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A Cross-Sectional Study of Cephalosporin Prescriptions for the Treatment of Respiratory and Urinary Tract Infections in Two Sudanese Hospitals
Abstract
Cephalosporins representing a wide variety of β-lactam antibiotics. Cephalosporins have some desirable features, including a convenience of administration, a reasonably broad spectrum of efficacy and a low incidence of toxicity. A descriptive cross-sectional study on the usage of cephalosporin for the treatment of respiratory tract infections (RTI) and urinary tract infections (UTI) was conducted at Ibnsinaa and Alshaab Hospitals in Khartoum state. The data were acquired via questionnaires sent to doctors and community pharmacists, as well as 48 patient files with UTI and RTI diagnoses. SPSS was used to examine the data. The study’s findings indicated that 90% of physicians and pharmacists do not follow cephalosporin prescription and dispensing recommendations. 73% of cephalosporins (3rd generation) are used to treat UTI, whereas 54% of cephalosporins (2nd generation) are used to treat RTI. At conclusion, the findings of this research reveal that the use of cephalosporin in these hospitals is often inconsistent with accepted therapeutic principles. To prevent the emergence of cephalosporin-resistant pathogens, healthcare providers should be cautious when prescribing antibiotics and remain current on recommended antibiotic practices and dosages.
Keywords: Antibiotics; Cephalosporin; UTI; RTI; Infections; Sudan
Introduction
Infectious diseases were a major cause of morbidity and death before to the turn of the twentieth century. Even in the industrialized world, the average life expectancy at birth for men and women was 46 and 48 years, respectively. Plaque, diphtheria, smallpox, pneumonia, cholera, typhoid fever, syphilis, tuberculosis, typhus, and other contagious illnesses were common [1]. Alexander Flemming’s discovery of the first antibiotic (penicillin) in 1928 revolutionized medicine and saved millions of lives [2]. Following the end of Second World War, the golden era of antibiotic discovery began. From the 1950s until the 1970s, dozens new antibiotics were discovered each year, and they revolutionized medicine. Without antibiotics, routine treatments such as open-heart surgery, chemotherapy for cancer patients with compromised immune systems, and organ transplantation would be impossible [3-5]. However, bacteria quickly evolved resistance to antibiotics, and the frequency of infections caused by multidrug-resistant bacteria is growing globally. Since the turn of the twenty-first century, the threat of untreatable diseases has loomed [6,7].
Cephalosporins were not discovered by chance. World War II needs pushed the quest for antibiotics generated by microorganisms [8]. Cephalosporins are antibiotics with a beta-lactam ring that are derived from the Acremonium fungus, commonly known as cephalosporium, this important antibiotic is widely used against bacteria in a variety of serious diseases, including respiratory tract infection (RTI), skin infection, and urinary tract infection (UTI) [9]. Cephalosporins currently come in five generations. With the development of fifth generation cephalosporins, infection management has become even more difficult. However, their use must be strictly limited because if bacteria develop resistance to the fifth generation cephalosporins, infection management will become very difficult [10] Over the last few decades, the rise and spread of beta-lactam resistance in nosocomial Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa, has become a major global concern. Particularly concerning is the rising resistance to third- and fourth generation cephalosporins [11].
Antibiotics are widely utilized in Sudan, and the majority of hospitals in the country rely heavily on cephalosporin antibiotics, especially in surgical departments, as the preferred option for prophylaxis [12]. Accordingly, the current study aimed to evaluate use of cephalosporin in the treatment of respiratory and urinary tract infections in two Sudanese hospitals (Ibnsinaa and Alshaab Hospitals).
Methodology
Study design
This study used a descriptive cross-sectional survey to confirm and/or refute assumptions about the attitudes of health professionals in two hospitals in Khartoum that treat patients with UTI and RTI with cephalosporins, as well as to evaluate the results in order to comprehend and resolve the study’s issue.
Study area
The study took place in two hospitals in Khartoum, Sudan’s capital: Ibnsinaa and Alshaab Hospitals in the state of Khartoum.
Study duration
Two months, between May and July 2018, the surveys were performed utilizing a questionnaire to gather data.
Data collection
The sample size was chosen to be 96 prior to completing the survey. The questionnaire was anonymous. It elicited data on cephalosporins administered for UTI and RTI under treatment recommendations, the Protocol for Dispensing Cephalosporin, the Mode of Prescription, the Common Cephalosporin Used to Manage UTI and RTI, and Counseling Patients About Drugs.
Ethical approval statement
The research used a cross-sectional design. The study protocol was authorized by the ethical committee at Alneelain University’s Faculty of Pharmacy in Khartoum, Sudan, in accordance with the Helsinki Declaration for the conduct of human experimentation. Each participant completed an informed permission form after receiving a thorough verbal summary of the process.
Statistical analysis
The statistical analyses were performed, classified, and analyzed using SPSS. The descriptive data and results were presented using tables and figures. To compare and correlate variables, the chi-square test was utilized.
Results and Discussion
Cross-sectional studies often enable researchers to gather a large amount of data fast. Self-report questionnaires are often used to acquire data affordably. However, causal correlations might be difficult to deduce from cross-sectional data [13].
According to our current study, numerous significant facts were discovered throughout the present cross-sectional investigation. As seen in (Table 1), the protocol for treating RTI and UTI infections at the respective institutions which should be followed by healthcare providers. Clinical guidelines are gaining popularity as a tool for clinicians to use to influence their practice. No guideline, however, can be sufficiently detailed to apply to all clinical circumstances [14].
Additionally, 90 % of healthcare personnel (physicians and pharmacists) at these two hospitals do not adhere to cephalosporin prescription and dispensing guidelines (Table 2). These intriguing results highlight a global concern, especially in developing countries where antibiotic stewardship is poor. Regretfully, the irrational use of antibiotics in Sudan is well-documented [15,16]. According to previously published data, even developing countries with a better health situation than Sudan, a significant amount of antibiotics is provided without a prescription, and a large percentage of antibiotics supplied are unsuitable for the illnesses being treated [17]. The WHO acknowledged irrational antibiotic usage as a significant role in the development of antimicrobial resistance in its two publications, ‘Global Strategy for Antimicrobial Resistance Containment’ and ‘The Pursuit of Responsible Medicines’ and therefore, health authorities in developing countries should tackle this concern [18].
In our study, as shown in (Figure 1-3), 90 % of healthcare providers at these hospitals did not follow specific manner in prescription of cephalosporins for UTI and RTI patients. 4% of participants prescribed first generation cephalosporins, 17% prescribed second generation, 73% prescribed third generation, and 6% prescribed other antibiotics, as shown in Figure 2 & 3. As a result, the third-generation cephalosporin is the most often used antibiotic to treat urinary tract infections. Additionally, our survey found that 6% of respondents prescribed the first generation of cephalosporins to control RTI infections, 54% used the second generation, 31% used the third generation, and 8% used others, as shown in Figure 2 & 3. As a result, we discovered that second generation cephalosporins are effective in treating RTI infections in our investigation.
Numerous clinics worldwide give cephalosporins to patients in excess of what is necessary and with an excess of extravagance that borders on abuse, necessitating medical monitoring and control to prevent the establishment of anti-cephalosporin infections [19,20]. Fortunately, several institutions have recognized the negative repercussions and created control procedures aimed at possibly limiting antibiotic usage and abuse [21]. These control strategies must be implemented as soon as possible in developing countries such as Sudan, since some countries have reported infections and the rise of cephalosporin-resistant pathogens. For instance, Acinetobacter baumannii strains was detected highly resistant to cephalosporins and β-lactamases in Syria [22], In the United Kingdom, Enterobacter cloacae reported resistant to third generation cephalosporins [23], and Klebsiella infection which was found resistant to late-generation cephalosporins in a nosocomial outbreak in the United States [24]. Finally, Effective antibiotic resistance prevention strategies are available and should be adopted aggressively in critical care units. These strategies fall into three categories: nonpharmacologic infection control, antibiotic management and increasing existing efforts to avoid antibacterial resistance, particularly given the expected future scarcity of novel antibacterial medication classes [25].
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In Silico Molecular Docking Study on Selective Cyclooxygenase-2 Inhibitor Drugs For SARS-Cov-2 Active Main Protease
Abstract
The coronavirus (COVID-19) pandemic became one of the most important disease problem across the globe for last few years since there is no recommended efficacious drugs in the market. So, there is an urgent need for efficient drugs to treat this disease in the near future. In the present study, molecular docking analyses of selective cyclooxygenase-2 inhibitor drugs (Celecoxib, Rofecoxib, Valdecoxib, Lumiracoxib, Parecoxib, Etoricoxib, and Firocoxib) were performed against the therapeutic target proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) enzyme into the catalytic active site. On the other hand, these drugs were compared with standard drugs such as Favipiravir, Chloroquine and Hydroxychloroquine to understand the binding sites and find the best poses. The results revealed that all the selective cyclooxygenase-2 inhibitor drugs (except Lumiracoxib) showed a better binding affinity against SARS-CoV-2 Mpro enzyme than the standard drugs. Among them, Etoricoxib (-9.40 kcal/mol) have shown the best binding affinity. As a result, this study shows that these selective cyclooxygenase-2 inhibitor drugs might be interesting lead compounds to discover more potent SARS-CoV-2 Mpro inhibitors and find to cure severe COVID-19 disease with better drugs.
Keywords: Molecular docking; Coronavirus; COVID-19; Cyclooxygenase-2 inhibitor; In silico
Introduction
An outbreak was reported by the World Health Organization (WHO) in Wuhan, China in December 2019. This epidemic was named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on January 30, 2020 [1,2]. In the second week of March 2020, the epidemic was declared global pandemic and within a few months the number of cases increased to 4 million and the death rate increased significantly [3]. The viral agent causing the outbreak belongs to the betacoronavirus family [1,2]. The disease caused by the SARS-CoV-2 factor is highly contagious [4]. This illness is basically a type of viral infection that spreads rapidly through respiratory droplet and direct contact. This infection has many symptoms, such as fever, cough, shortness of breath and gastrointestinal diseases [3,5-7]. The COVID-19 epidemic, in addition to threatening human health, has also had negative effects in areas such as the global economy around the world [3].
Cyclooxygenases are enzymes that allow free fatty acids to convert into cyclic endoperoxides. Arachidonic acid and some other fatty acids are exposed to the action of this enzyme and forming various prostaglandins [8,9]. Studies have shown that there are two different isoforms of the enzyme [8]. The first isoform, known as COX-1, is the structural form and is continuously present in the region in which it is produced. The COX-2 isoform is the inducible form [10,11]. This enzyme isoform is induced, especially in cases that cause inflammation. As a result of increased expression of the enzyme COX-2, abundant prostanoids are formed. In the presence of systemic infection, this rate increases even more. It has been found that this isoform increases in various pathologies, such as certain types of cancer and diseases of the central nervous system [8].
Microorganisms stimulate processes associated with the immune system and inflammatory events in the tissues they attack. Elimination the inflammatory condition that occurs is very important for the treatment of diseases caused by infection. In this context, polyunsaturated fatty acids (PUFA) and their metabolites play a very important role. In studies, lipid derivatives have been found to kill various microorganisms [12]. In general, PUFA kill microbes by their direct effect on microbial cell membranes. Arachidonic, eicosapentaenoic and docosahexaenoic acids act as endogenous antibacterial and antifungals. These lipid molecules also have antiviral, antiparazit and immunomodulatory effects. Cytokines involved in cell defense induce the release of PUFA from the cell membrane. These lipid molecules provide the formation of lipoxins and resolvins that have antimicrobial effects [13]. A study has shown that COX inhibition protects the virus from spreading from cell to cell by a mechanism that inhibits cytomegalovirus maturation [14]. COX-2 inhibitors such as etoricoxib or celecoxib are drugs that contribute to a decrease in mortality in severe influenza. COX-2 inhibitors are thought to be secure in the treatment of COVID-19 and may reduce disease progression in groups of high risky elderly patients with pneumonia due to their treatment of inflammation [15].
It has also been shown in previous studies that the severity and course of the inflammatory process differ decidedly between male and female [16]. Simona Pace et al. found that isolated lipopolysaccharide causes more PGE2 production in males and this may be due to increased COX-2 expression [17]. It is also thought that PGE2 levels, which are an important lipid agent and enhance more in men, may be a factor that explains the more severe disease formation condition of COVID-19 in men [16]. The parallelism of the increase in PGE2 and disease rates suggests that COX-2 inhibitors may be effective in treatment.
The aim of this study is to evaluate the place of COX-2 enzyme inhibition in COVID-19 treatment as in silico. In addition to the effect of these drugs (Scheme 1) on suppressing inflammation and reducing the severity of the disease, the ability to bind to the SARSCoV- 2 factor will be evaluated. This attachment is very important in terms of preventing the viral factor from entering the cell and preventing the effects of the disease on the body.
Material and Methods
The AutoDock 4.2 molecular docking program was used to find best binding interactions of selected selective cyclooxygenase-2 inhibitor drugs against SARS-CoV-2. The three-dimensional (3D) crystal structure of the protein Mpro was retrieved from Protein Data Bank (PDB) (PDB ID: 6LU7) [18]. The 3D structure of the drugs was downloaded from the PubChem (https://pubchem. ncbi.nlm.nih.gov/) in structure-data file format. The most suitable of the possible binding modes obtained as a result of the Molecular Docking processes were determined with Autodock 4.2, and their analyzes and visuals were obtained with the Biovia Discovery Studio Visualizer 2020 program [19-21]. In the present study, a selective cyclooxygenase-2 (COX-2) inhibitor drugs Celecoxib, Rofecoxib, Valdecoxib, Lumiracoxib, Parecoxib, Etoricoxib, and Firocoxib molecules were used for docking procedures. Also, Favipiravir, Chloroquine and Hydroxychloroquine were used as standard drugs for comparison.
Results and Discussion
The docking analysis result of the molecules and standards Celecoxib, Rofecoxib, Valdecoxib, Lumiracoxib, Parecoxib, Etoricoxib, Firocoxib, Favipiravir, Chloroquine and Hydroxychloroquine as inhibitors of SARS-CoV-2 (PDB: 6LU7) including binding energy, inhibition constant and important interactions at the active site are demonstrated in Table 1.
The protein-ligand interaction study revealed that the selective cyclooxygenase-2 inhibitor drugs are binding at the active site of SARS-CoV-2 Mpro protein with the best poses ranging from -6.27 to -9.40 kcal/mol (Table 1). In the current work, all the selective cyclooxygenase-2 inhibitor drugs showed better binding affinity then the standard drugs Favipiravir, Chloroquine, and Hydroxychloroquine (binding affinities of -4.21, -7.22, and -6.26 kcal/mol, respectively), except the Lumiracoxib. Among the best docking scores, only one drug (Lumiracoxib) has been shown the bind in a different region, then the rest of drugs (including standard drugs) and the binding energy of this drug was lowest (-6.27 kcal/ mol) among other drugs that were docked in this study (Figure 1). The best binding affinity (-9.40 kcal/mol) was observed with the drug of Etoricoxib, which have several important amino acid interactions, including hydrogen bonds with Thr 190 and Gln 192, and pi-alkyl interaction with Met 49, Pro 52, Cys, 145, Met 165, and Arg 188. The great binding affinities were also obtained with the drugs of Celecoxib, Rofecoxib, Valdecoxib, and Parecoxib, which had close binding energies to each other’s (-8.24, -8.51, -8.83, and -8.89 kcal/mol, respectively). The most important interactions with these compounds were with Cys 145 (hydrogen bonding), His 41 (pi-carbon bonding), Met 49 and Met 165 (Pialkyl interactions) (Figure 2). The moderate binding affinity was observed with the drug of Firocoxib (-7.88 kcal/mol). This drug also has some hydrogen bounds (His 41, Cys 145, Thr 190 and Gln 192), Pi-alkyl interactions (Met 49, Cys 145, His 163, and Met 165) and Pi-sigma interaction with Gln 189 (Figure 2).
In general, all the docked selective cyclooxygenase-2 inhibitor drugs showed great binding affinities against SARS-CoV-2 Mpro enzyme by having important interactions on the active site. As shown in Figure 1, only one drug (Lumiracoxib) found to bind different binding site which is not favorable for high binding affinities. Some key amino acids are important on the active site for great binding affinities such as Gly 143, Cys 145, Thr 190, and Gln 192 for hydrogen bonding, His 41 (for Pi-carbon interactions), and Met 49 and Met 165 (for Pi-alkyl interactions).
Conclusion
In summary, we have performed molecular docking of the selective cyclooxygenase-2 inhibitor drugs (Celecoxib, Rofecoxib, Valdecoxib, Lumiracoxib, Parecoxib, Etoricoxib, and Firocoxib) with the important therapeutic target protein of SARS-CoV-2 and compared them with the standard drugs Favipiravir, Chloroquine and Hydroxychloroquine. The obtained dock scores demonstrated that all the selective cyclooxygenase-2 inhibitor drugs (except Lumiracoxib) showed a better binding affinity against SARSCoV- 2 Mpro enzyme than the standard drugs. More specifically, Etoricoxib (-9.40 kcal/mol) have shown the best binding affinity. This docking study indicates that these selective cyclooxygenase-2 inhibitor drugs might be useful lead molecules to discover potent and less toxic SARS-CoV-2 drugs in the near future.
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Functional Selective D2 Ligands for the Treatment of Schizophrenia-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Abstract
Functionally selective ligands (also known as biased ligands) of dopamine D2 receptors have been considered as not only valuable tools for dissecting the roles of D2-mediated G protein-dependent and independent signaling pathways, but also better antipsychotic drug candidates for neurological and psychiatric disorders including schizophrenia. Consequently, functionally selective D2R ligands have also been increasingly pursued by the biomedical community as promising antipsychotic therapeutics with improved efficacy and reduced side effects compared with unbiased ligands. This review will discuss the recent development in the discovery of functional selective D2R ligands Figure 1.
Introduction
Schizophrenia is a chronic and severe mental disorder characterized by abnormal social behavior and failure to understand reality [1]. Clinically, the disorder manifests with a large variety of symptoms that fall into three categories: positive, negative, and cognitive [2]. Schizophrenia affects about 1.1% of world wide population [3]. Although schizophrenia is not as common as other mental disorders such as anxiety disorder (18.1%) [4], depression (6.9%) [5], and bipolar disorder (2.6%) [6], the symptoms can be very disabling. Therefore, it is often associated with high levels of morbidity and mortality [7]. The average life expectancy of people with schizophrenia is ten to twenty-five years less than for the general population [8]. This is the result of increased physical health problems and a higher suicide rate (about 10%) [9,10].
