▶ Pre-clinical and clinical development of drug substances and drug products for diseases of national importance, international relevance and public health needs;
▶ Provision of services to the pharmaceutical industry, especially micro, small and medium enterprises and public sector manufacturers in areas of clinical trials, regulatory toxicology, safety pharmacology, pharmaceutics and pharmacokinetics;
▶ Continued engagement with drug regulation and pharmaceutical policy making in India as well as internationally

Generation and compilation of data required for regulatory approval

Documentation of the physicochemical properties of drug candidates and formulations was carried out in the format specified by the New Drugs and Clinical Trial Rules, 2019. Regulatory data on specifications of physico-chemical properties (Chemistry, Manufacturing and Controls) of four CSIR-CDRI candidate drugs: S007-867, S-007-1500 and S-011-1793 were compiled. Investigational New Drug Applications to the Drugs Controller General of India for clinical testing of these candidate drugs are being prepared before submission.

Development, validation and deployment of methods of pharmaceutical analysis

Analytical methods were developed and validated according to the New Drugs and Clinical Trials Rules, 2019, for several new as well as known drugs. This year, pharmaceutical analysis of 23 different kinds of samples of synthetic compounds, plant products and industrial production batches were analysed; about half the samples analysed in the previous year. Another set of about 1000 samples were analysed for drug content, content uniformity, drug release, stability and impurity profiling in formulation development activities. There are 14 different active projects on pharmaceutical analysis in progress currently. The average time from receipt of sample to filing an analytical report this year was 36 days, up from 27.9 days in the previous year.

Inhalable particles containing anti-tuberculosis agents

The grant application for funds required for a Phase-1 b was reviewed and revised as per expert comments. The clinical testing plan has been submitted to the Institutional Ethics Committees of CSIR-CDRI and King George’s Medical University. The trial has objectives of determining safety, pharmacokinetics and early measurement of drug activity.

Inhalable particles containing drugs used in multi-drug resistant (MDR) tuberculosis

In collaboration with Prof. Gareth W Griffiths at the University of Oslo, Prof. Andrew Thompson from The University of Auckland, inhalable particles containing sutezolid, TBA-354 and Compound 32625 were prepared and evaluated against M. tuberculosis strain H37Rv in THP-1 derived macrophages. Pharmacokinetics following inhalation of micron-sized particles and intranasal instillation of nanoparticles of identical composition were established in Guinea pigs. Evaluation in a mouse model of tuberculosis is in progress at National JALMA Institute of Leprosy and Other Mycobacterial Diseases, Agra.

Transient transfection of the respiratory epithelium with gamma interferon to provide host-directed therapy in pulmonary tuberculosis

Pulmonary tuberculosis (PTB) is caused by intracellular pathogens like Mycobacterium tuberculosis (Mtb) and congeners in a fraction of the population that is infected by the bacteria. Nebulized gamma interferon (IFN-) has shown clinical efficacy against multiple drug resistant (MDR) PTB. Because IFN- protein is expensive, thermolabile and requires a cold chain for field deployment, it is not suitable for limited resource settings. We prepared a dry powder for inhalation (DPI) containing DNA constructs polyplexed with poly (ethyleneamine) (PEI) to permit transient transfection of the lung and airway epithelium with IFN- or with fluorescent proteins to enable imaging. The median diameter of the DPI particles by electron microscopy was 1.2 µm and the mass median aerodynamic diameter (MMAD) was 2.85±1.8 µm GSD. DNA incorporated in the particles remained intact. A549 cells exposed to particles showed transient expression of green fluorescent protein (GFP) from 6-24 h, after which the protein was observed to co-localize with lysosomes. Mice receiving inhalations were live imaged for red fluorescent protein and expression of IFN- in bronchio-alveolar lavage fluid. Both signals peaked at about 24h after inhalation and declined by about 36h. Particles inhaled by Mtb-infected mice bearing granulomatous lesions induced extensive autophagy. We concluded that the formulation provides controlled release of functional IFN- in a time-window suitable for investigation of preclinical efficacy and safety as a host-directed therapy of pulmonary TB.

