Isolation And Characterisation Of Rapamycin, Temsirolimus Regio Isomer (MONOESTER) And Temsirolimus Diester In Temsirolimus Drug

Objective: Separation and identification of the process impurities in the manufacture of temsirolimus drug viz., rapamycin, temsirolimus regioisomer (monoester) (TS monoester), and temsirolimus diester (TS diester). Methods: During the process development of temsirolimus (TS), three process impurities-rapamycin, temsirolimus regioisomer (monoester) and temsirolimus diester-were detected by high-performance liquid chromatography (HPLC). Impurities were isolated by medium pressure liquid Chromatography (MPLC) and characterized by ESI-MS/MS, 1H NMR, FT-IR spectral data. Results: These impurities are characterised with the help of ESI MS/MS, 1H NMR, and FT-IR data. The impurities are identified and characterised as the process impurities. One of them is the starting material i.e. rapamycin and the other two are formed during the manufacture of the drug. This method offers advantages over using photodiode-array UV detection (LC-PDA) for the determination of peak purity, viz. components with similar UV spectra can be distinguished. Conclusion: The structures of these impurities were characterized as rapamycin, TS Monoester, and TS Diester. Out of these process impurities, rapamycin has been previously identified while the other two are previously unreported.

Pre-clinical toxicity & immunobiological evaluation of DNA rabies vaccine & combination rabies vaccine in rhesus monkeys (Macaca mulatta).

Background & objectives: Pre-clinical toxicology evaluation of biotechnology products is a challenge to the toxicologist. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed recombinant DNA anti-rabies vaccine [DRV (100 μg)] and combination rabies vaccine [CRV (100 μg DRV and 1.25 IU of cell culture-derived inactivated rabies virus vaccine)], which are intended for clinical use by intramuscular route in Rhesus monkeys. Methods: As per the regulatory requirements, the study was designed for acute (single dose - 14 days), sub-chronic (repeat dose - 28 days) and chronic (intended clinical dose - 120 days) toxicity tests using three dose levels, viz. therapeutic, average (2x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in monkeys. The selection of the model i.e. monkey was based on affinity and rapid higher antibody response during the efficacy studies. An attempt was made to evaluate all parameters which included physical, physiological, clinical, haematological and histopathological profiles of all target organs, as well as Tiers I, II, III immunotoxicity parameters. Results: In acute toxicity there was no mortality in spite of exposing the monkeys to 10XDRV. In sub chronic and chronic toxicity studies there were no abnormalities in physical, physiological, neurological, clinical parameters, after administration of test compound in intended and 10 times of clinical dosage schedule of DRV and CRV under the experimental conditions. Clinical chemistry, haematology, organ weights and histopathology studies were essentially unremarkable except the presence of residual DNA in femtogram level at site of injection in animal which received 10X DRV in chronic toxicity study. No Observational Adverse Effects Level (NOAEL) of DRV is 1000 ug/dose (10 times of therapeutic dose) if administered on 0, 4, 7, 14, 28th day. Interpretation & conclusions: The information generated by this study not only draws attention to the need for national and international regulatory agencies in formulating guidelines for pre-clinical safety evaluation of biotech products but also facilitates the development of biopharmaceuticals as safe potential therapeutic agents.

Isolation, speciation, and antibiogram of clinically relevant non-diphtherial Corynebacteria (Diphtheroids).

Purpose: Coryneform or the non-diphtherial Corynebacterium species largely remains a neglected group with the traditional consideration of these organisms as contaminants. This concept, however, is slowly changing in the light of recent observations. This study has been done to find out the species distribution and antibiogram of various members of the clinically relevant Coryneform group, isolated from various clinical materials. Materials and Methods: One hundred and fourteen non-duplicate isolates of diphtheroids from various clinical isolates were selected for the study. The isolates were identified to the species level by using a battery of tests; and antimicrobial susceptibility was tested by using a combination of Clinical and Laboratory Standards Institute (CLSI) and the British Society for Antimicrobial Chemotherapy (BSAC) guidelines, in the absence of definitive CLSI guidelines. Results: Corynebacterium amycolatum was the predominant species (35.9%) in our series followed by the CDC Group G organisms (15.7%). Each of the remaining 19 species comprised of less than 10% of the isolates. More than half the total isolates were resistant to the penicillins, erythromycin, and clindamycin; while excellent activity (all the strains being susceptible) was shown by vancomycin, linezolid, and tigecycline. Chloramphenicol and tetracycline also had good activity in inhibiting more than 80% of the isolates. Multiply drug resistance was exhibited by all the species. Conclusion: This study was an attempt to establish the clinical significance of coryneform organisms. The high level of resistance shown by this group to some of the common antibacterial agents highlights the importance of processing these isolates in select conditions to guide the clinicians towards an appropriate therapy.