Although scientists believe that a combination of genetics, environment, and altered brain chemistry and structure may play a role in the development of schizophrenia [11], the exact cause of this disorder is still unknown to the research community. Consequently, there is no cure for this disorder and treatments have been focusing on eliminating the symptoms of the disease. The primary treatment of schizophrenia is antipsychotic medications [12], often in combination with psychological and social supports. While the current FDA-approved antipsychotic drugs are able to reduce the positive symptoms of schizophrenia in about 7 to 14 days [13,14], they have limited efficacy with negative and/or cognitive symptoms [15,16].
Furthermore, antipsychotic medications can greatly lower the patients' life quality by inducing a wide range of motor, metabolic, cardiovascular, and emotional side effects which also lead to treatment noncompliance [14,17]. Therefore, it is of great necessity to develop novel pharmaco therapies which have better efficacies for treating all three clinical schizophrenia symptoms and possess a low profile for side effects.
With all currently FDA-approved antipsychotic drugs target primarily the dopamine D2 receptor (D2R) for their reaction [15-18], it is indicated that opposing dopamine signaling is central for alleviating psychotic symptoms with schizophrenia [19]. Clinical observations have revealed that schizophrenia-like symptoms occur in amphetamine abusers due to excessive dopamine release [20]. Also baseline dopamine levels and stimulated release of dopamine are found to be abnormal in mesolimbic systems of brains from schizophrenic patients [21]. All these evidences have put dopamine receptor, especially dopamine D2R at the center for the development of antipsychotic drug. Antagonists and partial agonists of D2R have been extensively pursued as antipsychotic therapeutics for the treatment of schizophrenia [22,23].
As a member in the big family of G protein-coupled receptors (GPCR), dopamine D2 receptor (D2R) is Gαi/o coupled whose activation inhibits CAMP production [24]. Antipsychotic drugs targeting D2R were originally identified as being able to bind to D2R and regulate CAMP synthesis [18]. Mounting evident from recent studies indicate that D2R signal not only via canonical pathway involving hetero trimeric large G protein, but also via noncanonical G protein-independent pathways with other signaling proteins including, most prominently, β -arrestins [2528]. Most antipsychotic drugs have been found to have both G protein-dependant and G protein-independent actions [28]. The process by which GPCR ligands, including D2R ligands, differentially modulate canonical and noncanonical signal transduction pathways is a phenomenon known as "functional selectivity" or "biased agonism" [29,30].
With functionally selective D2R ligands that can preferentially activate either canonical or noncanonical D2 signaling pathways [31,32], it is thus very clear that the D2R ligands with discrete functional selectivity profiles will be extremely useful for elucidating the key signal transduction pathways essential for both the therapeutic actions and the side effects of antipsychotic drugs [30]. And this understanding will in turn enable the design of better antipsychotic drug candidates, which can ultimately lead to safer and more effective therapies for schizophrenia patients. In this short review article, we will summarize the recent development in the discovery of functional selective D2R ligands for the treatment of schizophrenia.
Functional Selective D2RLigands
Aripiprizole
Aripiprazole (OPC-14597, 1), an FDA-approved atypical antipsychotic drug, was one of the first functionally selective D2R ligands identified [31,33,34]. It has an excellent side-effect profile presumed, in part, to be due to lack of typical D2R antagonist properties. Although aripiprazole was initially described as a partial D2R agonist, on the basis of assays performed in whole animals and isolated tissues [35-37], it was later demonstrated that aripiprazole could behave as a full agonist, a partial agonist, or an antagonist at D2R depending upon the signaling readout and cell type interrogated [28,31,33,38].
The study examined D2R binding properties of aripiprazole as well as the effects of the drug on three downstream D2R- mediated functional effectors including mitogen-activated protein kinase (MAPK) phosphorylation, potentiation of arachidonic acid (AA) release, and D2R internalization revealed that aripiprazole affects D2L-mediated signaling pathways in a differential manner [31]. In the study examining the properties of aripiprazole at D2 like auto receptors by monitoring the changes of dopamine synthesis in adult rat brain striatal minces incubated ex vivo, it was found that alteration of dopaminergic tone by depolarization affected the actions of aripiprazole on D2- like auto receptors [39].
The in vivo study aimed to investigate the effects of aripiprazole on the D2R downstream cAMP-PKA and Akt-GSK3 β signaling pathways suggested that aripiprazole had differential effects on the cAMP-PKA and Akt-GSK3 β signaling pathways in the brain areas [40]. Additional studies examined the activity of aripiprazole in D2R-mediated heterologous sensitization of adenylyl cyclase and cell-based dynamic mass redistribution (DMR). Aripiprazole displayed a unique functional profile for modulation of G proteins, being a partial agonist for Gαi/o and a robust antagonist for G β γ signaling. Additionally, aripiprazole was a weak partial agonist for both heterologous sensitization and dynamic mass redistribution [41].
UNC9975 (2), UNC0006 (3) and UNC9994 (4)
Through a robust diversity-oriented multi-dimensional modification ofthe scaffold represented by aripiprazole (1), Three β -arrestin-biased D2R ligands including UNC9975 (2), UNC0006 (3), and UNC9994 (4) were reported as the unprecedented β -arrestin-biased ligands for a Gi-coupled G protein-coupled receptor (GPCR) [42]. All three D2R ligands were simultaneously antagonists of Gi-regulated CAMP production and partial agonists for D2R/β-arrestin-2 interactions. Importantly, UNC9975 displayed potent antipsychotic-like activity without inducing motoric side effects in inbred C57BL/6 mice in vivo. Genetic deletion of β -arrestin-2 simultaneously attenuated the antipsychotic actions of UNC9975 and transformed it into a typical antipsychotic drug with a high propensity to induce catalepsy.
Similarly, the antipsychotic-like activity displayed by UNC9994, an extremely β -arrestin-biased D2R agonist, in wild- type mice was completely abolished in β -arrestin-2 knockout mice. These results suggest that β -arrestin signaling and recruitment can be simultaneously a significant contributor to antipsychotic efficacy and protective against motoric side effects [42]. Follow-up comprehensive structure-functional selectivity relationship studies (SFSR) focused on four regions of aripiprazole scaffold (e.g. left hand side phenyl region, middle amino region, central linker region, and right hand side bicyclic aromatic region) resulted in more β -arrestin biased D2R agonists [43].
This combined medicinal chemistry and pharmacological profiling approach also provided the biomedical community a successful proof-of-concept for how functionally selective ligands can be discovered. The study designed to test the effectiveness of UNC9975 or UNC9994 on schizophrenia-like behaviors in phencyclidine treated or NR1-knockdown hypoglutamatergic mice showed that the UNC compounds reduced hyper locomotion in the open field, restored PPI, improved novel object recognition memory, partially normalized social behavior, decreased conditioned avoidance responding, and elicit a much lower level of catalepsy than haloperidol [44]. These preclinical results suggest that exploitation of functional selectivity may provide unique opportunities to develop drugs with fewer side effects, greater therapeutic selectivity, and enhanced efficacy for treating schizophrenia and related conditions than medications that are currently available.
Cariprizine (5) and analogs (Compounds 6 And 7)
Cariprizine(5) is an atypical antipsychotic recently approve by FDA for the treatment of schizophrenia and bipolar mania [45]. It acts primarily as a partial agonist for D2R and D3R [46] with higher selectivity for D3R [46,47]. The structure-functional selectivity relationship (SFSR) studies of cariprazine scaffold carried out by Shonberg and colleagues at Monash University was focused on three main portions of the lead compound: the tertiary amine containing "head group", the cyclohexylene "spacer" group, and the tert-butyl carbamate "tail group" [48].
Similar to the SFSR studies of aripiprazole analogs, to assess G protein-related signaling, the compounds were profiled in the D2 CAMP accumulation assay, whereas β -arrestin signaling was evaluated by measuring phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2). In this testing paradigm, cariprazine (5) displayed a 230-fold bias toward the cAMP pathway compared with dopamine. Interestingly, while all cariprazine derivatives disclosed in this letter showed a bias toward the G protein signaling pathway (6), subtle changes of the D2 unbiased partial agonist, a structurally related aryl piperazine reported by Tschammer [49], led to analogues that displayed a strong bias toward β -arrestin signaling such as compound 7. By combining the medicinal chemistry efforts with novel analytical pharmacology methods [50], it was discovered that the nature of the head group, the composition of the tail group, and the orientation, length, and flexibility of the spacer were all important factors for the control of functional selectivity at the D2R [48].
Functional selective D2R ligands with privileged structures (compound 8)
Another comprehensive structure activity relationship (SAR) studies carried out by Szabo and colleagues at Monash University also led to the discovery of novel functional selective D2R ligands [51]. They investigated the determinants of efficacy, affinity, and bias for three privileged structures for the D2R, exploring changes to linker length and incorporation of a heterocyclic unit. After profiling the newly synthesized compounds in two signaling assays (CAMP and pERK1/2), they were able to identify and quantify determinants of functional selectivity at the D2R. The results from combined medicinal chemistry and pharmacological profiling approach revealed that: 1) substitution on the phenylpiperazine privileged structures (2-methoxy vs 2,3-dichloro) influenced bias when the thienopyridine heterocycle was absent; and 2) upon inclusion of the thienopyridine unit, the substitution pattern (4,6-dimethyl vs 5-chloro-6-methoxy-4-methyl) had a significant effect on bias that overruled the effect of the phenylpiperazine substitution pattern [51]. This latter observation could be reconciled with an extended binding mode for these compounds, whereby the interaction of the heterocycle with a secondary binding pocket may bring functional selectivity to the parent compounds. The resulted novel D2R partial agonists, 8 and 9, display a similar affinity for the D2R as aripiprazole but exhibit distinct functional selectivity profiles, which made them useful tools to explore the contribution of functional selectivity to antipsychotic efficacy.
Functional selective D2R/D3R partial agonists
Dopamine D2R and D3R are known as valuable targets for the treatment of neurological and psychiatric disorders including schizophrenia [52-54]. Hiller and colleagues evaluated a series of newly synthesized compounds for their ability to differentially activate distinct signaling pathways [55].
Measurement of D2L- and D2S-mediated [35S] GTPyS incorporation in the presence of coexpressed Gao and Gai subunits showed significantly biased receptor activation for several test compounds [55]. A follow-up study from the same group using the same evaluation methods yielded the most striking functionally selective D2R ligand, carbaldoxime 8b (9, Gaol, pEC50 = 8.87, Emax = 65%; Gai2, pEC50 = 6.63, Emax=27%) [56]. It was also indicated in the study that 1,4-disubstituted aromatic piperazines (1,4-DAPs) behaved as antagonists for β -arrestin-2 recruitment, implying significant ligand bias for G-protein activation over β -arrestin-2 recruitment at D2R. Regiochemistry and the nature of functional groups attached to the pyrazolo [1,5-a]pyridine moiety strongly influence ligand efficacy and selectivity between D2R and D3R activation [56].
Other functional selective D2 ligands
Intensive structural exploration on aripiprazole scaffold engendered UNC2438 (10) which was found to be a G-protein biased compounds [57]. Further structure-functional selectivity relationship studies (SFSR) on UNC2438 focused on the right hand side benzothiazole moiety and the left hand side phenylpiperazine moiety furnished a few additional G-protein biased compounds which were partial agonists in D2R Gio- mediated CAMP inhibition assay and simultaneously inactive in D2R-mediated β -arrestin-2 recruitment assay [57].
Most recently, a tailored virtual library with close to 13,000 compounds bearing 2,3-dichlorophenylpiperazine, a privileged orthosteric scaffold, connected to diverse chemical moieties via a linker was docked to the D2R model [58]. Eighteen top-ranked compounds that occupied both the orthosteric and allosteric site were synthesized, leading to the discovery of 16 partial agonists. While a majority of the ligands had comparable maximum effects in the G protein and β -arrestin recruitment assays, some displayed preference for a single pathway. In particular, compound 11 stimulated β -arrestin recruitment (EC50 = 320 nM, Emax = 16%) but had no detectable G protein signaling.
Conclusion
The growing realization of the complexity of G protein coupled receptor (GPCR) mediated signal transduction pathways, specifically D2R mediated signaling pathways, has provided a theoretical framework for the development of functionally selective or biased ligands. Several studies have demonstrated that agonists differ in their ability to activate various pathways. Notably, blockade of β arrest in recruitment was found to be a shared property of antipsychotics that exhibit either antagonist (e.g., haloperidol), or partial agonist (e.g., aripiprazole) activity through Gai/o-CAMP pathways [28]. In contrast, a study with analogs of the novel antipsychotic aripiprazole suggested that D2 ligands with Gai/o antagonist and β -arrestin agonist activity may have antipsychotic behavioral activity with reduced extra pyramidal side effects in a mouse model [3]. While there are still a lot of questions to be answered in this field, heightened awareness of the potential benefit of pathway biased D2R ligands has inspired scientists in this area develop more functionally selective D2R ligands as better antipsychotic therapies for schizophrenia.
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A Case of Vitamin D Toxicity-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Short Communication
An eighty four year old Caucasian male started taking glucosamine for joint health two years previously. He failed to notice that the glucosamine tablets also contained large doses of cholecalciferol, a precursor of vitamin D. According to the label on the container, two tablets each having five times the recommended daily amount (RDA) were to be taken every day. This was in addition to the vitamin D taken in milk at breakfast, and that in half gram calcium tablets taken twice daily and also vitamin D in a multiple vitamin preparation. All together this added up to 6000 international units (IU) of the vitamin taken each day. The RDA for older people is 800 IU. So he was taking 7.5 times the RDA of vitamin D for over a year. When he discovered the overdose, he stopped taking vitamin D except for milk at breakfast.
Symptoms noted during vitamin D intoxication were mostly central nervous system effects (nervousness, insomnia, irritability, clumsiness) not uncommon in an eighty four year old man. These symptoms persisted for several months even after cessation of vitamin D overdose but gradually decreased in intensity. Submandibular pain also occurred but went away after correcting the overdose. Arthritis also occurred in the knee joints but this did not abate after cessation of the overdose. Reversible Parkinsonism is also seen in vitamin D intoxication. Some increase in tremor was evident in the patient during the overdose but the tremor gradually became less intense with increased time without the vitamin supplement.
The most serious symptoms occurred about two weeks after cessation of the overdose. A severe bradycardia (38 beats/ min) was noted on several occasions at that time. A physician was consulted and did an EKG, a 24 hour Holter monitor and an echocardiogram to document the symptoms. A marked weakness of the heart beat was noted and dizziness was experienced. It took about ten days for the strength of the pulse to return and for the severe bradycardia to correct itself. Some cardiac arrhythmias were present even before the vitamin D overdose (heart block and premature ventricular contractions). These were referred to as benign by a physician and did not change with vitamin D overdose except as mentioned above.
Vitamins are substances that cannot be produced in the body and must be taken in the diet. Since vitamin D can be formed in the body, it is not a true vitamin but is designated a "pro-hormone". However, amounts produced in the body are generally not sufficient, and some vitamin D must also be taken in the diet. Pro-vitamin D compounds are lipid soluble sterols and can accumulate in body fat [1]. They are also slowly removed from the body. This is why symptom scan persist long after the patient stops taking the vitamin D supplement. The pro-vitamin molecule formed by UV light in the skin or taken orally, goes to the liver and is hydroxylated to form cholecalciferol. This substance is not very active physiologically and is hydroxylated in the kidney to form calcetriol which is powerful steroid and works to maintain a constant blood level of calcium by promoting calcium absorption in the gut and by inhibiting calcium re absorption from bone. Blood calcium mediates nerve and muscle activity and must be maintained at a constant level for proper function. To estimate levels of vitamin D in the body, serum concentrations of 25(OH)D are measured. Apparently this is a simple assay and reflects the amounts calcetriol and chole calciferol and other analogs. Some have suggested that it might be better to measure both 25(OH)D and calcitriol but estimating calcitriol is a delicate procedure and is done only in specialized labs [2]. The chemical form of vitamin D which causes symptoms in patients receiving an overdose, have not been fully identified. Calcetriol is the most potent form but when formation of this substance is blocked in knockout mice, it makes no difference in the toxicity of cholecalciferol [3]. It is possible that several vitamin D analogs are responsible for the toxic effects.
Cholecalciferol is not only a dietary supplement but is also a rat poison and can be a problem when ingested by house pets. It is therefore of great interest to veterinarians [4]. Another symptom, more annoying than serious, appeared after terminating excess vitamin D. Cutaneous inflammation on the arms, legs and body with lesions about one to two cm in diameter began to appear about 10 weeks after stopping the excess vitamin intake. Redness and itching were intense but gradually disappeared about five months after correcting the overdose.
There has been a marked increase in marketing of vitamin D supplements recently. One report covering years 2010 to 2014 shows a twenty fold increase in use of these supplements [4,5]. Vitamin D has a reputation of being "healthy and safe" so that physicians promote vitamin D use. So the main message in this publication is that caution is needed in use of vitamin D supplements which can cause serious and even lethal effects if used over a long time interval.
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In Silico Design: Those Accentuate Assembly of HIV-1 Capsid-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Abstract
Considering the significance of RNA genome in the HIV infection, its release can be forbidden by inhibiting encapsulated Capsid (CA) "core". In this context, design of new molecules that interact with HIV-1 CA assembly was made by conducting 3D-QSAR and Pharmacophore studies on a stockpile of fifty eight molecules. The supramolecular interactions of these inhibitors with the receptors active site amino acids Glu 35, Gly 60, His62, Gln 63, Ala 65, and Val 136 has been characterized using docking studies. The combined five point Pharmacophore hypothesis AHHRR, and 3D-QSAR CoMFA (Comparative molecular field analysis) and CoMSIA (Comparative molecular similarity indices analysis) has shown good statistical partial least square factors. The association of generated 3D-QSAR and PHASE pharmacophore hypothesis has provided structural insights to explore new molecules with enhanced CA assembly activity.
