Basic Medical Sciences
Research in basic medical sciences is supported by the Council in all its permanent institutes/centres as well as through extramural funding to various research institutions, medical colleges, universities etc. The results of studies carried out in the area of pathology (at the Councilís Institute of Pathology, New Delhi), pharmacology, biochemistry, molecular biology and toxicology are given below.
To study the regulation of tumour suppressor protein p53 by estrogen and antiestrogens in breast cancer cells, estrogen receptor positive breast cancer cells were cultured in media containing 5% foetal calf serum and the cell proliferation and level of p53 expression were studied. Exogenous addition of 17b-estradiol (E2) in the medium had no significant effect on either cell proliferation or p53 expression. However, a significant decrease in cell number and p53 expression was found on addition of tamoxifen in the media. On culture of these cells in growth media devoid of serum but containing hydrocortisone, cholera toxin, sodium, insulin and ascorbic acid, cell number and p53 levels declined but the addition of 17b-estradiol to the medium increased cell proliferation and p53 expression.
In the project on genetics of breast cancer in the Indian population, preliminary work done on mutation analysis of BRCA1 and BRCA2 breast cancer susceptibility genes showed presence of mutations in both the genes in approximately 20% Indian breast cancer patients. Characterization of the mutation spectrum is being further pursued.
Study of oncogenes, tumour suppressor gene and growth factor status in premalignant and malignant prostate enlargement was carried out at the IOP, New Delhi. Forty patients with prostate enlargement due to adenocarcinoma (20), prostatic intraepithelial neoplasia (PIN) (10) and benign hyperplasia (10), studied for expression of tumour suppressor protein p53 and ras onco-protein p21 showed that the lower grades of prostate cancer were associated with higher p53 nuclear reactivity as compared to p21 positive patients who showed inverse correlation. Out of the 10 patients of PIN, p53 positivity was found in 40% while none of these patients showed positive p21 reaction.
Indian Childhood Cirrhosis
In continuing studies on the experimental model of Indian childhood cirrhosis (ICC) three of the four mice kept on herbal diet with garlic and borax showed evidence of mild to moderate fibrosis in liver tissue. Mice kept on garlic and hing (Ferula asafoetida) showed moderate degree of definite hepatic fibrosis.
Immunohistochemical localisation of metal-lothionein by ABC (avidin biotin complex) method on liver biopsy sections from patients of clinical ICC and from animals in ongoing studies on an experimental model of ICC showed positive results in all the cases tested so far. Further studies are in progress.
A guinea pig model for vitiligo to evaluate the therapeutic potential of new treatment modalities has been developed at IOP, New Delhi. A vitiligo like depigmented patch was produced on the dorsal aspect of black/brown ear lobe of coloured guinea pig by the application of a cream of 4-hydroxy anisole once a day for about two and half months. The ear lobes were divided into matched pairs of equal extent of depigmentation.
The repigmentation completion status at the end of 11 weeks of photochemotherapy revealed that 68.75% (11/16) of psoralen ultra violet A (PUVA) treated ear lobes were completely repigmented as against 25% (4/16) in control group (Fig.18).
The depigmentation produced experimentally in this study not only resembled human vitiligo both histologically and clinically but also responded to a comparatively successful and more popular photo-chemotherapeutic modality, PUVA suggesting that the guinea pig model could be a very useful tool for screening the efficacy of unknown/unscreened medications for vitiligo.
A study was supported at the Industrial Toxicology Research Centre, Lucknow on the toxicological and immunotoxicological potential of commonly used plastics and the identification of leachates. Fifty eight brands of plastic biomedical (40 samples) and food packaging (18 samples) products collected from different major cities of UP were evaluated. Of these six brands of intravenous sets showing temperature dependent global migration above the permissible limit, were evaluated for detailed in vitro and in vivo studies. The main parameters studied were neurobehavioural alterations, changes in clinical biochemistry, immunoglobulin profile and histopathology. Migration of Zn from all the six brands of i.v. sets was observed above the permissible limits in simulatory solvents viz. sodium carbonate (5%), acetic acid (3%) and ethyl alcohol (8%). The migration of Mn and Cd was above the permissible limits only in simulating solvent sodium chloride (0.9%) and sodium carbonate (5%). Subchronic oral toxicity studies with two brands of i.v. sets containing Mn beyond the permissible levels, revealed significant effect on some of the parameters of the immune system, especially with higher doses (0.8ml/kg b.wt./day). Out of 58 samples, six samples of i.v. sets (i.e. 10% of the sample) did not meet the requirement of physico-chemical parameters. Of the six samples subjected to detailed toxicological investigations, only two did not meet the safety requirements. Hence, there is a need of proper checking to ensure that the prescribed standards are being met by the manufacturers.
