ESM. These samples were dissolved in 10 ml of methanol in stopper conical flasks. The sealed flasks were agitated on a rotary shaker for 1 hour. The solution was diluted with methanol, filtered and analysed for ESM content using the above described HPLC method. The yield of formulation was calculated using the following equation:

Where: Q weighed is the amount of drug weighed at the beginning of SD preparation. QHPLC is the amount of drug detected at the end of SD preparation.

In vitro

dissolution

The In vitro dissolution studies were carried out for 1 hour in triplicate for crude ESM, PMs and SDs using Copley (Copley Scientific, UK) dissolution test apparatus Type II (Paddle) at rotation speed of 75 rpm. The dissolution medium was 1000 ml phosphate buffer 0.2M at pH=7.5. The temperature was maintained at 37±0.3°C throughout the study [21]. Predetermined weights of crude ESM, PMs and SDs equivalent to 300 mg of ESM were taken for test. At predetermined time intervals (0, 15, 30, 45 and 60 minutes), samples of (5 ml) were withdrawn and replaced with an equal amount of fresh dissolution medium. The withdrawn samples were filtered through 0.45 μm nylon filters and analysed by HPLC at 232 nm (method in section 2.3). Dissolution profiles were plotted as the mean percentage of released drug versus time. The percentage of dissolution enhancement percentage (DE%) was calculated using the following equation:

Where: QSD is the amount of drug released from its solid dispersion after 15 minutes of dissolution test commencing. QPD is the amount of drug released from its crude drug powder after 15 minutes of test dissolution test commencing.

Differential scanning calorimetry (DSC)

The thermal behaviour of crude ESM, empty carriers, PMs and SDs, were evaluated using differential scanning calorimetry (DSC, model PT10, Linseis, Germany). DSC measurements were performed at a heating rate of 10°C /min within a range of 25- 310°C using aluminium pans without seals. An empty aluminium pan was used as a reference. Samples of 3-10 mg of crude ESM, empty carriers, PMs, and SDs were accurately weighed, placed in aluminium pans and tested to determine melting point (Tm) for the crystalline form and Glass Transition Temperature (Tg) for amorphous form of ESM.

Fourier transform infrared spectroscopy (FT-IR)

The infrared spectra of crude ESM, empty carriers, PMs and SDs were recorded using FT-IR (Bruker, Germany) Samples were prepared using KBr disk method with around 5 mg of sample mixed with KBr at 2%, the scanning was performed between 400 to 4000 cm-1. OPUS Spectroscopy Software was used for the normalization and treatment of the obtained spectra.

Microscopic studies

A sample of SDs prepared with PEG3350 in ratio (1:2) was taken to study the shapes and diameters of SDs using Olympus CX31 (Olympus Corporation, Japan) photos were compared to those of crude ESM.

Statistical analysis

One Way Analysis of Variance (ANOVA) was used to determine the presence of any significant differences in dissolution rates (p<0.05) among the test groups. All other figures were created using Microsoft Excel 365 software (Microsoft, USA).

Results and Discussion

Eprosartan mesylate assay

HPLC is the method choice for the quantification of active substances in formulation due to its ability to separate excipients by chromatography [22]. The developed method was selective for ESM in the presence of other SD formulation excipients and the retention time for ESM was 15.8 minutes. The method was linear for ESM over a concentration range from 10 to 200 μg/ml with a correlation coefficient of r2=0.99999. Table 2 shows the precision of the method with RSD% of less than 1.5% recorded at all tested concentrations (n=3). The intra-day and inter-day precision was also established and all RSD% values were less than 1.5%. Finally, method accuracy was investigated by injecting a reference concentration of ESM and compared the results obtained to the true value Table 3. shows accuracy values, where the percentage yield falls in the range of 99.8 to 101.0 %. Based on the below data, the developed HPLC method can be regarded as a suitable method for ESM analysis in the subsequent steps of the study (Table 2,3 and Figure 2).

Table 2: Intra-day and Inter-day precision values for eprosartan mesylate HPLC detection method. Data reported as mean ± SD (n=3).

Table 3: Accuracy values for eprosartan mesylate HPLC detection method. Data reported as mean ± SD (n=3).

Eprosartan mesylate formulation yield

ESM was successfully formulated in different SDs, the drug content of formulations was found to be in the range (94 -102 %) by HPLC method (section 2.3 and 3.1). Thus, the proposed solvent method which was used to prepare the SDs in this study appears to be a good and reproducible method to prepare a stable and homogenized SDs.

In vitro

dissolution studies

Dissolution profiles of the solid dispersions of ESM prepared with either PEG3350 or PEG 6000 alone and in combination with Tween 80 added at constant concentration (0.1%, w/w) in relation to the drug itself are shown in Figure 3. In vitro dissolution test exhibited a considerable improvement in the amount of released drug from solid dispersions in comparison to that from crude drug in the dissolution medium used (p<0.05). The percentage of released drug after 15 minutes from dissolution testing of the crude ESM and its SDs and the enhancement ratios (ER) are shown in (Table 4). The percentage of released drug from the crude drug was around 70% of the dose after 15 minutes and 86% after 60 minutes of the dissolution testing (Figure 3a). Applying the solid dispersion technique using polymers of PEG 3350 or PEG 6000 lead to a significant dissolution enhancement ranged between 19.5% and 31% in comparison to that from the crude drug (p<0.05) (Figure 3a,3b). Using PEG 3350 and PEG 6000 polymer as carriers in ESM-SD enhanced ESM dissolution rate independent of polymer ratio (p>0.05). Within the first 15 minutes of the study. The highest dissolution enhancement ratio was obtained at (drug: carrier) of 1:5 and with PEG 3350 and was 31%, while the lowest was 19% and was observed with PEG 6000 at drug: carrier ratio of 1:1. After one hour in the dissolution medium, more than 90% of ESM was released from all formulations indicating a complete drug release. The SD 20 formulation with PEG 6000 and Tween 80 enabled the highest drug release with highest dissolution enhancement percentage of 40% (Figure 3d). This can be brought back to the reduction of particle size and the increase of formulation wettability by the addition of Tween 80 [12,23]. Surprisingly, the lowest DE% (24%) was observed with SD 15 which differs from SD 20 only by the grade of PEG used (3350 with SD 15 and 600 with SD 20). In general, SDs formulated with PEG 6000 and Tween 80 showed the fastest and the highest dissolution rate for ESM (Figure 3 and Table 4).

Table 4: In vitro drug release of eprosartan mesylate (ESM) from solid dispersions (SDs) prepared with PEG 3350, PEG 6000 with or without Tween 80 in the first 15 minutes of dissolution testing. DE% stands for dissolution enhancement percentage and PEG stands for polyethylene glycol. Data reported as mean ± SD (n=3).

Differential scanning calorimetry (DSC) studies

The molecular interactions between the drug and the polymers used in preparation of the solid dispersions are evaluated by conducting a thermal analysis on the fabricated solid dispersions using DSC. Thermograms of crude ESM and ESM-SDs are shown in Figure 4. The thermograms of crude Eprosartan Mesylate (ESM) and its solid dispersions (SDs) with PEG 3350 and PEG 6000 alone and in combination with Tween 80: (a) SDs with PEG3350 (b) SDs with PEG 6000 (C) SDs with PEG 3350 + Tween 80 and (d) SDs with PEG 6000 + Tween 80. PEG stands for polyethylene glycol (Figure 4).

Thermal study of SDs prepared with PEG 3350: The thermal study of ESM and PEG3350 are presented in Figure 4a. The thermogram of ESM showed a sharp endothermic peak at 256°C corresponding to the melting point of the drug [24]. Regarding SDs prepared with PEG3350, a wide peak appeared around 60°C which is the melting point of PEG 3350 [25]. Physical mixtures thermograms exhibited both the drug and the polymer peaks, but broadened ones with reduced intensity Figure 4a. This could be attributed to the complete miscibility of molten drug in the polymer, as a result of homogeneous distribution of drug in the surface of the polymer [26]. With PEG 3350 SDs, only formulation with ratio of (1:1) showed a small peak at 247°C. and this can be explained by part of ESM is still in a crystalline form [27]. Whereas all other thermograms of PEG 3350 SDs showed only the peak of PEG3350 with the absence of ESM peak. This indicates that the whole drug has changed to amorphous form [28].

Thermal study of SDs prepared with PEG 6000: Thermograms of SDs of ESM prepared with PEG6000 are presented in (Figure 4b). PEG 6000 thermograms show and endothermic peak at around 70°C which is also observed elsewhere and attributed to the oxidation of PEG 6000 [29]. SDs thermograms showed the presence of PEG 6000 peaks and a very small peak related to the melting point of ESM and shifted towards lower temperatures. This peak is clearly observed in the curve of SD (1:1) at 251°C (Figure 4b). This could be explained by some amount of ESM still in crystalline form and not molecularly dispersed in the polymer matrix [30].

Thermal effect of Tween 80 addition to SDs prepared with PEG 3350 and EPG 6000: For SDs prepared from ESM mixed with PEG 3350: Tween 80 (Figure 4c). and PEG 6000: Tween (Figure 4d). respectively. The DSC studies showed that the thermal behaviour of a PEG-Tween 80 mixture is similar to that of the PEG`s alone in SDs formulations. This is a further confirmation to what was observed by Morris et al, that Tween 80 up to 50% doesn’t influence PEG melting point and Tween 80 is incorporated into the amorphous region of PEG solid structure [31]. ESM peaks were absent from the thermograms of ESM-SDs which indicates that the drug has been transformed into amorphous form with SDs [28].

FTIR spectroscopy was carried out to further elucidate the interaction between ESM and the carriers in the SDs. The FTIR spectrum of crude ESM is shown in (Figure 5), ESM has three characteristic bands for the OH groups which were found at 2931, 2957 and 3105 cm−1. The peaks corresponding to two CO groups are found at 1693 and 1714 cm−1. The peak for the SO2 group appeared at 1217 cm−1. The peaks come in agreement to previous reports on ESM IR spectrum [32]. The ESM-SDs (1 to 20) are shown in (Figure 6), the spectra of PEG3350, and PEG6000 are similar due to their similar structure and exhibited a broad OH stretching vibration from 3200 to 3650 cm−1 (Figure 1,6a,6b). The peaks obtained with our sample are different from previously reported [33,34]. This can be explained by the source of PEG, the machine type, preparation method of sample and residual and impurities found in PEG [35]. In the spectra of SDs the absorption bands of OH groups were changed or disappeared. It could be attributed to hydrogen bonds formed between the OH groups of ESM and the oxygen atoms in the polymers [36]. In addition, a broadening of transmittance occurred also for CO and SO2 groups in ESM (Figure 1). These observations might indicate the presence of hydrogen bonds between the drug and the carriers resulting in reducing in drug recrystallization [37], these types of hydrogen bond extending the stability of the amorphous form of Griseofulvin in the solid dispersions [38]. It is worth noting that previous report on ESMSD prepared with polyvinyl alcohol (PVP K 30) as carrier did not show any interaction between ESM and PVP [32]. The addition of Tween 80 to SDs did not show any clear change in the recorded FTIR spectra (Figure 6c,6d) when compared to SDs without Tween 80 (Figure 6a,6b). The broadening of some of ESM characteristics peaks and the absence of others in SDs formation suggest that ESM is in its amorphous state with the solid dispersion. the amorphous form of ESM presents a higher dissolution ability than the crude drug [24] (Figure 5,6).

Microscopic studies

The higher in vitro dissolution rate of ESM from SD can also be attributed to the lower particle size obtained for ESM after incorporation into SDs. Microscopic images of crude ESM and ESM-SD 2 proofed the reduction of particle size of ESM in the SD compared to crude form (Figure 7a,7b). The lower particle size leads to higher surface area and higher dissolution rate according to Noyes–Whitney equation [39], (Figure 7).

Conclusion

Eprosartan mesylate, a hypotensive agent, suffers from poor water solubility and reduced oral bioavailability. The present study investigates solid dispersions of eprosartan mesylate prepared with the water-soluble carriers PEG 3350, PEG 6000 individually and in combination with Tween 80 using solvent evaporation method. The use of solid dispersion strategy showed significant increase in vitro dissolution rate of eprosartan mesylate. All formulations doubled the release rate of eprosartan mesylate compared to the crude drug within the first 15 minutes of the study. At the end of the in vitro dissolution study, the released eprosartan mesylate was 10% higher in SDs prepared with PEG 6000 and Tween 80 than crude drug. This observation could be interpreted by the amorphous nature of eprosartan mesylate inside solid dispersion formulations confirmed by FTIR studies and the increased wettability of formulation by the addition of Tween 80 and the hydrophilic polymer (PEG). Also, microscopic images showed a decrease in particle size between the crude drug and the solid dispersion which increased the surface area between the formulation and the dissolution medium and lead to an enhanced drug release rate. This formulation strategy can be used to improve the oral bioavailability of eprosartan mesylate. Further studies are required to define the correlation between the observed in vitro results and the in vivo situation.

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Considerations in the Quality Control towards Translation of Extracellular Vesicles Derived from Eukaryotic Prokaryotic Cells

Author by Jin Gao

Introduction

According to concept of ISEV (The international Society for Extracellular Vesicles), “extracellular vesicles (EVs) are defined as the particles naturally released from cells that are comprised of a lipid bilayer membrane [1]. Acting as important mediators between cells that regulate both physiological and pathological conditions in the living bodies, EVs are nanosized spherical compartments and contain lipids, proteins and various nucleic acids of their source cells [2,3]. Based on their biogenesis and sizes, EVs are generally categorized into three types, including exosomes, microvesicles and apoptotic bodies. In general, sizes of EVs vary within the range 30- 5000 nm [4,5]. Besides EVs derived from eukaryotes, prokaryotes also secrete EVs. It has been reported that Gram-positive and Gramnegative bacteria can both generate EVs [6-8]. The size of the outer membrane-derived vesicles (OMVs) from both Gram-positive and Gram-negative bacteria was reported to be around 20–100 nm in diameter [8,9]. Inspired by the generation of naturally secreted EVs, scientists are also seeking to prepare biomimetic EVs by physical [10,11], and chemical [12] methods in recent years. In general, the artificial EVs exhibit similar properties to the natural ones. As a supplementary to the natural EVs, biomimetic EVs possess some advantages in some aspects, such as purity, yield, targeting ability, etc. Either EVs or OMVs can perform as vaccines without loading. They also can be loaded by bioactive ingredients as drug delivery carriers. In the past decades, a lot of efforts had been made to translate EVs for clinical use. However, there are many challenges existing at each stage of commercialization of EVs. One of them is quality control. Generally, to control the quality of EVs as drug carriers or vaccines, the following aspects should be taken into consideration.

