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, 28 th 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.

Effect of chlorination on the development of marine biofilms dominated by diatoms.

This study addressed the antifouling efficiency of commercially available chlorine at different concentrations (0.5%, 1%, and 2%) and exposure times (0.5 min, 1 min, 5 min, and 15 min). The rapid and non-destructive FIRe (fluorescence induction and relaxation) technique was used to evaluate the effects of the biocide on diatom dominated biofilms. The efficiency of chlorine in removing diatoms from the developed biofilms increased with an increase in concentration and exposure time. The fluorescence measurements revealed low F(v)/F(m) and high σ(PSII) values for chlorine-treated Navicula and Amphora biofilms indicating that chlorination was efficient in damaging the photosystem-II reaction centers. Chlorination also caused mortality of diatom cells by damaging the cell body. In natural biofilms, the biocidal effect of chlorine was species specific; species of Amphiphrora, Navicula, Cylindrotheca, and Coscinodiscus showed an increase in the density of the population, but species of Pleurosigma, Amphora, and Thalassionema did not increase in density after chlorine treatment. It was also demonstrated that diatoms can colonize, grow and photosynthesize on chlorine-treated surfaces. Under pulse chlorination (treatment every 6 h), irrespective of chlorine concentration, the development of biofouling decreased with an increase in exposure time. Differences between exposure times of 1 to 15 min were not significant. Additionally, transmission levels of the control (non-chlorine-treated) fouled coupons reduced significantly (∼20%) compared to the chlorine-treated fouled coupons (<2%). These results suggest that chlorine can be used as a biocide to control the development of diatom biofilms.

Lipid peroxidation and activities of antioxidant enzymes in iron deficiency and effect of carcinogen feeding.

Iron deficiency has been implicated in increasing the risk of GI tract cancers in humans. Among various mechanisms of carcinogenesis, oxidative damage to DNA is well known and, hence, the present experimental study was undertaken to investigate lipid peroxidation and activities of different antioxidant enzymes in iron deficiency to explain the higher risk of tumorigenesis. Two groups of male weanling Fischer rats maintained on iron sufficient (C) or iron deficient (D) diets for a period of 32 weeks were subdivided, from 3 weeks onwards, into two subgroups each. The carcinogen, dimethyl hydrazine was fed at a dose of 30 mg/kg/week IG for a period of 9 weeks to groups that were designated as (C+) and (D+). The other two subgroups (C-) and (D-) served as controls. After the experimental period, hepatic assays for lipid peroxidation (MDA production) and activities of various antioxidant enzymes were carried out. The results showed that MDA production was elevated by 50% and activity of superoxide dismutase significantly depressed in carcinogen-fed, iron-deficient group (D+) by 28% compared to deficient (D-) group. There was an increase in hepatic selenium-dependent glutathione peroxidase activity in iron-deficient and iron-deficient, carcinogen-treated groups to the extent of 57 and 59%, respectively, as compared to controls; however, induction of enzyme in response to carcinogen feeding, observed in the control group, was not evident in iron deficiency. Liver catalase was not altered between control and deficient groups. These results suggest that prolonged iron deficiency superimposed with carcinogen ingestion may render the host susceptible to a greater risk of tumorigenesis through oxidative stress.

Functional significance of growth retardation in malnutrition.

Various functional parameters involved in resistance to infection were investigated in children suffering from varying grades of protein-calorie malnutrition. It was observed that the phagocytic function was impaired in children whose weights were below 80% of the Indian Council of Medical Research standard, whereas the cell-mediated immune response was altered in those with weights below 70% of the standard. Antibody response to typhoid antigen was impaired in children with severe protein-calorie malnutrition, while the response to diphtheria and tetanus toxoids was normal in all. These observations suggest that malnourished children whose weights are below 80% of the Indian standard are likely to suffer from at least one functional handicap which may increase the risk of infection. In any action-oriented program, priority should, therefore, be given to this group of children.

Interrelationship between vitamins E and A: a clinical study.

Plasma vitamin A and vitamin E levels were determined in 45 children. Seven normal children and 7 children with vitamin A deficiency were given daily supplements of 100 mg vitamin E for two weeks. Seven others received a placebo and served as controls. The mean levels of plasma vitamin E and A were 694 microgram/dl and 21 microgram/dl respectively. There was no correlation between plasma levels of the two vitamins. Administration of vitamin E resulted in a significant increase in plasma vitamin A concentration both in normal children and in those with vitamin A deficiency, while there was no change in the control group.

