Safety pharmacology of git

SAFETY PHARMACOLOGY STUDIES AND DRUG DISCOVERY AND GIT SAFETY PHARMACOLOGY AND BIOMARKERS OF GIT SYSTEM AND GASTRIC EMPTYING AND MOTILITY AND TRANSIT TIME AND ADVERSE EFFECT ON GIT SYSTEM AND SCALE OF GI ADVERSE EFFECTS AND NEERAJ KUMAR

1. SAFETY PHARMACOLOGY OF GASTROINTESTINAL TRACT PRESENTED BY: NEERAJ KUMAR M. PHARM. (PHARMACOLOGY) DEPT OF PHARMACEUTICAL SCIENCES BBAU LUCKNOW

2. INTRODUCTION Safety pharmacology is a branch of pharmacology specializing in detecting and investigating potential undesirable pharmacodynamic effects of new chemical entities (NCEs) on physiological functions in relation to exposure in the therapeutic range and above. Safety pharmacology studies are required to be completed prior to human exposure (i.e., Phase I clinical trials), and regulatory guidance is provided in ICH S7A and other documents . Primary organ systems (so-called core battery systems) are: Central Nervous System Cardiovascular System Respiratory System Secondary organ systems ( supplemental studies) are: Gastrointestinal System Renal System

3. Types of Pharmacology Primary Pharmacology- Effects of a substance in relation to its desired therapeutic target (Mechanism of Action; MOA). Secondary Pharmacology- Identifying/understanding “off-target effects”. Effects of a substance not related to its desired therapeutic target (Formerly called “General Pharmacology”) can lead to a new therapeutic indication. Rogaine ® (minoxidil); Viagra ® (sildenafil) (initial intended therapeutic effect- antihypertensive) Safety Pharmacology- Investigates the potential undesirable pharmacodynamic effects of a substance on physiological functions in relation to exposure in the therapeutic range and above.

4. Distinctions Between IND-enabling Toxicology & Safety Pharmacology Usually repeat dose studies Effects of chronic/cumulative exposure up to a maximum tolerated dose (MTD) Major endpoints are structural changes to tissues collected at necropsy Organ weights Haematology Biochemistry Postmortem data: Histo Morphologic & Histopathology Area Under the Concentration-time curve (AUC) driven Toxicology Safety Pharmacology Usually acute (single dose) studies Effects of a single exposure to a dose that produces moderate adverse effects Endpoints are usually short term, reversible, functional effects recorded from conscious animals Evaluate the onset, duration and magnitude of effects “C max -driven” (C max is the maximum serum concentration that a drug achieves in a specified compartment or test area of the body after the drug has been administrated and before the administration of a second dose )

5. Drug Development Cycle (Pugsley et al., 2008: Br. J. Pharmacol.) Supplemental: GIT, Renal

6. Regulatory guidelines for assessing GI liability Supplemental Safety Pharmacology Studies ……. ‘ Effects of the test substance on the gastrointestinal system should be assessed . For example, gastric secretion , gastrointestinal injury potential , bile secretion , transit time in vivo , ileal contraction in vitro , gastric pH measurement ICH S7A Safety Pharmacology Studies for Human Pharmaceuticals Supplemental Safety Pharmacology Studies……. ‘ Effects of the test substance on gastrointestinal transit should be assessed . Intestinal transit time should be investigated ; gastric emptying should also be examined if appropriat e Effects of test substance on secretion of gastric juice, saliva , bile on the motility , in vitro study of stomach and intestine motility of the gastrointestinal tract in situ on gastroduodenal mucous membrane should all be assessed in light of results of studies in category A’ Japanese Guidelines for Nonclinical Studies of Drugs Manual, 1995

7. Phase ‘Nonclinical’ Phase I Phase I-III Phase III/ M arke t i ng Post- M arke t i ng Post- M arke t i ng In f o r m at ion: Causes of attrition Serious ADRs Causes of attrition ADRs on label Serious ADRs Withdrawal from sale Source: Car (2006) Sibille et al. (1998) Olson et al. (2000) BioPrint® (2006) Budnitz et al. (2006) Stevens & Baker (2008) Sample size: 88 CDs stopped 1,015 subjects 82 CDs stopped 1,138 drugs 21,298 patients 47 drugs Cardiovascular: 27% 9% 21% 36% 15% 45% Hepatotoxicity: 8% 7% 21% 13% 0% 32% Haematology/BM: 7% 2% 4% 16% 10% 9% Nervous system: 14% 28% 21% 67% 39% 2% Immunotox; photosensitivity: 7% 16% 11% 25% 34% 2% Gastrointestinal : 3% 23% 5% 67% 14% 2% Re p r o t o x : 13 % % 1 % 10 % % 2% M u sc ulo ske l eta l: 4 % % 1 % 28 % 3 % 2% Res pi rat o r y : 2 % % % 32 % 8 % 2% Re n a l: 2 % % 9 % 19 % 2 % 0% Ge n et ic t o x : 5 % % % % % 0% Carc inog e ni c i t y : 3 % % % 1 % % 0% Ot h er : % % 4 % 16 % 2 % 2% Adapted from Redfern WS et al. SOT, 2010; SOT 2011 0% 1-9% 10-19% >20% Impact of adverse effects of drugs by organ function throughout the pharmaceutical life cycle    2010 Update (lower number of CDs): Trials halted/delayed/ development stopped 2010 (18 CDs) GI related – 11% (↑) Prescribing re st r i c t i on s/ l ab e lli n g 2010 (40 CDs) GI related – 5% (↓) W i t hd r a w al from sale 2010 (8 CDs) GI related – 0% (↓) The various toxicity domains have been ranked first by contribution to products withdrawn from sale, then by attrition during clinical development.

