Effect of ethyl gallate and propyl gallate on dextran sulfate sodium (DSS)-induced ulcerative colitis in C57BL/6 J mice: preventive and protective.

OBJECTIVE AND DESIGN: Inflammatory bowel disease (IBD) is an idiopathic inflammatory condition of the digestive system marked by oxidative stress, leukocyte infiltration, and elevation of inflammatory mediators. In this study, we demonstrate the protective effect of ethyl gallate (EG), a phytochemical, and propyl gallate (PG), an anti-oxidant, given through normal drinking water (DW) and copper water (CW) in various combinations, which had a positive effect on the amelioration of DSS-induced ulcerative colitis in C57BL/6 J mice. MATERIALS AND METHODS: We successfully determined the levels of proinflammatory cytokines and anti-oxidant enzymes by ELISA, tracked oxidative/nitrosative stress (RO/NS) by in vivo imaging (IVIS) using L-012 chemiluminescent probe, disease activity index (DAI), and histopathological and morphometric analysis of colon in DSS-induced colitis in a model. RESULTS: The results revealed that oral administration of ethyl gallate and propyl gallate at a dose of 50 mg/kg considerably reduced the severity of colitis and improved both macroscopic and microscopic clinical symptoms. The level of proinflammatory cytokines (TNF-α, IL-6, IL-1ß, and IFN-γ) in colonic tissue was considerably reduced in the DSS + EG-treated and DSS + PG-treated groups, compared to the DSS alone-treated group. IVIS imaging of animals from the DSS + EG and DSS + PG-treated groups showed a highly significant decrease in RO/NS species relative to the DSS control group, with the exception of the DSS + PG/CW and DSS + EG + PG/CW-treated groups. We also observed lower levels of myeloperoxidase (MPO), nitric oxide (NO), and lipid peroxidation (LPO), and restored levels of GST and superoxide dismutase (SOD) in DSS + EG-DW/CW, DSS + PG/DW, and DSS + EG + PG/DW groups compared to DSS alone-treated group. In addition, we showed that the EG, PG, and EG + PG treatment significantly reduced the DAI score, and counteracted the body weight loss and colon shortening in mice compared to DSS alone-treated group. In this 21-day study, mice were treated daily with test substances and were challenged to DSS from day-8 to 14. CONCLUSION: Our study highlights the protective effect of ethyl gallate and propyl gallate in various combinations which, in pre-clinical animals, serve as an anti-inflammatory drug against the severe form of colitis, indicating its potential for the treatment of IBD in humans. In addition, propyl gallate was investigated for the first time in this study for its anti-colitogenic effect with normal drinking water and reduced effect with copper water.

An innovative method of nonclinical efficacy and toxicological evaluation of recombinant Salmonella typhi Ty21a expressing HPV16 and 18 L1 proteins.

Advancement in technology led to development of live attenuated Salmonella typhi Ty21a as enteric vector for expression of foreign proteins. Such vector platform is inevitable for development of vaccine candidate against human papilloma virus (HPV), the etiological agent of cervical cancer with high prevalence in developing nations. The high risk HPVs like type 16 and 18 contributes to 70% of cervical cancer, hence Indian Immunologicals Limited (IIL), Hyderabad, India developed a recombinant HPV vaccine by introducing HPV 16 and 18 L1 protein coding genes into attenuated S. typhi Ty21a vector. Being a genetically engineered enteric vector vaccine, it would be less expensive, with an ease of oral administration, instead of injectable that needs trained personale, is an added advantage for low socioeconomic setup compared to existing HPV vaccines. Establishing the nonclinical efficacy and safety/toxicity as per the national/international regulatory guidelines has become major constrain for such recombinant S. typhi HPV (rSt.HPV) vaccine. Since, the intended clinical mode of rSt.HPV is through oral route, whereas the live attenuated S. typhi Ty21a doesn't colonize in gut of laboratory animals to be used for nonclinical experiments. Hence, an alternate and unconventional method of 'intranasal drug testing', was followed for nonclinical efficacy and toxicity evaluations. An array of parameters specified by regulatory agencies were investigated in mice, rat and rabbits administered with rSt.HPV through, intra-peritoneal, intranasal and oral routes, the intended clinical route.•Current unconventional and innovative nonclinical testing procedures helps in exploring the alternate methods by pharmacologist/toxicologist.•Ultimately, such new drugs developed through technology must serve the humankind justifying the guidelines of regulatory agencies.