Impurities. Safety is always the priority when quality is taken into consideration. Deriving from parent cells, EVs will unavoidably inherit some ingredients of their source cells. Obviously, some impurities, such as DNA [13] and RNA [14], are bioactive and may impair the functions of cargoes. Other potential impairments also need keeping an eye on include enzymes, kinases and some potential carcinoma-inducing agents especially when prepared from cancer source cells. Except endogenous impurities, the exogenous pathogens also need to be monitored when prepared from prokaryotic cells.

Cargo content. When cargos, either small molecules, such as chemicals, or large molecules, such as proteins and RNAs, were loaded into EVs, they have to be assessed to guarantee the necessary bioactive ingredient levels. As regard to the cargo loading methods, there are some reviews can be referred [9,15].

Targeting ability. As drug carrier, the content of targeting ligands [15] on the EVs should be evaluated. The targeting capability of EVs also need assessment both in vitro and in vivo.

Phospholipid concentrations and diversity. As main ingredients to maintain the structure of vesicles [16], phospholipid concentrations need to be measured. Also, the category of phospholipid needs to be identified and contents of each phospholipid need measurement.

Protein contents and diversity. Similar to phospholipids, the general protein contents should be measured. Furthermore, the diversity of proteins should also be studied, and this work can be done via proteomics analysis [10].

Vesicle identification and stability. This issue is not very sensitive when natural EVs are assessed but should be taken special consideration when biomimetic EVs are used. External force may help cell membrane assembly into vesicles, but the efficiency is a problem. Some small cell membrane debris, other than vesicle, may exist. Also, after vesicles formed, the stability is a problem.

Toxicity. The last concern about the clinical application of EVs is the possible immunotoxicity induced by EVs. In the previous studies, we found the EVs generated from nitrogen cavitation can activate HUVECs, while the function can be mitigated by the loaded anti-inflammatory drug. The following studies also demonstrate that the immunotoxicity may be caused by the exposed phosphatidylserine, originally locating on the inner surface of cell membrane, due to the external forces [16]. To avoid the impairment from immunotoxicity, antiinflammatory therapy may be necessary and helpful in some cases.

In the past decades, the research on EVs indicates that EVs are transforming the traditional nanoparticle-based drug delivery because EVs have the unique features. EVs have the excellent biocompatibility, long circulation time and low immunogenicity, and most importantly, they maintain their parent cell features that make them excellent drug carriers. Different to the conventional drug carrier, some EVs themselves perform as bioactive drug for disease therapy. Some special tissues, such as brain, can also be targeted by EVs, and no special targeting ligands are required. The above features confer EVs some outstanding advantages to be a drug carrier or disease treating agent. In summary, this review summarized the current most eminent challenges facing quality control towards commercialization of EVs. With advances in nanotechnology and immunology and genetic engineering, industrialization of EVs may become more and more realistic and eventually realize, and EVs will eventually turn to be a powerful tool for therapy of many human diseases.

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Antiurolithiatic Activity of the Leaf Extracts of Maerua Angolensis

Author by Atul Kaushik*

Abstract

A wide range of medicinal plants have been used conservatively for urolithiaisis due to their fewer side effects and because they contain copious phytochemicals that show advantageous effects in urolithiasis. Maerua angolensisis traditionally used in the treatment of urolithiasis in Eritrea. It is a small African tree growing west to Senegal, in Somalia, Ethiopia, and south to Natal. Maerua angolensis (keremo) was selected as there are no scientific reports available on the plant for anti-urolithiatic activity. In vitro anti-urolithiatic activity of the plant extracts was tested in terms of inhibition of calcium oxalate nucleation, aggregation and growth assays in the presence as well as absence of inhibitors. Three different concentrations (10mg/ml, 20mg/ml, and 30mg/ml) of the plant extracts were tested in each assay. Phytochemical screening revealed the presence of alkaloids, flavonoids, amino acids, tannins, phenols, saponins, resins, carbohydrates and proteins. Among all the extracts of M. angolensis, the highest percentage nucleation inhibition was obtained from water extract at a concentration of 30 mg/ml (95.5%). The aqueous, chloroform and ethanol extracts (10 and 30 mg/ml) were found to possess significant anti-urolithiatic activity when compared to standard Cystone (10 and 30 mg/ ml). In the aggregation assay, the highest percentage aggregation inhibition was obtained from chloroform extract at a concentration of 30mg/ml (196.25%). The chloroform extracts (10, 20, 30 mg/ml) was found to have anti-urolithiatic activity with significant difference when compared with cystone (standard drug). As revealed in the comparison of the percentage growth inhibition by the three extracts of M. angolensis, the chloroform extract at the concentration of 30 mg/ml was found to be the most effective. The highest percentage dissolution of calcium oxalate of Maerua angolensis was found from ethanol extract at the concentration of 30mg/ml (55.73%). The aqueous extract (10 and 20 mg/ml) was statistically different when compared to Cystone standard (10 and 20 mg/ml). The study concludes that the leaf extracts of Maerua anglensis have inhibitory effect on calcium oxalate for crystal nucleation, aggregation and crystal growth. It also showed great efficacy in the dissolution of calcium oxalate crystals. Thus these extracts may be valuable resources for treatment of urolithiasis.

Keywords: Urolithiasis; Maerua angolensis; Nucleation assay; Aggregation assay; Growth assay

Abbreviations: UV: Ultraviolet; MAA: Maerua Angolensis Aqueous; MAE: Maerua Aangolensis Ethanol; MAC: Maerua Angolensis Chloroform

Introduction

The term urolithiaisis (Greek- ‘ouron’ for urine, ‘oros’ for flow, and ‘lithos’ for stone) refers to the formation of solid concretions consisting of both protein and crystalline materials in the lumen of urinary tract attached to the uro-epithelium [1]. Urolithiasis is a major health problem that affects 12% of the global population [2]. It is more prevalent between the ages of 20 to 40 in both sexes, with a male to female ratio of 2:1 [3]. Urinary calculi may have various compositions which include: calcium oxalate (monohydrate or dihydrate), struvite (magnesium ammonium phosphate), uric acid, calcium phosphate, and cystine. There are other less common stones, including xanthine and drug-related stones as well. Globally, calcium oxalate is considered as the main constituent in the renal calculi and represents about 80% of all the cases [4,5]. A wide range of medicinal plants have been used in different countries and cultures as a prophylactic and curative agent for urolithiasis [6,7,8]. They have been used conventionally for urolithiaisis due to their cost effectiveness, fewer side effects and because they contain numerous phytochemicals that show beneficial effects in urolithiasis [9]. Maerua angolensis Figure 1 is traditionally used in the treatment of urolithiasis in Eritrea. It is a shrub or small tree, usually growing 5-6 m tall but with reports of some trees up to 10m tall. The plant is often rather rambling, with spreading or drooping braches. The bole is rarely straight. It is a small African tree growing west to Senegal, in Somalia, Ethiopia, and south to Natal. It is used in the treatment of various diseases such as asthenia and anorexia, rheumatism, epilepsy, headache, pus-filled abscesses, hydrocoele, influenza, toothache in addition to its use as an analgesic [10]. By keeping all the points in mind the study has been designed and undertaken to evaluate the phytoconstituents and anti-urolithiatic potential of the leaf extracts of Maerua angolensis using in vitro methods. Maerua angolensis (keremo) was selected as there are no scientific reports available on the plant for anti-urolithiatic activity (Figure 1).

Materials and Methods

Collection and processing of plant materials

The plant was collected from Shiketi (Menguda) (Figure 1), southern region of Eritrea, from June, 22nd -29th, 2018. The plant was authenticated by a botanist, Department of Botany, Eritrean Institute of Technology, Minefi, Eritrea. The leaves of Maerua angolensis (Keremo) were washed with tap water to eliminate any dust or other foreign particles. The plant leaves were then shade dried for two weeks and coarsely powdered. The powdered materials were passed through a 4mm sieve and stored in clean containers until the time of use.

Preparation of various extracts

Cold extraction method was used to prepare various extracts from the leaves of Maerua angolensis using water, ethanol and chloroform. 150g of the leaf powder was weighed and transferred in to Erlenmeyer flasks. 1000ml of each solvent was added to each flask and the flasks were closed tightly. Each flask was kept in a Stuart Scientific shaker at 150 revolutions per minute (rpm) for three days. Content of the flasks were filtered through a single layer of muslin cloth, and then the final filtrate was collected by passing it through a Whattman grade 1 filter paper in a Buchner funnel under vacuum to obtain pure liquid extract. All extracts were then concentrated by using a rotary evaporator. The concentrated extracts obtained were left to dry at room temperature. All the extracts were kept till further use. All the prepared extracts were subjected to qualitative chemical tests to detect the presence of different classes of phytoconstituents namely alkaloids, flavonoids, steroid, glycoside, reducing sugar, proteins and amino acids, resins, quinones, tannins and saponins.

Evaluation for anti-urolithiatic activity

Turbidometric method: The in vitro anti-urolithiatic activity of the plant extracts was tested in terms of inhibition of calcium oxalate nucleation, aggregation and growth in the presence of inhibitors (standard drug and extracts) and absence of inhibitors. A UV/Visible spectrophotometer (Jasco V-630) was employed to measure the turbidity changes in each assay. Three different concentrations (10mg/ml, 20mg/ml, and 30mg/ml) of the plant extracts were tested in each assay.

Nucleation assay: Solutions of calcium chloride and sodium oxalate were prepared at the final concentrations of 5 mmol/L and 7.5 mmol/L respectively in a buffer containing Tris 0.05 mol/L and NaCl 0.15 mol/L at pH 6.5. 950 μL of calcium chloride solution was mixed with 100 μL of plant extract at different concentrations (10mg/ml, 20mg/ml and 30mg/ml). Crystallization was started by adding 950 μL of sodium oxalate solution and the temperature was maintained at 37 °C. The optical density of the solution was monitored at 620 nm. The rate of nucleation was estimated by comparing the induction time in the presence of the extract with that of control. Cystone tablets were used as standard solution (11). Percentage inhibition of nucleation was calculated using the following formula: % Inhibition of nucleation = [(C-S) /C] × 100. Where, C is the turbidity without extract and S is the turbidity with extract.

Aggregation assay: COM crystals were prepared by mixing calcium chloride and sodium oxalate at 50 mmol/L. Both solutions were equilibrated to 60 °C in a water bath for 1 h and then cooled to 37 °C overnight. The crystals were harvested by centrifugation and then evaporated at 37 °C. The CaOx crystals were used at a final concentration of 0.8 mg/ml, buffered with Tris 0.05 mol/L and NaCl 0.15 mol/L at pH 6.5. The experiments were conducted in the absence and presence of the plant extract after stopping stirring. Cystone tablets were used as standard drug solution [11]. The percentage aggregation inhibition rate (Ir) was calculated by comparing the turbidity in the presence of the extract with that obtained in the control using following the following formula: Ir = (1−Turbiditysample/Turbidity control) × 100. Where, Ir is the percentage aggregation inhibition rate.

Growth assay: The percentage inhibition of calcium oxalate crystal growth was evaluated in presence and absence of plant extracts.4mM calcium chloride and 4mM sodium oxalate of 1ml each were added to a 1.5ml of solution containing sodium chloride (10mM) buffered with Tris (10mM) at pH 7.2. To this 30μl of calcium oxalate monohydrate crystal slurry (1.5mg/ml acetate buffer) was added. Consumption of oxalate begins immediately after COM slurry addition and was monitored for 30 minutes for disappearance of absorbance at 214nm.100 μl of each plant extracts were separately added into the reaction mixture. The depletion of free oxalate ions will decrease if extract inhibits calcium oxalate crystal growth. Rate of reduction of free oxalate was calculated using the baseline value and the value with or without extract after 30 minutes of incubation [11]. The relative inhibitory activity was calculated by the following formula: % relative inhibitory activity = ((C-S)/C) × 100. Where, C is the rate of reduction of free oxalate without any extract and S is the rate of reduction of free oxalate with drug extract.

Titrimetric method

This method was used to evaluate the activity of the plant extracts in dissolving the already formed stones in the kidneys. The steps involved in the process were as follows:

Step 1: Preparation of experimental kidney stones (Calcium oxalate stones) by homogenous precipitation

1.47gm of calcium chloride dihydrate was dissolved in 100ml distilled water and 1.34gm of sodium oxalate was dissolved in 100 ml of 2N H2SO4. Both solutions were mixed equally in a beaker to precipitate out calcium oxalate with stirring. The resulting precipitate was calcium oxalate which was freed from traces of sulfuric acid by treating with ammonia solution. Finally, it was washed with distilled water and dried at a temperature of 60 °C until all the water was evaporated [12].

Step 2: Preparation of the semi permeable membrane from farm eggs

The semi-permeable membrane of eggs lies in between the outer calcified shell and the inner contents like albumin & yolk. The preparation included the following steps: Apex of eggs was punctured by a glass rod in order to squeeze out the entire content. Empty eggs were washed thoroughly with distilled water and placed in a beaker consisting 2M HCl for an overnight, which caused complete decalcification. Then, the semi-permeable membranes were washed with distilled water, placed in ammonia solution for neutralization of acid traces in the moistened condition for a while and was then rinsed with distilled water. Finally, the egg membranes were stored in 2% ammonia until used [13].