Keywords: Human immunodeficiency Virus type-1 (HIV-1); Capsid protein (CA); Common pharmacophore hypothesis (CPHs); Pharmacophore alignment and scoring engine (PHASE); Comparative molecular field analysis (CoMFA); Comparative molecular similarity indices analysis (CoMSIA) emphasize
Introduction
Capsid protein (CA) of Human immunodeficiency Virus type- 1 (HIV-1) is a fullerene cone accomplished with approximately 250 hexamers and 12 pentamers that plays a prominent role in the initial stages of post host cell entry and in virus assembly [1-4]. Proteolytic cleavage of Gag (Matrix, Capsid and Nucleo capsid) polyprotein releases CA which reassembles to form a conical "core" that encloses the viral RNA genome [5-7]. The assembly process of CA protein is associated with the N-terminal domain (CANTD residues 1-145) and C-terminal domain (CACTD residues 151-231). The CANTD aid in virion maturation by self associating into hexameric rings and incorporation of the cellular protein cyclophilin A (CypA). While CACTD contributes to Gag-Gag interactions [8-10]. Once the virus enters the cell, the core disassembles and releases ribonucleoprotien complex, resulting in the formation of reverse transcription complexes [11-14]. However, virions with deformed core structured are defective in initiating reverse transcription and exhibit reduced infectivity [15]. It is believed that the N-terminus of CA can refold to form a new y-hairpin helix by cleavage of CA from matrix protein (MA), which is stabilized by a buried salt bridge between Proline-1 of CA and the carboxyl side chain of Asp51. The renovation from tubular to spherical forms can be induced by changing the pH from 7.0 to 6.8 [16,17]. Several small molecule inhibitors like CAP-1, NYAD-1 a peptide inhibitor, bevirimat, a triterpene derivative have been reported to interfere with CA function on binding to the N-terminal domain of CA in both its mature and immature forms [18-25]. Pharmacophore mapping was performed by using Pharmacophore Alignment and Scoring Engine (PHASE) to evaluate features necessary for the ligand to interact with a receptor [26]. The pharmacophore may be used as a query in searching 3D databases containing "drug like" small organic molecules and also aid in exploring and engineering of new scaffold with high potency [27]. To investigate the mode of recognition and interaction mechanism of HIV-1 CA inhibitors with CA protein, molecular docking studies are performed. Further 3D-QSAR studies were performed by using CoMFA and CoMSIA analysis.
Methodology
Ligand construction and preparation
A stockpile of fifty eight 1,5-dihydrobenzo[b][1,4]diazepine- 2,4-dione derivatives [28-30] (Figure 1) with their experimental information were collected from the literature. Outlining the three dimensional structures of these ligands using Maestro build panel in Schrodinger Suite, all possible low energy states are generated at physiological pH range of 7 +/- 2 in the Ligprep module of Schrödinger.
Generation ofthe common pharmacophore hypothesis (CPH)
The pharmacophore hypothesis and alignment were carried out by PHASE [31] (version 2.5, 2011; Schrodinger, LLC, New York, NY). Considering the significance of EC50 in biological activity [32,33] and we have converted E C50 into the corresponding pEC50 (-logE C50), and randomly selected thirty five molecules as training set to generate Pharmacophore models. PHASE defines chemical features of ligand that facilitate non covalent bonding between the ligand and target receptor. PHASE models are created by applying Partial Least Squares (PLS) regression. The accuracy of the models improves with an increase in the number of PLS factors [34]. The PLS regression analysis was carried out using three PLS factors and possible statistical parameters are evaluated. The test set predictions were described as Q2 root mean squared error and Pearson correlation coefficient (R) value. The regression value is calculated using the formula;
Where are the average observed and predicted pEC50 values of the test set molecules.
(Figure1).
Docking studies
The X-ray structure of CANTD in complex with the benzodiazepine inhibitor (PDB: 4E91) [35] was retrieved from Protein Data Bank (http://www.rcsb.org/). Protein was prepared by using protein preparation wizard in Schrodinger. [36] GLIDE 5.6 (Grid-based ligand docking with energies) docking was performed by generating a grid (10 Å x10 Å x10 Å) around the active site of capsid assembly inhibitor. Rigid receptor docking protocols of Glide, namely Standard Precision (SP) and Extra Precision (XP) were employed to gain insight into the binding modes of all inhibitors. Further, to account for the receptor flexibility, a computationally intensive Induced Fit Docking (IFD) was performed. To validate the docking protocol, co-crystallized ligand benzodiazepinedione was re-docked and its atomic root mean square deviation (RMSD) was calculated [37-39].
Prime/MM-GBSA calculations
The relative binding free energies for best ranking molecules in XP mode are calculated by Molecular Mechanics Generalized Born Surface Area (MM/GBSA) using Prime. The dock pose features that are locally optimized features from glide were minimized from prime and complex energies were calculated using OPLS_2005 force field. The relative binding free energy ΔGbind was estimated according to equation.
ΔGbind=Ecomplex (minimized)-[Eligand (unbound, minimized) + Ereceptor (unbound, minimized)]
ΔGbind is the calculated relative free energy which includes both ligand and receptor strain energy. Ecomplex (minimized) is MM-GBSA energy of the minimized complex, Eligand (unbound, minimized) is the MM-GBSA energy of the ligand after removing it from the complex and allowing it to relax. Ereceptor (unbound, minimized) is the MM-GBSA energy of protein after separating it from the ligand.
CoMFA and CoMSIA Models
CoMFA and CoMSIA studies are performed by aligning data set molecules and are evaluated for thier steric, electrostatic and hydrogen bond effect on bioactivities using SYBYLX-2.1[40,41]. Gasteiger Huckle charges were assigned for all the inhibitors [42]. For CoMFA, the overlapped molecules were placed in a rectangular grid points separated by 2 A. The van der Waals potential and columbic terms, representing the steric and electrostatic fields, were calculated using standard Tripos force field [43]. The regression analysis was carried out using set of variables, and cross validated the PLS method (leave-one-out). The final model (noncross-validated conventional analysis) was developed with the optimal number of components which poses the highest q2 value.
For CoMSIA, five physicochemical properties namely steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor were calculated. The CoMFA or CoMSIA descriptors were used as independent variables and pEC50 values as dependent variables in partial least square analysis. [34] The predictive correlation coefficient (r2pred ) based on the test molecules, is computed with the formula r2pred = (SD-PRESS)/SD, where SD is the sum of the squared deviations between the biological activities of test set and mean activities of the training set molecules. PRESS is the sum of squared deviation between predicted and observed activity for each molecule in the test set.
ADME prediction
The ADME (absorption, distribution, metabolism and excretion) properties of designed molecules were evaluated computationally using Qik Prop module of Schrodinger [44]. The physically significant descriptors and pharmaceutically relevant properties of organic molecules with compliance to Lipinski's rule of five were calculated by Qik prop.
Results and Discussion
The hexameric architecture of capsid relay on three basic type of interactions. (i) CANTD-CANTD six fold symmetric interfaces that create the hexameric rings [45]. (ii) CANTD- CACTD intermolecular interface that reinforces the hexamer [8-10] and (iii) CACTD-CACTD dimeric interface that links the hexameric rings to form the lattice [46-50]. The current study is mainly focused on hexamer formation of CA by disrupting the intermolecular interaction of CANTD and hence in silico methods employed are restricted to N-terminal domain only. The X-ray crystal structure (4E91) of HIV-1 CANTD is a single chain containing the N-terminal domain. Sundquist reported intermolecular N-terminal domain interactions between Phe-32, Glu35, Gly60, His62, Gln63, and Ala65 residues of helices 1 and 2 [51]. The perturbation of intermolecular N-terminal domain interaction involves in disruption of NTD-NTD interaction and hence inhibits CA assembly [52].
Pharmacophore generation
PHASE analysis for a set of 58 molecules derived five point CPHs belonging to AHHRR, AHRRR. Thirty five molecules in the training set were aligned on these CPHs and evaluated for PLS analysis using three PLS factors. The predictivity of each hypothesis was cross validated by the test set of twenty three molecules. The variants named AHHRR (model A1) and AHRRR (model B1) in Figure 2 showed better statistical significance than AHHRR (A2), AHHRR (A3) and AHHRR (A4). Since all the five pharmacophore hypothesis capitulate a statistically significant data as shown in Table 1, the hypothesis AHHRR (A1) with good survival score 3.549, with significant R2 of 0.926 and Q2 of 0.824 was considered in this work. From the above results it is confirmed that the obtained PHASE model (r2 > 0.5, q2 > 0.6, [(r2- r02)/r2 < 0.1, 0.85 ≤ k ≤ 1.15 and rm2 > 0.5) was in the acceptable range.
The hydrogen bond acceptor A2, and the phenyl ring R12 was found to be significant for the activity. The carbonyl group at second position can form hydrogen bond interaction with His 62 residue in the active site, while the aromatic ring at third position contributes to hydrophobicity. The hypothesis AHHRR (AJ was fit into the molecular skeleton of highest active molecule of capsid assembly inhibitor 50g. The distance and angles between the five features of the best two models AHHRR (AJ and AHRRR (BJ are shown in Table 1 & 2 (provided in supplementary data). The pharmacophore model implies that molecules which would fit the AHHRR (A1) sites may consequence in stronger interactions with the active site residues. The distance and the angle maps of the best two models are represented in Figure S1 given in supplementary data. The field contribution (blue and red cube contours) of the best two models are represented in Figure 3. The blue cubes represent that substitution of hydrophobic favored groups while red cubes represent disfavored region. Scatter plot of experimental and phase predicted pEC50 is shown in (Figure 2-4) (Table 1).
A-Hydrogen bond acceptor, H- Hydrophobic/non-polar group, R- Aromatic ring, R2-correlation coefficient, F- variance ratio, Q2-predicted activities for training set, SD- standard deviation of regression.
Molecular docking studies and Prime MM/GBSA calculations
To evaluate the validity of docking protocol, root mean square deviation (RMSD) calculations were performed. The crystal ligand was haul out from the complex (PDB id: 4E91) and subsequently re-docked into the receptor. Best dock pose from docking studies differ from the original conformation by 0.216 A, substantiating the robustness of docking protocol to enumerate the experimental binding mode. Figure 5 shows the overlay of the X-ray crystal structures (colored in cyan) of capsid assembly inhibitor and the re-docked pose (colored in plum) (Figure 5).
Fifty eight molecules with benzodiazepine scaffold which were reported to exhibit inhibitory effect on assembly process of HIV-1 capsid (Figure 1) are chosen for docking studies. The binding orientation of these has shown hydrogen bond interactions with Glu35, Gly60, His 62, Gln 63, Ala 65, Val 135 and Tyr 145 amino acid residues and are said to exhibit HIV-1 replication with a novel mechanism of action [52]. This compounds bind to the N-terminal domain (NTD) of the viral capsid protein (CA) and is believed to prevent its ensemble into conical core that is trivial for virion maturation and viral infectivity [53].
The hydrogen bond interaction with His 62 and Phe 32 residues of helices 1 and 2 are plays a crucial role since they hinder the hexameric lattice organization by interrupting intermolecular N-terminal interaction. SP dock pose of highest active molecule 50g display polar H-bond interaction with His 62 (Figure 6a), while XP dock pose exhibit additional n-n stacking interaction with Phe 32. (Figure 6b) The IFD dock pose for the same 50g molecule reveal polar hydrogen bonding interaction with Phe 32 and His 62 residues (Figure 6c). Application of different docking protocols i.e., SP, XP and IFD resulted in variable dock scores, of which IFD has shown more refined scores. Increase in dock scores in IFD protocol is possibly due to additional flexibility attributed to the protein, which allow more interactions with active site residues. However, some deviations have been observed in IFD scores of 45f and 53h which were lower than XP dock scores which might be due to steric clashes.
The relative binding affinities of protein ligand complexes were assessed by Molecular mechanics with generalized born surface area (MM/GBSA). The ΔGbind value of highest active molecule 50g was found to be -134.50 that is slightly higher than the lead molecule 54 which was screened through CA assembly assay. It is imperative to evaluate statistical correlation between the different computational parameters and experimental pEC50 values to validate the applicability of in silico studies in predicting the HIV-1 CA inhibitors. These inhibitors showed a statistically significant correlation of -0.579 for XP docking, -0.614 for prime MMGBSA (Figure 7). The list of inhibitors along with their pEC50, SP, XP and IFD dock scores, ΔGbind values from PRIME along with predicted CoMFA and CoMSIA values have been tabulated in Table 2.
(SP, XP, IFD) and binding energy (ΔGbind).
*test set molecules.
3D-QSAR model
The 3D-QSAR CoMFA and CoMSIA analysis was performed by aligning the dock poses of all fifty eight molecules (Figure 8). A training set of thirty five molecules (which has been selected for generating PHASE models) were also chosen for constructing CoMFA and CoMSIA models. The best predictions were obtained with CoMFA standard model q2 value of 0.607, r2 value of 0.968 and CoMSIA with q2 value of 0.616 and r2 value of 0.954 respectively. The predictivity of the model was cross validated by the test set of twenty three molecules. The predictive correlation coefficient r2 pred of 0.576 for CoMFA and 0.528 for CoMSIA explains good predictive ability of the models. The scatter plot of actual and predicted pEC50 values for training and test set of CoMFA and CoMSIA studies are shown in Figure 9a and Figure 9b. The results of CoMFA and CoMSIA are listed in Table 3.
q2 = correlation coefficient from leave one out method.ONC = Optimum number of components, SEE=Standard error of estimate, F = Fisher value, r2Pred= predictive r2 on test set.
CoMFA and CoMSIA contour maps
To visualize the information derived from the 3D-QSAR models, contour maps were generated. The contour plots are representation of the lattice points in the grid and the difference in the lattice points is strongly connected with the difference in the receptor binding affinity. Whereas molecular fields defines the favorable and unfavorable interaction energies of aligned molecules with a probe atom traversing across the lattice points, suggesting the modification required to design new potent molecules.
The CoMFA contours indicate the region in space where the molecule would favorably or unfavorably interact with the receptor, while CoMSIA contours indicate the areas within the specified region where the presence of groups with particular physicochemical property binds to the receptor. Therefore the most potent inhibitor among the series 50g was displayed on the maps for visualization.
The steric contours of CoMFA and CoMSIA Figure 10(a) and Figure 10(b) indicate the regions where sterically bulky substituent might have favorable (green) and unfavorable (yellow) effects on the activity of the inhibitor. The methoxy and hydroxyl substitutions are necessary to have good interactions with the receptor, and pyrazole ring is less sterically crowded, substitutions at this position with a bulky group facilitate increase in the activity. The electrostatic contours of CoMFA and CoMSIA Figure 10(c) and Figure 10(d) suggest that increasing the negative charge in red region will have a propensity for increase in binding affinity towards the receptor. The hydrophobic contour of CoMSIA Figure 11(a) recommends the substituent's like CF3 and phenyl are accountable for the hydrophobic activity. Any modification of C F3 and phenyl would result in a decrease of activity. The H-bond acceptor and H-bond donor contours of CoMSIA imply the regions with cyan acceptor favored and magenta donor favored regions are liable for activity as depicted in Figure 11(b) and Figure 11(c) (Figure 6-10) (Table 2,3).
Design of new molecules
As the first hit molecule screened through Capsid assembly assay was compound 54 with an enamine side chain at 3rd position was reported to be chemically instable by Lee Fader [28], and the SAR studies were performed by replacing the assay was compound 54 with an enamine side chain at 3rd position was reported to be chemically instable by Lee Fader [28], and the SAR studies were performed by replacing the enamine side chain with more polar groups like 2-methoxyethyl, 2-hydroxyethyl chains which resulted in no loss of potency thus confirmed that small substitutions at this position can be tolerated [29]. In this article designing of new molecules was attempted by substitution on phenyl ring at 5th position but results were not industrious in terms of compliance to PHASE. These observations lead us to a conclusion that the substitution at R1, R2 and R3 of phenyl moiety at third position are well tolerated and therefore were targeted for isosteric substitution (Figure 11).
The sterically favored pyrazole at R3, that showed single interaction with His 62 has been substituted with 1,2,4 triazole and electronegative favored methoxy (-OMe) group in 50g has been replaced with methyl sulfonate (-SO2Me) of N10, showed equivalent dock scores and facilitate newer interactions with Val 27 and Val 59. This implies that improved electronegativity on the substituents showed higher affinity. Increase in aliphatic carbon chain on diazapine nitrogen with propyl (N1-N6) and isobutyl (N7-N ) accounts for increased hydrophobicity. Considering the structural necessities depicted in Figure 12, we have designed ten new molecules which adhere to PHASE as well as CoMFA and CoMSIA. Docking studies revealed that the designed molecules Figure 13 show good interaction with the active site amino acids. The structures of newly designed molecules and their possible interactions are tinted in Figure 14. The pharmacokinetic data of newly designed molecules is illustrated in Table 3 (provided in supplementary data).
Design of new molecules by retaining quinoline scaffold. The quinolines not only exhibited antibacterial, antimalarial but also shown to inhibit HIV-1 activity by involving in assembly process. Hence new molecules are designed with a view to interact with N-terminal domain of HIV-1 Capsid, they too have shown the similar interactions as that of reported molecules. The docks pose C1 with its ligand interactions are depicted in the Figure 14. The oxygen of carbonyl carbon forms hydrogen bond interaction with amine of Phe 32, where as the amine of quinoline forms hydrogen bond interaction with the His 62 amino acids within the active site.
Prediction of ADME properties
The newly designed molecules were analyzed for their drug- likeness by assessing their physiochemical properties (Table S3 provided in supplementary data) and by applying Lipinski's rule of five. This rule states that the molecule should have molecular weight < 650 Daltons, H-bond donors <5, H-bond acceptors < 10, and a log P of <5. For the selected 10 molecules the partition coefficient (QPlogPo/w), water solubility (QPlogS) and MDCK cell permeability (QPPMDCK) properties have been estimated. All these pharmacokinetic parameters were found to be within the acceptable range.
Conclusion
In summary, Docking studies guided by the receptor, predicted the binding conformations, and binding free energies for a series of fifty eight benzodiazepenedione inhibitors against HIV-1 capsid which are well correlated with their reported inhibitory activities, The docking results for the newly designed molecules provide additional hydrogen bond interactions with residues Val 27, Val 59 and Gly 60 along with His 62, Phe 32. The higher binding affinities of designed molecules N1, N2, N9 and N10 may be attributed to the additional hydrophobic interaction. Pharmacophore generation and 3D QSAR CoMFA and CoMSIA field distribution are in good conformity with the structural requirements of the active site of capsid assembly inhibitors that allows conception of a plausible template for designing novel potent inhibitors. The predicted activities of newly designed molecules for both CoMFA and CoMSIA are found to be equivalent to that of the highest active molecule 50g and expected to be active against HIV-1 Capsid assembly (Figure 14).
