Anuradha K. Gajjar

Drug Solubility: Importance and Enhancement Techniques

Solubility, the phenomenon of dissolution of solute in solvent to give a homogenous system, is one of the important parameters to achieve desired concentration of drug in systemic circulation for desired (anticipated) pharmacological response. Low aqueous solubility is the major problem encountered with formulation development of new chemical entities as well as for the generic development. More than 40% NCEs (new chemical entities) developed in pharmaceutical industry are practically insoluble in water. Solubility is a major challenge for formulation scientist. Any drug to be absorbed must be present in the form of solution at the site of absorption. Various techniques are used for the enhancement of the solubility of poorly soluble drugs which include physical and chemical modifications of drug and other methods like particle size reduction, crystal engineering, salt formation, solid dispersion, use of surfactant, complexation, and so forth. Selection of solubility improving method depends on drug property, site of absorption, and required dosage form characteristics.

Pharmacological review on Centella asiatica: a potential herbal cure-all.

In recent times, focus on plant research has increased all over the world. Centella asiatica is an important medicinal herb that is widely used in the orient and is becoming popular in the West. Triterpenoid, saponins, the primary constituents of Centella asiatica are manly believed to be responsible for its wide therapeutic actions. Apart from wound healing, the herb is recommended for the treatment of various skin conditions such as leprosy, lupus, varicose ulcers, eczema, psoriasis, diarrhoea, fever, amenorrhea, diseases of the female genitourinary tract and also for relieving anxiety and improving cognition. The present review attempts to provide comprehensive information on pharmacology, mechanisms of action, various preclinical and clinical studies, safety precautions and current research prospects of the herb. At the same time, studies to evaluate the likelihood of interactions with drugs and herbs on simultaneous use, which is imperative for optimal and safe utilization of the herb, are discussed.

Isolation and structure elucidation of major alkaline degradant of ezetimibe.

This work presents the isolation and characterization of the alkaline degradant of Ezetimibe. Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, was subjected to alkaline degradation. Ezetimibe was reacted with 0.1M methanolic sodium hydroxide solution for 10min at 80°C to yield alkaline degradant to an extent of 90% of initial amount of the drug taken. This degradant was detected by high performance liquid chromatography (HPLC) at relative retention time (RRT) of 1.48 with respect to Ezetimibe. HPLC method involved an isocratic elution on a Waters Symmetry C(8) 150mm×4.6mm, 5μm column using ammonium acetate buffer (pH 4.5, 50mM) - acetonitrile (50:50, v/v) as the mobile phase at a flow rate of 1.0mL/min and UV detection at 242nm. The degradant was isolated by preparative HPLC. Purity of the isolated solid was found to be more than 99%. Structure of alkaline degradant was confirmed by LC-MS, (1)H and (13)C NMR and IR spectroscopy. On the basis of spectral data, the structure of the degradant was confirmed as 5-(4-fluorophenyl)-2-[(4-fluorophenyl amino)-(4-hydroxyphenyl)methyl]-pent-4-enoic acid. The route for the formation of this degradant is also proposed. Determining the structures of degradation products arouse during stress testing can be useful for preclinical discovery efforts.

Mechanisms of Resistance: Useful Tool to Design Antibacterial Agents for Drug - Resistant Bacteria