Toxicological data of rats and rabbits: Sub-chronic testing of recombinant HPV vaccine through oral and intranasal routes.

Current article illustrates the data of body weight, biochemical, haematological profile, and organ weights of rats and rabbits administered with recombinant human papilloma virus (HPV) vaccine, along with genotoxicity effect. The data was collected from nonclinical safety/toxicity and immune response evaluations of recombinant Salmonella typhi expressing the HPV 16 and 18 L1 proteins as vaccine. The intended clinical route of vaccine administration is through oral route, whereas it is established fact that attenuated S. typhi could not colonize in laboratory animals. In view of this it is challenging to undertake the nonclinical safety/toxicity evaluations following the regulatory guidelines. Hence sub chronic safety/toxicity testing was carried out in rat and rabbits by administration of HPV vaccine through oral (intended clinical route) and innovative intranasal routes. The prophylactic dose derived from adult human clinical dose (2 × 109CFU/70 kg) was administered to SD rats (PD: 0.18 × 109CFU/kg) and New Zealand White (NZW) rabbits (PD: 0.09 × 109CFU/kg) through oral and intranasal routes. Similarly, average dose (AD:5xPD) was administered to rats (AD:0.9 × 109CFU/kg) and rabbits (AD: 0.45 × 109CFU/kg) through intranasal route only. The repeated doses were administered on 3rd and 5th days of post-exposure of 1st dose through specified routes and test compound effects in relation with time of exposure was assessed by euthanizing animals and data collection at different time points i.e. 15th (25% of animals), 29th (25% of animals) and 93rd days (50% of animals) of post-exposure of 1st dose. The retro-orbital plexus blood was collected before euthanizing animals to unveil the biochemical and haematological profile. The data on genotoxicity effect of test compound, if any, was obtained by assessing the bone-marrow micronucleus assay. The immune response and allergenicity in terms of specific IgG and IgE levels against HPV 16 and 18 L1 proteins were determined in mice. The raw data of various parameters collected at different time points were compiled and computed according to the groups. The haematological profile and organ weights data can be used as reference data for SD rats and NZW rabbits for future studies.

Nonclinical safety evaluation of oral recombinant anti-human papilloma virus vaccine (RHPV 16 & 18): Regulatory toxicology studies in mice, rats and rabbits - An innovative approach.

AIM: The human papilloma virus (HPV) type 16 and 18 causes nearly 70% of uterine cervical cancers. Oral administration of live Salmonella typhi Ty21a, expressing major capsid proteins (L1) of HPV 16 and 18 is a potential choice for immunization in adolescent girls under low resource settings. Present study aimed to assess the nonclinical safety of recombinant S. typhi expressing HPV 16 and 18 (rStHPV) proteins. METHODOLOGY: The acute toxicity of rStHPV was tested by intranasal single dose administration, of 10 and 50 folds higher than clinical prophylactic dose, in mice and rat followed by monitoring their survival for 14 days. Sub-chronic toxicity was evaluated in rats and rabbits with prophylactic and 5 times (average) to clinical prophylactic dosages on scheduled days (1st, 3rd & 5th day) through oral and intranasal routes. The immune/allergic response of rStHPV was assessed in mice through intranasal and intra-peritoneal routes. Experimental animals were daily monitored for live phase, and clinical chemistry, haematology, immunotoxicology, immunogenic response and histopathological examination of vital organs on 15th, 29th and 93rd days. RESULTS: No abnormal changes were noticed in live phase activity, clinical chemistry and haematology profile. The gross necropsy, organ weights and histopathology were found to be normal. No immunotoxicity was recorded as evaluated by tier I tests. Allergic immune response, as evaluated with IgE levels was also negative irrespective of test routes. On the other hand, a significant (P < 0.01) increase of anti-HPV IgG levels was noted in mice exposed through intranasal route. Though the pre-terminal mortality was noted in mice (6-15%), rats (10%) and rabbits (15%), the autopsy revealed no signs of toxicity related to rStHPV, as the changes neither significant nor dose dependent; and even noted in vehicle control also. CONCLUSION: The study results suggested 'no observable adverse effects' of rStHPV even at higher dosages (5, 10 & 50 folds) than intended clinical dose. A significant increase of anti-HPV specific IgG suggests the immunogenicity of vaccine. The innovative approach of current study is nonclinical toxicology evaluation of vaccine through intra-nasal route, an alternate route apart from stipulated regulatory guidelines.