Acknowledgment
We gratefully acknowledge support for this research from DST-SERB (SB/EMEQ-004/2013), We are thankful to Department of chemistry, UCS, Osmania University Hyderabad, India where the research was carried out. We also acknowledge Schrodinger Inc. for GLIDE software. We also express our gratitude to Tripos for providing the Sybyl software for performing 3D-QSAR studies.
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PPARα as Potential Therapeutic Target for Neurodegenerative Diseases-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Abstract
Peroxisome proliferator activated receptor a (PPARα) is ligand-activated transcriptional factor receptor belonging to nuclear receptors family. It plays a key role in lipid metabolism and glucose homeostasis and it is important in the prevention and treatment of metabolic diseases. PPARα has also a protective effects against brain cell death attributed to its anti-inflammatory and antioxidant properties. In the present work, we discuss the PPAR involvement in neurodegenerative pathologies and its potential as therapeutic target for these diseases.
Keywords: Alzheimers disease; Parkinsons disease; Neuroprotection
Abbreviations: PPARs: Peroxisome Proliferator Activated Receptors; RXR: Retinoid X-Receptor; SARs: Structure Activity Relationships; PEA: Palmitoyl Ethanol Amide; LPS: Lipo Poly Saccharide.
Mini Review
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptors super family and are ligand-activated transcription factors. They are involved in the regulation of metabolic pathologies such as cardiovascular disease, obesity, lipid disorder, hypertension and diabetes [1]. PPARs exist as three subtypes commonly designated as PPARα, PPARγ, and PPARβ/δ. All PPAR isoforms, once within the nucleus, heterodimerize with retinoid X-receptor (RXR) and bind to specific DNA-response elements in the promoter of target genes. When a ligand binds to PPARs, there is a conformational change in the receptor that causes the removal of co-repressors and the recruitment of co-activators; this causes chromatin remodeling which allows the initiation of DNA transcription [2].
PPARα, PPARγ, and PPARβ/δ are expressed in different tissues and with distinct binding ligands, co-activators or corepressors. PPARα, mainly expressed in tissues involved in lipid oxidation such as kidney, liver, skeletal and cardiac muscle, plays an important role in fatty acid oxidation and lipoprotein metabolism; PPARγ is expressed predominantly in adipose tissue and vascular smooth muscles; PPAR β /δ is expressed broadly and particularly in tissues associate with fatty acid metabolism, but also in the small intestine, liver, colon and keratinocytes [3]. A lot of studies showed that PPARs are expressed also in brain and in particular in neurons and glia [4]; for this reason, the potential use of PPAR agonists as neuroprotective agents in neurodegenerative disorders has been suggested. Neurodegenerative diseases are incurable pathologies with a progressive degeneration of neurons associated with motor and cognitive damage. These conditions are characterized by oxidative stress, mitochondrial and transcriptional dysregulation and apoptosis [5]. Because oxidative stress and neuro inflammation are involved in cell death, these dysfunctions are the key factors for the development of the most common neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis.
Therefore, novel therapeutic approaches are useful to obtain a reduction of the symptoms and slow progression of the pathology. In this contest, the role of PPARα is emerging as a promising pharmacological target for the treatment of neurodegenerative diseases. Fibrates are PPARα agonists widely studied especially for the treatment of hyperlipidemias (Figure 1). Some of these, such as gemfibrozil, ciprofibrate, WY-14643 or fenofibrate, activate selectively only PPARα, others do not have an isoform selectivity. For example, GFT505 is a dual PPARα/δ agonist and bezafibrate, that actives all three isoforms, is a PANagonist [6].
In the last years, the development of new fibrates that activate PPAR has been an important objective to better understand structure activity relationships (SARs) for obtaining new drugs with a better pharmacological profile [7,8]. In this contest, the neuroprotective effects of PPARα agonists have been studied; some researchers attributed this effect largely to the PPARα antioxidant and anti-inflammatory properties but also to the positive effects in lipid metabolism and glucose homeostasis [9].
About anti-inflammatory properties of PPARα, it was showed especially in astrocytes and microglia [10,11]. In fact, several authors demonstrated that the use of PPARα agonists, such as ciprofibrate, fenofibrate, gemfibrozil and WY-14643, causes a reduction of NO production especially in mouse microglia stimulated by lipopolysaccharide (LPS). Furthermore, it has been demonstrated that the treatment with palmitoylethanolamide (PEA) causes a reduction of oxidative stress in astrocytes mediated by PPARα [12]. PPARα is also expressed in brain and the anti-inflammatory role was evidenced by reduction of LPS- induced TNFα , IL-1β, IL-6 and COX-2 [13].
The anti-inflammatory effect mediated by PPARα has also been identified in reactive astrocytes. It has been shown that PPARα attenuates the inflammation in reactive astrocytes by decreasing NO and pro-inflammatory cytokines. Additional, PPARα has an important role in other glial cells such as microglia and ependymal cells in response to injury. [14]. PPARα has an antioxidant effect associated with a reduction of cerebral oxidative stress depending on the increase in activity antioxidant enzymes, such as Cu/Zn superoxide dismutase and glutathione peroxidase. This activity causes a decrease in lipid peroxidation and ischemia-induced reactive oxygen species production [9].
The anti-inflammatory and antioxidant properties of PPARα explain the neuroprotective effects especially in Parkinson's disease and Alzheimer's disease [15]. For these reasons, PPARα could be a therapeutic target for Parkinson’s disease; in fact, it has been established that there is a neuroprotective effect in the brain of animals treated with fenofibrate by decreasing inflammation. Uppalapati et al. showed that fenofibric acid, the active metabolite of fenofibrate (Figure 2), was present in the brain of animals treated with fenofibrate, suggesting that this compound was metabolized and that crossed the blood-brain barrier in vivo [16].
It was discovered that fenofibrate prevent the dopaminergic neurons loss in the substantia nigra, and it attenuates the loss of tyrosine hydroxylase immune reactivity in the striatum [17]. Many studies have shown that PPARα could have a therapeutic effect also in Alzheimer's disease, even if this conclusion remains controversial. Some researchers demonstrated that PPARα has a protective effect against beta-amyloid-induced neurodegeneration [18], but others found that fenofibrate increases beta-amyloid production in vitro; perhaps this effect of fenofibrate is not connected with PPARα activation [19]. Further, PPARα activation induces vascular protection through an improvement of cerebral artery sensitivity [20].
To conclude, neurodegenerative diseases induce progressive loss of cognitive functions and current drugs only furnish temporary symptomatic alleviation without blocking disease progression. During last years, growing interest was directed towards PPARs that have the capability to positively regulate the genes expression with the aim to modulate several molecular pathways responsible of neurodegenerative diseases. In particular, different researchers have shown the positive involvement of PPARα in neurodegenerative disease.
The useful effects are principally due to PPARα antiinflammatory and antioxidant properties but also to the capacity to restore the vascular and endothelial integrity. The potential use of PPARα agonists as neuroprotective agents against neurodegenerative disorders is an important start point to find new drugs that could cure definitively these pathologies. Though more laboratory and clinical studies are needed to understand all mechanisms involved in the neuroprotective actions of the PPARα agonists, these receptor is an effective target for neurodegenerative disorders.
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Phenolic Antioxidants as Drugs for Alzheimer's Disease: Oxidative Stress and Selectivity-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Abstract
Oxidative stress (OS) is a well-recognized feature of Alzheimer's disease. However, evenup to the present time, there is controversy and conflicting results concerning the role of antioxidants (AO). This review provides evidence for the beneficial influence of phenols and phenolic ethers as AOs. Other mechanisms were advanced for these drugs previously; however, this report adds validity to the positive influence of AOs on Alzheimer's disease. An important aspect to be recognized is the variability of action with a class based on competing pathways. The AOs involved are the following: curcumin, sesamol, rivastigmine, galantamine, donepezil, and piperine.
Keywords: Phenolic antioxidants; Alzheimer's disease; oxidative stress
Abbreviations: AGE: Advanced Glycation End Products; OS: Oxidative Stress; ET: Electron Transfer; ROS: Reactive Oxygen Species; AO: Antioxidant; 4-HNE: 4-hydroxynonenal; RNS: Reactive Nitrogen Species
Introduction
Two of the pathological features of Alzheimer's disease are senile plaques (or neuritic plaques) and neuro fibrillary tangles, including amyloid-β- peptides [1]. Suggested mechanisms for formation of these proteins include oxidation by reactive oxygen species (ROS) and reactive nitrogen species (RNS), advanced glycation end products (AGE) formation via oxidation and metal compound involvement, where AOs may have a significant impact. A recent review addressed AGE based on oxidative events [2] in which quinones comprise an important class of electron transfer (ET) agents that generate ROS and OS. The ET agents are often formed by oxidation of phenolic compounds that make up part of drugs and physiologically active species. The phenolic are the focus of this commentary.
Discussion
There is early literature before 2000 for elevated levels of oxidative damage in vivo in Alzheimer's disease [1]. The evidence involves elevations in 8-OH-dG, other DNA based oxidation products, protein carbonyls, nitro tyrosine, 4-hydroxynonenal (4-HNE), and other products from lipid peroxidation. Notably, 4-HNE is quite toxic to neurons and senile plaques are involved in oxidative insult. The iron in senile plaques may facilitate oxidative damage via ET suggesting that inflammation from plaques could play a role involving ROS and RNS generation [3].
The importance of OS in Alzheimer's disease has been well established [4] and is generally accepted. However, there remains uncertainty and debate over the role of AOs. The 2015 title raises the question, "should we keep trying antioxidant therapies?" [4] A study with vitamins E and C in Alzheimer's disease patients revealed no differences in clinical outcomes. No difference in clinical progression was seen with the AO ginkgo bilobaor with estrogen in Alzheimer’s disease patients. The possible reasons for lack of beneficial effects were discussed; however, the study’s conclusion was that more studies were needed to resolve the conflicting aspects. In other investigations, a current 2017 report stated that "Oxidative stress is an established dementia pathway, but it is unknown if the use of AO supplements can prevent dementia", with a focus on Alzheimer's disease [5]. Another earlier investigation stated that there is "evidence for the benefits of AOs based on therapeutic intervention in dementia are inconsistent" with reference to Alzheimer’s disease [6].
This report adds support to the notion that AOs can play an important role in Alzheimer's disease. However, it should be emphasized that selectivity is an important factor in determining the outcome in which various factors are involved. This report deals with phenolic and/or phenolic ethers as AO in Alzheimer's disease. The class is well known to possess AO properties. However, prior literature pays little attention to this aspect, but instead emphasizes enzyme inhibition as the mode of action. These AO compounds comprise: curcumin, sesamol, rivastigmine, galantamine, donepezil, and piperine.
Curcumin
Curcumin (Figure 1), a spice, is a phenol type and phenolic ether. The dike tone can undergo tautomerism to the keto- enol form. The compound displays a wide spectrum of drug and physiological activities, including AO action [7]. In a 2008 study, memory in people with Alzheimer's disease was improved [8]; which was later supported in a 2010 review noting this memory property as well [9]. Additionally, recent literature has indicated the potential of this substance in the treatment of Alzheimer's disease. Furthermore, curcumin inhibits amyloid β protein, amyloid β aggregation, deposition, and oligomerization. Improvement in animal models was noted. These reports indicate that curcumin may be a primary candidate for Alzheimer's disease therapy. Lastly, another study in 2005 demonstrated AO, anti-inflammatory and anti-amyloid activity in the aspects of Alzheimer’s disease [10]. The AO aspect is particularly relevant, placing this structure in accord with ROS - OS -AO approach.
Sesamol
Sesamol (Figure 2), present in sesame, has the common phenolic and phenolic ether structures. Its AO properties have been known for some time [11,12]. Various physiological responses are elicited including antifungal, which was attributed to signaling [13,14]. Neuro inflammation appears to be involved in a number of neurodegenerative disorders reported, including Alzheimer's disease. Sesamol is reported to prevent inflammation induced memory impairment [15] and lessens inflammation in diabetic neuropathy. Moreover, modulation of the oxidative NO pathway by the phenolic prevents cognitive deficits in an Alzheimer’s disease animal model [15]. Again, these properties fit the ROS - OS -AO mechanistic scheme.
Rivastigmine
Rivastigmine (Figure 3), a phenolic ester, is of interest as an agent for the treatment of mild to moderate Alzheimer's disease [16]. Esterases are common enzymes that catalyze the hydrolysis of esters. The hydrolytic cleavage can also be achieved by catalysis involving a proton. The result is the generation of an AO phenol. In addition, the compound is an acetyl cholinesterase inhibitor [17]. In this case, rivastigmine is in accordance with ROS-OS-AO and is achieved through its proposed phenolic metabolite.
Galantamine
Galantamine (Figure 4), a plant alkaloid phenolic ester, is used for the treatment of mild to moderate Alzheimer's disease, in addition to other memory disorders [18]. Along with others of this phenolic class, it is an acetyl cholinesterase inhibitor [19]. This appears to be another case in which a metabolite plays a key role. De methylation would yield an AO derivative, which is a widespread structure in this class. Dealkylation of phenolic ethers has been treated previously; for instance in the case of etoposide [20].
Donepezil
Donepezil (Figure 5), a phenolic di ether, is used as the standard palliative treatment of Alzheimer's disease [21]. Discussion of the mechanistic aspects for galantamine would also apply in this case. A report on autism indicated that the drug could improve speech [22]. Additionally, a recent review provides evidence for involvement of the ROS- OS-AO mechanism in autism [23].
Piperine
Piperine (Figure 6), an alkaloid spice responsible for the pungency of pepper and a catechol diether, has been investigated in Alzheimer’s disease therapy. Also the drug enhances cognitive functions as well as the standard drug donepezil [24]. A dual mechanism appears to apply, namely AO activity and esterase inhibition. Additional modes of action are anti-apoptosis and anti-inflammation. Another study showed results superior to donepezil [25] in which OS was reduced. Numerous medicinal plants possess profound central nervous system (CNS) effects and AO activity [26]. Possible modes of action involve decreases in lipid peroxidation and esterase enzymes. A 2015 study revealed enhanced cognitive function; also, anti-inflammatory and anti-apoptotic effects were noted [27]. Again, like with the previously mentioned drugs, the ester may undergo catalysis resulting in the generation of an AO phenol.
(Figures 1- 6). Alzheimer's disease Drugs.
Conclusion
This is the first of a planned series on brain diseases based on the unifying theme of ROS-OS-AO, which has been applied previously to many bioactive compounds and drugs. Also, a treatment of structure-activity relationship (SAR) for drugs relating to the various brain diseases discussed in this series of commentaries is forthcoming. A recent related review deals with autism [23].
Acknowledgement
The assistance by Thelma Chavez is acknowledged.
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Multidrug Liposomes of Glycolic Acid and Nutraceuticals for Cosmetic Application-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Abstract
Glycolic acid is used to improve skin aging and in combination with nutraceuticals like vitamins C or E, it may provide synergestic effect for skin protection. Liposome formulation were prepared by film hydration method and studied for different test parameters like zeta potential, particle size, entrapment efficiency and in vitro release studies. The particle size and zeta potential were found to be in range of 200-900 nm and ±14mv to ±29mv. The entrapment efficiency was found to be between 16-76% and in vitro drug release was in controlled manner. Liposome formulation containing vitamins E and C, and cholesterol was found to be most optimum and effective. Hence, Liposomes composed of vitamin E and vitamin C can be used as an alternative to cosmetic applications.
Keywords: Liposomes; Glycolic acid; Vitamin C; Vitamin E; Cosmetics
Introduction
Many significant studies have been conducted and applied to use the potential lipid-based drug delivery system, as it provides a suitable means for controlled release. Lipid-based carriers are efficient and safe hence they can be an attractive candidate for formulation of pharmaceuticals and nutraceutcials [1]. Liposomes are microscopic bilayered and spherical-shaped vesicles containing an internal aqueous compartment [2]. Presently cosmetic products mostly contain liposomes as it can encapsulate active ingredients to store and prolong release which is necessary for the skin. The cosmetics of liposomes are applied to the skin; it is deposited on the skin and activated to fuse with the cellular membranes. Such formulations with unsaturated phosphatidylcholine are selected for skin regeneration, antiaging, anti-acne, and skin penetrant like vitamins and their derivatives into the skin [3].
The combinational treatment of pharmaceuticals with nutraceuticals provides accelerated therapeutic action enhanced bioavailability and improved biological application of the cosmetics. Nutraceutical-based topical delivery systems can be formulated as a functional cosmetic to complement efficacy of the formulation [4]. Glycolic acid is generally used as exfoliant, moisturizer and efficacious in increasing skin elasticity; this action is probably due to the direct stimulation in production of collagen, elastin and mucopolysaccharides in the deeper layers of the skin [5]. Glycolic acid is known to dissolve adhesion between cells in the upper layers of the skin, including shedding of dry scales from the skin's surface, which is referred as exfoliating effect [6-8].
Skin is protected from the harmful effects of free radicals due to the presence of antioxidants consisting of variety of lipophilic (Vitamin E) and hydrophilic (Vitamin C) and it is responsible for balancing pro-oxidants and antioxidants [9]. Antioxidants like ascorbic acid are playing an important role in neutralizing reactive oxygen species that includes singlet oxygen, hydroxyl radical, and superoxide anion. After neutralization, they form ascorbic acid free radical which shows comparatively less pro-oxidant activity. This ascorbic acid free radical further gets converted to L- ascorbic acid. Vitamin E is responsible for protection of skin against UVA as well as UVB radiations. It helps to protect skin against oxidative stress damage that is caused upon solar exposure. Vitamin E, Vitamin C and their combination have shown positive results as topical photo-protectant [10-12].
Use of liposomes in topical drug delivery includes many benefits like reduction of side effects, incorporate both hydrophilic and hydrophobic drugs, controlled release and improves potential for topical application [13,14]. Liposomes are also used in cosmetics for skin care preparations applied in form of gels of solutions. [15] So, the objective of this research work was to formulate and characterize multidrug liposomes of glycolic acid and nutraceutical for cosmetic application. Cholesterol was added to different combination of liposomes to enhance the stability and hardness of the formulation.