Drug-resistant bacteria are now a global health threat. In the last 5 years the WHO, The House of Lords (UK), the Centre for Disease Control (USA) and many more agencies have presented reports on the scale of this problem. Microorganisms multiply very rapidly and have adapted to fill almost every available environmental niche (Rapidly growing species of bacteria under ideal conditions of growth can multiply in about 20 minutes). All members of the chemically related beta-lactam class act at the same phase in cell wall synthesis; as a result, a bacterial cell resistant to one agent is often resistant to all other analogues. The beta-peptide has two promising characteristics that distinguish it from traditional antibiotics. Firstly, bacteria may have trouble developing resistance to the beta-peptide since bacterial defenses may not recognize its unnatural amino acids. Secondly, the magainins that the beta-peptides mimic have been around for millions of years, yet bacteria have not become resistant to them. All classes of antibiotics are subject to resistance by an efflux mechanism mediated by more than one type of pump within the same organism. The bacterial cell may have a membrane pump capable of pumping a class or several classes of antibacterial agents back out of the cell. Other mechanisms of drug resistance include destruction of beta-lactam ring by beta-lactamases, impermeability of the drug into the bacterial cell wall, alteration of targets within the bacterial cells and the by-pass mechanism (bacterial cell may have acquired an alternative mechanism for achieving the essential function).

Impurity Profiling: A Case Study of Ezetimibe

Impurity profiling includes a description of the identified and unidentified impurities present in new drug substances or drug products. Isolation and elucidation of the structures of degradation products are typically collaborative research involving knowledge of analytical, organic and physical chemistry with spectroscopic information. Stability testing guidelines issued by International Conference on Harmonization (ICH) require the reporting, identification and characterization of degradation products (DPs). The alkaline degradant was detected by high performance liquid chromatography (HPLC) at relative retention time (RRT) of 1.48 with respect to Ezetimibe. This degradant was isolated by preparative HPLC. Purity of the isolated solid was found to be more than 99%. Structure of alkaline degradant was confirmed by LC-MS, 1 H and 13 C NMR and IR spectroscopy. On the basis of spectral data, the structure of the degradant was confirmed as 5-(4-fluorophenyl)-2-((4-fluorophenyl amino)-(4-hydroxyphenyl)methyl)-pent-4-enoic acid. An understanding of the parts of the molecule that are susceptible to degradation can help in the design of more stable analogs. Determining the structures of the major degradation products can reveal whether or not a known carcinogen or toxic compound is or might possibly be formed.

Combiphore (Structure and Ligand based Pharmacophore) Approach for Design of GPR40 Modulators in Management of Diabetes.

Pharmacophore mapping and molecular docking can be synergistically integrated to improve the drug design and discovery process. A rational strategy, combiphore approach, derived from the combined study of Structure and Ligand based pharmacophore has been described to identify novel GPR40 modulators. DISCOtech module from Discovery studio was used for the generation of the Structure and Ligand based pharmacophore models which gave hydrophobic aromatic, ring aromatic and negative ionizable as essential pharmacophoric features. The generated models were validated by screening active and inactive datasets, GH scoring and ROC curve analysis. The best model was exposed as a 3D query to screen the hits from databases like GLASS (GPCR-Ligand Association), GPCR SARfari and Mini-Maybridge. Various filters were applied to retrieve the hit molecules having good drug-like properties. A known protein structure of hGPR40 (pdb: 4PHU) having TAK-875 as ligand complex was used to perform the molecular docking studies; using SYBYL-X 1.2 software. Clustering both the models gave RMSD of 0.89. Therefore, the present approach explored the maximum features by combining both ligand and structure based pharmacophore models. A common structural motif as identified in combiphore for GPR40 modulation consists of the para-substituted phenyl propionic acid scaffold. Therefore, the combiphore approach, whereby maximum structural information (from both ligand and biological protein) is explored, gives maximum insights into the plausible protein-ligand interactions and provides potential lead candidates as exemplified in this study.