A study was undertaken at the Central Drug Research Institute (CDRI), Lucknow to design and synthesize novel oligopeptides as antiasthmatic/antiallergic agents. Synthesis of all the peptides and various amino acid derivatives was done using standard procedures. The peptides were tested for anti-PCA (passive cutaneous anaphylaxis) mast cell stabilising and bronchodilator activity in male rats. A convenient route for the solid phase synthesis of IgE related hexapeptide having a troublesome sequence Asp-Ser and Asp-Gly was standardised and an Indian patent filed. Thirty three new congeners of IgE-Fc hexapeptide were synthesized; 96/199 was identified as the most potent compound exhibiting high order of antiallergic activity and was found to be more potent than at least the clinically used drug disodium chromoglycate in experimental animals. A suitable formulation of 96/199 for administration by the nasal route has been prepared and evaluated.
At the Jamia Hamdard, New Delhi, a study was undertaken to investigate the neuropsychopharmacological properties of metallic preparations used in the Indian systems of medicine and study the rationale of their therapeutic action so as to establish their LD50/minimum therapeutic dose and therapeutic index. Calcined preparations of gold and silver used in Ayurveda (swarna bhasma, raupya bhasma) and Unani Tibb (Kushta Tila Kalan, Kusha Nugra), thin silver leaves used in India on sweets and betel and on tonic pills (chandi warf), and oral gold preparation (Auranofin) used in modern medicine were subjected to a battery of >30 screening tests for general neuropsychopharmacological effects, cognitive functions, anti-depressant, anxiolytic, neuroleptic and serenic activities as also for the effects on endurance and fatigue. Both gold and silver preparations showed moderate to marked analgesic effects which appear to be mediated through opioidergic mechanisms. Other interesting effects included reduction of haloperidol-induced catalepsy in rats (in both gold and silver preparations), nootropic effects in rats and mice against both active and passive avoidance models, anxiolytic effects observed by Vogelís conflict test and elevated plus maze studies (in gold preparations) and antiaggressive action (in silver preparations). Some pro-convulsant effects were observed with silver preparations, which need further evaluation.
BIOCHEMISTRY AND CELL & MOLECULAR BIOLOGY
A project aiming at elucidating the biochemical mechanisms involved in Ca2+ release from mitochondria of the bovine pulmonary vascular smooth muscle tissue under oxidant stimulation has been completed at the University of Kalyani, Kalyani. The results indicated that H2O2 mediated stimulation of lipid peroxidation in mitochondria of bovine pulmonary vascular smooth muscle occurs predominantly via hydroxyl ion (OH). Iron release caused by H2O2 from the mitochondria plays an important role in this process. The reactive OH is generated, at least partly, extra-mitochondrially and the anion channels appear to play an important role in mediating the response produced by H2O2 on lipid peroxidation in the mitochondria. H2O2 induced Ca2+ efflux from bovine pulmonary artery smooth muscle mitochondria does not occur primarily via non-specific permeability alteration of the mitochondrial inner membrane, instead it appears to occur via a Ca2+ selective, oxidant dependent release pathway(s). OH plays an important role in H2O2 induced stimulation of Ca2+ release. Treatment of bovine pulmonary artery smooth muscle mitochondria with H2O2 stimulated oxidation of GSH and NAD(P)H alongwith an increase in Ca2+ release. Addition of oxaloacetate to mitochondrial suspension stimulated Ca2+ release and oxidation of NAD(P)H while GSH level remained unchanged. Subsequently, addition of b-hydroxybutyrate which reduced mitochondrial pyridine nucleotides caused reuptake of the released Ca2+ without causing appreciable alteration of GSH level. These results indicate that the redox state of pyridine nucleotides, but not glutathione, regulates Ca2+ release from the pulmonary smooth muscle mitochondria under oxidant stimulation.
Elevated levels of lipoprotein (a) [Lp(a)] have been regarded as an independent risk factor for coronary, peripheral and cerebral atherosclerosis. The enormous inter-individual variation in the plasma concentration of Lp(a) is almost entirely controlled by the apolipoprotein (a) [apo(a)] gene locus on the chromosome 6q26-27. In a study conducted at the All India Institute of Medical Sciences(AIIMS), New Delhi 127 angiographically assessed male coronary artery disease (CAD) patients (aged 30-70 yr) and 133 age and sex matched controls were analysed. The apo (a) molecule exists in multiple, genetically determined isoforms, which differ from each other in the number of K-4 type-2 repeats. These variations may be responsible for different promoter activity and thereby variation in Lp(a) concentration. For analysis of the penta nucleotide repeat (PNR) polymorphism, PCR amplified products were loaded on 12% polyacrylamide gel for electrophoresis. Apo(a) K-4 size polymorphism was studied by SDS-agarose gel electrophoresis and immunoblotting. The important observations of the study are: PNR alleles 8 & 9, in general associated with high Lp(a) levels, were the wild ones whereas alleles 10 & 11 associated with low Lp(a) levels, were rare. 12 PNR allele was also detected and confirmed by DNA sequence analysis. A trend for high Lp(a) levels was observed in subjects with PNR sum<20 and low in those having PNR sum>20. The number of K-4 type 2 repeats varied between 12-50 in the subjects. The most frequent allele among the patients was 23K-4 repeats whereas in controls it was 32. An inverse correlation was found to exist between the size of K-4 repeats and serum Lp(a) levels. Subjects with small (<21 K-4 repeats) and intermediate allele (21-28 K-4 repeats) showed high levels of Lp(a) (>40mg/dl) when compared with the subjects with large allele (>28K-4 repeats). K-4 type 2 polymorphism has a profound effect on Lp(a) levels as compared to PNR polymorphism. Although an inverse correlation has been observed between the number of K-4 type 2 repeats and plasma levels of Lp(a), there were also exceptions to this finding.