Step 3: Estimation of Calcium oxalate by Titrimetry

Exactly 5 gm of calcium oxalate and 10 mg, 20 mg and 30 mg of each plant extract were packed together in the semi-permeable membrane of egg. These were allowed to suspend in a conical Erlenmeyer flask containing 100 ml of 0.1M Tris buffer. All the conical flasks were placed in water bath at 37 °C for an overnight. Then, the semi permeable membranes along with the inside contents were removed from the conical flasks. 2 ml of 1N sulfuric acid was added to the contents remaining in the flask and was titrated with 0.1356 M of KMnO4 till a light pink color was obtained. Each first group containing only 5mg of calcium oxalate served as blank. The second group containing 5 mg of calcium oxalate and along with the 10, 20, 30 mg/ ml of standard formulation that is, Cystone served as a positive control. The 3rd and 4th groups contained 5 mg of calcium oxalate and 1 ml of aqueous and ethanolic extracts at a concentration of 10, 20 and 30 mg/ml. All the experiments were performed in duplicate. Finally, the amount of calcium oxalate dissolved was calculated using the mole concept.

Results and Discussion

Results of Phytochemical screening

Table 1: Results of Phytochemical screening.

Phytochemical screening Table 1 revealed the presence of alkaloids, flavonoids, amino acids, tannins, phenols, saponins, resins, carbohydrates and proteins. Different activities observed in the plant extracts might be due to the presence of these phytochemicals. For example, Tannins and polyphenols inhibit CaOx crystal formation as well as dissolve the preformed CaOx crystals by aiding calcium complexation. Flavonoids also possess calcium oxalate crystallization inhibition and dissolution potency [14]. Therefore, the anti-crystallization, anti aggregatory and crystal growth defying activity of the plants would have been an outcome of these phytoconstituents present in the plants (Table 1).

Anti-urolithiatic activities of the leaf extracts of

Maerua angolensis

The aqueous, ethanol and chloroform extract of Maerua angolensis, inhibited crystal formation as well as promoted crystal dissolution in a dose dependent manner and were comparable to the activity of Cystone. From all the extracts, the highest percentage nucleation inhibition was obtained from water extract at a concentration of 30 mg/ml (95.5%). The aqueous, chloroform and ethanol extracts (10 and 30 mg/ml) were found to possess significant anti-urolithiatic activity when compared to standard Cystone (10 and 30 mg/ml). These findings were consistent with the activity of the aqueous extract of Bauhinia variegate which was 94.2% at 1000 mcg/ml, as demonstrated in the study by Vani Mamillapali et al, at the Vijaya Institute of Pharmaceutical Sciences for Women, in 2016. They also indicated that the plant was effective in inhibiting aggregation and growth using turbidity assays [15] (Table 2 & Figure 2).

Table 2: Results of anti-urolithiatic activities of the leaf extracts of Maerua angolensis.

In the aggregation assay of different extracts of Maerua angolensis, the highest percentage aggregation inhibition was obtained from chloroform extract at a concentration of 30mg/ ml (196.25%). The chloroform extracts (10, 20, 30 mg/ml) was found to have anti-urolithiatic activity with significant difference when compared with cystone standard (10, 20 and 30 mg/ ml). (Figure 3) As revealed in the comparison of the percentage growth inhibition by the three extracts of Maerua angolensis, the chloroform extract at the concentration of 30 mg/ml was found to be the most effective. However, there was no significant difference in the anti-urolithiatic activity as compared with Cystone. (Figure 4) Pronounced anti-urolithiatic activity was found in in-vitro anti-urolithiatic model for calculating percentage dissolution of kidney stone when titration method was performed. The highest percentage dissolution of calcium oxalate of Maerua angolensis was found from ethanol extract at the concentration of 30mg/ml (55.73%). The aqueous extract (10 and 20 mg/ml) was statistically different when compared to Cystone standard (10 and 20 mg/ml). These results were comparable to the results of ethanolic extracts of Gossypiumherbaceum (87%) in the study done by Niharika M. et al., in 2018 at the Vishnu institute of pharmaceutical education and research, India. In addition, they found that the aqueous extract of Gossypiumherbaceumwas also effective with a percentage dissolution of 64% [16]. An additional study done by Vennila etc al., at DGGA College in India in 2015, revealed a high percentage of dissolution (72.72 %) by the aqueous extract of Meliadubia. The acetone extract, however, had the greatest activity at a percentage of 98% [17] (Figure 3,4 &5).

Conclusion

Findings of the present study clearly demonstrate antiurolithiatic potential of Maerua angolensis against calcium oxalate urolithiasis in vitro. The plant showed prominent inhibition of all the phases of calcium oxalate stone formation which include nucleation, aggregation and growth. It also showed great efficacy in the dissolution of calcium oxalate crystals. This plant is used in ethnomedical practice in the treatment of urinary problems and renal stones. The study has given a basic scientific evidence for the traditional uses of the plant in the prevention and treatment of urolithiasis. Therefore, this plant could be a potential source of new drug molecules with anti-urolithiatic activity.

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-The Collagen Challenge: Preventing Myocardial Fibrosis Is the Hype Reflective of the Potential?

Author by John D Rozich*Mini Review

Mounting evidence has suggested that the cardiac extracellular matrix (ECM) has a nuanced but determinative role in myocardial remodeling in response to pathological insult and subsequent recovery [1-3]. These alterations in the structure and actual composition of ECM may actually reflect many of the welldescribed phenotypic cardiac properties typical of common diseases states including diabetes, hypertension, obesity and ischemia. Current thought is that dysregulatory influences result in a maladaptive remodeling process that begins an inevitable progression to irreversible and often untimely cardiac failure [1,2]. This potentially injurious perturbation in the dysfunctional ECM response is complex involving profibrotic collagen synthesis and post-translational modification including increased crosslinking and reduced degradation [1,4]. The result is a stiffening noncompliant and impaired myocardium that underpins observed clinical changes causing the heart failure syndrome. Sampling changing serum levels of distinct proteins or molecular fragments are now being used to assess collagen turnover. This includes using the N-terminal propeptide of procollagen type III and C-terminal propeptide of procollagen type I considered directly correlative to histologically proven fibrosis [1,5]. Other biomarkers have been proposed to be associated with pathology involving collagen metabolism, (tissue inhibitor of metalloproteinases-1 (TIMP-1), matrix metalloproteinase [MMP]-2, MMP-9, galectin-3 [Gal-3], etc) but are not yet validated as directly representative of histologically confirmed fibrosis [1,6].

Very recent publications have raised the intriguing possibility that a certain pharmacological class of drugs, exemplified by entresto [sacubitril/valsartan], may be antifibrotic linked to an improved response in examined cohorts [7-9]. There are clearly limitations with the data examined thus far that must temper any premature conclusive enthusiasm, but the potential of selectively influencing the adverse outcomes associated with myocardial hypertrophic growth is indeed remarkable. It begs the question as to whether cohorts shown to have disproportionate hypertrophy in the setting of previously mentioned chronic disease states should be explicitly targeted for use of entresto or like agents. Additionally, should different hemodynamic conditions causing excessive hypertrophic growth, including the pressure overloaded state of progressive aortic stenosis also be considered?Foreseeably, there are numerous remaining questions including whether the sacubitril/valsartan affect is mechanistically distinct from other agents, based on its combined (perhaps additive) molecular targets [3,4,10]. Alternatively is the collagen impact secondary to a family of drugs, including enalapril, (since a subset of the PARADIGMHF data were examined); all able to impact fibrosis but though mechanism(s) not presently understood [1,2,5,6]? Also under consideration is whether different forms of phenotypic hypertrophy, as dictated by different variables among hosts, demonstrate fundamentally different responsiveness. Keys to the prospect of enhanced specific therapy directed at reigning in the maladaptive fibrotic process are the following. First, clear evidence must emerge showing use of collagen biomarkers is an acceptable substitute to direct histopathological evidence of fibrosis in different forms of phenotypically expressed myocardial hypertrophy [1,7,8]. Not all forms of hypertrophy responsive to distinct triggers may respond uniformly even when appearing similar [11,12]. And existing literature is replete with false or minimally relevant surrogate indices creating disappointing time-consuming blind alleys [13].

Second, it will be important to address whether there are any important unanticipated factors impacting the serum levels reflecting collagen turnover [11]. This concern, while closely related to the first point, differs in a subtle but key aspect. If sacubitril/valsartan or other similar agents substantively alter or dynamically influence renal clearances for example, volume of distribution and bioavailability may also be impacted. Thus, assigning determinative insights to variations in referent serum levels may still reflect valid collagen turnover metrics, but imbedded adjustments will be requisite to ensure validity of global fibrotic impact. Finally, the entire evolving menu of serum biomarkers supporting collagen turnover must be individually examined in their response to other well-characterized nonfibrotic biomarkers to ensure that a form of “external quality control” exists. The use of BNP, troponin and N-terminal proBNP have reflected this need and are believed important, but other markers may demonstrate prescient relevance [3,4]. The demographics of aging, obesity, hypertension and diabetes reveal a growing threat of heart failure in its various forms [14,15]. The ability to slow the progression of fibrotic injury to the vulnerable myocardium will potentially be a significant tool but will require accompanying detail in the actual mechanisms of cardiac transformation from health to disease. Entering the “game” too late, for individuals or populations at risk may falsely demonstrate no real impact on the metrics of survival. But a temporal understanding of the natural progression of fibrosis and scar will undoubtedly raise hope for ideal intervention precluding emergence of the low output state as well as threatening dysrhythmias. Despite the gaps in existing knowledge, there is excitement in the air as clinicians begin to anticipate optimizing therapy linked to metrics of improved survival. Much work needs to be done but already there are populations at risk that some have proposed may disproportionately benefit from this therapy.

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Effects of Antipsychotic Drugs on Neuroactive Steroids Brain and Plasma Levels in Humans and Animals: A Systematic Review of the Literature

Author by Emerson A Nunes*

Abstract

Objectives: The present study aims to review systematically the studies that investigated the effects of antipsychotic drugs on neuroactive steroids brain and plasma levels in animal and human studies

Methods: We conducted a review of the Medline databases, including articles published in English, Spanish and French, describing the results of controlled original animal and human studies that evaluated the effects of antipsychotics on brain and plasma neuroactive steroids levels.

Results: It was identified 291 studies through our search strategy. Of these studies, we selected 20, with one more study included after further searching throughout the reference list. Of these 21 studies, eight studies evaluated neuroactive steroids levels in humans, and 13 were animal studies. The antipsychotic used in the studies were: haloperidol, sulpiride, clozapine, olanzapine, risperidone, quetiapine and aripiprazol. Among the neuroactive steroids evaluated, we found studies evaluating levels of progesterone, allopregnanolone, DHEA, DHEA-S, TDHOC, pregnenolone and 3a,5a-THP. The human studies demonstrated diverse and non-conclusive alterations on the plasma levels of progesterone, no changes on allopregnanolone and TDHOC levels after clozapine use. Among the animal studies, nine of these evaluated progesterone levels, with no changes in three studies, and increased plasma levels in six studies. The same researchers in two studies investigated pregnenolone brain levels in rats, showing that clozapine and olanzapine acutely increased the levels of pregnenolone in rats. Four studies evaluating allopregnanolone showed increased brain levels after the use of olanzapine and clozapine. No changes with haloperidol or risperidone. The only animal study that investigated DHEA/DHEA-S levels, showed that clozapine decreased brain levels of DHEA-S, unlike haloperidol. Increased TDHOC levels after use of clozapine were observed in the only study that measured this neuroactive steroid. Two studies from the same group showed increased brain levels of 3a,5a-THP after olanzapine use.

Conclusion: The antipsychotic effects observed on neuroactive steroids levels reinforce their potential role in the pathophysiology of mental disorders, particularly schizophrenia. In the case of progesterone, the studies are in sufficient number to demonstrate the effect of antipsychotic on changes in their levels in animal but human studies. For the other neuroactive steroids, the inconclusive data points to the need for more well designed studies.

Introduction

Schizophrenia follows as one of the most challenge mental disorders, despite the highly available pharmacological treatment options, currently represented mainly by antipsychotic medications [1,2]. Most of these antipsychotic medications have in common some level of antagonism of dopamine type 2 receptors (D2) and also blockade of 5HT2A serotonin receptors is present in some of these compounds [3,4]. Even with the different types of currently available antipsychotics, all of them as stated earlier, have in common some blockade of D2 receptors. This pharmacodynamics effect is in accordance to the main theory that tries to explain the physiopathology of schizophrenia, the dopaminergic theory (CARLSSON AND LINDQVIST, 1963). Albeit reasonable improvement can be achieved with the use of antipsychotic to treat schizophrenia symptoms, especially when we consider the positive symptoms (hallucinations and delusions), almost one third of all patients is completely refractory to pharmacological approaches [1,5]. Therefore, the need for new drugs that target other neurotransmission systems other than the dopamine transmission is urgent [6] and the neuroactive steroids, like pregnenolone glutamate has attracted great attention in the last decade (MARX et al., 2011). Neuroactive steroids are endogenous molecules derived from cholesterol metabolism, which exert actions in the central nervous system. They may also suffer biotransformation in the central nervous system, only then reaching biological action, when they are called neurosteroids (NOORBAKHSH et al., 2011). Neuroactive steroids, including testosterone, dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) might play an important role in the pathophysiology of schizophrenia [7]. Many of the neuroactive steroids demonstrate important pleiotropic actions that significantly change brain function in animal models, these include modulation of GABAergic inhibitory and excitatory glutamatergic systems (Wu et al., 1991; Paul and Purdy, 1992; Belelli AND Lambert, 2005; Belelli et al., 2006), increased neurogenesis (MAYO et al., 2005), dendritic growth (FONTAINE-LENOIR et al., 2006), and myelination (KOENIG et al, 1995;. Azcoitia et al. 2003; GHOUMARI et al. 2003. LIAO et al, 2009), among other functions (MARX et al, 2011) [8]. Given the pleiotropic range of actions performed by different neuroactive steroids, demonstrated in animal models, it is assumed that probably deregulation of such substances should be relevant to the pathophysiology and treatment of various mental disorders, and that neuroactive steroids can be seen as potential new therapeutic agents.