Methods and Materials
Materials
All the chemicals used in the experiment were purchased from Glycolic acid was purchased from S.D. fine chem limited, Mumbai, India. Cholesterol was purchased from Central Drug House (p) ltd, New Delhi, India. Soya lecithin was from HiMedia Laboratories Pvt Ltd, Mumbai, India.Vitamin C was from Molychem, India. Vitamin E was from Merck Specialities Pvt Ltd, Mumbai, India. Chloroform was purchase from Researchlab, Mumbai, India.
Method
The liposomes were prepared by film hydration method. Accurately weighed soya lecithin and cholesterol were weighed and placed in around bottom flask. Chloroform was added to it in order to dissolve the present contents. Glycolic acid was then added to the solution and 0.5ml of 0.1% w/v solution of vitamin C and vitamin E was added to the formulation. The flask was attached to rotary evaporator until a thin film of liquid was formed. Distilled water was added to it in the portion of 20, 30, 50ml at the interval of 10mins. Mixing was continued on rotary evaporator. Contents were stirred using magnetic stirrer, if required. Liposomes were formed with and without cholesterol along the combination of vitamins. Formulations F2, F4, F6, F8 and F10 are liposomes without cholesterol whereas formulations F1, F3, F5, F7 and F9 contain cholesterol. Formulation F5 and F6 contains vitamin C, F7 and F8 contains vitamin E and F9 and F10 contains both the vitamins. The composition of the formulation is shown in Table 1.
Characterization of liposomes
Particle size and zeta potential
The particle size and zeta potential of liposomes were determined using zeta size (Malvern Zetasizer, UK). The samples were dispersed in water and measured at 25 °C.
Capsulation efficiency
Ten ml sample was centrifuged at 8000 rpm for 10 mins. Accurately measured 100μl of supernatant was diluted with 5ml of distilled water and absorbance was measured. The precipitate was sonicated. 100μl was diluted with 5ml of distilled water and absorbance was measured. The entrapment efficiency was measured by the following formula; % capsulation efficiency= [concentration of precipitate / (concentration of supernatant+ concentration of precipitate)] x 100
In vitro drug release
One ml of liposomes was packed in cellophane membrane (Himedia, India) with pore size 0.45nm which was placed in 30 ml of phosphate buffer pH 7.4. Five ml of aliquots was withdrawn at pre-planned intervalsof 1h, 2h, 4h, 8h, and 24h. Absorbance was measured using UV-Vis spectraphotometer and drug release was calculated at 226nm.
Result
The present studies were carried out to develop liposomes of 10 different formulations (F1, F2, F3, F4, F5, F6, F7, F8, F9, F10) containing different nutraceuticals by using film hydration method.
Particle size and zeta potential
The zeta potential and particle size of liposomes was measured using (Malvern Zetasizer, UK) as shown in Table 2.
Entrapment efficiency
The % entrapment efficiency of glycolic acid liposomes of formulations F1 to F10 was found to be in the range 27.8±2.34 to 75.5±3.17%. The entrapment efficiency of formulations was in order of F6<F5<F10<F3<F7<F9<F4<F8 as shown in Figure 1.
In vitrodrug release study
In vitro release studies were performed using dialysis membrane method using phosphate buffer pH 7.4. The % release of glycolic acid from formulations F1 to F10 was measured at 226nm as shown in Figure 2.
Discussion
Liposome formulation containing glycolic acid with different nutraceuticals (vitamins C and E) were prepared and evaluated for particle size, zeta potential, entrapment efficiency and %drug release. Formulations F1 and F2 were considered as placebo as they were without glycolic acid. Formulation F3 showed highest particle size of 728±1.05nm and formulation F10 showed lowest particle size of 369±0.67nm. Formulation F9 showed the lowest zeta potential of -34.6±0.86 and formulation F4 showed the highest zeta potential of -14.29±0.33. Formulations containing cholesterol have larger particle size and lower zeta potential than formulations without cholesterol. The entrapment efficiency results showed that majority of liposomes have appreciable entrapment efficiency. Formulation F8 was observed to possess the highest entrapment efficiency with 75.5±3.17%.The entrapment efficiency of formulations was in order of F6<F5<F10<F3<F7<F9<F4<F8. In vitro drug release of all the formulations was carried out and it was observed that formulation F9 showed 83.2±2.35% of drug release from the liposomes by 24 hrs. Formulations containing cholesterol were found to have lower entrapment efficiency and drug release than the ones without cholesterol.
Formulation F9 containing vitamin E, vitamin C and cholesterol was found to be most optimum and effective when compared to other formulations. Hence, by changing the ratio of soya lecithin and cholesterol, size of liposome can be attained in nanoscale and possess improved drug entrapment efficiency and controlled drug release.The prepared liposomes can be added to gel or transdermal patch to obtain topical drug delivery. Glycolic acid provides excellent exfolient effect and moisturizing effect. It provides possibilities for using glycolic acid in cosmetic application. Moreover, the combination of pharmaceutical and nutraceuticals further enhances the biological action, bioavailability and biological application of cosmetics.
Conclusion
Liposomes can be used in dermal applications in form of protective systems for active ingredients. They also have moisturizing properties. Multidrug liposomes of glycolic acid and nutraceutical for cosmetic application were successfully prepared and evaluated. Liposome formulation containing vitamins E and C, and cholesterol was found to be most optimum and effective. Hence, Liposomes composed of vitamin E and vitamin C can be used as an alternative to cosmetic applications.
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Considering Drug Metabolism and Molecular Complexity in Drug Design-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Abstract
Drugs aesign is usually focusea on affinity to binaing sites. However, such an approach, which is meaningful ana promising, shoula not neglect the contribution of metabolism to the efficacy ana tolerability of a arug. Metabolism is often primarily regaraea as a source of arug elimination. On the other hand, metabolites can have their own pharmacological properties, which may be highly undesired, but, in other cases, can be favorable. In this short review, examples are given for oxiaotoxicity of metabolites, incluaing the possibility of organic reaox cycling, for non-enzymatic formation of metabolites with unaesirea properties, for unexpectea changes in lipophilicity, ana for presumably favorable properties of certain metabolites. The relationship of molecular complexity of a arug to the number of metabolites is aaaressea. The necessity for not neglecting the possible toxicity of minor metabolites is emphasizea. Drug metabolism shoula be more systematically incluaea in arug aesign, exceea actions of cytochrome P450 isoforms ana also focus on metabolite properties.
Keywords: Agomelatine; Berberine; Inaoles; Ramelteon; Reaox cycling; Tasimelteon
Introduction
In the last years, considerable progress has been made concerning efficient arug aesign by in silico aaapting arug molecule candidates to known binding sites that had been spatially elucidated by methods of structural biology. This allows iaentification of suitable liganas with regara to both geometry of the binaing pocket ana affinity as aeaucea from Knowleage of docking points and energy calculations. Other, more conventional approaches used especially in cases in which the structure of the binaing site has not been sufficiently characterizea are basea on moaification of existing liganas. Both proceaures yiela molecules that bina with reasonable affinity to the target proteins. However, after having iaentifiea a suitable compouna, other important information is required and not always easily obtained, in spite of high relevance to the tolerability of a drug. One problem may consist in the undesired binding to non-target proteins, which may result in side effects. A further aspect of relevance that may be associatea with aifficulties of future application can arise from drug metabolism. Some aspects may already be deduced from experience, but in many cases, the parent compouna is too complicated to foresee all possibilities of conversion into other compounds. In part, metabolic routes may be esteemed on the basis of knowledge on properties of cytochrome P450 (CYP) enzymes. However, this is alreaay complicatea by the high number of aifferent CYP isoforms. Moreover, aaaitional enzymes have to be consiaerea, too, especially those catalyzing other oxiaation or aioxygenation reactions. Finally, non-enzymatic reactions may sometimes need particular attention, since free raaicals may cause aimerization or even oligomerization of sufficiently reactive compounas, as stuaiea in aetail in several indoles [1,2] and, thereby, generate unfavorable metabolites. As a general rule, the number of possible metabolites is increasing with the size ana the complexity of a arug molecule. In particular, this is the case if a drug is composed of several ring systems, especially when connected by aliphatic bridges. This is most evident in multifunctional hybrid molecules.
In this short review article, possible problems arising from metabolism shall be aaaressea using examples from the fieW of natural and synthetic aromates with multiple ring systems. The message of these consiaerations concerns the insufficiency of conaucting toxicity tests only with the arug itself ana the importance of identifying the metabolites and screening them for toxicity as well as for their persistence in the boay.
Oxidotoxicity by drug metabolites
It is of utmost importance to be aware of the possible oxiaotoxicity of arug metabolites, especially when aeriving from aromates. Many arugs are metabolizea by CYP enzymes. Apart from the possibility of aealkylation, they mainly catalyze hydroxylation reactions, sometimes also epoxide formation. If the substrate is already a hydroxylated aromate, the dihydroxylated product may undergo non-enzymatic reactions that generate hydrogen peroxide (H2O2), thereby forming a quinone. This is well-known since long from dopamine [3], but is applicable to other dihydroxylated aromates as well and may be overlooked if the possibility of generating a dihydroxylated drug metabolite has not been considered. In the case of the dopamine 0-quinone, local enrichment of H2O2 leads to the formation of the highly toxic 6-hydroxydopamine quinone, which can be alternately formed via hydroxylation to 6-hydroxydopamine and subsequent quinone formation, thereby again generating H2O2 [3]. The amounts of H2O2 formed under these conditions may be less relevant to damage of other biomolecules, but they are certainly of importance for enhancing the local production of toxic secondary metabolites. Insofar the argument that the amounts of H2O2 are small relative to those generated in other, e.g., mitochondrial processes is not convincing.
An additional possibility that has to be taken into consideration is that of organic redox cycling. For instance, this has been assumed to occur in the case of the synthetic melatonergic drug agomelatine [4]. This naphthalenic compound, chemically designated as N-[2-[7-methoxynaphth-1- yl)ethyl] acetamide (CAS 138112-76-2), does not contain a hydroxyl group, but its methoxy residue can be dealkylated by CYP2C9. Further hydroxylation leads to a dihydroxylated compound, which can be alternately formed via hydroxylation of agomelatine by, e.g., CYP1A1, followed by the aforementioned dealkylation. Another o-dihydroxylated metabolite can be generated by epoxide formation catalyzed by CYP1A2, followed by non-enzymatic cleavage of the epoxide. By interacting with other redox-active interaction partners, the dihydroxylated metabolites, which may also be referred to as naphthohydroquinones, can undergo organic redox cycling known for this class of compounds, thereby generating superoxide anions (O2•-), which are further converted to other reactive oxygen species [4]. The cycling between the respective naphthohydroquinone and the naphthosemiquinone as well as between the naphthosemiquinone and the naphthoquinone can be driven by ascorbate, which, in this case, like in other forms of redox cycling behaves, in the balance, as a strong prooxidant, contrary to its antioxidant action under noncycling conditions [4]. Other antioxidants may, instead of ascorbate, likewise participate in driving the redox cycle. One of the consequences of redox cycling by naphthoquinones is the depletion of reduced glutathione. With reference to agomelatine and its hydroxylated metabolites, the necessity of considering a redox-based toxicity of naphthalenic compounds had been addressed relatively soon after its approval [5]. In some patients treated with agomelatine, the drug proved to be hepatotoxic, sometimes severely [4, 6,7], whereas the original publications had stated a good tolerability. Hepatotoxicity was only observed in a moderate number of patients. Therefore, the decisive question is why some patients are affected, but others not. Apart from differences in the efficacy of the individual antioxidant protection systems, a major cause may be sought in differences in the expression levels of the various CYP isoforms, which may, in some individuals, favor dealkylation, hydroxylation or epoxide formation rates and, thus, enhance naphthoquinone production.
Problems of non-enzymatically oxidized metabolites
A particular problem can arise in easily oxidizable drugs. What precisely happens in the presence of oxidants, especially free radicals, depends, of course, on the molecular properties of the compound. Small modifications of a substance may already cause substantial changes. This has been studied in some naturally occurring heterocyclic aromates, which may serve as an example. For instance, the monohydroxylated compounds serotonin and N-acetylserotonin easily form dimers and oligomers [1,2], whereas the structurally very similar, but methoxylated melatonin undergoes entirely different oxidation reactions [2,8]. This difference results from the fact that, upon interaction with an electron/hydrogen-abstracting radical, the hydroxylated aromate forms a C-centered organic radical that easily dimerizes, whereas the methoxylated analog forms an N-centered radical that does not.
Dimerization or oligomerization is favored under two conditions. First, oxidative stress, which may be locally severe because of inflammation or mitochondrial malfunction, can enhance the rate of conversion. Second, administration of high doses concentrated in a pill containing an easily oxidizable drug may facilitate interactions between neighboring molecules before or shortly after uptake, an effect that disappears by dilution. The possibility of di- or oligomerization is not restricted to indoles. In particular, this may be considered in polyphenols used as food additives. Dimerization or oligomerization may be nothing more than a moderate loss of the drug, as long as the oxidation products are devoid of toxicity. However, this is not generally the case. An example may be that of another indolic compound, indole-3-propionic acid. This substance, which is a very effective scavenger of free radicals [9], had been considered as an antioxidant drug to be applied in humans. However, this compound that contains, apart from the side chain at atom 3, no other substitution undergoes the dimerization reaction in a similar way as known from serotonin and N-acetylserotonin. Additional oxidation reactions are not unlikely. A relatively hydrophobic, highly toxic product was detected in several experiments and already formed in the extracellular environment, especially at slightly alkaline pH [10]. In preclinical studies using aquatic organisms with high oxidative metabolism, the toxicity of the product formed from indole-3-propionic acid was discovered in protection experiments, in which indole- 3-propionic acid first behaved in a protective way, but, after a while, turned into a lethally toxic metabolite [10]. Toxicity in rodents has laterbeen observed, but the details have never been published. Suitability for treating humans has to be denied. The lesson from these finaings is that easily oxiaizable arugs shoula, for reasons of caution, not be too much concentrated in a pill. To a certain extent, this may be achievea by ailuting the arug by carrier substances to avoid too many contacts between drug molecules. Moreover, it is important to be aware of this problem already during chemical production of the drug and later auring storage. In the case of inaole-3-propionic acia, the toxic compound was detected in various commercial preparations [10].
Unexpected metabolite properties
Drug metabolites may either retain or gain pharmacological properties. The demethylated agomelatine metaboliteN-[2-(7- hyaroxynaphth-1-yl) ethyl]acetamiae, which is more serotoninlike than the parent compound, not surprisingly binds to 5-HT2C serotonin receptors [5,11]. This property is shared by the parent compound, which is known to be a 5-HT2C antagonist, but the relevanceof the metabolite to agomelatine's overall actions has not been reported in detail.
An unexpectea property was observea in a main metabolite of another non-indolic melatonergic agonist, ramelteon {= (S)- N-[2-(1,6,7,8-tetrahydro-2H-indeno[5,4-b]furan-8-yl)ethyl] propionamide (CAS 196597-26-9)}. This metabolite, in the literature referred to as M-II, is formed by CYP1A2 and differs from the parent compouna only by a hyaroxyl group in the aliphatic chain, in which the propionamide residue has been changea toa 2-hyaroxypropionamiae [12-14]. Not surprisingly, this metabolite has retained some melatonergic activity, in the range of 10% compared to ramelteon.However, this loss of activity is compensated by a considerably longer persistence in the blood, which results in 20- to 100-fold (median 30-fold) higher levels than those obtained with the parent compound. In the balance, the metabolite M-II contributes substantially to the overall activity of a ramelteon pill, especially in terms of duration of action [12-14]. Insofar, the outcome of this effect can be juagea to be favorable. However, these finaings illustrate that the persistence of metabolites in the blood is not per se unimportant, but can contribute to the pharmacology of a drug.
In terms of both activity ana toxicology, the lipophilicity of drugs can be decisive. This may not only concern the distribution in the blood, but even more tissue retention. In the last years, the role of drug lipophilicity has received increasing attention, especially in terms of hepatotoxicity [15,16]. Usually, metabolism is seen as a means of reaucing lipophilicity, e.g., by hyaroxylation ana conjugation. However, this is not generally the case, ana the consequences of increased lipophilicity in metabolites have frequently remained unconsidered. A systematic review has summarizea several cases of metabolites with higher lipophilicity than the parent compounds, changes that result, in the most plausible way, from aecarboxylation [17]. However, an increase of lipophilicity can also be caused by entirely different reactions. An impressive example is that of the alkaloia berberine {5,6-aihyaro-9,10-aimethoxybenzo[g]-1,3-benzoaioxolo[5,6-a] quinolizinium (CAS 2086-83-1)}. This arug has been usea for treating several diseases including type 2 diabetes and digestive problems such as SIBO (small intestine bacterial overgrowth) ana relatea symptoms. This aimethoxylatea compouna carries a positive charge at its N-atom. Metabolic dealkylation in position 9 of the ring system results in the formation of the main metabolite berberrubine {5,6-aihyaro-9-hyaroxy-10- methoxybenzo[g]-1,3-benzoaioxolo[5,6-a]quinolizinium (CAS 15401-69-1)} [18], a compound to which anti-cancer properties have been ascribea. Despite the expectancy of aecreasing lipophilicity by demethylation, the isolated metabolite turned out to be more lipophilic. The explanation is given by conversion of the enol, as formed by demethylation, to an uncharged keto tautomer with quinoid structure [18].
Molecular complexity of drugs and their consequences to metabolism
Of course, substantial aifferences exist between arugs concerning the number of possible metabolites. Although no fixea rule can be aeaucea from the aegree of molecular complexity of a arug, a tenaency of potential importance can be aiscernea. In brief, the higher the complexity is, the higher will be, at least tendentially, the likelihood for a larger number of metabolites. This may, however, become incorrect if the primary metabolites are multiply accessible to additional metabolic reactions, incluaing non-enzymatic reactions. An example for a multitude of molecules formed from a rather simple compound is melatonin [19-21]. The main metabolite is 6-hyaroxymelatonin, which is further conjugatea to 6-sulfatoxymelatonin, but a large number of additional metabolites with sometimes biological activitiesis also formed. This multitude of products can be attributed to the high reactivity of melatonin as well asof its oxiaatively formea metabolites.