Inhalable D-cycloserine and ethionamide to overcome pharmacokinetic interaction and impart bactericidal efficacy to drugs considered as bacteriostatic agents

Pharmacokinetics and efficacy of a combination of D-cycloserine (DCS) and ethionamide (ETO) via oral and inhalation routes were studied in mice. The plasma t1/2 of oral ETO at human-equivalent dose reduced from 4.63±0.61 h to 1.64±0.40 h when DCS was co-administered. AUC(0-t) reduced to one-third. Inhalation overcame the interaction, restoring primary pharmacokinetic parameters to expected values. Inhalation, but not oral doses reduced lung CFU/g of Mycobacterium tuberculosis H37Rv from 6 to 3 log10 in four weeks, indicating bactericidal activity of inhaled combination; whereas the two drugs are classified as bacteriostatic agents by the WHO.

Formulations and Targeted Delivery

Targeted co-delivery of the aldose reductase inhibitor epalrestat and chemotherapeutic doxorubicin via a redox-sensitive prodrug approach promotes synergistic tumor suppression

Rapidly growing evidence suggests a strong dependence of a polyol pathway enzyme Aldose Reductase (AR) in cancer progression and invasion. Thus, inhibiting the AR through therapeutic inhibitors has a potential application in cancer treatment. Epalrestat (EPR) is the only marketed AR inhibitor with proven safety and efficacy in the management of complications like diabetic neuropathy. However, its short half-life and highly hydrophobic nature restrict its use as an anticancer agent. In the present study, we first developed a redox-sensitive prodrug of EPR by conjugating Tocopherol Polyethylene Glycol Succinate (TPGS) which can form a self-assembled micellar prodrug (EPR-SS-TPPGS). Subsequently, to achieve synergistic chemotherapeutic efficacy Doxorubicin (Dox) was co-loaded into the EPR-SS-TPGS micelles where the system is disrupted in a tumor redox environment and co-delivers Dox and EPR in a ratiometric manner. We then employed TPGS conjugated vitamin-B6 as a targeting moiety and prepared the mixed micelles to facilitate VTC receptor-mediated uptake. The encapsulation of Dox and EPR with the developed prodrug approach showed significant synergies with increased intracellular accumulation and redox triggered release in MDA-MB-231 and 4T1 cell lines leading to superior cell cycle arrest, mitochondrial membrane potential, and apoptosis. Prolonged circulation half-life and tumor site bioavailability were achieved for both the drugs with the developed approach. Surprisingly, EPR and Dox combination significantly down-regulated the CD44 receptor expression which is the main contributing factor of tumor metastasis. Furthermore, in vivo evaluation demonstrated a significant reduction in Dox-induced cardiotoxicity. In summary, this nanoencapsulation paradigm of AR inhibitors with chemotherapeutic agents lays the foundation of new opportunities in combination chemotherapy. Biomater. Sci., 2019, 7, 2889

Multifunctional hybrid nanoconstructs facilitate intracellular localization of doxorubicin and genistein to enhance apoptotic and antiangiogenic efficacy in breast adenocarcinoma