Podophyllotoxin and Rutin Modulate M1 (iNOS+) Macrophages and Mitigate Lethal Radiation (LR) Induced Inflammatory Responses in Mice.

Accidental exposure to lethal doses of Gamma radiation leads to the systemic inflammatory syndrome which causes mortality. In view of this, management of hemopoietic syndrome by modulating pro-inflammatory response in clinically manageable time period seems to be the most appropriate strategy for encountering radiation induced damage and recovery. As both tissue and peripheral macrophages are critical for the management of radiation induced injuries, we have unraveled the immunomodulatory potential of radioprotective formulation (G-003M) on peripheral macrophages populations in this study. G-003M inhibited lethal radiation induced NO and Th1 effector cytokines in the exposed macrophages indicating its M1 dim polarizing capacity. In similar lines, conditioning of mice with G-003M before lethal irradiation (LR) inhibited LR induced titre of Th1 effector cytokines in both serums as well as in lung, small intestine, and spleen tissue confirming its immunomodulatory potential. G-003M potentially down modulated inflammatory response in LPS induced inflammatory model and enhanced M2 polarization of iNOS+ M1 effector macrophages providing a molecular hint on G-003M mechanism of action on macrophages. These observations revealed that G-003M potentially modulate pro-inflammatory programming of macrophages and mitigate radiation-induced inflammatory stress which is believed to contribute significantly to radioprotective attribute of G-003M. In this study, we demonstrate that Rutin and Podophyllotoxin drive M1dim/M2 polarization of LR primed macrophages apart from protecting DNA from radiation. These drugs have the capacity to programme innate immune cells like macrophages which may be involved in homeostasis during recovery.

Sphingosine-1-Phosphate (S-1P) Promotes Differentiation of Naive Macrophages and Enhances Protective Immunity Against Mycobacterium tuberculosis.

Sphingosine-1-phosphate (S-1P) is a key sphingolipid involved in the pathobiology of various respiratory diseases. We have previously demonstrated the significance of S-1P in controlling non-pathogenic mycobacterial infection in macrophages, and here we demonstrate the therapeutic potential of S-1P against pathogenic Mycobacterium tuberculosis (H37Rv) in the mouse model of infection. Our study revealed that S-1P is involved in the expression of iNOS proteins in macrophages, their polarization toward M1 phenotype, and secretion of interferon (IFN)-γ during the course of infection. S-1P is also capable of enhancing infiltration of pulmonary CD11b+ macrophages and expression of S-1P receptor-3 (S-1PR3) in the lungs during the course of infection. We further revealed the influence of S-1P on major signaling components of inflammatory signaling pathways during M. tuberculosis infection, thus highlighting antimycobacterial potential of S-1P in animals. Our data suggest that enhancing S-1P levels by sphingolipid mimetic compounds/drugs can be used as an immunoadjuvant for boosting immunity against pathogenic mycobacteria.

Exploring antiurolithic effects of gokshuradi polyherbal ayurvedic formulation in ethylene-glycol-induced urolithic rats.

Gokshuradi Yog (GY) is a polyherbal ayurvedic formulation used traditionally for several decades in India for the treatment of urolithiasis. The aim of the present study was to determine the underlying mechanism of GY action in the management of calcium oxalate urolithiasis. The effect of Gokshuradi polyherbal aqueous extracts (GPAEs) was studied on various biochemical parameters involved in calcium oxalate formation by employing in vitro and in vivo methods. GPAE exhibited significant antioxidant activity against 1, 1-diphenyl-2-picrylhydrazyl free radical and inhibited lipid peroxidation in the in vitro experiments. The rat model of urolithiasis induced by 0.75% ethylene glycol (EG) and 1% ammonium chloride (AC) in water caused polyuria, weight loss, impairment of renal function, and oxidative stress and decreased antioxidant enzyme activities in untreated control groups. However, GPAE- (25, 50, and 100 mg/kg) treated groups caused diuresis accompanied by a saluretic effect and revealed significant increase in antioxidant enzyme activities along with decreased oxalate synthesizing biochemical parameters at higher doses. This study revealed the antiurolithic effect of GPAE mediated possibly through inhibiting biochemical parameters involved in calcium oxalate formation, along with its diuretic and antioxidant effects, hence supporting its use in the treatment of calcium oxalate urolithiasis.