Data from different studies in animal models have demonstrated the potential of such steroids as important factors in the pathophysiology of different mental disorders. We can mention the effects of allopregnanolone, a neuroactive GABAergic steroid, which has shown positive results in animal models of schizophrenia (reversing cognitive effects of MK-801, an animal model of schizophrenia) (ROMEO et al., 1994; Cheney et al. 1995), as well as in studies with animal models of other mental disorders such as post-traumatic stress disorder, depressive disorder and pain models (Pibiri et al, 2008; PINNA, 2010; MATSUMOTO et al, 2005; AGÍS- BALBOA et al, 2007; Meyer et al, 2011). Pregnenolone, a neuroactive steroid from the cholesterol metabolism, a progesterone precursor, have demonstrated positive properties on memory and learning in animal models in mice (Flood et al, 1992). Alopregnenolona, another neuroactive steroid from the same metabolic chain, has shown potentiating action on the action of GABA A receptors, exhibiting neuroprotective properties in animal models [9]. Thus, it is suggested that the pathways for biosynthesis involving the formation of pregnenolone, alopregnanolone and sulfate alopregnanolone, should be relevant to the pathophysiology and treatment of schizophrenia, worth note that these substances are interconverted, becoming complex understanding what to expect when trying to manipulate the concentrations of these molecules when administered exogenously, or while trying to accurately measure their serum or brain levels (these in animal models) (Marx et al., 2011). Therefore, antipsychotic drugs already used to treat schizophrenia may possibly have mechanism of action not only influencing directly the dopamine and serotonin receptors, as was recognized earlier, but also influencing GABAergic action, interfering with brain and serum levels of neuroactive steroids. Changes in the levels of neuroactive steroids (progesterone and alopregnenolona) have been observed after the use of olanzapine and clozapine in rats [9], reinforcing the theory of an influence of neuroactive steroids in the mechanism of action of drugs that target the symptoms present in schizophrenia. Thus, the present study aims to systematically review the studies that investigated the effects of antipsychotic drugs on neuroactive steroids serum and brain levels in animal and human studies.

Methods

A systematic review of the Medline database was conducted using a search strategy that combined the following keywords: (antipsychotic* or neuroleptic* or ziprasidone or haloperidol or thioridazine or clozapine or olanzapine or quetiapine or sulpiride or amisulpride or risperidone or fluphenazine or chlorpromazine) and (neurosteroid* or neuroactive steroid* or pregnenolone or “pregnenolone sulfate” or pregnanolone or dehidroepiandrostenedione or “dehidroepiandrostenedione sulfate” or progesterone or allopregnanolone or “allopregnanolone sulfate“) and (“brain levels” or levels or “plasma level*” or “tissue levels” or “serum levels” or “cerebrospinal fluid level*” or “CSF levels”). It was included articles published in English, Spanish and French, describing the results of original human and animal studies that evaluated the effects of antipsychotics on serum and brain levels of neuroactive steroids. Also it was required as an inclusion criteria the presence of a control group. The publication period included articles published in the last 20 years. Review articles, letters to the editor, case reports and other publications that present no direct empirical results were all excluded.

Result

Table 1: Local Government Areas and the Areas sampled.

It was identified a total of 291 studies through our search strategy. Of these studies, 20 were included in our review. A further search throughout the reference list yielded one more study. In the end, a total of 21 studies were included in our review, with further details available in Table 1. Of these 21 studies, 13 were animal studies, with the rest evaluating neuroactive steroids levels in humans. Three of these studies performed HPLC associated with another essay to measure amino acids (e.g. spectroscopy), and the other 14 studies used only HPLC analysis to measure the amino acid levels. Five studies used post mortem material for the analysis, with the other 12 studies analyzing microdyalisated samples of in vivo specimens. Only one study didn’t presented a placebo control group, utilizing a basal measure to control their data. Fourteen studies used Sprague-Dale rats, one study utilized Hooded Lister rats, another study used wistar rats and only one study didn’t stated which type of rat was used. The number of rats per group varied from 6 to 17 for each group. The time from drug initiation and tissue analysis varied from 60 minutes to 8 months (Table 1).

Antipsychotic agents

A total of seven different antipsychotic agents were evaluated in the studies included in the present review (haloperidol, sulpiride, clozapine, olanzapine, risperidone, quetiapine and aripiprazol.), although some studies enrolled patients in use of antipsychotics that weren’t specified. All the human studies used the medication orally. Among the animal studies, nine administered the antipsychotic through intraperitoneal (i.p.) injections, two through subcutaneous (s.c.) injections, one through oral administration (gavage) and one used intrathecal injections (i.t.).

Neuroactive steroids

Throughout the studies selected to take part of this review, a wide range of neuroactive steroids were studied. We found studies that evaluated the following neuroactive steroids: progesterone, allopregnanolone, DHEA, DHEA-S, TDHOC, pregnenolone and 3a,5a-THP.

Human studies

Changes in basal neuroactive steroids levels after antipsychotic use. It was observed a wide variation of changes among the different studies present in this review, above all among the human studies. At least part of this variation was related to methodological issues, for instance, the lack of control over the type of antipsychotic used by the patients enrolled in the studies. Only two of the total (eight) of human studies reviewed here controlled the type of antipsychotic used, one using clozapine and the other study sulpiride (MONTENLEONE et al., 2004; BAPTISTA et al., 1997). Therefore, it is hard to make clearly conclusions with the data from these human reviewed here, with these data had demonstrated diverse and non-conclusive alterations on the plasma levels of progesterone, no changes on allopregnanolone and TDHOC levels after clozapine use. Three human studies evaluated DHEA/DHEA-S levels, with no alterations in two of these, and decreased levels in the third study, although there was no control regarding the type of antipsychotic used by the patients enrolled in these studies.

Animal studies

Changes in basal neuroactive steroids levels after antipsychotic use. All animal studies controlled the type of antipsychotic used, different form the majority of human studies. However, the difference among the studies regarding how the medication was administered apparently influenced the observed results. Among the animal studies, nine administered the antipsychotic through intraperitoneal injections (IP). Two used subcutaneous injections (SC), one used medication given orally by gavage and the last study utilized intracerebral injections. Of 13 animal studies that evaluated the levels of neuroactive steroids, nine of these measured progesterone levels, with no changes in three studies, and increased plasma levels in six studies. Among the three studies with negative results, the antipsychotic used were sulpiride in lower doses (1mg / kg, 20mg / kg), risperidone, haloperidol and olanzapine, this last one administered through intracerebral injections (FREYE AND SELIGA, 2003). The average duration of antipsychotic use was 15,75 days. Among the six studies that showed increased levels of progesterone, the antipsychotic used were: sulpiride in high dose (40mg / kg), clozapine and olanzapine (IP gavage, SC). The average of antipsychotic use was 10.6 days. The same researchers investigated in two studies the pregnenolone brain levels in rats, showing that clozapine and olanzapine acutely increased the levels of pregnenolone in rats [10,11]. Four studies evaluating allopregnanolone showed increased brain levels after the use of olanzapine and clozapine. No changes with haloperidol or risperidone were observed [3,9,12,13]. The only animal study that investigated DHEA/DHEA-S levels, showed that clozapine decreased brain levels of DHEA-S, unlike haloperidol [14]. Increased TDHOC levels after use of clozapine were observed in the only study that measured this neuroactive steroid [5]. Two studies from the same group showed increased brain levels of 3a,5a-THP after olanzapine use [15,16].

Discussion

There is emerging preclinical and clinical evidence supporting a role for neurosteroids in the neurobiology and therapeutics of schizophrenia, with a primary focus on progesterone, pregnenolone and selected pregnenolone metabolites—specifically pregnenolone sulfate, a neurosteroid that positively modulates excitatory glutamatergic NMDA receptors and allopregnanolone, a positive modulator of inhibitory GABAA receptors. The positive modulation of NMDA receptors by pregnenolone sulfate could potentially contribute to the amelioration of hypothesized NMDA receptor hypofunction in schizophrenia (JAVITT, 2004; MILLAN, 2005; RUJESCU et al., 2006; JAVITT, 2007) [17]. Metabolism to allopregnanolone could impact the GABA neurotransmitter system, which also appears to be dysregulated in schizophrenia (BENES AND BERRETTA, 2001; LEWIS et al., 2003, 2004, 2005; GUIDOTTI et al., 2005; BENES et al., 2007), as allopregnanolone potentiates GABAA receptor responses more potently than benzodiazepines or barbiturates (MAJEWSKA et al., 1986; MORROW et al., 1987, 1990). Different patterns were observed throughout the studies in our review, concerning the alterations of neuroactive steroids brain and serum levels after the use of antipsychotic agents. It is of great value to carefully analyze this issue since the increasing attention over neuroactive steroids in schizophrenia pathophysiology and treatment has gained increasing attention in the past decade (MARX et al., 2011). Multiple lines of evidence suggest that alterations in peripheral neuroactive steroids may be linked to brain functions and that changes in steroids metabolism could play a role in the pathogenesis of psychiatric disorders, like schizophrenia [18]. Even with all this attention over these alterations involving neuroactive steroids, we didn’t find enough data among the studies included in our review that support consistent changes with the use of antipsychotics in neuroactive steroids content in human serum samples. Among the 08 studies here reviewed, only two have controlled the type of antipsychotic used, one using clozapine and the other sulpiride (MONTENLEONE et al., 2004) [19]. Therefore, it is hard to make clearly conclusions with the data from these human studies, with these data had demonstrated diverse and non-conclusive alterations on the plasma levels of progesterone, no changes on allopregnanolone and TDHOC levels after clozapine use. Another point of interest among researchers is the hypothesis that neuroactive steroids changes after antipsychotic use could explain the differences among the different classes of these agents, believing that not only dopamine and serotonin changes explain the atypical profile of some antipsychotics, and their lower propensity to cause motor side effects. Also noteworthy is the particularly important role of progesterone in psychosis, with recent data pointing to progesterone levels being inversely associated with the severity of positive symptoms in first episode psychosis [20-26]. It was of great value the observation that the progesterone levels measured in the animal studies here reviewed had showed a clear differentiation between first and second generation antipsychotic medications, with the latter showing the capability of change the progesterone levels, what wasn’t possible with the use of haloperidol, for example [9, 27-39].

Conclusion

The description of the findings of the studies included in this review, suggest that there are sufficient studies to help demonstrate a relationship between antipsychotic use and changes in brain and serum levels of neuroactive steroids, mainly when we consider animal studies. Nevertheless, the heterogeneity of findings and lack of consistency in the observed changes difficult the formulation of definitive conclusions about this issue, pointing to the need of more studies that help to elucidate the remaining answered questions about the role of antipsychotic medications in changing neuroactive steroids content in humans. The antipsychotic effects observed on neuroactive steroids levels reinforce their potential role in the pathophysiology of mental disorders, particularly schizophrenia. In the case of progesterone, the studies are in sufficient number to demonstrate the effect of antipsychotic on changes in their levels in animal and human studies. Both clozapine and olanzapine showed to be capable of altering the levels of progesterone. For the other neuroactive steroids, the inconclusive data points to the need for more well designed studies, with better controlled variables, regarding type of antipsychotic used for example, or studies using the medication for longer period of time (more than 30 days).

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Relation of Pulse Rate with Flu Medication Treatment

Author by Bushra Khalid

Abstract

Influenza is caused by Influenza virus which has many types A, B, C and D. Each type is divided into subtypes like Influenza A (H1N1 and H3N2) are most common. Its symptoms have resemblance with cold but if it is not treated properly then many nearby healthy individual also affected by flu. Vaccination is best way for prevention but some people prefers medication while other prefer home remedies for flu treatment. Heart is muscular organ which is responsible for blood pumping moreover a healthy pulse rate declare that heart is at right state. Abnormal pulse rate is associated with many other illnesses. To measure the pulse rate, hold the hand as its palm side up and by placing the two fingers on wrist feel pulse rate and count it for one minute by using stopwatch. Finally, here is significant relation is observed for flu treatment with pulse rate.

Introduction

Influenza virus leads influenza or flu which is highly contagious disease. Its symptoms are related to cold but it differs in some aspect like causing agent Rhino virus for flu and influenza virus for influenza. Influenza virus has four types A, B, C and D further A and B cause seasonal influenza. Among them H1N1 and H3N2 are common subtypes of influenza type A and other subtypes lead sporadic human infection [1]. Vaccine provide protection against influenza A (H1N1) and Influenza B rather than influenza A (H3N2) [2]. To understand the host pathogenicity relationship, structural composition of Influenza virus is noticed mean Influenza virus is RNA enveloped and belong to Orthomyxoviridae family. It consists helical capsid mediated by matrix protein M1 and M2, neuraminidase and hemagglutinin act as glycoprotein and involved in enveloped formation. It is easily penetrated in epithelial cells of human respiratory tract. Its structural evolution via antigen drift and antigenic shifting facilitate in its pathogenicity and scientists need revision in vaccine formulation each year [3]. Heart is muscular organ which is present in the center of chest near left side and responsible to pump oxygenated blood to all body parts and receive deoxygenated blood and pump toward lungs to receive oxygen. Healthy heart is responsible to deliver oxygen at right amount irrespective of activity. Pulse rate and heartbeat declared the health of heart but both are confusing term but pulse declared the expansion and contraction of arteries in response to heart pumping, so both are considered equal [4]. Pulse rate increase in many diseases and it can measure at upper arm, wrist and neck. Slow pulse rate with dizziness helped to identify internal bleeding and other serious conditions while fast pulse rate is in the case of high blood pressure and cardiovascular problems. A healthy pulse rate is between 60 and 100 beats per minutes in adults. Infants have up to 160 bpm which decrease with the passage of age. A healthy and fit person has slow pulse rate as compared to those persons who exercise less and suffered with some health issues [5].

Materials and Methods

Materials

Stopwatch, notepad

Methodology

There are many places where heartbeat can measure like top or inner side of feet and behind the knees etc. Arteries runs closely to the surface of skin especially wrist and neck. To measure the pulse rate firstly turn one hand over in such a way palm-side up. Gently placed two fingers in the groove of forearm. By placing fingers at right side feel pulse rate and at the same time set the stopwatch for one minute and measure the pulse rate as per minute. Another method in which gentle place the fingers at soft groove on either side of windpipe. Count the pulse rate in one minute and note down on the page.

Results and Discussion

We counted pulse rate by using stopwatch for 60 seconds. Here Graph show the number of individuals whose pulse rate were noticed with respect of flu treatment with and without medicine. Out of 30 males and 70 females only 10 males and 25 females treat flu with medicine while they treat flu without medication (Table 1). shows the average value with standard deviation Further T-test is also performed to check its significance (Figure 1) (Table 1).

p<0.1 = Non-significant

Table 1: Relation of Pulse rate with flu treatment with and without medicines

Conclusion

There is significant relation observed for flu treatment with pulse rate.