Effect of food restriction on benzo(a)pyrene binding to DNA in Wistar rats.

A study was carried out to assess the binding of the carcinogen benzo[a]pyrene to DNA in different tissues under in vivo and in vitro conditions in Wistar rats which have been subjected to different levels of food restriction. The results showed that there was a significant increase in the binding of benzo[a]pyrene to hepatic DNA in food restricted animals in in vivo experimentation although this was not observed under in vitro conditions. There was a decrease in binding to pulmonary DNA and no change for renal DNA.

Immune studies in oral contraceptive users.

Some of the parameters of immune status-percentage of B and T lymphocyte subpopulation, PHA-induced lymphocyte transformation measured by 3H-thymidine incorporation (PILT) and levels of total haemolytic complement (CH5O) were studied in Indian women from low income group who were using estrogen-progestogen combination pills. There were no differences in percentage of B and T lymphocyte subpopulation or PILT between OC users and the control group. However, CH5O levels were significantly lower in OC users. The depression in circulating complement levels in OC users is intriguing and suggests the possibility that complement system and circulating immune complexes may be altered in OC users.

PIP: Studies have shown that estrogens and progestogens can cause immuno-depression. Oral contraceptives (OCs) have also been linked with viral and urinary tract infections. In developing countries, women have higher risks of developing these infections because of undernutrition and anemia. This study evaluates cell-mediated immunity (CMI) and one parameter of hormonal immunity--serum hemolytic complement--in undernourished Indian women using OCs. The study population consisted of a random sample of urban women of low income status, between 20 and 30 years of age, mean weight of 45.2 kg. and mean height of 151.2 cm., and mean hemoglobin level of 13.2 g/dl (none had hemoglobin levels below 8 g/dl). 52 of the women were taking OCs (150/ug. of d. norgestrol and either 30 or 50/ug. ethinyl estradiol). The T and B lymphocytes, hemolytic complement in serum (CH50), PHA-induced lymphocyte transformation (PILT) were measured. Results revealed a wide scatter of values in all 4 parameters--% of T cells; % of B cells; 3H-thymidine incorporation, and CH50 levels even in the control group. No significant differences in % of B and T lymphocytes subpopulation or PILT were observed between OC users and the control group. CH50 levels were significantly lower, however, in OC users. Generally, clinical experience of pill users suggests that there is no need for undue concern for potential immune-associated problems in OC users.

Effects of riboflavin deficiency and riboflavin administration on carcinogen-DNA binding.

A study was conducted to assess the effects of riboflavin deficiency and riboflavin supplementation on carcinogen-DNA binding. After 12 wk on a riboflavin-sufficient or a riboflavin-deficient diet male Wistar rats were administered 3H-labelled benzo[a]pyrene (BP) ip. [3H]BP was given either at a uniform dose of 450 muCi/rat irrespective of body weight or at a dose adjusted to body weight. After 17 hr the animals were killed, various organs were dissected and the level of [3H]BP bound to DNA was quantified in organs that are known to be the seats of drug metabolism (i.e. the liver, lungs and intestinal mucosa). In a separate experiment, the effect of riboflavin supplementation on BP-DNA binding was also investigated. When [3H]BP was administered at 450 microCi/rat, BP-DNA binding was markedly increased in the livers and intestinal mucosae of the pair-fed and deficient groups compared with controls. With the administration of [3H]BP adjusted to body weight, no differences in BP-DNA binding between groups were observed in any tissue. However, on administration of riboflavin there was a decrease in the level of [3H]BP bound to DNA in almost all tissues, especially in the lungs, where the reduction was significant. The results suggest that undernutrition/riboflavin deficiency may increase the risk of carcinogenesis by way of an increase in carcinogen binding, which however can be reversed by riboflavin supplementation.

Immune studies with T-2 toxin: effect of feeding and withdrawal in monkeys.