The progressive development of tumors leading to angiogenesis marks the advancement of cancer which requires specific targeted treatment preferably with combination chemotherapy. However, there is still a long way to go to develop an efficient delivery system that could overcome the tumor microenvironment to achieve efficient delivery. Herein, we have developed spermine (SPM) tethered lipo-polymeric hybrid nanoconstructs with two tier strategies, cell surface heparan sulfate proteoglycans (HSPG) specificity for higher intracellular localization and pH dependent charge reversal in the tumor microenvironment (below pH 5.8) to facilitate Doxorubicin (Dox) and Genistein (Gen) release in a synergistic combination. We have observed the specific uptake of SPM anchored hybrid nanoconstructs by receptor-mediated endocytosis in human breast cancer cells (MDA-MB-231) through the HSPG receptor. The SPM-D + G/NPs induced a higher rate of apoptosis in MDA-MB-231 cells via disruption of the mitochondrial membrane potential and also exhibited a stronger anti-angiogenic effect governing the inhibition of VEGF pathway modulation, proliferation, invasion and migration of HUVECs in in vitro and in vivo Balb/c mouse models. The involvement of Akt/Hif1α/VEGF dependent signal cascading and its down-regulation with a pro-apoptotic drug Dox and an anti-angiogenic agent Gen was evident as demonstrated by an in silico docking study and subsequently proven by RT-PCR and western blotting. Altogether this study highlights the potential role of SPM in targeting HSPG receptors and synergistic delivery of Dox and Gen as a promising strategy to effectively inhibit BAC progression and these findings could open a new window to deliver combinations of chemotherapeutic agents along with anti-angiogenic ligands using hybrid nanoparticles. Biomaterials Science, 2020, DOI: 10.1039/C9BM01246J

Induction of mitochondrial cell death and reversal of anti-cancer drug resistance mediated via nanocarrier composed of triphenylphosphonium derivative of TPGS

We have devised a nanocarrier using “tocopheryl polyethylene glycol succinate (TPGS) conjugated to triphenylphosphonium cation” (TPP-TPGS) for improving the efficacy of doxorubicin hydrochloride (DOX). Triphenylphosphonium cation (TPP) has affinity for an elevated transmembrane potential gradient (mitochondrial), which is usually high in cancer cells. Consequently, when tested in molecular docking and cytotoxicity assays, TPP-TPGS, owing to its structural similarity to mitochondrially directed anticancer compounds of the “tocopheryl succinate” family, interferes specifically in mitochondrial CII enzyme activity, increases intracellular oxidative stress, and induces apoptosis in breast cancer cells. DOX loaded nanocarrier (DTPP-TPGS) constructed using TPP-TPGS was positively charged, spherical in shape, sized below 100 nm, and had its drug content distributed evenly. DTPP-TPGS offers greater intracellular drug delivery due to its rapid endocytosis and subsequent endosomal escape. DTPP-TPGS also efficiently inhibits efflux transporter P glycoprotein (PgP), which, along with greater cell uptake and inherent cytotoxic activity of the construction material (TPP-TPGS), cumulatively results in 3-fold increment in anticancer activity of DOX in resistant breast cancer cells as well as greater induction of necroapoptosis and arrest in all phases of the cell cycle. DTPP-TPGS after intravenous administration in Balb/C mice with breast cancer accumulates preferentially in tumor tissue, which produces significantly greater antitumor activity when compared to DOX solution. [Molecular Pharmaceutics, 2019, 16, 3744]

Analytical method development and validation utilizing QBD approach for oral sustained-release formulation of Fenofibrate intended to control alcohol –induced dose dumping

The peformulation and formulation development studies have been performed, to develop oral sustained-release tablet formulation of fenofibrate intended for controlling alcohol induced dose dumping. Further, a robust analytical method was developed for estimation of fenofibrate deliberated for content uniformity test, blend uniformity test, multimedia in-vitro studies, and dose dumping studies. The analytical method development has been done utilizing a novel and rational quality by design approach.

Development of self emulsifying delivery system bearing Moringa oleifera extract

This particular work involves formulation of self emulsifying drug delivery system intended for oral delivery of extract of Moringa oleifera. The formulation involves selection of surfactant, co-surfactant and oil phase in accordance with solubility study of extract in different vehicles involving FDA approved oil, surfactant, and co-surfactant. The preformulation studies suggested compatible sets of excipients that can best accommodate the herbal extract providing a large window for dose adjustment. The compatibility study was conducted in 14 different ratio of oil and surfactant-cosurfactant mixture. The selected excipients were subjected for various formulation trials to get most favouring formulation on the basis of particle size, polydispersity index, emulsification volume & time in simulated gastric fluid, simulated intestinal fluid and water. The formulation was rationally optimised using quality by design approach i.e. involving response surface methodology using Box–Behnken design. After development, formulations were characterized for various physicochemical and pharmaceutical attributes.