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Mikania Cordata (Burm.F.) B.L. Rob. – A Bangladesh Folk Medicinal Plant for Gastric Disorders

Author by Mohammed Rahmatullah*

Abstract

Mikania cordata (Burm.f.) B.L. Robinson belongs to the Asteraceae family of plants and is a rapidly growing creeping perennial vine found in Bangladesh and other tropical regions. In Bangladesh, the plant is known by a number of names like ‘refugee lota’, ‘libuji lota’, ‘assam lota’, and ‘gastric gach’, the latter meaning gastric plant. In English, the plant is known as bitter vine, climbing hemp vine, and American rope. The plant is considered an important folk medicinal plant in Bangladesh and is used in the north central districts of Bangladesh (like Jamalpur) for treatment of gastric disorders. Other medicinal uses of the plant in Jamalpur district, Bangladesh include use as poultice for treatment of swelling, itches and wounds. This paper attempts to correlate the folk medicinal uses of the plant in Bangladesh versus ethnic uses reported from other countries along with reported relevant phytochemicals and pharmacological activities.

Keywords:Folk medicine; Mikania cordata; Ethnic uses; Jamalpur; Bangladesh

Introduction

Plants produce a bewildering variety of phytochemicals, otherwise known as secondary metabolites. Secondary metabolites are compounds, which are not necessary for a plant cell to live but are necessary for a plant to survive biotic or abiotic stresses [1]. Secondary metabolites can produce pharmacological responses when introduced into humans, which in turn can be toxic or serve therapeutic purposes. A number of such secondary metabolites have been isolated and have found uses in medicine like aconitine, L-hyoscyamine, camptothecin, tetrahydrocannabinol, and tubocurarine, to name only a few [2]. Although the existence of secondary metabolites were possibly not known or understood by ancient hominids, plants have always played a role in the treatment of diseases from the beginning of humankind. Medicinal plant material has been found in a 60, 000 year old Neanderthal grave in Iraq [3]. Early Asian and Egyptian texts (since the discovery of writing and writing materials) mention medicinal plants and their uses [4]. Use of plants as medicines since time immemorial gradually led to the establishment of distinct forms of traditional medicinal practices like the Ayurveda in India, Unani in Greece, Kampo in Japan, and other systems throughout the world [5]. Besides these ‘ritualized’ forms of traditional medicinal systems, there also exists folk medicine (FM), tribal medicine (TM, same as FM but practiced by tribal people instead of the mainstream population), and home remedies. Allopathic medicine has borrowed heavily from traditional medicinal practices and more than a hundred allopathic drugs are plant-derived [6]. It has been said that approximately 70- 80% of primary health care throughout the world is based on plant materials [7].

To discover a new allopathic drug starting from traditional medicinal practices, an excellent place to start is folk medicine or ethnomedicinal reports on any given plant. The more the number of reports and agreement on the plant’s medicinal use in those reports, the better probability is that a new drug can be discovered from the plant. So the first thing to do is perform an ethnobotanical survey and compare it with other ethnobotanical surveys. During the past ten years, we had been doing ethnobotanical surveys in Bangladesh in an extensive manner [8-37], for ethnobotany is still at its infancy in this country. On the other hand, the country has a rich floral diversity with around 5500 floral species. So the probability of new drug discovery is higher in countries like Bangladesh with a rich diversity of flora, which remains to be scientifically studied, but which plants have been medicinally used from time immemorial but the data lies scattered among the folk medicinal practitioners (FMPs) or other types of traditional medicinal practitioners. So the primary objective of any scientist dealing with drug discovery from natural products is to analyze the ethnobotanical data from different angles. Mikania cordata (Burm.f.) B.L. Robinson is a vinous perennial plant found in tropical regions like Bangladesh and belongs to the Asteraceae family. In Bangladesh, the plant is known by a number of names like ‘refugee lota’, ‘libuji lota’, ‘assam lota’, and ‘gastric gach’, the latter meaning gastric plant. In English, the plant is known as bitter vine, climbing hemp vine, and American rope. The plant is considered an important medicinal plant in Bangladesh. It was our objective to compare the ethnic uses of the plant and check out the scientific validation of some uses. The plant was chosen because one of its local names is ‘gastric gach’, suggesting that the plant may have a long history of folk medicinal use for alleviating gastric disorders.

MethodsPreliminary information was obtained from a FMP practicing in Ramnagar village in Jamalpur district, Bangladesh. Prior informed consent was initially obtained from the FMP. The FMP was informed the reason for our visit and consent obtained to disseminate any information provided both nationally and internationally. Actual interviews were conducted in the Bengali language, which was spoken fluently by the FMP as well as the interviewers, the language being the mother tongue of FMP, villagers and the interviewers. The FMP mentioned the name ‘gastric gach’ and described its various uses. He also took the interviewers to show a sample of the plant. The plant was photographed and plant specimen brought to Dhaka for identification by a competent botanist at the University of Development Alternative (UODA). A voucher specimen was deposited at the Medicinal Plant Collection Wing of UODA. Secondary information on the ethnobotany, pharmacological properties and phytochemicals of the plant used by the FMP were obtained from papers in PubMed, SCOPUS and Google Scholar abstracted journalszResults and DiscussionThe plant shown by the FMP was identified as

Mikania cordata

(Figure 1). The FMP used the leaves of the plant to treat primarily gastric symptoms. Other uses by him were for treatment of swelling, itches and wounds, where the plant was used as a poultice. However, for gastric disorders, aqueous extract of leaves (one spoon) was taken orally in the morning on an empty stomach. This was done till gastric disorder was cured. By gastric disorder the FMP meant acidity, stomach pain, bloating, bleeding with stool and heartburn, all of them being possible symptoms of gastric ulcer. In Barisal district (located in the southern part of Bangladesh), the leaves of the plant are used to treat wounds and gastric disorders [38]. In Shitol Para village of Jhalokati district, Bangladesh, the leaves are used to stop bleeding [39]. The leaves of the plant are used by the Ilongot-Eģongot community of Bayanihan, Maria Aurora, Aurora, Philippines, for treatment of toothache [40]. The Tonchongya tribe of Bandarban district, Bangladesh, uses leaves of the plant to stop bleeding from cuts and wounds [41]. The Sundanese people at the Bodogol area, Gede Pangrango Mountain National Park, West Java, Indonesia, use the plant to treat coughs [42]. Leaves are used as antiseptic on wounds and to stop bleeding by tribal people residing in South and West district of Tripura State, India [43]. Leaves of the plant are used to stop bleeding from cuts and wounds by various communities residing in Garo Hills of Durgapur, Bangladesh [44]. The people residing in Surigao Del Sur Mountain Region, Philippines use the plant for genitor-urinary disorders [45]. A Tonchongya tribal healer in Cox’s Bazar district, Bangladesh reportedly used leaves of the plant to treat cuts and wounds to stop bleeding and to treat piles, indigestion, diarrhea, and gonorrhea [46]. The Meetei healers of Manipur, India use leaves of the plant to treat snake bites [47]. The FMPs residing around the Rema-Kalenga Wildlife Sanctuary, Habiganj district, Bangladesh use leaves of the plant to stop bleeding from cuts and wounds [48]. The Chorei tribe of Southern Assam, North Eastern India uses the leaves of the plant to treat cuts and wounds [49]. The leaves of this plant are used by inhabitants of Batak Simalungun, North Sumatra, Indonesia, to cure gastrointestinal disorders. The plant has a bitter taste caused by the presence of tannins in the plant, which compounds can form a thin layer on the lumen thus reducing irritation [50] (Figure 1).From ethnobotanical reports, it appears that the most common ethnic use of the plant is to stop bleeding from cuts and wounds. However, a recent review has pointed out that Mikania genus plants such as

M. glomerata, M. laevigata and M. cordata

are active against disorders of the digestive system including inhibition of gastric ulcers [51]. Sesquiterpene compounds are abundant in

Mikania genus

plants [51] and sesquiterpenes like alpha-cubebene, caryophyllene oxide, alpha-bisabolol, gamma-curcumene, betapinene, copaene, alpha-cedrene, and spathulenol are present in

Mikania cordata

[52]. The anti-ulcer activity of sesquiterpenes has been reported [53].ConclusionLeaves of

Mikania cordata

are used by a FMP of Jamalpur district, Bangladesh for treatment of gastric symptoms. Isolated ethnobotanical reports also demonstrate the use of the leaves of the plant against gastric disorders though most reports indicate ethnic use against cuts and wounds. Perusal of various scientific reports suggests that the plant is rich in tannins and sesquiterpenes, which compounds are beneficial in gastrointestinal tract disorders like diarrhea and gastric ulcer.

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Expanding the Kidney Donor Pool through Use of Hepatitis C-Infected Donors: is it Time to Dive in?

Author by Dinesh Kannabhiran

Abstract

The survival benefit of kidney transplantation for patients with end-stage renal disease (ESRD) is well established. However, the demand for kidney donor organs greatly exceeds the current supply. The use of hepatitis C infected donors could increase the number of kidneys available for transplantation. The use of highly effective second-generation direct acting antivirals (DAAs) has been recently studied for the prevention of chronic hepatitis C virus (HCV) infection in kidney transplant recipients who are HCV negative and receive HCV infected kidney allografts.

Introduction

The survival benefit of kidney transplantation for patients with end-stage renal disease (ESRD) is well established. However, the demand for kidney donor organs greatly exceeds the current supply. The use of hepatitis C infected donors could increase the number of kidneys available for transplantation. The use of highly effective second-generation direct acting antivirals (DAAs) has been recently studied for the prevention of chronic hepatitis C virus (HCV) infection in kidney transplant recipients who are HCV negative and receive HCV infected kidney allografts. Small, open-label trials have demonstrated the feasibility of using DAAs as either pre- and post-exposure prophylaxis or as treatment after detection of HCV transmission. Short term outcomes illustrate 100% prevention of chronic HCV infection with renal function and allograft survival that are comparable to recipients of non-HCV infected kidney donors. Long-term allograft and patient outcomes are required to determine whether the use of HCV infected organs should be considered for all patients with ESRD waiting for kidney transplant. The survival benefit of kidney transplantation for patients with end-stage renal disease (ESRD) is well established [1,2]. However, the demand for kidney donor organs greatly exceeds the current supply which encourages organ procurement organizations and transplant centers to look for innovative strategies to increase the donor pool. Recently, the opioid crisis has increased the number of overdose deaths exponentially [3]. Additionally, the number of hepatitis C virus (HCV) seropositive donors increased from 452 organs per year to 1506 per year between the years of 2000 and 2016 but only 57% of the HCV seropositive kidneys were transplanted in 2016 [4]. The use of hepatitis C infected donors could increase the number of kidneys available for transplantation but this strategy has historically been avoided because of risk of HCV transmission as well as inadequate treatment response and risk of rejection with interferon-based regimens. The advent of highly effective secondgeneration direct acting antivirals (DAAs) has increased viral cure of patients with chronic HCV infection to more than 96% [5-7]. Ongoing research is investigating whether DAAs can be prescribed post-transplant to HCV negative recipients receiving HCV infected donors to increase the donor pool.

The Transplanting Hepatitis C Kidneys Into Negative KidnEy Recipients (THINKER) trial, was a prospective, open-label, nonrandomized trial that included a total of 20 kidney transplant HCV negative recipients who received hepatitis C infected kidneys, determined by HCV nucleic acid testing (NAT) [8,9]. Recipients were included if they were expected to have prolonged times on the wait list while being maintained on dialysis. Recipients could not have any contraindications to liver transplantation or evidence of liver disease detected by FibroScan imaging or serologic testing. Only HCV genotype 1 infected donor kidneys were included. Patients were assessed for HCV transmission on post-op day 3 and were started on elbasvir-grazoprevir (ELB-GRZ) upon detection of HCV infection. Participants with genotype 1a HCV were tested for baseline nonstructural protein 5A (NS5A) resistance mutations. Patients with genotype 1a/1b were treated with ELB-GRZ for a total of 12 weeks while patients with genotype 1a with NS5A resistance mutation were started on ELB-GRZ and ribavirin for 16 weeks total. Outcomes included HCV cure determined by sustained viral response at 12 weeks (SVR12) and adverse events attributable to HCV infection or DAA therapy with 1year follow-up. All patients received rabbit antithymocyte globulin induction therapy and triple immunosuppression maintenance with tacrolimus, mycophenolate mofetil, and prednisone. Twenty patients were included in the trial, of which 19 became HCV PCR positive by post-operative day 3 (range 2-4). The last patient became HCV PCR positive on day 5 after transplant. Seventeen donor allografts were infected with HCV genotype 1a and only 3 donor allografts had NS5A mutations. All patients had undetectable HCV PCR within 4 weeks of therapy. Renal function at 12 months was similar between patients who received a hepatitis C infected kidney donor and matched recipients who did not accept hepatitis C infected kidney donor. No episodes of allograft rejection occurred during treatment. Four patients developed low-level de novo donor specific antibodies (DSAs) that were detected during routine surveillance. Five patients experienced increased but transient elevations in aminotransferase levels which returned to baseline without intervention. One patient with ESRD due to IgA nephropathy was diagnosed with focal segmental glomerulosclerosis post-transplant with no evidence of recurrent IgA nephropathy. Of interest, 50% of donor organs were excluded in THINKER because of HCV infection that was non-genotype 1 [10]. For this reason, a second trial started, called Exploring Renal Transplants Using Hepatitis C Infected Donors for HCV Negative Recipients (EXPANDER), to utilize these discarded organs [11]. Overall, the methodology of EXPANDER was very similar to THINKER, however, EXPANDER included kidney donor organs infected with genotype 1–4 and DAA therapy was administered as pre- and post-exposure prophylaxis. One dose of ELB-GRZ was administered pre-operatively as pre-exposure prophylaxis. After transplant, patients with genotype 1a/1b and 4 were treated with ELB-GRZ for 12 weeks total. Donors with HCV genotype 1a were tested for NS5A mutation and recipients who received a HCV genotype 1a with NS5A mutation kidney allograft were prescribed ELB-GRZ and ribavirin for 16 weeks total. Patients who received genotype 2 or 3 organs were treated with ELB-GRZ and sofosbuvir (SOF) for 12 weeks total. If the donor allograft genotype was unable to be determined due to insufficient viral load, the recipient was treated with ELB-GRZ for 12 weeks.