Ingestion of T-2 toxin, a product of Fusarium fungi, has been reported to have a variety of effects leading to morbidity and mortality in animals and humans. Semi-purified T-2 toxin was given to monkeys by gastric intubation at a level of 100 microgram/kg body weight/day for 4-5 wk and the haematological and immune parameters were studied before and after the treatment. Leucocyte counts were depressed at the end of wk 4 of treatment. The immunological studies studies showed suppression of the bactericidal activity of neutrophils, of cell-mediated immune status as assessed by T-cell number and lymphocyte transformation, and of humoral immunity as reflected in B-cell number and IgG and IgM levels. However serum complement (CH50) did not show any change. Investigations carried out 5 months after withdrawal of the toxin indicated that these parameters had returned almost to the initial, pretreatment levels. These data suggest that the greater incidence of infection seen in mycotoxin-ingesting animals may be due to immune suppression. Withdrawal of the mycotoxin results in improvement of haematological and immune functions.

Effect of long term iron deficiency on the activities of hepatic and extra-hepatic drug metabolising enzymes in Fischer rats.

Male Fischer rats were maintained for a period of 17 weeks on an iron-deficient diet along with suitable controls. The effect of long term deprivation of iron on xenobiotic metabolism was studied by the activities of various drug metabolising enzymes in both liver as well as extra-hepatic tissues like lungs, kidneys and intestinal mucosa (I.M.). The results show that among the Phase I (activating) enzymes, the hepatic activities of benzo(a)pyrene hydroxylase (AHH) and microsomal epoxide hydrolase (mEH) are significantly reduced in iron deficiency. The other parameters of the activating system, namely cytochrome P450, aminopyrene demethylase (ADM) and aniline hydroxylase (AH), are not altered. Of the two Phase II (conjugating) enzymes studied, only uridine diphospho glucuronyl transferase (UDPGT) is found to be depressed, but not glutathione S-transferase (GST) in liver in iron deficiency. Activities of Phase I enzymes are markedly lowered in extra-hepatic tissues compared to liver; such depression is not observed in conjugating enzymes. Iron deficiency does not seem to make much impact on the enzyme activities of extra-hepatic tissues. Overall, the hepatic results suggest a defect in detoxification mechanisms in iron deficiency. Such impairment may very well predispose an iron-deficient host to an increased risk of carcinogenesis.

Serum complement levels in normal pregnancy and pregnancy-induced hypertension.

Serum complement assay (CH50) was carried out in urban low-income women belonging to the following groups: (i) non-pregnant and non-lactating women; (ii) pregnant women in different periods of gestation; (iii) women suffering from pregnancy-induced hypertension. Serum CH50 titers showed significant increase in the second and third trimester pregnancies as compared to non-pregnant, non-lactating women. There were no differences in CH50 levels between women suffering from pregnancy-induced hypertension and those with normal pregnancy of comparable period of gestation. Nutritional status did not seem to have any influence on complement titers.

Study of activating and conjugating enzymes of drug metabolism in zinc deficiency.

A study was undertaken to investigate the activities of certain enzymes of drug metabolism in zinc deficiency. For this purpose, an experimental model for zinc deficiency was produced in a NIN/Wistar strain of rats by feeding an egg albumin-starch based diet. Of the two enzymes of Phase I pathway of drug metabolism studied, Benz (alpha) pyrene hydroxylase was altered in zinc deficiency and food restriction; the other one microsomal epoxide hydrolase was unchanged. The activity of glutathione-S-transferase, a key enzyme in conjugation reaction was significantly lowered in zinc deficiency as well as food restriction. These alterations in the activities of xenobiotic metabolising enzymes are discussed with reference to toxicity manifestation in zinc deficiency.

Nonclinical toxicology study of recombinant-plasmid DNA anti-rabies vaccines.

The absence of standard guidelines from National and International regulatory agencies for the safety evaluation of biotechnology products challenges the ingenuity of toxicologists. At present, the development of standard pre-clinical toxicology protocols for such products is on an individual case basis. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed DNA based anti-rabies vaccine in India. The test compounds were DNA rabies vaccine [DRV (100 microg)] and combination rabies vaccine (CRV (100 microg DRV and 1/50 dose of cell culture vaccine)), intended for clinical use by intramuscular route on 1, 7, 14 and 28 day. As per the regular mandatory requirements, the study has been designed to undertake acute (single dose--10 days), sub-chronic (repeat dose--28 days) and chronic (intended clinical dose--120 days) toxicity tests using three dose levels viz. therapeutic, average (2 x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in Swiss Albino mice. The selection of the rodent model viz. Swiss Albino mice is based on affinity and rapid higher antibody response during the efficacy studies. Apart from physical, physiological, clinical, hematological and histopathology profiles of all target organs, the tier-I immunotoxicity parameters have also been monitored. There were no observational adverse effects even at levels of 10x therapeutic dose administration of DRV and CRV. The procedure also emphasizes on the designing of protocols for the products developed by recombinant technique.