CoMFA, CoMSIA and HQSAR Analysis of 3-aryl-3-ethoxypropanoic acid derivatives as GPR40 modulators

Human GPR40 receptor, also known as free fatty-acid receptor 1, is a G-protein-coupled receptor that binds long chain free fatty acids to enhance glucose-dependent insulin secretion. In order to improve the resistance and efficacy, computational tools were utilized. To build the 3D-QSAR models, CoMFA (Comparative Molecular Field Analysis), CoMSIA (Comparative Molecular Similarity Indices Analysis) and HQSAR (Hologram Quantitative Structure Activity Relationship) were performed on series of 3-aryl-3-ethoxypropanoic acid derivatives. 3, pharmacophore and docking based alignments were performed from which distill alignment was found to be reliable and predictive CoMFA and CoMSIA models were obtained with good statistical results. The results obtained from CoMFA analysis q2, r2cv and r2pred were 0.693, 0.69 and 0.992 respectively and in CoMSIA analysis q2, r2cv and r2pred were 0.668, 0.648 and 0.990. Contour maps of CoMFA (lipophilic and electrostatic), CoMSIA (lipophilic, electrostatic, hydrophobic, and donor) and HQSAR (positive & negative contribution) provides significant insights i.e. favoured and disfavoured regions or positive & negative contributed fragments with R1 and R2 substitutions, which gave hints for the modification required to design new molecules with improved biological activity. Molecular docking studies were carried out on designed molecules using Sybyl-X software. Therefore such a methodology, whereby maximum structural information (from ligand and biological target) is explored, gives maximum insights into the plausible protein-ligand interactions and is more likely to provide potential lead candidates as exemplified from this study.