Ten hepatitis C negative patients were transplant with hepatitis C infected kidneys. Each patient received one dose of ELB-GRZ prior to transplant. Post-transplant, 7 patients received 12 weeks total of ELB-GRZ for genotype 1,4, or unknown genotypes. No patients received an organ infected with genotype 1a with N5SA mutation. The remaining 3 patients had non-genotype 1 or 4 HCV and were treated with ELB-GRZ and SOF. SVR12 for all patients was 100%. No adverse events occurred related to HCV infection or DAA therapy. Like THINKER, one patient experienced increased aminotransferase levels which increased to 5 times greater than the upper limit of normal (ULN) and then returned to normal without clinical symptoms or intervention. While renal function of HCV infected recipients was not compared to non-HCV infected, matched controls, the median creatinine level at week 12 was 1.05 mg/dL (IQR: 0.9 – 2.0 mg/dL) and GFR was 63.5 mL/min (IQR: 47.8 – 69.9 mL/min). THINKER and EXPANDER both illustrated the feasibility of using highly effective DAA therapy as post-exposure prophylaxis in HCV negative recipients who receive HCV NAT positive donors. The participants of both trials were carefully selected under IRB and the DAA was supplied by pharmaceutical companies. A recent, single center experience by Molnar et al reported outcomes for the use of hepatitis C infected donors for hepatitis C negative recipients when used as standard of care treatment [12]. The standard of treatment was offered to all waitlisted recipients regardless of time of the waiting list or dialysis. Allograft donors were considered if they were HCV NAT-positive, donor age of 45 years or younger, and had a donor biopsy with less than 10% glomerular sclerosis. Recipient HCV PCR was not tested until 4–8 weeks after transplant. Once HCV RNA was positive, HCV genotype was tested and patients were started on glecaprevir-pibrentasvir (GLE/PIB), sofosbuvirvelpatasvir (SOF/VEL), or sofosbuvir/ledipasvir (SOF/LED) for at least 12 weeks as determined by hepatology. The hospital planned to pay for treatment if the third-party payor denied any patient HCV treatment. Standard immunosuppression was rabbit antithymocyte globulin induction with triple immunosuppression of tacrolimus, mycophenolate mofetil, and prednisone maintenance. Between March 1, 2018 and December 31, 2018, the program transplanted 53 hepatitis C negative kidney transplant recipients with HCV NAT positive donors. All patients had undetectable HCV RNA by 12 weeks of treatment. Renal function was similar to THINKER trial at 6 months post-transplant with eGFR 66 mL/min (IQR: 54–79). Forty-seven patients (89%) were treated with GLE/PIB, 5 patients (9%) were treated with SOF/VEL, and 1 patient (2%) was treated with SOF/LED. All insurance plans approved treatment within a median of 5 days of insurance application (IQR: 2–8). Eleven patients originally were denied by the third-party payor but were approved after appeal. Ten patients had AST > 3 times upper limit of normal and 8 patients had ALT > 3 times the ULN. All returned to baseline without intervention. One patient developed fibrosing cholestatic hepatitis. This patient was started immediately on DAA therapy and experienced compete resolution. Thirty-two (60%) of patients experienced low level CMV infection without tissue invasive disease. Eighteen patients (34%) developed BK viremia and 2 patient developed BK nephropathy on biopsy. Sixteen patients (30%) developed de novo DSA. Three patients had acute cellular rejection (ACR) and 1 patient had an episode of combined antibody-mediated rejection and ACR.

The short-term outcomes for kidney transplantation of HCV negative recipients using HCV infected kidney donors are excellent. The renal function and allograft survival in clinical trials are comparable to recipients of non-HCV infected kidney donors. Furthermore, the use of HCV infected kidney donors for transplantation decreases the patient’s wait time on dialysis [8- 12]. Emerging evidence is addressing some of the initial concerns about the use of HCV infected donors including cost-effectiveness of this strategy [13,14]. The study performed Molnar et al further illustrates that third-party insurance companies are paying for the therapy with approval of DAA therapy occurring in less than a week in most cases [12].

While initial studies increase the collective understanding of managing this patient population, some questions remain unanswered [8-16]. First, what is the optimal timing of DAA therapy? EXPANDER was the first study to attempt pre- and postexposure DAA prophylaxis strategy [11]. While the number of patients in this study was small, less patients in EXPANDER had elevated transaminase levels compared to either THINKER or the Molnar et al studies leading to the conclusion that avoidance of HCV infection through pre- and post-exposure prophylaxis may help to decrease transaminitis associated with HCV transmission [8-9,11-12]. Moreover, one patient in the Molnar et al. study developed fibrosing cholestatic hepatitis. The authors mention that DAA therapy was started early for this patient and the fibrosing cholestatic hepatitis resolved completely but no additional information was documented. Originally, the treatment protocol for this study started DAA therapy between 4–8 weeks posttransplant but evolved over time to start DAA therapy < 3 weeks after transplant. Further studies will help to elucidate if pre- and post-exposure DAA therapy is associated with less transaminitis compared to waiting to treat after confirmed HCV transmission. Another interesting finding of the Molnar et al study that is not mentioned in either THINKER or EXPANDER is the development of low level CMV viremia in 60% of patients and BK viremia in 34% of patients [8-9,11-12]. Two patients with BK viremia developed biopsy proven BK associated nephropathy (BKAN) but no patients had allograft loss during follow-up. It is unclear whether HCV viremia contributed to the concurrent CMV and BK viremias or whether this population of patients were at higher risk of these infections at baseline.

The detection of DSAs was noted in both THINKER and the Molnar et al study [8-9,11-12]. While no rejection episodes occurred during DAA treatment in the THINKER trial, 4 episodes of rejection were documented in the Molnar et al study; 3 episodes of ACR and 1 episode of mixed ACR and antibody mediated rejection. The grades and timing of these rejections were not discussed. It is unclear whether the rejections occurred during or after DAA therapy but it should be noted that the overall incidence of ACR for this cohort is similar to national outcomes [17]. Patients received rabbit antithymocyte globulin for induction therapy with a mean total dose of 4.9 mg/kg (±0.9 mg/kg). Target tacrolimus goal levels post-transplant and goal mycophenolate mofetil doses were not noted. Future studies should continue to monitor the development of DSAs and rejection in this patient population.

Overall, the utilization of HCV infected kidneys for HCV negative recipients is a promising strategy to increase access to transplantation and should continue to be studied. Current evidence suggests that HCV negative recipients who receive pre- and postexposure DAA prophylaxis or DAA therapy upon detection of HCV transmission can be transplanted with HCV infected kidneys with no documented chronic HCV transmission to date and excellent shortterm renal outcomes. Future studies should investigate the optimal timing of DAA therapy to decrease adverse events described in the literature including the development of DSAs, CMV and BK viremia, and elevated transaminase levels. Long-term allograft and patient outcomes should also be collected to determine whether the use of HCV infected organs should be considered for all patients with ESRD waiting for kidney transplant.

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Do Folk Medicinal Practices of Bangladesh Have any Scientific Value? an Appraisal of Phytotherapeutic Practices of a Rural Folk Medicinal Practitioner

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Abstract

Folk medicine is generally considered as phytotherapy practiced by an individual or a group of individuals, who do not need to obtain any institutional training or approval to practice, and who can practice on a regular basis or as a hobby. In Bangladesh, folk medicinal practitioners (FMPs) are a varied lot using a bewildering variety of plants to treat almost every ailment suffered by human beings. It is the general opinion of allopathic doctors and the affluent section of the Bangladeshi population that FMPs practice nothing but quackery and their main objective is to deceive people. It was the objective of the present study to document the phytotherapeutic practices of a village FMP of Bangladesh and to search through the scientific literature to determine whether the plants used by the FMP had any scientific validation behind their uses. Our Results and Discussions clearly demonstrate that folk medicinal plants have substantial scientific validations, which possibly has come through practice of folk medicine and honing of such practices over thousands of years and transmission of acquired knowledge to successive generations initially orally and then through written methods.

Keywords: Folk medicine; Scientific validation; Phytotherapy; Kushtia; Bangladesh

Introduction

Folk medicine (FM) is practiced by part-time or full-time folk medicinal practitioners (FMPs) in Bangladesh, utilizing for the most part plant-based remedies as their modus operandi for treatment of practically all ailments suffered by the Bangladesh people. FM is not unique to Bangladesh; it is present in practically every country of the world under different names or guises like home remedies, herbal remedies, etc. With time, FM can even take on a more formal form in which cases they are known as Ayurveda and Siddha (in India), Unani (in Greece) or Kampo (in Japan). People of Thailand are said to use herbal remedies since the Sukothai period (1238- 1377) [1]. However, the use of plants as medicines dates back to much earlier times. Radiocarbon dating shows that plants were cultivated in ancient Babylon (present Iraq) more than 60,000 years ago [2]. It is possibly safe to say that human beings have suffered from ailments since their very advent and have tried to cure such ailments possibly from the earliest human ancestors about 6-7 million years ago – the Australopithecines [3]. It is to be taken into account that the great apes and other animal species instinctively partake of some plants for medicinal purposes [4], and the earliest hominids could have easily caught onto this ‘cure’ system.

Ethnomedicine is still somewhat a new concept in Bangladesh even though the country possibly has more than a hundred tribes and over 5500 plant species. With the emergence of new diseases, drug-resistant vectors, and adverse effects of allopathic medicines, even modern doctors and scientists are giving FM a second look. There is a desperate need for new drugs to treat diseases like malaria and antibiotic-resistant microorganisms; plant kingdom, which has always been a source for new drugs [5], can also be the sources for novel drugs at present and in the future. In this instance, FMs can play a key role in guiding scientists to plants with strong potential as sources of novel and efficacious drugs, for from constant practice they can be the most knowledgeable persons on therapeutic properties of plants. Another possible advantage of FMs and tribal medicinal practitioners (TMPs, really tribal FMs) is that due to regional diversity or simply knowledge-based diversity, there can be huge variations between the selections of plants to treat the same disease from one FM to another, even though both FMs may be practicing quite close to one another [6-35]. This opens up alternate sources of discovering possible new drugs with less or no adverse effects. FM is generally dismissed by the allopathic doctors and the affluent section of the population as ‘quackery’. Our objective was to select at random a FMP, and see whether the FMP’s therapeutic uses of plants are supported by available scientific reports on the pharmacological activities of the plant and reported phytochemicals in the plant with appropriate therapeutic benefits. As such we chose a rural FMP for such FMPs, if any, may be more prone to such ‘quackery’.

Methods

The FMP was named Shahidul Islam, male, middle-aged, that is around 45 years of age. He practiced in the villages of Daulatpur Upazila (sub-district) in Kushtia district, Bangladesh. The subdistrict had a total number of 246 villages with an area of 468.76 square kilometer and a population density of 946 per square kilometer. Kushtia district is located in between 23°42’ and 24°12’ north latitudes and in between 88°42’ and 89°22’ east longitudes with an area of 1621.15 sq km. The practice of the FMP was to travel in the various villages searching for prospective patients and also practicing from home at Gobrapara village, Kushtia district. Prior informed consent was initially obtained from the FMP. The FMP was informed the reason for our visit and consent obtained to disseminate any information provided including his name both nationally and internationally. Actual interviews were conducted in the Bengali language, which was spoken fluently by the FMP as well as the interviewers, the language being the mother tongue of FMP, villagers and the interviewers. The interviews were conducted with the help of a semi-structured questionnaire and the guided field-walk method of Martin [36] and Maundu [37]. In this method the FMP took the interviewers to spots from where he collected medicinal plants for therapeutic purposes. (In Bangladesh every village will have fallow land, secluded spots, small water bodies, or a strip of forest from where FMPs and folk herbalists collect their medicinal plants.) The FMP showed the interviewers a number of plants and described their therapeutic uses. All plant specimens shown by him were collected on the spot, pressed, dried and brought back to Dhaka for identification by a competent botanist. Voucher specimens were deposited with the Medicinal Plant Collection Wing of the University of Development Alternative. Secondary information on the pharmacological properties and phytochemicals of the plants used by the FMP were obtained from papers in PubMed, SCOPUS and Google Scholar abstracted journals.

Results and Discussion

The FMP used a total of twelve plants in monoherbal and polyherbal formulations to treat fever, loss of appetite, rheumatism, snake bite, female hormonal disorders, gastritis, anemia, neck pain, nerve sprain, sleep paralysis, ear infection and as a galactagogue. The results are shown in Table 1. Female hormonal disorders are taken in rural Bangladesh to mean either infertility or menstrual problems. Nerve sprain is pain in any portion of the body due to sudden twisting of that body part; in most cases, it indicates neck pain caused from not using proper support for the head and neck during sleeping. Sleep paralysis is inability to speak or move after waking up or while falling asleep; it can be temporary or last longer if it happens from extreme fear, which in rural areas happens when a person imagines to have seen something evil from the spirit world in dream or while waking up suddenly during sleeping. Andrographis paniculata was used by the FMP to treat fever and loss of appetite. The latter can be an individual problem or arising out from fever. The plant contains the anti-pyretic compound, andrographolide, which has been shown to reduce fever in Brewer’s yeast induced pyrexia in rats [38]. Improvement of appetite has been seen with infective hepatitis patients following treatment with the plant [39]. Lasia spinosa and Zingiber officinale have been reported to possess analgesic and anti-inflammatory properties [40,41]; the reports validate the use of the FMP of these two plants to treat rheumatism. The leaves of Carica papaya have lactagogue effect [42]; the FMP used the fruits of the plant (Table 1).

Table 1: Medicinal plants and formulations of the FMP from Kushtia district, Bangladesh.