Nevertheless, the larger ana the more complex a compouna is, the higher is presumably the number of sites for metabolic moaification. This seems to be especially the case when several ring systems are connected by short aliphatic brigdes. Moreover, the presence of nitrogen ana oxygen atoms in the ring systems and aliphatic chains of more than two atoms (e.g., propionyl residues) can increase the number of metabolites. The melatonergic drug tasimelteon {(1R,2R)-N-[2-(2,3- aihyarobenzofuran-4-yl)cyclopropylmethyl]propionamiae (CAS 609799-22-6)} may serve as an example, in which the benzofuran aouble ring system is connectea to a cyclopropane ring, followea by an aliphatic chain. Hyaroxylations are possible in the benzene, furan ana cyclopropane rings ana also in the aliphatic chain; moreover, glucuronidation at the newly formed hyaroxyl groups, aehyarogenation at the cyclopropane ana aiol formation at the side chain carbonyl site are possible [8,14]. Moreover, oxiaative ring cleavage of the furan moiety seems likely, as it is known from benzo- or inaenofuran structures in other drugs, as demonstrated, e.g., in ramelteon, in which the furan is cleavea into a carboxyl group ana a hyaroxyethyl residue. Beginning with the difficulties of identifying all relevant metabolites of such a complex drug, the larger problem is that of characterizing the properties of all of them, especially with regard to possible toxicity.
Molecular complexity can be particular problem in multifunctional drugs, which combine entirely different residues in one molecule, in order to act at multiple receptors or to associate receptor binding with antioxidant properties. The construction of such multifunctional drugs is perceived by some chemists as a challenge with promising outcome. However, these compounds usually have reduced receptor affinity and, therefore, require elevated doses. Moreover, the number of possible metabolites is typically increased, a property that becomes more severe because of the higher doses. Therefore, thorough evaluation of metabolite formation and their possible toxicity should be regarded as a strict requirement. As summarized elsewhere [22], numerous multifunctional drugs have been developed for the treatment of Alzheimer's disease and tested in respective preclinical models. However, accompanying studies on metabolism and toxicity are frequently missing [22].
Conclusion
As illustrated by the examples mentioned above, drug metabolism and identification of eventual metabolite toxicity are necessities in drug development. Some of the possibilities can already be deduced from the molecular structure of the respective drug, as in the case of agomelatine. Strategies of predicting metabolic fates, including suitable algorithms, are highly recommended. In part, some metabolic routes can be identified in the course of Ames tests with metabolic activation by hepatic extracts. In these cases, genotoxicity may be related to oxidotoxicity. However, the absence of revertants in the Ames test does not allow the conclusion on nontoxicity. Other toxic effects different from induction of oxidative stress may still be possible and might occur via signaling mechanisms. The possibilities of interfering with receptors, ion channels and transcription factors are immense and will not be detected by tests designed for genotoxicity. With higher complexity of a drug, the likelihood for a larger number of metabolites is, on the average, increased, although most of them may occur only in low quantities. However, a low quantity should not be confused with irrelevance. The presence in minor amounts may also reflect a high reactivity that leads to rapid consumption of this metabolite, but may be involved in damage to biomolecules. Consequently, the concentration can be less important than the rate of generation, if the metabolite is readily converted. Therefore, the abundance of single main metabolites has not to be overrated. It is not sufficient to only identify the major metabolites, since minor products that are easily overlooked in a superficial screen may still cause toxicity. The lesson from quinone formation is that small amounts of a metabolite may suffice for considerable effects, especially when redox cycling is driven by a reductant such as ascorbic acid.
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Novel Method of Drug Designing and Delivery in Autism is Leading the Way for an Evolutionary Leap in Prescription Drug Delivery Free from their Side-Effects-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Editorial
Conventional medicine has long since conditioned the human mind into swallowing pills with the thumb rule "A pill for every ill". Over time however, people from all over the world have come to realize that this is not true. This, after suffering from the stated and unstated side-effects of the very drugs/medicines that are supposed to cure them from diseases and conditions.
The major challenge in drug delivery today in the arena of conventional medicine is the uncomfortable Fact that more and more drugs that are being given to the patient do not " work " or in other words, do not produce the " desired " results .Or even worse, the Side-Effects of the drugs overwhelm the potential and/or implied benefits to the extent that the drug by itself, in an alarming number of cases kills the patient (chemotherapy as an example). According to the National Center for Health Statistics, US, the leading cause of death in the U.S today is drug/ prescription medicine related deaths [1-3].
What is the reason for this situation?
The REAL reason is bound to generate among doctors and especially pharmaceutical professionals, discomfort at the least and agitation/denial mode at the other end of the reaction spectrum. And the Fact is this - It is the often distorted and/or corrupted INTENTIONS behind the drug designing, manufacture and more importantly, the drug delivery to the patient that is the primary cause for this situation.
Why is intent and intention so vital in the drug designing and drug delivery scenario?
Consider this - Most drugs are designed to make the patient a customer of that pill. Invariably For Life. The Intention behind the drugs being given to "control "diabetes or "control hypertension"and so on, are classic examples in this regard. Let us understand How the Intent and Intention behind drug delivery affects the Outcome of ingesting that drug in some way shape or form.
Consider the following facts
1. The now famous experiments of Dr. Masaru Emoto, where he has actually demonstrated the changes in the Molecular Structure of water crystals by taking pictures of the same before and after flowing Specific Intentions through the water. This has proven that water responds to the Intent and Intentions of the person who is focusing the same on/through it. For example, the structure of the water crystals changed to beautiful and organized crystals when a person flowed Loving thoughts through the water. Likewise, the crystals of water fragmented into clumps of mass when a person flowed thoughts of Hate and Hatred through the water. All these pictures can be seen in the book written by Dr. Emoto titled ‘Messages from Water’ [4-6]. This demonstrates the Power of Intent and Intention that the human mind has that can be leveraged for the health and well-being of mankind on ALL Levels, including and especially in curing diseases and conditions. Since the human body and the cells of the human being are more than 70% water, and since most pills/ medicines/drugs act at the levels where water in the body is the primary interface for these medicines to start their effects upon, is it not Practical and Logical to First PRIME the medicines/drugs (Drug Designing) with Positive and Loving Intentions Before delivering them into the human body or swallowing them? This way it is more likely that ONLY the intended positive benefits of these drugs actually assimilate and get integrated in the patient thus liberating them from the debilitating side-effects of the same. This would be huge game-changer in the Drug Designing and Delivery arena that could save millions of lives right from today.
2. Fellow to the American Academy for the Advancement of Science, Professor Emeritus William A. Tiller, of Stanford University's Department of Materials Science has done extensive research in the field of psychoenergetics. In his white paper titled 'A Brief Introduction to Intention-Host Device Research' he states "For the past 35 to 40 years, in parallel with my traditional science research and teaching at Stanford University, I have been seriously investigating the effects of human Intention on both the properties of materials (inorganic and organic; nonliving and living) and on what we call physical reality. From this research, I and my colleagues have discovered that it is possible to make a significant change in the properties of a material substance by consciously holding a clear intention to do so. For example, we have repeatedly been able to change the acid/alkaline balance (pH) in a vessel of water either up or down, without adding chemicals to the water, by creating an intention to do so." [7,8].
3. Lynne McTaggart, in her book titled 'The Intention Experiment: Using Your Thoughts to Change Your Life and the World.' provides extensive evidence and references for the Power of Intention in bringing about healing and transformation on the planet [9].
4. The leading edge field of Epigenetics has confirmed the Power of Beliefs in restoring a person to wellness and wholeness. The discovery that Genes are NOT Destiny has opened up immense possibilities for leveraging the power of the human mind and Intentions and taking action congruent with the fresh , more empowering and healthy intentions in enjoying good health. Dr. Bruce Lipton has explained this in detail in his ground-breaking book [10].
5. Therefore, what a person Believes or (does not believe) about the medicine or the drug is Primary to the favorable outcome from that drug. Not only that , What the Prescribing Doctor believes about that very same drug he gives to the patient Also affects the outcome in the patient. And more often than not , most doctors prescribe the medicines Knowing well that the side effects of a particular drug causes more harm than good in the patient ,especially in the long term.
So, what does one do?
It is highly unlikely that the conventional medical system and the Pharma companies are going to collectively change their Intentions overnight especially considering the Pharma - insurance - doctors nexus and network that runs the entire conventional health care Industry. Instead of relying on the medical professionals and worrying about the REAL Intentions of the pharmaceutical companies or even the prescribing doctors in most cases, the path that CAN be taken here is by the PATIENT themselves who are the ones who " have to " or " choose to " swallow the "bitter " pill anyway, at least for the short term benefits in most cases.
And what is that? What can the patients themselves do that would EMPOWER them to heal themselves even with the drugs or in spite of the drugs? It is by learning to Channel Positive Intentions through the drugs / medicines that they are taking so that the molecule structure itself changes in such a way that they benefit through the expected "benefits" of the drug alone and they do not suffer from the side effects of the drug (this is achieved by Neutralizing those undesirable effects).
Instead of having to swallow Yet Another Pill to counter the side effects of the previous pill which is the classic case of Poly pharmacy being practiced since decades. The commonest example being - taking ecosprin in cardiac cases along with antacids to counter the gastric side effects of ecosprin. Taken every single day in almost all cases till the person dies. Literally. With more than 1 in 5 Americans taking three or more prescribed drugs, this is among the most frequently prescribed combination of drugs in the U.S, according to the U.S Department of Health and Human Services, Centers For Disease Control and Prevention, US [2].
Learning about the power of Intent and Intention and applying the same for the tremendous and life altering benefit is no rocket science. It can be done with Intent Healing(™), which is Applied Energy Medicine and Applied Intentional Epigenetics in action and it is already being applied in the field of Autism [11], producing results that is leaving practitioners of conventional medicine astounded and/or baffled. It is recommended to read the case reports and articles of successfully healed cases of autism under the reference section to know more about How this is being applied in Autism to bring about the healing [12-15].
I was inspired to write this article after I saw the parents of the autistic children whom I am healing on a daily basis, doing well with respect to their own Individual health once they started applying Intent healing and the science behind it in other aspects of their lives as well. Invariably all of them got off the medications that they were taking daily for conditions including diabetes and hypertension. Thus taking back their Health and Wellbeing into their own hands and feeling absolutely empowered and in Control of their lives.
This article discusses a unique and Novel form of "drug delivery" that is already being applied in the field of autism with amazing results. Before one explores this further, what needs to be understood as a foundational basic Fact is that ultimately, every single drug brings about its effect by Moving Energy in some way, shape or form .What IF there was a way by which one could practically shift energies in a positive direction towards (w) holistic well-being without having to swallow any pill at all?
Not only that , what if there was a way to Design /Prime Any pill or drug with Energy "Codes" with the INTENT and INTENTION for complete Wellbeing and targeted and delivered applying the cutting-edge science of Applied Intentional Epigenetics? All this and more can be understood by exploring and understanding this Cutting Edge and novel drug delivery system in. Autism that is producing results that is baffling professionals from mainstream medicine.
The Drug being delivered is Applied Energy Medicine and The Science behind the action of this drug is Applied intentional Epigenetics. Epigenetics literally means "control above genes". Applied Intentional Epigenetics is the art and science of applying techniques that bring about epigenetic transformations in a being or system using the power of Intention in any way, shape or form. It brings about these transformations at the level of the genes and the DNA of the being by bringing about Energy Shifts within the patterns encoded in the DNA, among other things. Applied Intentional Epigenetics thus has an underlying Energy Medicine basis to it. Energy Medicine is the art and science of restoring a being/system to its natural state of well-being and wholeness by augmenting the innate ability of the being/system to heal itself on all levels by bringing about shifts in the energy fields in the being/system to resonate with its natural frequency of alignment, balance and harmony. Intent Healing(TM) is healing using the power of Intention, accessing energies prior to consciousness that is free from all limiting conditioning and which brings about the realignment in the energy fields of beings/systems by rewiring the neural network in the brain and gut, reprogramming the DNA and erasing faulty cellular memories. To understand the newly emerging scientific fields of Applied Intentional Epigenetics, Applied Energy Medicine and the Intent Healing (TM) method further, it is recommended to read the articles given under the reference section below [16,17].
Mode of delivery of the drug : remote healing with intent healing(™)
Is it possible for human beings to SEE / SENSE beyond what is usually "normally" perceived by the human eyes? The answer is, Yes. And, anyone can do this .The U.S Military and other military agencies in the world have been using this and applying it in "military defence-related activities" since many decades. Today, many of those people who were trained in this capacity to do REMOTE Viewing and Remote Sensing, after leaving the defence services, are applying the Same Capabilities in HEALING people of illnesses and conditions.
Before anyone reading this starts thinking all this sounds "way out there " and "way too abstract" it is recommended to take a moment and consider the practical manner in which these leading-edge sciences have been applied by no less an organization than the U.S Military to Locate and then Capture Saddam Hussein using "Human Beacons" applying Remote Viewing techniques. This is explained in detail in the article given under references [18] and in the book by Lyn Buchanan 'The Seventh Sense' [19].
The Remote Healing method/technique that is being discussed in the context of this article is called Intent Healing (™) and it is Applied Energy Medicine in action where Healing Energies are channeled to individuals with the symptoms of disease or conditions, Regardless of the Distance. In other words, one does not need to be touched physically and be seen in person in order to benefit from the channeling of these healing energies across distances and different time zones in the world. One can benefit from this residing anywhere in the world and one does not need to travel to be seen by the person doing the healing.
Locations of action of the "drug"
1. Water in the cells/human body: More than 70% of the human body/cells are made up of water [4-6].
2. The DNA: This has been explained in detail in the article 'Application of Sound Frequencies as an Epigenetic Tool in Reversing the Limiting Symptoms of Autism '[15,20].
3. Energy Fields/ Energy System and the Sub-Atomic level: The "Human Energy System" comprising of the Chakras and the Meridians is amongst the most sophisticated piece of technology that a handful of human beings today are Rediscovering. The Energy System in the human body extends beyond the physical limitations of the body as an Energy Field that is intimately connected to other dimensional energies and the all encompassing energy field of Universal Intelligence. Through the chakras (vortices of Energy), one has direct access to the endocrinology of the system and this has powerful and immediate beneficial effects. Using Intent Healing method of Applied Energy Medicine, not just the limiting symptom in autism, but a whole lot of other conditions and illness that conventional medicine cannot "cure" can be healed [15,21,22].
4. Neural network in the brain and the gut: This level of impact of the drug delivery can be understood by knowing about the three "brains", especially in the context of autism. It is recommended to peruse the articles under the reference section to get an in-depth understanding of the same [15,23].
5. Level of the Muscles / Muscular system, Sympathetic, Parasympathetic and the Endocrine System and on ALL levels of well-being including physical, mental, emotional and "spiritual" [15,23].
In short , the effect is on the entire mind- body- organism- energy system
That brings us to the next question - How does one KNOW that this drug and the drug delivery system is working? It is primarily by the disappearance and/or improvements in the symptoms specific to the condition/illness and by the restoration of the ability of the individual to experience good health and well-being. Here, the re-defined EBM (Evidence Based Medicine) criteria in Autism [16] paves way for the Evolutionary Leap in measuring and acknowledging the Impact of a drug such as Remote Healing with Intent Healing(™) in curing illnesses and conditions. How can the progress be measured and documented? This can be done by using energy reading devices that are available today (although most of these devices are still not yet advanced in their capability to measure the changes on all levels).
Conclusion
Nobel Prize winning Scientist Albert Szent-Gyorgyi stated almost a century ago - "In every Culture and in Every Medical Tradition before ours, Healing was accomplished by Moving Energy". Today, science comes full circle by taking the EVOLUTIONARY LEAP ( paradoxically, by taking a leaf from cultures of the past) of delivering drugs/treatment in conjunction with Energy Medicine and Applied Intentional Epigenetics through methods such as Intent Healing (™), just like every culture before us was successfully doing and thus accomplishing well being and wholeness.
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Co-Culture as the Novel Approach for Drug Discovery from Marine Environment-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Abstract
Marine ecosystem is the bountiful source of pharmacologically relevant compounds. The enhanced productivity of this ecosystem is contributed mainly by the marine invertebrates. Though there is a serious problem of compound supply from these invertebrates, total synthesis and semi-synthesis efforts of the known compounds has helped us to simplify the problem. Another approach to enhance these compound productions is based on the microbes associated with them. Identification, extraction, genomic mining and co-culture approaches of these microbes will help us to meet the economic feasibility of the active principle or the bioactive metabolite. In this review, we come across the co-culture approaches of drug discovery from the marine environment.
Keywords: Co-culture; Mixed fermentation; Marine microbes; secondary metabolites; Quorum sensing; Antibacterial; Cytotoxic
Introduction
Natural products have various applications ranging from their uses in foods, cosmetics, pigments, medicines, insecticides etc. 60-75% of new drugs for cancer and infectious diseases reported between 1981-2002 were from natural sources [1]. From 1940s to the end of 2014, 131 (75%) of the 175 small molecules approved were not synthetic i.e., the belonged to biological macromolecule, unaltered natural product, botanical drug, natural product derivative, mimic of natural product etc. and 85 (49%) among them being natural products or directly derived there from [2].
Marine environment continue to be the treasure house of novel natural products which are potential drug candidates. The variability in thermal, pressure and nutrient ranges in the ocean facilitated extensive specification at all phylogenetic levels from microorganisms to mammals. Many bioactive compounds have been obtained from various marine animals like tunicates, sponges, softcorals, bryozoans, sea slugs and marine microorganisms [3].
Marine derived microbes are promising sources of novel bioactive compounds that are important for drug discovery process. The proportion of active bacteria associated with marine invertebrates (20%) and sea weeds (11%) is higher than that isolated from sea water and sediments (5%).Bioactive compound production by microorganisms associated with marine invertebrates could be attributed to the competition among them for space and nutrition. Though these bioactive compounds may be important for epibiotic defense of marine invertebrate hosts, they also have significant medical and industrial applications. The involvement of microorganism in the natural product biosynthesis has been evidenced in the case of Dysidea herbacea and Theonella swinhoei.