Computer aided docking studies of some antimicrobial agents

Mark S. Kindy Department of Neurosciences, Medical University of South Carolina, and the Ralph H. Johnson VA Medical Center, USA Recently, interest in semiallogeneic vaccines has been increasing, as shown by the publication of successful preclinical and clinical studies by us and others that validate this immunotherapeutic approach to cancer, viral diseases such as the acquired immunodefi ciency syndrome and neurological diseases. Th ese reports indicate that treatment with semiallogeneic vaccines can induce a specifi c immune response against various tumors and against the human immunodefi ciency virus. Experimental studies using inbred mice and their syngeneic tumors initially established that 1) inoculation of semiallogeneic cell hybrids (derived from the fusion of syngeneic tumor cells with an allogeneic cell line) protects the animal host from subsequent lethal challenges with unmodifi ed syngeneic tumor cells; 2) adoptive transfer of immunity required T lymphocytes; and 3) the enhanced immunity was not the result of an allogeneic eff ect per se, because the tumor-associated antigens and alloantigens needed to be on the same cell (the hybrid). Human studies with semiallogeneic cell hybrids have focused on the use of therapeutic vaccines for cancer. Because of the many technical problems associated with obtaining suffi cient amounts of autologous tumor cells for the preparation of patient-specifi c cancer vaccines, and because of evidence that allogeneic eff ects may enhance the antitumor immune response, it seemed reasonable to combine the best of both approaches (autologous and allogeneic), because semiallogeneic hybrids focus allorecognition and major histocompatibility complex self-restricted recognition on the same cell and, therefore, in the same microenvironment. Our studies demonstrate the feasibility of using semiallogenic vaccines to treat a variety of tumors and neurological disease.Daming Zhu Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Disease, National Institutes of Health, USA Malaria is one of most deadly infectious diseases in the world, with an estimated 350-500 million people suff ering from malaria episodes and nearly 1 million deaths per year. Most of these episodes are caused by Plasmodium falciparum and P. vivax parasites. Th e world is in urgent need of a vaccine to combat against malaria. Th e quality control is one of the most important processes in vaccine research and development because it directly relates to the safety and effi cacy of the vaccines. Th is presentation will focus on the quality control evaluation of malaria sub-unit vaccines. Th e biophysical/biochemical/biological methods involved in evaluating vaccine purity, identity, integrity, stability and potency will be discussed. Th ese methods should have general applications for the quality control evaluation of subunit vaccines.Olga Borges University of Coimbra, Portugal Th e development of oral and nasal formulations for under-utilized vaccines in order to simplify their transport, storage and administration in poor countries is considered a challenge [1]. In fact, for eff ective oral immunization, antigens must be protected from the acidic and proteolytic environment of the gastrointestinal tract, effi ciently taken up by cells of the gut associated lymphoid tissue (GALT) and an appropriate immune response must be induced [2]. With this in mind we have been working on the design of chitosan-based particles as adjuvant for mucosal vaccination and recently a new delivery system was evaluated as adjuvant for oral administration of the recombinant hepatitis B antigen (HBsAg) [3]. In this previous work we obtained some promising results, like high titers of anti-HBsAg IgG in serum and antiHBsAg sIgA in mice intestinal washings. However, a high percentage of non-responder mice were observed. Th erefore, the main objective of the group is to develop more sophisticated chitosan-based delivery systems associating to the particles not only the antigens but also an immunopotentiator. Th ree diff erent chitosan-based formulations were developed associating to them aluminum salts, a mast cell activator (c48/80 compound) and CpGODN. An overview of the work done by group on the development of a mucosal hepatitis B vaccine, as well as in vitro results of these new adjuvants will be present during the congress.Yasuyuki Ishi RIKEN Research Center for Allergy and Immunology, Japan Invariant natural killer T (iNKT) cells having Th e invariant T cell antigen receptor (TCR)  chain are a unique population to regulate the immune response between the innate and acquired immunity. iNKT cells interact with glycolipids presented via CD1d molecule on antigen-presenting cells (APCs), resulting in the activation of killing activity and the production of a variety of cytokines. KRN7000, alpha-galacosyl ceramide (-GalCer), among glycolipids functions as the powerful ligand for not only rodent but also primate iNKT cells. As iNKT cells potentially have two bi-directional functions, one is immune activation and the other is immune suppression, it was expected that KRN7000 would be utilized as an adjuvant for various vaccines of cancer, infectious and immune diseases. However, the pharmaceutical development of KRN7000 is not successful because of the bi-directional functions of iNKT cells. In our recent studies, two functions of immune activation and suppression are dissected by the in vivo delivery system of KRN7000. Dendritic cells (DCs), the professional APCs, could be used as delivery cells of KRN7000 for immune activation. Systemic administration of KRN7000-pulsed DCs preferentially enhanced protective immunity against tumors and infection by microbes. In contrast, liposomal formulation of KRN7000 could be delivered to the marginal zone B220positive cells in a spleen, induce regulatory T cells (Treg) and diminish immune responses in model animals of autoimmunity, allergic diseases and graft versus host diseases (GvHD).Immunology and Biotechnology Unit, Zoology Department, Faculty of Science, Tanta University, Egypt Th e success of anti-tumor immunity depends on the generation of functionally eff ective T cells. Adoptive cell therapy (ACT) of autologous tumor-reactive T cells aft er chemotherapy a nd followed by vaccination is a promising approach for generation of functional T cells for cancer immunotherapy. Th is ACT modality consists of in vitro stimulation of T cells from a host own peripheral blood or tumor and then infusing them back to the same host blood followed by vaccination regimen such as peptide or peptide-pulsed dendritic cells (DCs). Th e host is irradiated or treated with chemotherapeutic drug such as cyclophosphamide (CTX) prior ACT to induce lymphopenia. Th e cellular and molecular mechanisms underlying the benefi cial eff ects of lymphodepletion in the context of adoptive T cell therapy and vaccination, however, are not well understood. Defi ning these mechanisms would signifi cantly improve the application of lymphodepletion to ACT. Our recent studies have identifi ed expansion of DCs, the central player of immune response, as a potential mechanism. We have utilized the toll-like receptor 3 (TLR3) agonist poly(I:C), a synthetic viral mimic double-stranded RNA, to induce the full activation of DCs expanded in vivo aft er chemotherapy and during vaccination with defi ned tumor antigens, resulting in effi cacious therapeutic anti-tumor responses. Our results reveal that the combination of T cell therapy and vaccination in the presence of a potent adjuvant such as a TLR agonist at precise timing post chemotherapy opens a new avenue for cancer immunotherapy that can be translated into the clinical setting to cure diff erent cancers.