Evaluation of rabies virus neutralizing antibody titres induced by intramuscular inoculation of rabies DNA vaccine in mice and Bonnet monkeys (Macaca radiata).

A rabies DNA vaccine consisting of plasmid DNA expressing the rabies virus surface glycoprotein was injected (im) twice at two week interval to outbred swiss mice or Bonnet monkeys (Macaca radiata) and the levels of rabies virus neutralizing antibody (VNA) titres were examined over a one year period. In mice, the VNA titre was maintained above the minimum protective level (0.5 I.U./ml) up to 10 months after primary immunization, while in monkeys, the titre dropped below the protective level by 6 months. An anamnestic B cell response was seen in both mice and monkeys following the administration of a booster dose, 10 and 6 months after the primary immunization, respectively. These results indicate that im injection of rabies DNA vaccine induces VNA in nonhuman primates and mice unlike intradermal (id) immunization, which was shown to induce VNA only in mice but not in monkeys. This is the first report on the induction of VNA in nonhuman primates by im inoculation of rabies DNA vaccine.

Nucleolar organizer regions in smooth muscle and breast tumours.

Paraffin sections of formalin fixed tissues, obtained from patients with smooth muscle and breast tumours, were studied by the silver staining technique to quantitate the Nucleolar Organizer Regions (AgNORs) per nucleus and to assess its significance as an independent variable in predicting the behaviour of these neoplasms. Five benign and five malignant tumours of smooth muscle along with ten benign and ten malignant epithelial tumours of breast were studied. Normal myometrium and breast tissue served as controls. Control, benign and malignant tumours of smooth muscle showed mean AgNOR scores of 2.68, 3.89 and 12.50 per nucleus respectively. Control, benign and malignant tumours of breast showed mean AgNOR scores of 1.75, 7.45 and 12.72 per nucleus respectively. These results suggest that quantitative analysis of AgNORs per nucleus is capable of differentiating benign from malignant lesions of smooth muscle and breast.

Control of bancroftian filariasis by diethylcarbamazine medicated common salt in Karaikal, Pondicherry, India.

A trial of diethylcarbamazine (DEC) mixed in crystal common salt at a concentration of 0.15 to 0.2 per cent was carried out for 46 months (1982 to 1986) in Karaikal town and five commune panchayats of Pondicherry for control of bancroftian filariasis. Comparison of pre and post trial surveys showed 97.6 per cent reduction of microfilaria rate. No microfilaria or disease case was found in less than 5 years age group in the post trial survey. There was about 72 per cent reduction of disease rate. In the post trial substantial reduction of disease manifestation was found in 40 years and below age groups. Vector Culex quinquefasciatus density, filaria infection and infectivity rates were monitored only in Karaikal town. Infective vector mosquitoes were found in 1982 and 1983 only but afterwards no infective mosquito was encountered.

Isolation and identification of Micrococcus roseus and Planococcus sp from Schirmacher oasis, Antarctica.

Five cultures isolated from soil samples collected in Schirmacher oasis, Antarctica, have been identified as members of the family Micrococcaceae, with 3 belonging to the genus Micrococcus and two to Planococcus. The 3 Micrococcus isolates (37R, 45R and 49R) were red-pigmented and h a d ~ 75 mol% G + C in their DNA; they were identified as Micrococcus roseus. The two Planococcus isolates (30Y and Lz3OR) were yellow and orange in colour, and had 43·5 and 40·9 mol % G + C in their DNA respectively; they were identified as Planococcus sp.