Crataeva religiosa has medicinal uses in traditional medicinal system of India to treat snake bite [43]. Commelina benghalensis is used to treat infertility in women in many parts of tropical Asia [44]. The fruits of black pepper can be beneficial during gastritis; the fruits also stimulate digestive enzymes, which in turn can lead to amelioration of anemia by stimulating more food consumption; the active ingredient is known to be piperine [45]. Mature fresh leaves of Vitex negundo have analgesic and anti-inflammatory properties [46], so the leaves can be used for neck pain or sprain. The same applies to Cissus quadrangularis, Paederia foetida and Datura metel [47-49]. Thus the three plants in combination can be useful against neck pain and nerve sprain. However, their effectiveness against sleep paralysis needs to be scientifically determined. The analgesic and anti-inflammatory properties of C. quadrangularis can also make it an effective plant to be used against any pain and inflammation associated with ear infection. Overall, it can be seen from published scientific reports that the various plants used by the FMP are more or less scientifically validated in their therapeutic uses based on their reported pharmacological activities and phytochemical contents. FMPs and FM cannot then be describes as mere ‘quackery’, although there may be isolated cases of fraudulent practices.

Conclusion

Our findings suggest that the phytotherapeutic methods of the present FMP were quite consistent with available scientific reports on the pharmacological properties and phytochemical contents of the plants used by the FMP. As such, folk medicine can form the basis for herbal cures and the plants used in folk medicine can prove to be valuable sources of new drugs.

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Development and Validation of Novel Spectro- Chemometric, Chemometric and TLC-Densitometric Methods for Simultaneous Determination of Timolol and Travoprost in their Bulk Powders and Pharmaceutical Formulation

Author by Hesham Salem

,Abstract

Simple, sensitive and reliable spectro-chemometric, Chemometric and thin layer chromatography (TLC)-densitometric methods were developed for simultaneous determination of binary mixture of timolol (TIM) and travoprost (TRA) without prior separation in their bulk drugs and dosage forms. The speectro-chemometric methods used are derivative spectrophotometry and isosbestic point methods. Multivariate methods involves the application of three chemometric techniques; classical least square (CLS) principal component regression (PCR) and partial least-squares regression (PLS) and thin-layer chromatography (TLC)-densitometric methods were developed for the simultaneous determination of TIM and TRA in presence of each other in bulk powder or in dosage form. Separation was carried out on merck HPTLC aluminium sheets of silica gel 60 F254 using ethyl acetate-methanol-ammonia (5:3:0.5) as mobile phase. All variables influencing the spectrophotometric and HPTLC methods were carefully investigated and optimized. The developed methods were evaluated according international conference of harmonization (ICH) rules. The current methods were successfully implemented for direct the determination of these drugs in their trade pharmaceutical formulations. The obtained results were contrasted with those calculated after application of reference method. The data obtained from both methods were statistically analyzed by Fand t-tests. The results of such study showed excellent agreement in respect to the accuracy and precision of the suggested and reference methods.

Keywords: Derivative spectrophotometry; Isosbestic point spectrophotometry; Multivariate chemometry

Introduction

Timolol (TIM) (Figure 1a) is (S)-1-(tert-butylamino)-3-[(4- morpholin-4-yl-1,2,5-thiadiazole-3-yl) oxy] propan-2-ol. Timolol maleate is nonselective β-adrenergic receptors in the heart and decreases renin activity, all of which may play a role in reducing systolic and diastolic blood pressure. TIM is indicated for treatment of mild to moderate hypertension, reduction of mortality after myocardial infarction and migraine prophylaxis [1]. Travoprost (TRA) (Figure 1b) is chemically [1R-[1ᾳ(Z),2β(1E,3R),3ᾳ,5ᾳ]]- 7-[3,5-Dihydroxy-2-[3-hydroxy-4[3-(trifluoromethyl) phenoxy]- 1-butenyl]cyclopentyl]-5-heptenoic acid-1-methyl-ethyleste. Travoprost is used in the treatment of glaucoma [1]. Both drugs are still used as a binary mixture in some pharmaceutical preparations for treatment of reduction of elevated intraocular pressure with open-angle glaucoma. Several analytical procedures are available for the analysis of the cited drugs in their bulk forms, multi-component mixtures, dosage forms or biological mixtures. These procedures are UV-Vis spectrophotometric [1-8], HPLC [9-18], HPTLC [19- 21] and Chemometric [22,23] assay methods are reported in the literature for the estimation of TIM and TRA individually and in combination with other dugs. For the time being only few reports were found on the direct determination of binary mixture of TIM and TRA through HPLC and direct UV methods [1,15-18]. For this reason, this study describes for the first time the simultaneous determination of TIM and TRA by spectro-chemometric such as first derivative (1D) and isosbestic point methods, multivariate methods involves the application of three chemometric techniques; classical least square (CLS) principal component regression (PCR) and partial least-squares regression (PLS) as well as thin-layer chromatography (TLC)–densitometric method, for simultaneous determination of the current drugs without prior separation in their authentic powder and available dosage form (Figure 1a & 1b).

Experimental

Apparatus

Shimadzu-UV 1800 double beam UV-Visible spectrophotometer (Japan) with matched 1 cm quartz cells at 200-400 nm range was used for all absorbance measurements. Spectra were automatically obtained by Shimadzu UV-Probe 2.32 system software. The data points for the zeroorder spectra were then collected at every 0.1 nm. All computations were performed in Matlab for WindowsTM version 6.5. The PLS procedure was taken from PLS-Toolbox 2.0 for use with Matlab® 7.9. TLC-spectrodensitometric system: CAMAG TLC scanner 3 S/ N 130319 operated with winCATS software, Linomat 5 autosampler (CAMAG, Muttenz, Switzerland), CAMAG micro syringe (100 μL). TLC aluminum sheets (20x20 cm) precoated with silica gel 60 F254 (Merck KgaA, Darmstad, Germany) were used. Calculations were performed using the Microsoft Excel program. The centrifugation system: Laboratory Centrifuge, Sigma 2-16KL, Sigma 2-16KHL, with order number 10350, 10353.

Materials and reagents

Pharmaceuticals: The investigated drugs were obtained and were used as such without further treatments : TIM (E.I.P.I.CO., Cairo, Egypt), TRA (Novartus Co., Egypt) with general estimated purity 99.44% w/w and 99.18% w/w on dried basis for TIM and TRA [1], respectively.

Solvents and reagents: Methanol (S.D. Fine-Chem Limite, India) was of analytical grade. Ethyl acetate and ammonia 30% (El- Nasr Pharmaceutical Co., Abu Zaabal, Egypt). In addition, Whatman No. 1 filter paper 110 mm diameter (Sigma-Aldrich) was used for filtration of sample solutions.

Pharmaceutical formulations: The pharmaceutical preparation; Avatan-T® eye drops labeled to contain 40 μg and 5 mg TRA and TIM per mL., respectively, manufactured by (Alcon, Co., Belgium).

Methodology

Standard preparations: Standard stock solutions containing 0.5 mg.mL-1 of either TRA or TIM were prepared separately by dissolving the reference materials in methanol. Regarding the stability of solutions of drugs, stock solutions were stored at room temperature and were found to be stable for at least 10 days.

Working preparations: Preparation of working solutions was done by diluting the standard solutions with methanol to give concentrations in the range 0.4-50 μg.mL-1 for either TRA or TIM.

Synthetic mixtures: Accurate volumes of standard solutions of TRA and TIM were transferred into two sets each of three 10-mL volumetric flasks then completed to volume with methanol so that the final concentration of each drug was within its linearity range as cited in Table 1.

Sample preparation • Eye drops sample: Dilute an appropriate volume of the solution in a volumetric flask so as to contain 40 μg and 5 mg TRA and TIM, respectively. Proceed as directed under 4.3.1. and 4.3.2.

General procedure

Spectro-chemometric methods: Different working standard solutions were prepared with known concentrations within the calibration range specified for each drug as shown in Table 1. These ranges were confirmed to obey Beer’s law for each of the cited drugs in methanol. These concentrations were used to construct calibration matrix which can be used for the direct determination of the current drugs by the different used techniques; (First derivative spectrophotometric (1D) and isosbestic point methods. The obtained absorption data in the general range of 200-350 nm (digitalized every 1.0 nm), against reagent blank treated similarly Figure 2, were stored in PC apparatus program. The spectral data were transferred to Microsoft excel 2003 program by performing statistical manipulation and processing them with the Standard curve fit package and matrix calculations and then were subjected to statistical treatment specified for each technique (Table 1) (Figure 2).

Table 1: Analytical parameters of the proposed methods

a-Intercept, Sa-SD of intercept, r-correlation coefficient, r2-Determination coefficient.

b-Slope, Sb-SD of slope, LOD-Lower limit of detection, LOQ-Lower limit of quantitation

a) First derivative spectrophotometric (1D) method: 1D values of TIM were measured at the zero crossing point of TRA at 226 nm. The 1D value of TRA was measured at 292 nm. For each drug, the calibration graphs were plotted demonstrating the concentration values of 1D amplitude and the regression equations were computed which is further used for calculation of each drug in the sample preparation.

b) Isosbestic point method: The zero order absorption spectra of 10 μg mL-1of each of the studied drugs were recorded as shown in Figure 2. In order to relate the absorbance of the zero order spectra of TIM at their isosbestic points; 230.5 or 272 nm to its corresponding concentrations in μg mL-1. Calibration curves were plotted and the regression equation at each wavelength was derived, illustrated in Table 2. In order to analyze the studied drugs in their binary mixture, different volumes of TRA and TIM were precisely withdrawn from their corresponding working solutions and pipetted into 10 mL volumetric flask to make solutions containing different ratios of each two drugs in their binary mixtures as shown in Figure 2 and then the resultant solution was made up to volume with the same solvent. To calculate the concentration of the studied drugs in their binary mixture, the absorbance of the resulting solution at (1D) 226 nm represented the content of TIM only, which can be obtained using its derived equation at same wave length as shown in Table 2. The total content of each of the prepared binary mixture was determined at their isosbestic points; 230.5 or 272 nm by measuring their corresponding absorbencies at these points and then applied the derived regression equation at each points provided in Table 2. TRA concentration was calculated at each points by subtraction of the concentration of TIMA, determined at (1D) 226 nm, from the total concentrations of both drugs at their isosbestic points (Table 2).

Table 2: Evaluation of the accuracy and precision of the proposed methods

SD: Standard deviation, RSD: Relative standard deviation

Multivariate chemometric methods: Prepare twenty five mixtures of TIM and TRA standard solution; seventeen mixtures were used for building the calibration model, while eight mixtures were chosen to be used as an external validation set. The laboratory prepared mixtures of each of the TIM and TRA were prepared in a concentration range of (0.4-50 μg mL-1) to reach the concentrations listed in Table 3. The absorbance of these mixtures were measured between 200 and 400 nm at 1 nm intervals with respect to a blank of methanol. Several multivariate calibration models were constructed using the data obtained. The concentration of TIM and TRA were calculated by application of the developed models and to analyze the spectra of unknown samples. For construction of the models, to build the CLS, PCR and PLS models, feed the computer with the absorbance and concentration matrices for the training set, use the training set absorbance and concentration matrices using Matlab® (version 7.9), together with PLS-Toolbox 2.0 software for the calculations. The concentrations were calculated from the corresponding regression equations (Table 3).

Table 3: Concentrations of TIM and TRA in the calibration and validation set using PCR, PLS and CLS methods

A Mixtures of validation set

(TLC)-Densitometric methods: The TLC densitometric method was used for the determination of TIM and TRA by separation from each other, depending on the difference in Rf values. Complete separation of TIM from TRA was achieved using ethylacetate:methanol:ammonia (5:3.0:0.5, v/v/v) as the mobile phase. Densitometric scanning was performed at 274 nm with accepted results (Figure 3 & 4).

Results and Discussions

Optimization of variables

Spectro-chemometric method

Spectro-chemometric methods were considered the methods of choice for assay of two component mixtures [24-29]. In this work two spectro-chemometric method such as first derivative (1D) and isosbestic point procedures were developed for the direct determination of TIM and TRA in their dosage form preparation.

Selection of solvent used: Methanol was the solvent of choice for this mixture, where it was found to be the most suitable one for both TIM and TRA from their dosage form. In addition, significant decrease in the degree of overlapping between the two mixture components and higher sensitivity were also observed.

Stability study: The absorbance of the studied drugs was found to be stable for 60 min within 200-350 nm range.

First derivative spectrophotometric (1D) method: In this work, first derivative spectrophotometry was examined for the assay of two component drug mixture. Figure 2 showed that TIM absorption spectrum is overlapped with that of TRA, which in turn made them difficult to be determined simultaneously. In the corresponding 1D curve, provided in Figure 3, the TIM 1D values were located at the zero crossing point of TRA at 226 nm. TRA exhibited a 1D value at 292 nm where TIM first derivative value was nil (Figure 3).

Isosbestic point method: This method is based on the fact that the total concentration of the mixture was determined at their isosbestic points; 230.5 or 272 nm. As illustrated in Figure 2, Thus TIM concentration was determined by the application of first derivative spectrophotometric technique by measuring the absolute value of absorption spectra at its 1D, ʎmax 226 nm without any interference from TRA. Then the concentration of TRA in the accompanied mixture at 230.5 or 272 nm was calculated as explained under 4.4.1.2.

Multivariate Chemometric methods

Among the different regression method existed for multivariate calibration, the factor analysis based methods including partial least squares (PLS regression and principal component regression (PCR) have received considerable attention in the chemometrics literature [30-34]. Due to the overlapped spectra of the drugs, (Figure 2), the previous chemometric methods have been used to analyze this mixture. An appropriate choice of the number of principal components or factors is necessary for PCR, PLS and CLS calibrations. The number of factors should account as much as possible for the experimental data without resulting in over fitting. Various criteria have been developed to select the optimum number [35]. Cross-validation methods leaving out one sample at a time was employed [36]. The predicted concentrations were compared with the known concentrations of the compounds in each calibration sample. The root mean squares error of cross-validation (RMSECV) was calculated for each method for examining the errors in the predicted concentrations. The optimum number of factors was selected by following the criterion of Haal and and Thomas [37]. The selected model was that with the smallest number of factors such that RMSECV for that model was not significantly greater than RMSECV from the model with additional factor. A number of factors were found to be optimum for the mixture of TIM and TRA using PLS, PCR and CLS, Figure 5,6 respectively. To assess the prediction ability of the suggested models, an external validation set was used. The root mean squared error of prediction (RMSEP) values were calculated [38] Table 1. The percentage recoveries of the validation samples are shown in Table 2. The suggested methods were valid and applicable for the analysis of TIM and TRA in Avatan-T® eye drops Table 4. The validity of the proposed method is further assessed by applying the standard addition technique. The results obtained are shown in Table 5 (Table 4 &5 and Figure 5&6).

