Manash Pratim Pathak

Acute Dermal Irritation, Sensitization, and Acute Toxicity Studies of a Transdermal Patch for Prophylaxis Against (±) Anatoxin-A Poisoning

The skin irritating, sensitizing, and acute dermal toxicity potential of a novel combinational prophylactic transdermal patch, mainly composed of eserine and pralidoxime chloride as active pharmaceutical ingredients, against (±) anatoxin-a poisoning were investigated in rabbits, guinea pigs, and rats in compliance with the Organisation for Economic Cooperation and Development guidelines. In primary skin irritation test, rabbits were dermally attached with the therapeutically active transdermal patch or with a placebo patch for 72 hours. The transdermal patches did not induce any adverse reactions such as erythema and edema on intact skin sites. The active patch was classified as a practically nonirritating material based on the score in the primary irritation index. In the Buehler test, guinea pigs were sensitized by the active or placebo transdermal patches attached for 24 hours. The patches did not induce any sensitization reactions in contrast to a severe sensitization reaction that occurred in the positive control. Therefore, the active patch and placebo patch were both graded as weak in sensitization score and rate. Acute dermal toxicity test in rats did not produce any overt signs of toxicity following a 14-day treatment period. Taken together, these findings suggest that the transdermal patch does not cause skin irritation, skin sensitization, or dermal toxic effects following dermal application.

Exploration of ethyl anthranilate-loaded monolithic matrix-type prophylactic polymeric patch

Compromised stability of pharmaceutical formulations loaded with volatiles is a serious problem associated with devices designed to deliver volatile compounds. The present study has been focused to evaluate the stability potential of matrix-type polymeric patches composed of volatile ethyl anthranilate for prophylaxis against vector-borne diseases. Ethyl anthranilate-loaded matrix-type polymeric patches were fabricated by solvent evaporation method on an impermeable backing membrane and attached to temporary release liners. Stability testing of the polymeric patches was performed as per the International Conference on Harmonization (ICH) guidelines for 6 months under accelerated conditions. In addition, the quantification of residual solvents was also performed as per the ICH guidelines. After conducting the stability studies for 6 months, the optimized patches showed the best possible results with respect to uniformity of drug content, physical appearance, and other analytical parameters. Furthermore, the amount of residual solvent was found well below the accepted limit. Thus, the present report outlined the analytical parameters to be evaluated to ensure the stability of a certain devices consisting of volatile compounds.

Protection by a transdermal patch containing eserine and pralidoxime chloride for prophylaxis against (±)-Anatoxin A poisoning in rats

The prophylactic and neuroprotective impact of a transdermal patch containing eserine and pralidoxime chloride (2-PAM) against (±)-Anatoxin A poisoning was investigated using Wistar strain albino rats. Rats were smooth-shaved on the dorsal side, attached with a drug-in-adhesive matrix type prophylactic transdermal patch for 72 h and challenged with subcutaneous injection of three doses (1.0, 1.5 and 2.0×LD50) of (±)-Anatoxin A. The LD50 value of (±)-Anatoxin A was determined to be 1.25mg/kg, and at this particular dose (1.0×LD50) of toxin induced severe clinical symptom including extreme seizures in rats, resulting acute brain injuries in discrete brain regions, leading to 100% mortality within 5 min. The anticonvulsant effect, antiarrythmic effect, nerve conduction study, clinical observations and mortality, neuroprotective effect as well as skin histopathology of the prophylactic transdermal patch against (±)-Anatoxin A poisoning were investigated systematically. It was found that seizures, tachycardia, nerve damage, clinical symptoms, brain injuries and mortality induced by such lethal toxin were effectively prevented by the prophylactic patch treatment up to certain LD50 level. Hence, it could be a choice of potential therapeutic regimen against such lethal poisoning.

Acute and subchronic dermal toxicity of Vitex negundo essential oil.