Effect of administration of Aroclor 1254 on the activities of hepatic drug metabolizing enzymes in zinc deficiency.

In order to understand the mechanisms of carcinogenesis in zinc deficiency, a study was conducted in experimental animals to investigate the effect of administration of an inducer. After the production of zinc deficiency in NIN/Wistar strain of rats by feeding an egg albumin-starch based diet almost devoid of zinc, the animals were administered a potent inducer of mixed-function oxidases: Aroclor 1254 and various Phase I and Phase II enzymes of drug metabolism like benzo[a]pyrene hydroxylase, microsomal epoxide hydrolase, cytosolic epoxide hydrolase, and cytosolic glutathione-S-transferase studied in liver tissues. Control and pair-fed groups were also run alongside. The results showed that while the activities of various enzymes studied were low in the uninduced basal condition, these activities increased many-fold after induction. This induction was observed not only in the control group, but in the pair-fed and deficient groups as well. These results suggest that the ability to respond to a carcinogenic insult, though initially present in zinc deficiency, may not be adequate to counteract an excess or chronic exposure to carcinogen in the long run.

Effects of dietary zinc deficiency on the activity of enzymes associated with phase I and II of drug metabolism in Fischer-344 rats: activities of drug metabolising enzymes in zinc deficiency.

A study was undertaken to assay the various phase I and phase II drug metabolising enzymes in zinc deficiency. Male weanling Fischer rats were subjected to zinc deficiency for a period of 7 weeks. Zinc levels in the control and deficient diets were 30 mg and 1.1 mg/kg diet, respectively. At the end of the experimental period, the activities of various hepatic cytosolic and microsomal enzymes were estimated. It was observed that the activities of microsomal epoxide hydrolase (with benz(a)pyrene 4-5 oxide as substrate), uridine diphospho glucuronyl transferase (with 1-naphthol as substrate) and cytosolic glutathione-S-transferase (with chlorodinitrobenzene as substrate) were altered exclusively due to zinc deficiency. There was a change in the activities of the following enzymes, which could be due either to zinc deficiency and/or food restriction: 1) aryl hydrocarbon hydroxylase; 2) cytochrome b; 3) cytochrome c; and 4) cytochrome b5. Other enzymes studied, i.e., cytosolic epoxide hydrolases, microsomal EHSTO, and UDPGT testosterone were not different in the control and experimental groups. The results are discussed in relation to the activation of carcinogens and neoplastic formation in zinc deficiency.

Plasma tocopherol and lipid levels in pregnancy and oral contraceptive users.

Plasma tocopherol (vitamin E), cholesterol and triglycerides were estimated to normal non pregnant women and in women throughout pregnancy. All three constituents were increased in the later stages of pregnancy. There was a significant statistical correlation between tocopherol and cholesterol levels, but not between the vitamin and triglycerides. The data suggest that the increase in tocopherol may be associated with a corresponding increase in cholesterol since both are known to share a common transport system, but the association was evident only during pregnancy. Compared to controls, plasma vitamin E levels were not altered in pre-eclampsia nor in oral contraceptive users.

PIP: 246 women (54 nonpregnant controls, 56 women up to 12 weeks pregnant, 65 women between 14 and 28 weeks gestation, and 71 women over 28 weeks pregnant), had their plasma tocopherol (vitamin E), cholesterol, and triglyceride levels estimated. All women were of lower socioeconomic class and were Indian. All 3 levels increased in later stages of pregnancy. Tocopherol and cholesterol levels showed statistically significant correlation, but triglycerides and vitamin E did not. In correlation analyses, using pooled data for all pregnant groups, vitamin E vs. cholesterol was .66 (P .001). Using the same pooled data, vitamin E vs. triglycerides was .42 (P .001). 22 controls and 22 oral contraceptive users had the same constituents tested and were compared. Vitamin E (in mcg/dl) was 939 for controls and 983 for users; cholesterol (mg/dl) was 183 for controls and 177 for users; and triglycerides was (mg/dl) 89 for controls and 107 for users. None of these differences was significant. The data suggest that the increase in tocopherol may be associated with a corresponding increase in cholesterol since both share a common transport system. However, the association was only evident during pregnancy, not in preeclampsia sufferers or in oral contraceptive users.