Incorporation of Cestrum diurnum leaf improves intestinal Ca transport in broilers.

The economy of Ca utilization is under the control of vitamin D(3), particularly its active metabolite 1,25-dihydroxy cholecalciferol [1,25(OH)(2)D(3)]. In sufficient Ca absorption leads to tibial dyschondroplasia resulting in not attaining optimum body weight. Our earlier studies [T.P. Prema, N. Raghuramulu, Phytochemistry 37 (1994) 167] have shown that the Cestrum diurnum (CD) leaves contain vitamin D(3) metabolites. It was felt whether incorporation of CD as a source of 1,25(OH)(2)D(3) could improve the Ca absorption in broilers. Four groups of 60 birds each were fed with either normal diet or normal diet+0.25% CD or normal diet without vitamin D(3) or normal diet without vitamin D(3)+0.25% CD leaf powder for 45 days. In subsample of six birds it was observed that incorporation of CD leaves in the feed had the maximal effect on all the parameters studied. The results indicate that the intestinal Ca transport as represented by Serosa/Mucosa (S/M) ratio was found to be significantly (p<0.01) higher in broilers fed diet with CD leaf powder and the 1alpha hydroxylase activity in kidney is significantly (p<0.001) higher in negative controls. On the other hand the supplementation of CD leaves enhanced the serum Ca, body weight, tibia weight, density and strength resulting in the disappearance of tibial dyschondroplasia. No lesions of toxicity were observed in any of the soft tissue examined. The results suggest that the incorporation of CD leaf powder in poultry feed could be beneficial to the poultry.

Cestrum diurnum leaf as a source of 1,25(OH)2 Vitamin D3 improves egg shell thickness.

A continuing concern of the poultry industry is the high incidence (12%) of egg losses in the laying house due to poor egg shell quality. Calcium (Ca) homeostasis is a key factor in egg shell formation. The economy of Ca utilisation is under the control of Vitamin D(3), particularly its active metabolite 1,25-dihydroxy cholecalciferol [1,25(OH)(2)D(3)]. Supplementation of 1,25(OH)(2)D(3) has been shown to increase specific gravity, shell thickness and shell weight of the egg. However, commercially available synthetic 1,25(OH)(2)D(3) is very expensive. Earlier studies from our Institute [Phytochemistry 37 (1994) 677] have identified a cheap, natural and rich source of 1,25(OH)(2)D(3) in the leaves of Cestrum diurnum (CD), a member of the Solanaceae family. In this study, CD leaves were explored as a source of 1,25(OH)(2)D(3) in the feed of layer birds to improve the egg shell thickness. Fifteen-week-old white leghorn layers were divided into four treatments of 60 birds each and as follows: (I) normal diet with Vitamin D(3), (II) normal diet with Vitamin D(3) + CD, (III) normal diet without Vitamin D(3) and, (IV) normal diet without Vitamin D(3) + CD powder. CD leaf powder was incorporated in to the feed at 0.3% level. The experimental feeding was continued up to 72 weeks of age of the birds. Weekly food intake and daily egg production were noted throughout the experimental period and the specific gravity of the eggs, feed consumed to lay one egg and egg shell thickness were determined. Incorporation of CD leaves in the feed had the maximal effect on all the parameters studied. The feed consumed to lay one egg was 20 g less than the control group. The specific gravity of the egg was higher by 0.005, than the control egg, indicating a 5% decrease in the breakage of eggs in CD fed chicks. Also there was a significant increase (P < 0.001) in egg shell thickness. The data suggest that incorporation of CD leaf powder in the feed of poultry layers increased the egg shell thickness, which in turn could decrease the economic loss due to breakage of eggs.