Abstract Vitex negundo is a common herb in different herbal formulation. The potential acute and sub-chronic dermal toxicities were evaluated as per OECD (Organization for Economic Cooperation and Development) guidelines 402 and 411, respectively. Both sexes of Wistar rats were exposed to Vitex negundo oil of 2000 mg/kg body weight for acute dermal toxicity, whereas in the dermal sub-chronic toxicity study, rats were exposed to Vitex negundo oil 250, 500 and 1000 mg/kg body weight, respectively, for five times a week for 90 d. In acute and sub-chronic toxicity studies, all animals were normal without any behavioral, serum biochemistry, hematology, necroscopical and histopathological changes. The no observed effect level (NOEL) and no observed adverse effect level (NOAEL) of Vitex negundo oil were 250 and 1000 mg/kg/day, respectively. Vitex negundo oil is under the category 5 (Unclassified) according to the Globally Harmonized System, with an LD50 value of over 2000 mg/kg.

Safety assessment and toxicological profiling of a novel combinational sunprotective dermal formulation containing melatonin and pumpkin seed oil

&NA; Ultraviolet (UV) radiation exposure has been known to cause irreparable damages to human skin. The daunting risk of UV radiation exposure faced by military personnel led to the development of a sunscreen formulation which has superior sun protection factor combined with the ability to counteract reactive oxygen species. The present work deals with the preclinical safety evaluation of the sunscreen formulation comprising of four US FDA approved UV filters; namely avobenzone, octinoxate, oxybenzone, titanium dioxide along with melatonin and pumpkin seed oil, via OECD protocols of assessing acute oral and dermal toxicity; skin sensitizing; skin irritating; ocular irritating and genotoxic potential. Both oral and dermal LD50 values were found to be >2000 mg/kg body weight in adult Wistar albino rats using acute dermal and oral toxicity tests. The sunscreen formulation was found to be non‐sensitizing to the skin of guinea pigs and non‐irritating to both skin and eyes of rabbits. The sunscreen formulation was also found to be non‐mutagenic which was affirmed by a battery of genotoxicity and muagenicity assays. The results obtained from this preclinical study indicated that the sunscreen formulation is non toxic and safe in animal models. This study along with additional preclinical evaluations may serve as a basis for considering the formulation as a potential candidate for further trials to establish its efficacy, tolerability and applicability. Graphical abstract Figure. No caption available. HighlightsA sunscreen formulation using four US FDA approved ingredients along with melatonin and pumpkin seed oil was developed.Dermally, the sunscreen did not cause toxicity; irritation or sensitization in rats, rabbits and guinea pigs respectively.Instillation of the formulation into rabbit eye caused low adversities, indicating ocular safety and non irritancy.Oral administration of the sunscreen did not demonstrate mortality, gross toxicity or histopathological alterations in rats.A battery of genotoxicity assays confirmed that the formulation was non mutagenic.

Validated RP-HPLC/DAD Method for the Quantification of Insect Repellent Ethyl 2-Aminobenzoate in Membrane-Moderated Matrix Type Monolithic Polymeric Device

A simple, accurate and sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for the estimation of ethyl 2-aminobenzoate (EAB) in a matrix type monolithic polymeric device and validated as per the International Conference on Harmonization guidelines. The analysis was performed isocratically on a ZORBAX Eclipse plus C18 analytical column (250 × 4.4 mm, 5 μm) and a diode array detector (DAD) using acetonitrile and water (75:25 v/v) as the mobile phase by keeping the flow-rate constant at 1.0 mL/min. Determination of EAB was not interfered in the presence of excipients. Inter- and intra-day relative standard deviations were not higher than 2%. Mean recovery was between 98.7 and 101.3%. Calibration curve was linear in the concentration range of 0.5-10 µg/mL. Limits of detection and quantification were 0.19 and 0.60 µg/mL, respectively. Thus, the present report put forward a novel method for the estimation of EAB, an emerging insect repellent, by using RP-HPLC technique.

