PHARMACOKINETIC VALIDATION

PHARMACOKINETIC VALIDATION : 

PHARMACOKINETIC VALIDATION Submitted to ANU as a part of 1st M.pharm , 1st semester curriculum . by I.VENKATESWARLU (M.pharm) REG NO:Y10MPH08017 Department of pharmaceutics A S N Pharmacy college, Tenali . submitted to Mr. ASHOK . THULLURU, M.pharm ( Asst.Professor ) Department of pharmaceutics A S N Pharmacy college, Tenali .

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Pharmacokinetics : - Is defined as the kinetics of drug (ADME)and their relationship with the pharmacological, therapeutic or toxicological response in man &animals . Theoretical aspects Pharmacokinetic studies Experimental aspects Theoretical aspects :- which involves development of pharmacokinetic models to predict drug disposition after its administration. Statistical methods are commonly applied to interpret data asses various parameters . Experimental aspect :- which involves development of biological sampling techniques, analytical methods for measurement of drug and metabolites concentration in biological samples and data collection &evaluation. A.S.N

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Various applied branches of pharmacokinetics are :- Clinical pharmacokinetics Population pharmacokinetics Toxicokinetics . Toxicokinetics : - is defined as the application of pharmacokinetics to design conduct & Interpretation of drug safety evaluation studies . Preclinical drug safety evaluation or toxicological studies In these studies a minimum of two animal species are employed, as per regulation of FDA. 1) rodents- eg :-Rats, Mice. 2) non-rodents- e.g :- Dog A.S.N

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Specific and non specific enzymes participate in drug metabolism, primary in the liver, but also in the kidneys, lungs & GIT. If the first pass effect is to be avoided, other routes of administration my be used . Drug metabolism frequently results in the production of or more metabolites of the administration drug, which may be pharmacologically active others not. Drug metabolism may be essentially to convert prodrug to active compounds. For reasons of drug safety, it is important determine whether a drug’s metabolic products are toxic or non toxic to the animal &later to the human . A.S.N

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The ADME studies are performed through the timely collection and analysis of Urine, Blood, Faeces samples and Careful examination of an animal behavior Special studies are undertaken to determine the presence, if a test drug or its metabolites in the milk of lactating animals, the ability of drug to cross the placental barrier and enter the fetal blood supply or metabolites in the body. In studying the formation and disposition of metabolites a radio labeling is commonly incorporate into the administered compound & traced in the animal’s waste products & tissues. A.S.N

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TOXICOLOGY Toxicology is the area of pharmacology that deals with the adverse or undesired effects of drugs. The direct extrapolation of preclinical animal safety data to human is difficult because of species variation, different dose response relationship, and immunological differences in animals and for other reasons. Preclinical drug safety evaluation (DSE) or toxicology studies are under taken to determine :- The substances potential toxicology with short-term (acute effects) or long term use (chronic effects). The substances potential for specific organ toxicity. The mode, site & degree of toxicity. Dose response relationship for low, high & intermediate doses over a specified time course. Gender, reproductive teratogenic toxicities and the substances carcinogenic and genotoxic potential. A.S.N

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The toxicology profile includes:- 1 Acute/short-term toxicology studies (7-28days or 1 months duration) a)Acute oral toxicity b)Acute dermal toxicity c)Acute inhalation d)Skin sensitization 2 Sub-chronic toxicology studies (3or6 month’s duration) a)Sub chronic dermal toxicity (90 days studies) b)Sub chronic inhalation toxicity (90 days studies) c)One generation reproduction toxicity d)Two generation reproduction toxicity e)Neurotoxicity study in rodents A.S.N Reproduction/ teratogenicity studies

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3 chronic\long term toxicology studies (12 months duration) a) Chronic oral toxicity b) Chronic dermal toxicity 12 months duration c) Chronic inhalation d) Skin sensitization Long term test, studies also including a) Carcinogenicity or oncogenicity 18 to b) Mutagenicity studies or genotoxicity 24 months duration A.S.N

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Acute/short-term toxicology studies: Introduction: Peganum is a small genus belonging to the family Zygophylaceae and mainly distributed in the Mediterranean region. Peganum harmala is the only species found growing wild in the middle and Acute Toxicological Studies on the Extract of Iraqi Peganum Harmala in Rats northern parts of Iraq. The plant is rich in alkaloids and contains up to 4% total alkaloids (Abdel- Fattahet al, 1997; Chopra et al, 1958). The principle alkaloids present are harmaline , harmine , harmalol and peganine (Abdel-Fattah et al, 1995). It also contains fixed oils. There are several reports which indicated the great variety of pharmacological and biological activities of Peganum harmala such as antibacterial, antifungal and MAO inhibition (Abdel-Fattah et al, 1997) and to be effective in the treatment of dermatosis ( Saad and Rifaie , 1980), hypothermic (Abdel-Fattah et al, 1995) and cancer (Adams, 1983). A.S.N