▶ Analytical and bioanalytical method development, quality assurance and stability studies on drug substances and drug products;
▶ Preclinical pharmacokinetics and metabolism of synthetic compounds and natural products in rodents, small animals and monkeys;
▶ Bioanalysis and pharmacokinetic modelling for clinical pharmacokinetics and metabolism including bioequivalence and bioavailability studies for generic medicines;
▶ Pre-formulation, ‘Quality by Design’ (QbD) formulation and process development for conventional and novel drug substances;
▶ Safety pharmacology of CSIR-CDRI candidate drugs under certified “good lab practices” (GLP);
▶ Preclinical toxicology and toxicokinetics of candidate drugs as per international guidelines under GLP;
▶ Preparation of dossiers on new candidate drugs for regulatory filings;
▶ Protocol design, trial monitoring and coordination for Phase I to Phase IV clinical trials;
▶ Generation of information on mechanism of action, toxicity and metabolism of drugs and deployment of alternative model systems for assessing the efficacy of new chemical entities;
▶ Development of better drug delivery systems for CSIR-CDRI candidate drugs as well as known drugs;

Pharmacokinetic studies

Bioanalytical method development and intravenous pharmacokinetic study of S016-1271 in swiss albino mice

S016-1271 (LR8P) is a broad spectrum novel cationic antimicrobial peptide. Bioanalytical method of S016-1271 in mice was developed, in order to uncover its pharmacokinetic aspects. The chromatographic separation of S016-1271 (FR8P as internal standard) was achieved on a Waters™ X select CSH-C18 column (75 x 3.0 mm, 2.5 µ) using mixture of acetonitrile and triple distilled water (TDW) both containing 0.05% formic acid as mobile phase. A seven-minute linear gradient method was designed to separate analytes from ion suppression at a flow rate of 0.3 mL/min. The extraction of analytes from mice was performed through solid phase extraction technique using mixed mode weak cation exchange cartridge (Thermo SOLA WCX 10 mg 1CC) with an extraction recovery of analytes about 75%. Mass spectrometric detection of S016-1271 and FR8P was performed with optimized multiple reaction monitoring (MRM) transitions (Q1/Q3) at 658.8 [M+3H] 3+ / 653.2 [M+3H-NH3] 3+ and 443.4 [M+5H]5+ /434.7 [y12-NH3]4+, respectively in positive electrospray ionization (ESI) mode. The linearity in mice plasma was established over a concentration range of 7.81 to 250 ng/mL with regression coefficient (r2 > 0.99). The currently developed method was validated as per US-FDA guidelines and found to be within the acceptable limits. The method was successfully applied to intravenous (IV) pharmacokinetic study in mice wherein the levels were detected upto 24 hrs. The peptide demonstrated poor distribution characteristics as the volume of distribution at steady state was less than total body water of mice. The clearance of the peptide predominantly occurred through central compartment (central clearance is 25 fold greater than peripheral clearance). Also, the in vitro pharmacokinetic studies demonstrated the stability of S016-1271 in plasma and high plasma protein binding in mice and humans.

Oral Pharmacokinetic study of enrich fraction N-012-0001

Pharmacokientic study of biomarker of Terminalia Chebula was carried out by estimation of chebulinic acid in systemic circulation following oral administration at 100 mg/kg. A selective and sensitive bioanalytical method was developed and validated for estimation of chebulinic acid in plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument. Healthy, male SD rats, weighing 220±20 g were obtained from National Laboratory Animal Facility, CSIR-CDRI and animal study was conducted as per the guidelines of Institutional Animal Ethical Committee. Animals were housed in hygienic conditions under controlled temperature (23-25 οC) and humidity (50-70%) conditions with 12/12 h light/dark cycles for one week prior to the start of experiments. A standard chow diet was given to animals with free access to water. Oral pharmacokinetic studies of enriched extract was carried out at dose of 100 mg/kg body weight. The chebulinic acid content in enriched fraction was 59% w/w. Oral formulation of enriched extract was prepared in 0.5% w/v methyl cellulose suspension. Following oral administration at 100 mg/kg, blood samples were collected at 0.25, 0.5, 1, 2, 3, 4, 6,8, 10, 12, 18 and 24 hr. Plasma was separated and chebulinic acid content was analysed using LC-MS/MS.