Table 4: Determination of the studied drugs in their pharmaceutical dosage form using the proposed methods compared to reference method.

*Tabulated value for the proposed methods and reported HPLC method (n=4) at 95% confidence limit; t=2.45 and F=9.28.

Table 5: Determination of the studied drugs in their pharmaceutical dosage form using standard addition technique.

Table 6: Statistical analysis of parameters required for system suitability testing of TLC method.

(TLC)-Densitometric methods

The current work discussed the implementation of a newly developed HPTLC with regard to direct determination of TIM and TRA in their dosage form preparation without prior separation.

Optimization: The TLC densitometric method was used for the determination of TIM and TRA by separation from each other, depending on the difference in Rf values. Complete separation products was achieved using ethylacetate:methanol:ammonia (5.0:3.0:0.5, v/v/v) as the mobile phase. Densitometric scanning was performed at 274 nm with accepted results Figure 4.

Solution stability: The stability of TRA and TIM in their both standard and pharmaceutical preparation solutions was investigated. This was done by storing solutions of the two drugs at room temperature for different time intervals (1, 3 and 5 h) before being analyzed by the proposed method. No decomposition of the drugs was observed during development which was confirmed by the absence of any additional peaks in the chromatograms obtained throughout the analysis time together with the nearly unchangeable peak area of the drugs during stability studies (RSD % less than 2%) indicating stability of the drugs solutions for at least 5 h, which was enough for completing the analytical procedure.

Spot stability: The time for which the sample stands on the plate between spotting and chromatographic development can affect its stability and should be assessed for validation. Twodimensional chromatography was applied to study decomposition occurring during spotting and development. Appearance of peak (s) of decomposition product (s) in both the first and second development directions indicates decomposition during development. Using the proposed conditions, no decomposition was observed during spotting and development indicating stability of the four drugs in their solutions.

Validation of the current procedures

The suggested spectro-chemometric, multivariate chemometric and HPTLC procedures were validated according to USP [39] and ICH [40] guidelines.

Linearity and analytical parameters: Upon application of the optimized experimental conditions, for all the procedures studied, the least-squares regression analysis was applied to compute the regression equations for each drug under study as explained under the general procedure 2.4., and the results obtained are summarized in Table 1. In all cases, the analytical parameters such as linear range, the intercepts (a), slopes (b), correlation coefficients (r), determination coefficients (r2), limits of detection (LOD) and limits of quantitation (LOQ) are included. The slopes are used as measure of sensitivity of the suggested methods, while intercepts indicate the magnitude of background interferences. In all methods applied, the results listed in Table 1 indicate the high sensitivity and low background effects. The obtained values of correlation coefficients (r) and determination coefficients (r2) confirmed a linear relationship between the response measured at the chosen wavelength(s) and the selected concentrations of both drugs in the range specified.

Accuracy and precision: Accuracy and precision of the current procedures were studied at three various levels of the cited drugs concentrations. In this study each of the chosen concentration was analyzed six times by the proposed methods. Table 2 summarizes the results of such study. These results were quite satisfactory reflecting good accuracy of the current procedures. The computed relative standard deviations were found to be below 2% for all procedures studied proving perfect precision of all suggested methods. In addition, the suggested methods were used for the analyses of TIM and TRA in prepared laboratory mixtures with various ratios of both drugs ranged from 1: 1 to 1:125. The percentage recoveries in all cases were satisfactory and the calculated relative standard deviation values for both drugs were below 2 reflecting good accuracy and precision of the suggested procedures.

Specificity and interference: This study has been done by all the proposed methods through analysis of pharmaceutical preparation extract by standard addition method where the samples under study were spiked separately with certain concentration of the investigated drugs in different ratios. In all cases, satisfactory % recovery with relative standard deviation value for both drugs did not exceed 2 were obtained; indicating that no interference is observed from the matrix background

Analyses of available dosage form: The suggested procedures were used for the direct estimation of TIM and TRA in Avatan-T® eye drops and the results obtained were contrasted with those obtained upon application of reference method [1] as illustrated in Table 1. Statistical analysis of the obtained results using Fand t-tests shows that there is no remarkable differences at 95% confidence level. As can be seen, the percentage recoveries in all cases were satisfactory for both drugs reflecting good accuracy, precision and the applicability of the suggested methods to the pharmaceutical preparations.

Robustness: Robustness of the proposed methods was investigated by studying the effect of small change in the experimental parameter(s) on the analytical performance of the method. In this study the parameter under study was changed while the others were kept the same. In case of chemometric and HPTLC methods, variation of wavelength of detection ± 2 nm were tried. In all cases the general procedure was applied and the percentage recovery was computed. It was clear that small variation of the variables studied has no significant influence the efficiency of the method under study. Therefore the proposed methods are reliable with regard to the analyses of the chosen drugs in either bulk forms or their dosage forms.

Conclusion

The current research work describes simple, sensitive, reliable, accurate, reproducible, time saving and economic two spectro-Chemometric, three multivariate chemometric and HPTLC procedures with regard to the direct estimation of the studied drugs in their binary mixture and in the available dosage form. The most important features of these methods are no tedious sample preparation or treatment such as heating, derivatization which may needed especially for HPLC procedure. For HPTLC the cited drugs were detected simultaneously within a period of time less than 8 min. The current procedures were evaluated according to USP and ICH rules where acceptable regression values, RSD % and standard deviations were observed, thus make them versatile and valuable for simultaneous estimation of two drugs in their formulation. Data obtained after analysis of the studied formulations by the suggested methods were too close to label claims, which reflect absence of interference from the accompanied excipients during estimation of the cited drugs by the proposed procedures. Owing to the suitability of the suggested procedures with regard to the routine analyses of the dosage forms of these drugs, they may be used in quality control for these products.

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Is there any Relation Between Glucose in Urine with Music Listening During Travel?

Author by  Maria Rizvi

Abstract

Music can be found almost everywhere being used for entertainment. People also listen to music while traveling. Glucose appears in urine due to hyperglycemia. Basic purpose of this study is finding any relationship between urine glucose and music listening during travel. Urine test was performed for this purpose. 5% students have glucose in urine and like music in their journey. 77% students also like music but have no glucose in urine. 18% students don’t like music and have no glucose in urine. Glucose in urine has no relation with music listening during travel.

Keywords: Hyperglycemia; Music listening

Introduction

Music is used everywhere for entertainment purpose. It can be played at work, home, gym, restaurant, cafes and during travel. People listen to music even for hours. Recent technology of mobiles has made access to music easier than before. As now people can listen to music wherever they are going and these days individuals are spending more time and money on it [1]. Music listening while traveling is a popular activity which is increasing day by day. Approximately 90% of the transport has musical manifestation. It influences a person traveling in a stress condition. Soft music helps to get rid off from anxiety and feel relaxed. But hard rock or heavy metal music puts negative effect on one’s mood [2]. Renal damage of proximal tubules or hyperglycemia causes glucosuria. Normally, all the filtered glucose is reabsorbed by these proximal tubules. Diabetes mellitus and stress causes hyperglycemia. Glucose starts appearing in urine when ultrafiltrate concentration and blood concentration increase from the absorption level of proximal tubules. Detection of glucose is done by enzymatic reaction of glucose oxidase that is specified for glucose. Shelf life of reagent pads is limited. So sunlight protection should be given. If concentration of glucose is low and ascorbic acid is in high concentration in urine than ascorbic acid interferes with this test and false negative results are produced. Formaldehyde also produces false negative results. For false positive results, sample must be contaminated with hypochlorite, chlorine or hypochlorite [3]. Basic purpose of this study is finding any relationship between urine glucose and music listening during travel.

Materials and Methodology

Required equipment for this test is gloves, urine sample, test strips, watch, clean surface, tissue paper and waste bag. Test was performed on hundred students selected randomly. Gloves were put on before starting the test. Test strip was completely soaked in urine for 2 seconds and then it was extracted from the container having urine sample. To remove the excess urine from the strip it was supported on the container’s mouth. Strip was placed on tissue paper to avoid mixing color pads. Time was observed through watch. About after one minute, reactions on reagent strip were appeared. Then this strip was matched with colors of strip container and all the results were recorded. After that all the used materials were thrown in the waste bag and hands were washed completely. Positive results mean there is some abnormality in the person. While negative results mean the person is normal.

Result

Relation of music listening while traveling with urine glucose is provided in Table 1. About 2% male students and 3% females like music and have glucose in urine while14% males and 63% females also like music but they don’t carry glucose in their urine. On the other side, 5% males and 13% females don’t like music during travel and have no glucose in urine (Table 1).

Table 1: Relation of urine glucose with listening to music while traveling.

Conclusion

Urine glucose has no affinity with music listening while traveling.

Acknowledgement

None.

Conflict of Interest

No conflict of interests.

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-Folk Medicine in Bangladesh: Healing with Plants by a Practitioner in Kushtia District

Author by Mohammed Rahmatullah,

Abstract

Folk medicine or treatment by individuals without any formal training, supervision or registration is a common form of medicinal practice in Bangladesh and is generally done with whole plants or plant parts. Folk medicinal practitioners (FMPs) are a common feature in rural Bangladesh with practically every village having one or more FMPs. The unique feature of the FMPs is their remarkable diversity in the selection of plants for treatment. Since folk medicinal practice has been going on in Bangladesh for centuries, it follows that this system of practice has to be found beneficial by the patients or otherwise it would have disappeared a long time ago. As such, the various plants used by FMPs need to be documented for they can serve as important sources of novel drug discoveries. The objective of this study was to document the plant-based remedies of a rural FMP in Kushtia district, Bangladesh. The FMP was found to use a total of 12 plants in his treatment. The plants were distributed in twelve families and were used for the treatment of rheumatic fever, pain, piles, hormone disorders in male, skin disorders, leprosy, hernia, and antidote to poisoning. The advantage with herbal medicine lies in the availability and affordability of medicinal plants. If the FMP’s formulations prove to be scientifically sound, the plants can prove to be possibly a less expensive alternative to allopathic treatments. At the same time, these would create awareness and spur conservation of the plants, quite a few of which are rapidly becoming endangered in the wild.

Keywords: Folk medicine; Medicinal plants; Phytotherapy; Kushtia; Bangladesh

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Iris Publishers-Journal of Archives of Pharmacy & Pharmacology Research-The Impact of Peripheral Venous Catheter Procedural Kits on Improving Clinical Outcomes in Hospitalised Patients

Author By Kathryn Burnett

Abstract

Introduction: Peripheral Venous Catheters (PVC) are an essential component in modern healthcare and their inclusion into a Clinical Procedure Pack has been identified as an area of healthcare where an enhancement in design and implementation could improve practitioner performance and associated patient outcomes.

Aim: The aim of this study was to assess the clinical impact of two interventions;

i. procedural kits with an enhanced training programme

ii. a specific feedback mechanism on the rates of inappropriate removal of PVCs within Antrim Area Hospital. Methods: A time series design, using retrospective and prospective data was used. The study consisted of three phases. Phase A aimed to establish existing PVC adverse event rates. Phase B included the introduction and assessment of the first intervention, a PVC procedural kit and associated training package, to specific clinical areas. Phase C involved the introduction and assessment of the second intervention, a performance feedback mechanism.

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Iris Publishers-Archives of Pharmacy & Pharmacology Research-Tribal Medicinal Plants: Documentation of Medicinal Plants Used by a Mogh Tribal Healer in Bandarban District, Bangladesh

Authored by Mohammed Rahmatullah*

Abstract

Chittagong Hill Tracts region in the southeast portion of Bangladesh is considered a hotspot for medicinal plants. This hilly forested region is inhabited by a number of tribal communities, both large and small, and who still rely on their age-old traditional medicinal practitioners (TMPs) and their mainly phytotherapeutic practices for treatment and cure of various diseases. Since many important allopathic drugs have been discovered through documentation and studies of medicinal plants used by indigenous and tribal people, it was the objective of the present study to document the medicinal plants used by a Mogh (also called Marma) TMP, who claimed his original arrival in Bangladesh from Myanmar several decades ago. Information on ten plants was obtained from the TMP. The plants were distributed into ten families. The TMP used the plants to treat various disorders like tumor, gastrointestinal disorders, paralysis, pain, fever, convulsions, leucorrhea, and antidote to poisoning and as a muscle relaxant. Tumor, paralysis and convulsions (the latter may be due to epilepsy) are serious disorders. Gastrointestinal disorders can be chronic in Bangladesh and so needs effective treatment, which is also affordable. The medicinal plants used by the TMP merit scientific attention because further research can lead to discovery of lead compounds and new drugs.

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Iris Publishers-Archives of Pharmacy & Pharmacology Research-An Overview of Ocular Microbiology: Ocular Microbiota, the Effect of Contact Lenses and Ocular Disease

Authored by Mohammad AA Al-Najjar

Abstract

The human eye is an organ that is exposed to the environment continuously, which makes the ocular surface subjected to different types of pathogenic and non-pathogenic microorganisms. Different types of microorganisms can be found naturally in each layer of the eye, such as the Grampositive bacteria (i.e., the Coagulase-negative staphylococci including S. epidermidis and Bacillus sp), the Gram-negative bacteria (as Pseudomonas sp. including P. aeruginosa) and fungi. Apparently, bacteria are considered to be the major causative agent of ocular infections that are believed to be a global problem. Unfortunately, Ocular infections, if left untreated, may lead to damage in the structure of the eye with possible complications such as blindness and visual impairments. Scientists highlighted the importance of studying the ocular microbiome, which indeed has been accelerated recently. In this review, we aim to overview ocular microbiota and the factors that may affect it, the diagnosis of ocular infections, and the available treatment so far.

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ISSN 2641-2020 (Online) | Archives of pharmacy & pharmacology research

ISSN 2641-2020 (Online) | Archives of pharmacy & pharmacology research