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Materials and Methods: 1. Extract preparation The dry seeds of Iraqi Peganum harmala (100 g) were grinded and then were extracted with purified water for 24 hours in continuous ( Soxhlet ) apparatus. The extract was filtered, and water was removed by evaporation on a rotator evaporator under vacuum at 60ºC to a small volume. A small amount of diluted ammonia was added to make pH of 9. Subsequently, 100 ml of chloroform was added and slowly shake for several minutes until the alkaloids enter to the chloroform phase. This was repeated for three times and total chloroform phase was evaporated, yielding a total alkaloid extract. A.S.N

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2. Animals: Albino Wister rats of either sex, weighing 200-350 g were used throughout the study. They were fed a commercial chow, water was given ad libitum . The animals were divided into four groups designated as A, B, C, D. each group consists of 42 rats divided to 6 subgroups of 7 rats. Rats of group A treated with a single daily intramuscular (IM) LD50 dose of the aqueous extract. Rats of group B treated with a single daily (IM) 1/2 LD50 dose of the aqueous extract and group C treated with a single daily (IM) 1/4 LD50 dose of the aqueous extract. Group D taken as control treated with IM saline. Treatment was done for 6 weeks. The experiment protocol was revised and approved by the ethical committee of school of pharmacy, Petra University. A.S.N

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3. Hematological methods Total RBC count, WBC count and differential leucocytes count were performed visually according to the methods described in (Lewis et al, 2006). Hematological studies were done weekly for 6 weeks. Blood was withdrawn from the heart directly after anesthetizing the animals lightly with ether. Bone marrow examination was done on all animal remained alive after the end of the experiment. 4. LD50 determination LD50 (420 mg/kg) was calculated according to Litchifield and Wilcoxon (1949) method. The aqueous extract was given intramuscularly. Groups of 6 rats were used for each dose given. A.S.N

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5. Biochemical tests Biochemical tests include the measurement of GPT, GOT and ALP enzymes. Sodium, potassium, sugar, urea and creatinine were estimated in the plasma of some rats. The enzymes were estimated by autoanalyzer ( Technicon AA2). Other biochemical tests were carried out by autoanalyzer ( Technicon SMA-600). 6. Pathological methods Rat killed by exsanguinations. Tissue specimens (heart, lung, spleen, skin, testis, ovary, glands) were cut transversely into 1 cm thick slices. These slices were fixed with 10% formalin for 3-4 days, embedded in paraffin and cut at 4 thick. The sections were generally stained with haematoxylin and counter stained with eosin. A.S.N

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Carcinogenicity studies (or) oncagenicity studies Carcinogenicity testing is usually a component of chronic testing and is undertaken when the compounds has shown sufficient promise as a drug to enter human clinical trials. Carcinogenicity studies are usually carried out in a limited number of rat and mouse strains for which there is reasonable in formation on spontaneous tumor incidence. A.S.N

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For carcinogenicity studies, the high dose should only high enough to elicit signs of minimal toxicity without altering the animals normal life span due to effects other than carcinogenicity. Carcinogenicity studies are long term (18 to 24 months) with surviving animal’s scarified and studied at defined weeks during the text period. Data are collected and evaluated on the causes of animal death, tumor incidents. Type and site. A.S.N

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Mutagenicity or genotoxicity studies: Bacterial reverse mutation assay (with strains of salmonella.) Rodent dominant lethal test. Genetic toxicity tests DNA damaged repair. Mouse spot test. These studies are performed to determine if the test compound can effect gene mutation or causing chromosomal or DNA- damage. A.S.N

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Genotoxicity: Unequivocally genotoxic compounds, in the absence of other data, are presumed to be transspecies carcinogens, implying a hazard to humans. Such compounds need not be subjected to long-term carcinogenicity studies. However, if such a substance is intended to be administered chronically to humans a chronic toxicity study (up to one year) may be necessary to detect early tumourigenic effects. Assessment of the genotoxic potential of a compound should take into account the totality of the findings and acknowledge the intrinsic value and limitations of both in vitro and in vivo tests. The test battery approach of in vitro and in vivo tests is designed to reduce the risk of false negative results for compounds with genotoxic potential. A single positive result in any assay for genotoxicity does not necessarily mean that the test compound poses a genotoxic hazard to humans (note for guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals ). A.S.N

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References: ByNet : http:// www.ncbi.nlm.nih.gov/pubmed/9862198 . http:// www.aapsj.org/view.asp?art=ps060214 . http:// www.ncbi.nlm.nih.gov/pubmed/8350381 . http:// iccvam.niehs.nih.gov/methods/genetox/genetox.htm . By Books: Pharmaceutical Process Validation: An International Edition : by Ira R . Berr , Alfred Wachter , Robert Nash, Robert A. Nash. A.S.N

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