Determination of LCt50 of aerosolized paraquat and its pulmonary toxic implications in non-anesthetized rats

Abstract Paraquat (PQ), a highly popular agricultural herbicide, is a serious occupational hazard with lethality reported at doses as low as 35 mg/kg body weight with intoxication occurring via inhalation or dermal route. The main objective of this study was to determine the median lethal concentration (LCt50) of paraquat through whole body exposure in adult male Wistar rats. Aerosolized PQ dissolved in water was delivered in a dose-dependent manner, to fully conscious rats confined in whole body plethysmograph (WBP), in a nebulized form with concentrations ranging from 40–200 mg/kg of air over a 4 h exposure period. Animals were observed up to 24–48 h post-exposure to observe any lethality. LCt50 estimates (±95% confidence interval) were obtained from the sequential stage-wise experiments using probit analysis. Rat lungs were examined radiologically and histopathologically. Gas chromatography–mass spectrometry (GC-MS) analysis determined the correlation of PQ accumulation in the lungs with the actual exposed dose of PQ. The actual LCt50 was found to be 218 g·min/m3 whereas 57.9 ± 2.90 µg/g of PQ accumulated in the lungs of each lifeless animal. All animals exhibited severe respiratory changes and pulmonary abnormalities. This study demonstrated that when compared with the actually exposed dose, the amount of PQ that accumulated in the lungs was very low, but enough to cause death in 50% of animal population and cause pulmonary abnormalities in each of the experimental animal. The PQ exposure carried out in WBP also facilitated the dermal absorption of aerosolized PQ, which replicated the real-life situation in workers operating with PQ.

Safety evaluation of an oat grain alkaloid gramine by genotoxicity assays

Abstract Gramine is a natural indole alkaloid that has been isolated from different raw plants occurring mainly in Avena sativa, etc. The study was aimed to investigate the possible in vitro antioxidant, in vitro mutagenic, in vitro antimutagenic, and in vivo genotoxic activity of gramine using ferric reducing ability of plasma (FRAP) assay, Metal chelating, Ames bacterial reverse mutation test, and the mouse bone marrow micronucleus assay as well as chromosomal aberration. Four concentrations of gramine viz. 250, 500, 1000, and 2000 μg/mL were evaluated for its antioxidant activity in FRAP Assay and Metal Chelating Test. Four concentrations of gramine (1250 μg/plate, 2500 μg/plate, 5000 μg/plate, and 10 000 μg/plate) were employed in Salmonella typhimurium strains to study the mutagenicity in the presence and absence of standard mutagens, 2-aminofluorene (2-AF), sodium azide (SA), and 2-nitrofluorene (2-NF). Three doses, i.e. 0.1, 0.2, and 0.3 × the LD50 of gramine (i.e. 50 mg/kg, 100 mg/kg, and 150 mg/kg) were administered orally to either sex of Swiss albino mice for 48 h to study the genotoxic activity in micronucleus assay as well as chromosomal aberration. Gramine showed potent antioxidant activity in both the assay. Gramine at the given dose lacks mutagenicity as well as found to possess antimutagenic efficacy. Interestingly, S9 enzymes increase the antimutagenic activity in a dose-dependent manner. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs), as well as no significant difference in the percentage of chromosomal aberrations was observed between the gramine groups and the negative groups but percentage of polychromatic erythrocytes (PCEs) is found to be higher in all the gramine groups. These results indicate significant antioxidant, non-mutagenic as well as non-genotoxic activity of gramine in vitro and in vivo in the given doses.

STAT3 and NF-κB are common targets for kaempferol-mediated attenuation of COX-2 expression in IL-6-induced macrophages and carrageenan-induced mouse paw edema

Cycloxygenase-2 (COX-2) is the inducible isoform of cycloxygenase enzyme family that catalyzes synthesis of inflammatory mediators, prostanoids and prostaglandins, and therefore, can be targeted by anti-inflammatory drugs. Here, we showed a plant polyphenol, kaempferol, attenuated IL-6-induced COX-2 expression in human monocytic THP-1 cells suggesting its beneficial role in chronic inflammation. Kaempferol deactivated and prevented nuclear localization of two major transcription factors STAT3 and NF-κB, mutually responsible for COX-2 induction in response to IL-6. Moreover, STAT3 and NF-κB were simultaneously deactivated by kaempferol in acute inflammation, as shown by carrageenan-induced mouse paw edema model. The concomitant reduction in COX-2 expression in paw tissues suggested kaempferol’s role in mitigation of inflammation by targeting STAT3 and NF-κB.