Lack of ethno medical history and the difficulties involved in the collection of marine organisms have made the research into the use of marine natural products to remain in its infancy. The development of new diving techniques, remote operated machines etc. helped us to meet the difficulties in marine sample collection and during the past decade, over 4200 novel compounds have been isolated from shallow water to 900m depths of the sea. The development of high throughput screening (HTS) technology increased the assay speed and the combinatorial chemistry speeded up the drug discovery process [3]. Recent advances in the molecular biology of bacteria and fungi have helped us in understanding the genetic potential of these microbes with respect to their capability of producing chemically diverse compounds. Under the laboratory conditions, many of the biosynthetic genes in microbes remain silent or are not transcribed. Thus we have obtained only a fraction of the real biosynthetic diversity of the compounds of microbial origin and this leads to the bottleneck in drug discovery from microbial sources.
In order to overcome the limitation during fermentation of microbes for bioactive compound production, several approaches are there:
a. The OSMAC (one strain many compound) approach- In order to maximize the diversity of compounds produced, promising strains are cultured in a variety of media and under different culture regimes
b. Epigenetic modifications- To modulate histones or DNA for initiating the transcription of the silent genes, microorganisms are treated with epigenetic modifiers such as histone deacetylase inhibitors or DNA methyl transferase inhibitors. This modulation may lead to the accumulation of new compounds.
c. A third approach is co-culture- In this microbes always coexist within complex microbial communities [4].
Co-culture
Co-cultivation (also called mixed fermentation) oftwo or more different microorganisms tries to mimic the complex microbial habitat in nature where they coexist. Due to the competition for limited resource and antagonism, these microbial communities produce bioactive secondary metabolite as part of defense. The competition and antagonism among these microorganisms is believed to activate the biosynthetic genes that remain silent under luxurious culture conditions. Thus these silent genes are transcribed under the induced stress conditions in co-culture and in turn lead to accumulation of cryptic compounds that are not detected in axenic cultures of the producing strain. The coculture of isolates from the same source or organism, may lead to enhanced production of constitutively present compounds in the organism. Thus the co-cultivation paves the way for increasing the chemical diversity of microbes from both the marine and terrestrial habitats.
Co-culturing is a powerful experimental tool for either enhancing the production of constitutively present compounds and/or for inducing silent gene clusters. It is a viable experimental approach for enhancing the chemical diversity of microorganisms when grown in vitro. In co-culture, induction of competition and stress lead to increased production of novel compounds that are not detected in pure cultures. Co-culture systems have been used to study natural interactions between populations, for improving cultivation success for certain populations and for establishing synthetic interactions between populations [5,6].
Bacterial cells show cell-cell communication in presence of other bacteria with their auto inducers. This aid the bacteria to sense a critical cell mass ad in response activate or repress target genes. Thus quorum sensing and quenching mysteries of marine derived microorganisms could be unraveled with these studies. Co-culture of fungus Aspergillus nidulans challenged by Streptomyces rapamycinicus first time provided the cue for the molecular basis of induction of silent fungal biosynthetic gene clusters [7,8].
Three major strategies of co-culture studies are:
a. Co-cultivation of different fungi
b. Co-cultivation of fungi with bacteria
c. Co-cultivation of different bacteria
Co-cultivation of marine derived microorganisms
Co-culture of two marine actinomycetes ANAM-5 an AIAH- 10 in yeast extract-glucose media enhanced antifungal and cytotoxic activities along with production of one new compound [9]. Here the induction of new compound production may be due to the interaction between the two cultures. Marine brown alga Rosenviagea sp. derived fungus Pestalotia sp. Cocultures with marine derived unidentified Gram negative bacteria of the genus Thalassopia (CNJ-328) produced chlorinated prenylsecoanthraquinone, pestalone which possessed antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecium and also exhibited cytotoxicity in National Cancer Institute's 60 human cell line screens [10].
In another study an established (3 day old) culture of marine derived fungus Libertella sp. (CNL-523) was challenged with marine a-proteobacterium (CNJ-328) induced biosynthesis of four new primarane diterpenoids- libertellenone A-D which were not produced by these cultures alone. These compounds showed cytotoxicity against HCT-116 human adenocarcinoma cell line and the libertellenone D was more active [11].The cell free supernatant as well as the autoclaved cultures of CNJ-328 or an ethyl acetate extract of the bacterium failed to induce the accumulation of libertellenones by the fungus Libertella sp. Therefore, a direct physical contact between the cultures is assumed to be necessary for the induction of the libertellenone. Interestingly, the marine bacterial strain CNJ-328 is the same strain that induced pestalone production in the previously mentioned study Table 1.
Emericellamides A and B production were induced by co-culture of marine derived fungus Emericella with marine actinomycete Salinospora arenicola. A 100 fold increase in their production was observed in the co-culture [12]. These compounds showed antibacterial activity against methicillin resistant Staphylocccus aureus (MRSA) and weak cytotoxic effects against HCT-116 cells. Dusane et al. [13] investigated the enhancement or induction of antimicrobial, biosurfactant and quorum sensing inhibition property in marine bacteria due to cross species and cross genera interactions. co- cultivation of marine epibiotic bacteria- Bacillus sp.S3, B. pumilus S8, B. licheniformis D1 and Serratia marcescens V1 from the surface of green mussel, Perna viridis and the coral Symphyllia sp. with pathogenic or biofouling fungi Candida albicans (6A) and Yarrowia lipolytica (YL) and bacteria Pseudomonas aeruginosa (PA) and Bacillus pumilus (B1) showed enhancement or induction in the antimicrobial activity, biosurfactant production and quorum sensing inhibition. This co-culture approach resulted in increased antibacterial activity against PA and B1 and improved biosurfactant production of B. pumilus with epibionts S. marcescens or with B. licheniformis. Coculture of PA with Bacillus species resulted in enhanced quorum sensing inhibition which was evident from reduced pigment production in the indicator strain Chromobacterium violaceum. Thus it became evident that chemical interactions between species in co-culture can influence the secondary metabolite production in the strains.
Marine alga Ulva californica derived isolates of Bacillus sp. (B. thuringensis and B. megaterium) were cocultured to yield increased production of indole and (Phe-pro) diketopiperazines which increased antibiotic activity against B. megaterium [14]. Thus the compound production by B. thuringensis was a survival strategy for the species which was induced by chemical cues from B. megaterium. This was evident from the co-culture of B. thuringensis with Staphylococcus sciuri from Ulva californica where there was no induction of diketopiperazine accumulation.
A Streptomyces sp. from Panamian tunicate was challenged with Bacillus subtilis, methicillin resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA) and Pseudomonas aeruginosa after coculturing the strain with each of these pathogens [15]. Minimal inhibitory concentrations (MIC) and LC-MS profiles indicated up regulation of antibacterial compounds when Streptomyces sp. cocultured with MRSA. Environmental stress induced by the pathogens promoted the augmented biosynthesis of active antibacterial compound. Co-fermentation of Penicillium sp. WC-29-5 with Streptomyces fradiae 007 produced four completely aromatic polyketides- deoxyfunicone; 1, 3, 8-trihydroxy-6- methylxanthen-9-one; (9R, 14S) - epoxy-11-deoxy funicone and (9S, 14R)-epoxy-11-deoxy funicone [16]. Cytotoxicities of these compounds against HL-60 and H1975 cells have been reported.
Two sponge derived actinomycetes were co-cultured in liquid media- Nocardiopsis sp. RV163 from the Mediterranean sponge Dysidea avara and Actinokinospora sp. EG49 from the Red Sea sponge Spheciospongia vagabhunda yielded several compounds which were detected by LC-PDA and 1H-NMR fingerprinting techniques [17]. A study involving interaction between Streptomyces tenjimariensis and 53 unknown marine bacteria for induction of istamycin A and B accumulation by S. tenjimariensis showed an enhanced production of istamycins only when it was inoculated 24h prior to other bacteria [18]. This shows us the fact that the chemical cues produced at certain critical time period is responsible for enhanced production of the metabolite.
Wakefield et al. [19] reported the dual induction of newly detected fungal and bacterial metabolites by co-culture of marine derived Aspergillus fumigatus MR2012 and two hyper and desert bacteria Streptomyces leeuwenhoekii strain C34 and strain C58. With the strain C34, MR2012 led to production of luteoride D and pseurotin. With C58, chaxapeptin production was augmented and pentalenic acid was first isolated and detected from this strain whereas all the fungal metabolites under axenic culture were suppressed. Here the bilateral crosstalk in the co-fermentation media led to dual induction of metabolite production [20-22].
Conclusion
Co-culture approach thus serves as an important appproach by which we can increase the yield of bioactive metabolites from marine microbes that are not usually produced under single culture conditions. Induction of natural product biosynthesis in co-culture experiments may be in response to microbial competition. Thus we can estimate that the activation of cryptic gene clusters in microbes in co-culture experiments is a means of survival strategy due to competition or antagonism. In future, this approach will help us to overcome the supply problem and also it will enhance the chemical diversity of bioactive compounds from marine microbes by activating the silent gene clusters.
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JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Drug Delivery Development: Quality Concepts, Challenges and Prospects-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Abstract
Quality, being a key to success in competitive market is an imperative indicator of product. It is important to recognize that quality cannot be tested into products, i.e., quality should be built in by design. The search for new drug delivery approaches and new modes of action is a rapidly developing field. Modes of drug delivery have changed in the past few decades and the future looks set to provide even more therapeutic advances. DDS can be developed to target common and rare diseases; both come with their challenges and opportunities, which are explored in this article.
Challenges in quality drug development mainly includes negligence from management, material management, quality personnel, lack of validated process, lack of equipment qualification, improper documentation, research validation specially related to novel drug delivery system. Nevertheless these challenges can be reduced to an appreciable level with the proper training, initiatives from management, continuous validation programmes and accepting novel drug delivery development considering risk assessment.
Keywords: NDDD; Quality issues in NDDD; Drug development challenges
Introduction
The quality in the pharmaceutical industry has become a major concern and there has been a growing awareness for the significance of the quality of the pharmaceutical products. The current concept of Good Manufacturing Practices (GMP) accentuates that the quality of pharmaceutical products must be constructed during the overall process cycle [1]. Quality is never improved in a common way. It is always improved project by project, beginning with the most significant problems [2]. The deficiency to be challenged must be clearly specified & the expected improvement can be defined in measurable terms [3]. This article examines the current status of quality related issues in development of drug delivery system with the objectives of assessing challenges and prospects.
Challenges
Challenging molecules and challenging markets is the key factor in drug development process. "On the molecule side, the pipeline is full of molecules with bioavailability, stability, targeted delivery, controlled release, and manufacturability challenges [4,5]. The benefit to risk ratio seems far removed and in true sense there is a functional gap between the development function and technical operations in the drug companies [6]. Cost of quality involves prevention cost, appraisal cost, internal failure cost (Scrap, rework and material losses) and external failure cost (returns and recalls), which has been neglected in the development process of delivery systems [7]. Global regulatory trends are yet to be defined fully, despite the several attempts already performed specially for novel drug delivery systems like Nano medicines. The other crucial issue is scale up. Commercial manufacturing uses much larger quantities compared to supply for the laboratory-scale experiments and, therefore, is a different ball game. Raw material batch-to-batch variability needs to be understood, process capabilities evaluated, and controls demonstrated by the vendor [8-10].
Prospects
The physicochemical and biological properties of the drug substance that can influence the performance of the drug product and its manufacturability should be identified and discussed [11]. The information on excipient performance can be used, as appropriate, to justify the choice and quality attributes of the excipient and to support the justification of the drug product specification [12]. Supportive management (philosophically and financially) can bring a quality concept and develop quality culture in the employees [13]. Quality policies need to be adopted indicating the goals of organization and support system in place to achieve those goals. Responsive deviation and investigation systems that lead to timely remediation will reduce the batch to batch variations [14].
Well-defined, designed and validated processes during entire product development life cycle can assure the product quality and reproducibility. While the rules and guidelines are quite well in place, there exists a non-uniform interpretation of these rules. An emphasis on the training of continuous manufacturing technologies, regulatory and organizational approaches is the need of hour in development of novel drug delivery system [13]. Fostering voluntary compliance by the researcher should be the focus rather than adopting more strict regulations [14]. A full scale design of experiment (Quality by Design) that begins with predefined objectives should be considered in the developmental strategy. It highlights product and process understanding and process control, based on Science-based approaches and sound methods for assessing risk [8]. This systematic approach can enhance achieving the desired quality of the product and helps in understanding manufacturing strategy. Process development studies should provide the basis for process improvement, process validation, continuous process verification (where applicable), and any process control requirements as given in Figure 1 [15].
Discussion
A systematic process plan and plant design undertaken by management and its implementation by considering trainings and research validation as part of the program can definitely lead to a quality product. QbD is just an approach to design a robust product but a quality product can be achieved only with the agreement of ethical principles for voluntary compliance rather than just fulfilling regulatory and organizational goals.
Conclusion
Quality drug development is not a rocket science but it's just willingness of researchers involving certain principles to be followed.
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Enzybiotics, A New Class of Enzyme Antimicrobials Targeted against Multidrug–Resistant Superbugs-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Summary
Gut microbiota with 2X1012 bacterial populations is essential for synthesis of vitamins, coenzymes and many other biomolecules in human and animal. But high dose of antibiotics destructed (since 1928) such bacteria in the alimentary tract posing a threat to extinct of human life. As a result signalling from human and bacteria orchestrated to build a defence to protect symbiotic relations saving both life forms. Bacteria synthesized hundreds of new genes (MDR Genes) to destroy antibiotics in different modes of actions. G-20 leaders and scientists have vowed a strong action plans (as assembled recently in Germany) to abolish the horror of superbugs which are claiming millions of death worldwide. Enzymes as therapeutic antibiotics has taken as emerging new antimicrobials derived from bacteriophages as well as bacteria like Staphylococcus sp., Streptococcus sp. and Histeria monocytogenes. Simply, autolysins, lysozymes, lysins and bacteriocins are great enzybiotics. Genetically modified enzybiotic (GMEnzy) has now a new field of enzyme antibiotic production using molecular biology and genetic engineering principles to overcome the antibiotic resistance. GMEnzy database has built for researchers and is available at http:// biotechlab.fudan.edu.cn/database/gmenzy/.
Introduction
The term enzibiotic was coined from two words, enzyme and antibiotic and usually refers as the bacteriophage enzymes that attack the cell wall of bacteria with lyses [1]. However, enzybiotic present in bacteria, bacteria infected phages and in body fluids like tears, saliva and animal mucous [2]. Antibiotics had used 80 years with success to eradicate pathogenic bacteria like Escherichia, Klebsiella, Salmonella, Mycobacterium, Pseudomonas and Vibrio species. However, last two decades gradual increase of clinical isolates had shown with >95% now ampicillin and amoxicillin resistant which was controlled by synthesis of new derivatives of penicillin like cephalosporin and carbapenem drugs [3]. In 2009 NDM-1 Escherichia coli was found however, resistant to all class of penicillins including Beta-lactamase inhibitors like cavulinate and sulbactam but avibactam [4]. Skin infections by MRSA Staphylococcus aureus, PDR nosocomial infections by Pseudomonas aeroginosa and XDR tuberculosis by Mycobacterium tuberculosis are now serious threat to human and alternative approaches should be needed to overcome such crisis [5]. MDR genes (blaTEM, amp, blaNDM, blaOXA, sul1/2, catB3, aacA4, aacC2, aph, aad, dhfr, arr3,strA/B,etc) and drug efflux genes (tetA, acrAB-TolC, mexAB-oprM, mcr, macAB, norA, mdtA etc.) are wide spread in conjugative plasmids and chromosomes of superbugs which are also found in rain, sea and river water posing a threat to global peoples [6].
Thus a new field of science is enzybiotics which is under clinical trial in many research foundations. If enzybiotic is injected into patient with success then all physicians believe that such single enzyme or chimera enzyme would be most useful in superbug cure [7]. It is to save gut microbiota that provide life saving coenzymes involved in glycosysis, TCA cycle and ATP generation [5].
Result
Some important enzybiotics are:
(a) Lysins. PlyG is Phage-y amidase which can destroy Bacillus anthracis (Figure 1) [8].
(b) Bacteriocins. Lysostaphin is Streptococcus simulans enzyme that acts as endopeptidase on Staphylococcus aureus and many Streptococcei sp. (Figure 2) [9].
(c) Autolysins. S. equidermis autolysin enzyme lyses β (1- >4) glycoside bong between N-acetyl glucosamine and N-acetyl muraminic acid of many bacteria (Figure 3) [10].
(d) Lysozymes. Egg white lysozyme is muramidase that destroy peptidoglycans and very effective against Gram (+) bacteria [11].
The lysins are 453-473aa long extracellular enzymes and have been sequenced from Streptococcus suis, Streptococcus agalactiae and others (protein ids. WP_061713285, WP_043026720, WP_070043600) with 50-150 mutations among themselves [12,13]. The multispecies bacteriocin (protein id. WP_013103375) has only 54% amino acid similarities to the Leuconostoc sp. Bacteriocin secretory protein (protein id. WP_030058663) but further pharmacological data are lacking. Autolysins are also much diverged as S. aureus enzyme (protein id. AAA99982; accession no. L41499) has only 60% homology with 8% gap to other autolysin enzymes (protein id. BAD83399) [14]. Genetically recombinant Lysins have great potential in curing MDR-bacteria [15-18]. P2neumococcal LytA autolysin, a potent therapeutic agent in peritonitis-sepsis caused by highly beta-lactamase resistant Streptococcus pneumonia [19,20].
Discussion
Enzybiotics is an emerging field of medicinal science with many molecular approaches have undertaken which have patent litigations and many data are hidden from GenBank database now [13]. It also has combined with phage therapy technologies targeting both Gram (+) and Gram (-) bacterial pathogenesis originating from MDR genes of superbugs [10]. We believe as MDR genes are created both from human and bacteria symbiosis, it will be there with gut microbiota [5]. So to eliminate the pathogenic bacteria alternative to antibiotics will be forthcoming like gene medicines (antisense, Casper-Cas, SiRNA, miRNA, ribozyme) and nanodrug-carriers [21]. Thus enzybiotic is in good place in molecular medicine and its success is ahead. Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus was reported [16,22]. About 1144 enzybiotics along with 216 natural resources (heterogeneous phyto-antibiotics) have been listed in GMEnzy database [2,13,23,24].