The Council continued the centrally co-ordinated multicentric clinical trial on Vijaysar (Pterocarpus marsupium) for diabetes mellitus at the Madras Medical College, Chennai, Cuttack Medical College, Cuttack and Kottayam Medical College, Kottayam through the task force approach. Vijaysar has repeatedly shown promising results in newly diagnosed diabetic patients as evident from the data of the recently concluded 36 and 72 weeks study, a trend similar to that seen in the earlier 12 weeks study. In order to validate the findings in known uncontrolled diabetics, further multicentric trial has been initiated at the same centres. On account of the expected increase in demand for this plant drug, steps were taken for a dialogue between related Government agencies to conserve Vijaysar trees, prevent their misuse as timber and start their cultivation by using appropriate agro-technology.
For providing support to the Traditional Medicine (TRM) Programme, two Centres for Advanced Research were initiated, one for clinical pharmacology in traditional medicine at the Seth G.S. Medical College and KEM Hospital, Mumbai and the other for drug development from natural/plant sources at CDRI, Lucknow. For pre-clinical toxicological studies of new plant based drugs or chemical entities, studies are proposed to be undertaken at NIN, Hyderabad. The Central Biostatistical Monitoring Unit (CBMU) of the National Institute of Epidemiology (NIE) at Chennai earlier known as the Institute for Research in Medical Statistics (IRMS) continued to lend its support in designing the protocols and monitoring the multicentric trial.
As part of the expanded activities in traditional medicine, a closer interaction with the Department of Indian Systems of Medicine and Homeopathy (ISM & H) and the respective Councils was maintained. The National Institute of Pharmaceutical Education and Research (NIPER), Chandigarh has been identified for establishing a second Unit for Standardisation and Quality Control of Selected Herbal Remedies/Natural Products. For cultivation of selected medicinal plants and for agro-technology of endangered species of medicinal plants like Picrorrhiza kurroa, Institute of Himalayan Bioresource Technology, Palampur has been identified.
At University College of Medical Sciences, Delhi, four compounds with anti-diabetic activity have been isolated from Eugenia jambolana and for these Indian and international patents have been filed.
Standardisation, Quality Control and Formulations for Selected Natural Products/Herbal Drugs
The activities carried out in this area at the Advanced Centre at the Regional Research Laboratory (RRL), Jammu involved studies on microbial and heavy metal load of natural products or herbal drugs, characterisation of the active principles and isolation of biomarkers to facilitate preparation of monographs. Out of 13 markers isolated from Vijaysar, 6 have been reported. Two new markers VS-12 and VS-13 were isolated and the structure of VS-08 was revised. VS-08 and VS-12 possessed a very rare isoaurone skeleton. HPLC fingerprinting of the extract with quantitative estimation of the markers and their concentration ranges were established. In addition to six monthsí accelerated stability studies, the shelf life of Vijaysar capsules for 2 years at room temperature was also studied. VS-04 and VS-09 showed hypoglycaemic activity at 10mg/kg orally. General pharmacology showed no anti-inflammatory and antipyretic activities. The Centre continues to do further studies on the pharmacology of Vijaysar and preparation of formulations for the clinical trial.
Drug Development from Natural/Plant Products
Studies were carried out at the Advanced Centre at CDRI, Lucknow on Picroliv and plant products from Terminalia chebula and Centella asiatica. Picroliv is undergoing clinical trial as a hepatoprotective agent. Phase II multicentric trials have been completed and phase III trials are to be initiated. LC-MS fingerprinting of 7 compounds has been carried out besides identifying the two markers, picroside I and II. The HPLC method for estimating the active marker compound of T.chebula, a hepatoprotective plant and marker compounds asiaticoside (active) and madicassoside of C.asiatica, a wound healing plant drug was also standardised. Further work on the pharmacology of these products is being continued.
Clinical Pharmacology in Traditional Medicine
At the Advanced Centre at the Seth GS Medical College and KEM Hospital, Mumbai, controlled clinical trials are proposed to be undertaken on Azadirachta indica for wound healing and on medicated oral rehydration solution (containing in addition to the WHO recommended ORS, Zingiber officinalis, Cyperus rotundus and Cumminum cyminum) for diarrhoea. On the basis of fingerprinting, in-house standardisation of A.indica formulation for use in the study has been taken up. The Centre also proposes to study the mechanism of action of Vijaysar which has shown effective antidiabetic properties in various clinical trials undertaken.