Pharmacokinetic studies of antidiabetic compound S009-0629

S009-0629 [methyl-8-(methylthio)-2-phenyl-6-p-tolyl-4,5-dihydro-2H-benzo[e]indazole-9-carboxylate] is a novel antidiabetic agent with PTP1B inhibitory activity. In this study, we have investigated the in vitro metabolic stability, plasma protein binding, blood partitioning and oral pharmacokinetic study of S009-0629 in rats. The plasma protein binding, blood partitioning and metabolic stability were determined by HPLC method. The oral pharmacokinetic study was analyzed by liquid chromatography coupled mass spectrometry (LC-MS/MS) method. The plasma protein binding of S009-0629 using modified charcoal adsorption method at 5 and 10 µg/mL was 80.58±1.04% and 81.95±1.15%, respectively. The KRBC/PL of S009-0629 was independent of concentration and time. The in vitro half-life of S009-0629 at 5 and 10 µM using rat liver microsomes was determined as 273±24.46 and 281.67±26.53 min, respectively. After oral administration, S009-0629 exhibited Cmax 55.51±1.18 ng/mL at 18 h (tmax). S009-0629 was found to have the large apparent volume of distribution (1894.93±363.67 L/kg). Oral in vivo t1/2 of S009-0629 was found to be 41.23±5.96 h. A rapid and highly sensitive LC-MS/MS method was validated for S009-0629 in rat plasma. S009-0629 has high plasma protein binding and low hepatic extraction. S009-0629 has no affinity with human P-gp and BCRP in ATPase assay. After oral dosing, S009-0629 has slow absorption and elimination in rats.

Trans-Resveratrol formulation for Oral Administration

Trans-resveratrol (t-RES) a natural polyphenolic compound, with extensive therapeutic activities however its clinical application is circumscribed due to its poor solubility and low bioavailability. The purpose of this study was to prepare stable t-RES nanocrystals (t-RES-NCs) with different stabilizers to improve its oral bioavailability. t-RES-NCs were fabricated by probe sonication method and optimized by particles size, poly dispersive index and zeta potential. The pharmaceutical characterization of t-RES-NCs was further performed systematically. The in-vitro cellular efficacy and in-vivo pharmacokinetics of t-RES-NCs were also evaluated. The optimized NCs were successfully accomplished a submicron particle size (110.28±12.55 nm) with high ζ-potential (-32.96±3.85 mV) value. Scanning electron microscopy (SEM) image indicated that morphology of t-RES-NCs was regular and rod like in shape. Meanwhile, the result of in-vitro cellular efficacy against MDA-MB-231 cells revealed that developed t-RES-NCs were more efficacious and potent (p<0.05) than plain t-RES. Compared to plain t-RES, t-RES-NCs exhibited significant higher AUC0–t (3.5 folds), and Cmax (2.2 folds), demonstrating improved oral bioavailability of t-RES after grafting as NCs (p<0.05). The significantly increased oral bioavailability of developed t-RES-NCs represents an ideal vehicle for oral delivery of t-RES.