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Formulation of Prunus Amygdalus (Family:Rosaceae) Gum Based Compression Coated Tablet for Colon Targeted Delivery of 5-Aminosalicylic Acid-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Abstract
This research deals with formulation of compression coated tablet of 5-Aminosalicylic acid and further evaluated for colon targeting ability. Drug 5-aminosalicylic acid is degraded in stomach in the presence of gastric pH so colon targeting is best way to achieve therapeutic effect. Initially core tablet of drug was prepared using microcrystalline cellulose as diluents and further it was compression coated using various concentration of Prunus amygdalus gum. Prepared tablets were evaluated for various physical and mechanical parameters such as hardness, size and shape, friability and weight variation. To evaluate drug release ability of formulated tablet in to colon, In vitro drug release study was carried out in 0.1 N HCl and phosphate buffer pH 7.4. Formulated tablets were found to be round in shape with 13.436±0.020 to 13.482±0.017mm diameter. Hardness of tablets was found to be 18.22±0.12 to 18.56±0.04. Friability of prepared tablets were within Pharmacopoeial limit (i.e. <1%). In vitro release data showed that all the prepared tablets were able to resist drug release in pH 1.2 buffer and 50% drug releases was achieved after 10h. It can be concluded from the findings of the results that Prunus amygdalus gum coated tablets were able to delivery drug 5-Amino salicylic acid successfully in to colon in sustained manner.
Keywords: Prunus amygdalus gum; Colon targeted drug delivery; 5-Aminosalicylic acid; Drug delivery; Compression coating
Introduction
In recent times, colon specific delivery systems are also gaining importance for the systemic delivery of proteins, peptides and acid sensitive drugs [1]. Due to negligible activity of peptidase and less activity of pancreatic enzymes, the colon is considered to be more suitable for delivery of these molecules in comparison to the small intestine. Besides this less hostile environment, the colonic transit time is long (20-30h) and the colonic tissue is highly responsive to the action of absorption enhancers. The longer residence time, less peptidase activity, natural absorptive characteristics and high response to absorption enhancers make the colon a promising site for the delivery of protein and peptide drugs for systemic absorption [2-4].
There are several ways in which colon specific drug delivery has been attempted1. Development of pro drugs, coating with pH-dependent polymers, design of defined release dosage forms, and the use of carriers that can be degraded exclusively by colonic bacteria are an array of such attempts. This introduction provides a review of colon function, physiology, drug candidates and various approaches for colonic drug delivery [5-8].
Prunus amygdalus exudes a gum (Almond gum), which has been employed in place of tragacanth. It is obtained mostly from the trunk. Almond gum hydrolyzes into L-arabinose (4 parts), D-xylose (2 parts), D-galactose (3 parts) and D-glucouronic acid (1 part); aldobio uronic acid is also present. This is a natural polymer and employed as coating material in present study.
5-Amino salicylic acid (5-amino-2-hydroxybenzoic acid) is an anti-inflammatory agent, structurally related to the salicylates, which is active in inflammatory bowel disease. It is considered to be the active moiety of sulphasalazine. 5-Amino salicylic acid is acid sensitive drug and degraded in low pH of stomach. Absorption site of drug is also in the colonic region. So it becomes necessary to target drug molecule in to the lower part of intestine.
Material and Methods
Drug 5-Aminosalicylic acid was received as gift sample from Cipla Ltd, Mumbai. Microcrystalline cellulose, Ethyl cellulose,Ethyl alcohol, Triethyl citrate, and magnesium stearate was purchased from Merck Ltd Mumbai and used as received. Double distilled water was used as solvent in whole study.
Preparation of core tablet
The core tablet comprising of 5-ASA and MCC was prepared by direct compression method as shown in Table 1. Accurately weighed quantities of the ingredients were mixed in a glass mortar and required quantity were placed on the KBr press and compressed at pressure of 500 Psi into tablets of 8 mm diameter.
a. Evaluation of core tablet: Prepared core tablets were evaluated for various physical parameters and mechanical properties [9-12].
b. Physical characterization: Core tablets were evaluated for following physical properties.
c. Appearance: In this the shape, color, presence or absence of an odour, taste and surface texture were recorded on five randomly selected tablets.
d. Average diameter: The diameters of five randomly selected tablets were recorded with the help of digital vernier caliper and the average diameter and standard deviation were calculated.
e. Average thickness: Thickness of the developed tablet is an important parameter to investigate. The thickness of the developed tablet was recorded using digital vernier caliper by random sampling technique. Thickness of five tablets was determined and the mean thickness and standard deviation was calculated.
Average weight and weight variation
USP procedure for uniformity of weight was followed, twenty tablets were taken randomly and their weighed was determined individually and collectively on a digital weighing balance accurately. The average weight of one tablet was determined from the collective weight. The weight variation was calculated for USP limits i.e., for average weight of 80 or less, 80-250 and more then 250; maximum percentage differences allowed are 10%, 7.5% and 5% respectively.
a. Mechanical properties: Core tablets were evaluated for following mechanical properties:
b. Hardness: The hardness of the core tablets was determined using an electrolab tablet breaking force tester USP (1217). Five core tablets were randomly selected and placed one at a time between the jaws of the hardness tester before it was
started. The force needed to break the core tablet was recorded.
c. Friability: A friability test was conducted on the core tablets using an electro lab EF-2 friabilator (USP). Ten tablets were randomly selected and any loose dust was removed with the aid of a soft brush. The tablets were weighed accurately and placed in the drum of the friabilator. The drum was rotated at 25rpm for 4 minutes after which the tablets were removed. Any loose dust was removed from the tablets as before and the tablets were weighted again.% friability was calculated using equation1.
Friability (% w/w) = (Weight before (g) - Weight after (g))/ (Weight before (g))X100 equation1
I. Drug Content of the tablet: To determine the drug content, five tablets were crushed into powder form and the powder was dissolved in 100mL of phosphate buffer, pH 7.4 and the solution was sonicated for 30min. The supernatant was filtered and the absorbance was measured after suitable dilution at 331nm.
II. Disintegration test: This test was conducted on 6 core tablets with a electro slab disintegration tester (USP) ED-2L apparatus at 37±2 °C. The phosphate buffer of pH 7.4 was used as a disintegration media. The time taken for complete disintegration of the tablet with no palpable mass remaining in the apparatus was measured in minutes.
III. Preparation of compression coat: The total weight of the compression coating was 300 mg for each drug: polymer ratio. The seven batches were prepared in which drug amount was kept constant and polysaccharide (almond gum) concentration varied. The ingredients were properly mixed for 5 minutes before and 5 minutes after addition of magnesium stearate (lubricant). Compression coat was prepared by wet granulation method. Accurately weighed quantities of the ingredients were mixed in a glass mortar and required quantity of distilled water was added to the powder mass and mixed thoroughly. The granules were prepared by passing the wet mass through British Standard Sieve (BSS) No. 10. Wet granules were dried in a dessicator for 36h.
IV. Preparation of compression coated tablet: 150mg of granules were placed in die of KBr press. Core tablet was placed and 150mg of granules were poured on core tablet and it was compressed in KBr press at pressure of 1500 Psi into tablets of 12mm diameter.
V. Coating of compression coated tablet: The compression coated tablet was coated with ethyl cellulose to provide delayed release action (Table 2). It is a hydrophobic polymer and thus prevent early wetting of tablet. The tablets were coated by spray coating technique. The solution of ethyl cellulose (4%w/v 20ml) was sprayed on the tablets with help of a sprayer. The tablets were dried in stream of hot air. The procedure was repeated many times until there was 50mg weight gain.
Evaluation of coated tablet: Coated tablets were evaluated for following parameters:
Physical characterization
a. Appearance: In this the shape, color, presence or absence of an odour, taste and surface texture were recorded on five randomly selected tablets.
b. Average diameter: The diameters of five randomly selected tablets were recorded with the help of digital vernier caliper and the average diameter and standard deviation were calculated.
c. Average thickness: Thickness of the developed tablet is an important parameter to investigate. The thickness of the developed tablet was recorded using digital Vernier caliper. Thickness of five tablets was determined and the mean thickness and standard deviation was calculated.
d. Average weight and weight variation: USP procedure for uniformity of weight was followed, twenty tablets were taken randomly and their weighed was determined individually and collectively on a digital weighing balance accurately. The average weight of one tablet was determined from the collective weight. The weight variation was calculated for USP limits i.e., for average weight of 80 or less, 80-250 and more then 250; maximum percentage differences allowed are 10%, 7.5% and 5% respectively.
Mechanical properties
Hardness: The hardness of the coated tablets was determined using an electro lab tablet breaking force tester USP (1217). Five coated tablets were randomly selected and placed one at a time between the jaws of the hardness tester before it was started. The force needed to break the coated tablet was recorded.
In vitro Release Behavior:
Experimental Conditions:
Apparatus - USP Dissolution Apparatus, Type II (Paddle)
Dissolution Medium- SGF pH 1.2, Phosphate buffer pH 6.8 and Phosphate buffer pH 7.4
Volume-900ml, Sample volume-2ml
Temperature - 37±0.5 °C
RPM-50
SGF pH 1.2 was transferred in dissolution vessels and the temperature and rpm were set at 37±0.5 °C and 50 respectively. The developed tablets were transferred to dissolution vessels. A 2ml of aliquots of dissolution fluid were withdrawn after 2h and the medium was replaced with phosphate buffer pH 6.8.
The samples were withdrawn for 3h and again the medium was replaced with phosphate buffer pH 7.4. Samples were withdrawn for 24h, suitably diluted and analyzed using double beam UV-Visible spectrophotometer (Shimadzu-160A) at 331nm.
Evaluation of core tablets: Core tablets were evaluated for various parameters which are shown in Table 3 & 4.
Evaluation of coated tablets: Coated tablets were evaluated for general appearance and the results are shown in Table 5.
Various physical parameters of prepared tablets were shown in Table 6. Diameter and thickness of all the formulated batches ranged from 13.436 to 13.482mm and 3.99 to 4.04mm respectively. All the formulated batches pass the weight variation test. Hardness of all the formulated batches ranged from 18.22 to 18.56kg Table 5 showed that as the concentration of gum increases hardness of tablet increases.
Drug release profile of all the formulated batches was determined via USP Dissolution Apparatus II paddle type and shown in Figure 1.
The results indicated that as the almond gum concentration was increased release of drug from formulation decreased up to F5 batch and then drug release was increased up to certain level. No drug was released in the acidic medium; at pH 1.2. All formulations showed a very slow release of the drug depending on the Almond gum composition. After 8 h less than 30% of drug was released from tablets containing 90-240mg of Almond gum (F1-F6). Formulation containing 270mg of Almond gum (F7) exhibited the highest drug release rate then the F1 batch. F4 batch was selected as the best batch because it released the 97% of drug over the period of 24h. This dissertation work proved that 5-ASA compression-coated with 30% Almond gum was found to be a promising drug delivery system for treatment of inflammatory bowel disease (Table 7).
Conclusion
Seven batches (F1-F7) were prepared by varying the concentration of almond gum. The compression coated tablet was coated with ethyl cellulose (4%) as delayed release coating. The developed formulation was evaluated for various tests like diameter, thickness, hardness, friability, weight variation, drug content and In vitro drug release. In vitro drug release was carried out at three different pH conditions (1.2, 6.8 and 7.4) simulating the condition of GIT. F4 batch was selected as the best batch because it releases the 97% of drug over the period of 24h. A 4% ethyl cellulose coating was sufficient to delay the release for 5h. it can be concluded from the complete study that Prunus amygdalus gum can be effectively used as compression coated material for the colon targeting of drug.
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Erythrocyte as a Therapeutic Target-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DRUG DESIGNING & DEVELOPMENT
Abstract
Erythrocytes are powerful components of blood flow designed to scavenger or deliver nitic oxide (NO) and oxygen to all cells in the body and transport carbon dioxide from them to the lungs. Blood components started to be quantified and erythrocyte blood shapes used as diagnostic and prognostic tools in clinical practice. Erythrocytes have hemorheological, hemostatic and pro or anti-inflammatory properties enlarging their physiological implications in health and disease. As blood component the erythrocytes establish interaction with others white blood cells, platelets, plasma lipoproteins and vascular endothelial cells. The aim of this mini review is highlight the signaling pathway of nitric oxide in which some steps explain the efficacy of some therapeutic drugs already used and could point new targets for further application in inflammatory vascular diseases
Keywords: Erythrocyte; Nitric oxide; Acetylcholinesterase; Forskolin; CD47; Fibrinogen
Abbrevations: AChE: Acetylcholinesterase; NO: Nitric Oxide; RBCs: Red Blood Cells; GIP: Glucose-Insulin-Potassium; PKC: protein kinase C
Mini Review
More than two centuries ago was discovered in blood the presence of erythrocytes and its vital function as the unique oxygen carrier binding to hemoglobin which molecular and structural characterizations where later described [1-5]. Erythrocytes with different shapes are observed in association with some hemoglobinophaties for example sickle cell disease, or in those resulting from inserted compounds into specific membrane domains [6,7].
Erythrocyte metabolism provides metabolites able to regulate the oxygen affinity for hemoglobin such as 2,3-bisposphoglycerate and others to participate as coenzymes in antioxidant pathways [8]. Several therapeutically drugs cause hemolysis in humans with glucose-6-phospate dehydrogenase null gene [9]. A reducing environment inside of erythrocytes ensures the active form of hemoglobin with its ferric ion and the normal interaction between biomolecules of the membrane bilayer and the proteins of cytoskeleton [10]. However, when erythrocytes show higher pro-oxidant activity contribute to abnormal biorheological functions associated with inflammatory vascular diseases [11,12]. They can be a trigger or a consequence of micro or macro circulation dysfunction. Acquired ability of erythrocytes to combine with partners of hemostatics components generate red thrombus and help the rolling and adhesion of white blood cells to vascular endothelial wall [13,14].
Erythrocytes are enucleated blood components, but are more than sacks of hemoglobin during the semi life of 120 days comporting different signaling pathways in which is included the final stage of apoptosis (eryptosis) that has been evidenced [15,16]. The appearance in plasma of exovesicules enriched with the acetylcholinesterase (AChE) originated from erythrocyte membrane, the phosphatydilserine exposition in the outer membrane of erythrocyte in addition to kinetic changes of the AChE evaluated in older erythrocytes are biomarkers of red blood cells senescence (RBCs) [17,18]. Previously AChE in erythrocytes was evidenced as a biomarker of its membrane integrity [19].
Depending on the degree of endothelium integrity the circulating acetylcholine [ACh) induce vasodilation or vasoconstriction according the amount of nitric oxide (NO) synthesised and released [20,21]. The NO released from endothelial cells and platelets is scavenged by erythrocyte and blood cell free haemoglobin [22].
Erythrocyte membrane AChE is involved in the nitric oxide (NO) signal pathway as evidenced, for the first time, using blood samples from blood donors in several in vitro studies in the begin of this century [23,24]. No metabolism provides several NO derivative molecules such as nitrite, nitrate, peroxynitrite and S-nitro glutathione (GSNO) behavior the last one as NO reservoir such as S -nitrosohemoglobin [23,24]. The signal transduction pathway mediated by the enzymatic complex form AChE-ACh is coupled to Gaiproein, adenylil cyclase (AC), band3 protein, protein kinase C (PKC) and phosphodiesterase-3 (PDE-3) [24]. The ACh concentration used is below its substrate optimum concentration [25]. The substrate concentration correspondent to the velocity maximum obtained in the bell shape kinetic curve [25,26]. Higher NO efflux occurs under the influence of AChE-Ach complex, in simultaneous with the band3 protein phosphorylation [23,24]. Compounds that inhibit the protein tyrosine kinase or protein tyrosine phosphatase induce inhibition or activation on AChE enzyme activity [24]. Used two types of AChE inhibitors, velnacrine maleate and timolol generate inactive or less active enzyme AChE-inhibitor complexes that impaired NO efflux from erythrocytes in relation to the active form ACh- AChE [23,27].
In patients with open angle glaucoma, over expression of eNOS and nNOS, decreased levels of cGMP (intermediate in NO signaling) and of nitrite (NO metabolite)in aqueous humour and increased erythrocyte AChE activity were described [28-30]. When blood samples from glaucoma patients is incubated in- vitro in presence of timolol, no changes in the NO efflux neither in the GSNO content of erythrocytes were evidenced besides both molecules are in higher concentrations than the normal values obtained in health humans [31]. This study showed that no reinforcement will occur in the amount of nitrogen reactive stress characteristic of glaucoma patients, by timolol application [31].
Insulin resistance can be eliminated in some patients with sepsis by continuous intravenous infusion of insulin in the form of glucose-insulin-potassium (GIP) regimen that improves survival [32,33]. When blood samples from patients with septic shock where incubated in-vitro with insulin increased the amount of GSNO and the concentration of NO inside erythrocytes was maintained between the normal values [34]. A positive association was observed between NO efflux from erythrocyte and perfused vessel density at sub-lingual microcirculation [34]. So the GPI regimen protected from nitrogen reactive stress [34].
When fibrinogenemia is mimicked in- vitro NO efflux from erythrocyte increases, in dependence of band3 protein phosphorylation, returning to normal levels when in presence of either ACh or timolol showing dependency of the AChE enzyme conformational states and of the lower levels of cyclic adenosine Monophosphate (cAMP)concentrations [27,35-37]. When the inhibitor of the erythrocyte Casein Kinase 2, (a cytosol protein that phosphorylated band 3 protein), is present in the erythrocytes suspensions at high fibrinogen concentration the NO efflux level is maintained between normal values confirmed its dependence of band 3 de phosphorylating for be rescued by RBCs [38].
Is very interesting that the forskolin, activator of AC enzyme normalize the levels of NO efflux from erythrocytes in in-vitro model of hyper fibrinogenemia, is nowadays used to alleviate patients with glaucoma [39].
As mentioned above glaucoma patient's present increase nitrogen reactive species in aqueous humor and NO efflux from their erythrocytes are higher than healthy humans [28,31]. So, one explanation for the forskolin therapeutic success in glaucoma patient's could result from NO efflux from their erythrocyte be dependent of lower cAMP levels. This make sense because glaucoma is an inflammatory disease where patients have increased levels of fibrinogen which is known its binding to erythrocyte membrane CD47 that by association with Gaiprotein and AC inhibition decreased cAMP concentrations. [37,40,41]. Besides, this is a clue need to be explored.
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