Pharmacokinetics of novel DNA ligase I inhibitor S012-1332

S012-1332 is the first DNA ligase I inhibitor that demonstrated in vivo anti-breast cancer activity. The present study aimed to assess the in vivo pharmacokinetics of S012-1332 in rats and correlated them with in vitro findings. A sensitive and selective liquid chromatography-tandem mass spectrometry bioanalytical method was developed and validated for quantitation of S012-1332. Following oral administration, the absolute bioavailability was 7.04%. The absorption was prolonged which can be explained by low solubility in simulated gastric fluid and several folds higher solubility in simulated intestinal fluid. The effective permeability across the intestinal membrane in in-situ single pass perfusion study for S012-1332 was 5.58 ± 1.83 * 10-5 cm/sec compared to 5.99 ± 0.65 * 10-5 cm/sec for carbamazepine, with no significant difference, indicating S012-1332 has high permeability. It was rapidly partitioning into plasma in blood, where it was stable. Plasma protein binding was moderate which may have attributed to the rapid distribution out of the vascular compartment. The pharmacokinetics of S012-1332 was characterized by extensive clearance as seen with rat liver and intestinal microsomes. In vitro results elucidate the in vivo pharmacokinetic data. These findings provide crucial information for further development of S012-1332 as anti-breast cancer agent.

Toxicity studies

Attenuation of Diethylnitrosamine (DEN) induced hepatocarcinogesis by the enriched fraction of Kalmegh, Ratanjot and Parijat in experimental rat model

Hepatic cancer is well known, and leading cancer around the world and remain asymptomatic. The enriched fraction of Kalmegh, Ratanjot and Parijat possess the anti-proliferative, hepatoprotective property and used in the treatment of hepatic cancer. The current study deals to evaluate the chemoprotective and therapeutic property of the enriched fraction against diethylnitrosamine (DEN)-induced hepatic cancer. For the study, wistar rats were divided into four groups and hepatic cancer was induced with DEN. An oral dose of enriched fraction at a dose of 100 mg/kg body weight was given for 15 weeks. Histological evaluation, serum biochemistry, haematological profile and western blotting for VEGF were assessed to assess the therapeutic potential of the enriched fraction. Following are the results of the study: Nodules count-The gross examination of the liver showed 70% reduction of nodule count in the treated group as compared to the DEN group. VEGF Expression-The enriched fraction down-regulated the expression of VEGF (approximately 30%) in western blot analysis. Hepatic Serum enzymes - The level of hepatic serum enzymes were observed to be reduced in the treatment group. ALT (Alanine aminotransferase) was found to be reduced up to 35% while AST (Aspartate aminotransferase) was reduced up to 30% as compared to DEN group. Non-hepatic parameters- Parameters such as TLC, haemoglobin were elevated (76%), RBC (73%) in the enriched fraction group compared to DEN group. Masson's staining of liver showed that fibrosis level was reduced in the treatment group and lipids content was considerably low as indicated in oil red O staining with respect to the DEN group. Histopathological features also showed recovery of hepatic architecture in cancer-induced rats.

Regulatory pharmacology and toxicology GLP studies

All safety pharmacology and toxicology studies of CDRI compound S007-1500 is completed. The compound is found safe.

CNS safety studies on anti-leishmanial drug candidate 96/261:

The CNS safety of anti-leishmanial candidate drug 96/261 on was evaluated in Swiss albino mice as per “Schedule Y” guidelines. No adverse effect of this candidate drug on gross behavior and sensory systems at any tested doses (250, 500, 1000 and 2000 mg/kg) was observed. However, we observed that this compound induces hypothermia, decrease in locomotor response and attenuates neuromuscular coordination at highest dose 2000 gm/kg.

Single dose toxicity of 96/261 in Swiss mice and rat by oral route is completed. Based on acute studies ten day dose finding studies was done in rat. The data is being analysed.

90 day toxicity study of standardised fraction of Cassia occidentalis was conducted with 2.5 g, 1.25 g and 6.5 g/kg and the results are being analysed. No drug related adverse effects were seen in gross observation, biochemical parameters and haematology parameters. Histological data is being analysed.

Safety studies of compound S011-1793 has been completed.

Besides our own drug candidates, we also completed AMES assay of one candidate phytopharmaceutical from Murraya koenigii received from CSIR-IICB, Kolkata and a 28 days repeat dose toxicity study of a preparation from DIBER has also been conducted.

Clinical Trials

Phase 1 clinical trial permission by Drugs Controller General of India for antiplatelet molecule S007-867 has been granted.