8. Scale of clinical GI adverse reactions The % of total ADRs associated with the GI system Lewis , 1986 The number of drugs implicated in causing diarrhoea Chassany et al 2000 The % of all ADRs associated with diarrhoea Chassany et al 2000 In a study of Phase 1 clinical trial data (1986-95) 1 % of subjects reporting severe ADRs with GI-associated ADRs In a review of AZ portfolio Phase 1 (SAD) clinical trial data the % of compounds with at least one reported GI AE 1 ADR data referenced from Redfern WS et al 2010. The Toxicologist 114(S1):1081 The % of GI ADRs that are predictable Type A pharmacological reactions Gatenby et al 1995 The % of patients hospitalised with GI effects Lewis, 1986

9. Clinical GI adverse reactions Effects on mouth, gums, tooth discolouration, taste, ulcers Inflammation, ulceration, fibrosis Ulceration, inflammation, bleeding, colitis Pancreatitis Diarrhoea/constipation Nausea and vomiting The majority of GI effects are functional in nature, with fewer of pathological causation Abdominal cramps/discomfort Motor dysfunction

10. Anti depressants SSRIs and TCAs Nausea, diarrhoea, constipation Drug classes commonly reported to have GI side effects Drug withdrawal and GI effect Anti inflammatories NSAIDs Ga st rod u o d e n al ulceration Anti psychotics Chlorpromazin e Dry mouth Anti microbials Penicillin, erythromycin Diarrhoea Anti bacterials Doxycycline, tetracycline Oesophageal inflammation Cytotoxics 5-fluoro-2’-deoxyuridine Perforated ulcers Wide range of drug classes , therapy areas and chemical classes have GI AEs/ ADRs which can lead to: Reduced patient compliance Dose-limiting toxicity Not always primary pharmacology driven

11. Charcoal meal A number of methods are available which allow the assessment of drug-induced altered GI function at the membrane, cellular and whole animal levels In silico Available avenues for assessing altered GI function Endoscopy Measure of gastric acid secretion Imaging Wireless m o t ili t y / pH devices Faecal pellet assessment In vivo In vitro Histology M o r p ho m e t r y / stereology Cellular transport and flux studies El e ct rol y t e transport Solute uptake Motility studies Computer simulation and modelling Marker studies Emesis models

12. Methods to Assess Drug Effects on Gastrointestinal Function ASSESSMENT OF GASTRIC EMPTYING AND INTESTINAL MOTILITY Invitro : Ileum contraction method Invivo : Pe ll e t assess ment Wireless capsules X-ray Imaging 2. ASSESSMENT OF GASTRIC SECRETION : Invitro method Invivo method 3.MODELS OF GASTROINTESTINAL ABSORPTION Invitro method Everted small intestinal sac technique CELL CUTURE TECHNIQUES Invivo method : Direct method Indirect method 4.ASSESSMENT OF EMESIS

13. Methods to Assess Drug Effects on Gastrointestinal Function Assessment of Gastric Emptying and Intestinal Motility Regulatory guidelines (ICH S7A) indicate that findings from “ ligand binding or enzyme assay data suggesting a potential for adverse effects” should be considered in the “selection and design of safety pharmacology studies.” In vitro profiling to maximize detecting off-target interaction and potential safety liabilities of the NCE. Smooth muscle contraction has been studied Radiotracer or fluorescent techniques can be applied to monitor calcium ion mobilization and handling by ATPase -dependent Ca2+ pumps present at cell membrane and sarcoplasmic reticular membrane

14. C u r ren t m e thods /t echno l og i e s . : Pe ll e t assess ment Weight (g) Nu m b er Appearance Used as an indice of intestinal / colonic transit Number, weight and appearance of faecal pellets assessed Can be performed in metabolism cages, home cage, e.g. during condensed Irwin, or in WBP chambers Single snap shot or time course Non invasive Marks et al 2013 J Pharmacol Toxicol Methods 68(1):123-136

15. Current methods/technologies: Wireless capsules Animal orally administered a SmartPill ® capsule (26 x 13 mm) or Bravo capsule (6 x 5 x 25 mm) Temperature, pressure and pH data acquired and transmitted (wirelessly) to pc Increase in pressure and/or pH indicates time of entry into duodenum Drop in temperature indicates exit from rectum Multiple endpoints can be measured with SmartPill ® – GE , colonic transit and total transit times, antral and duodenal pressure changes and motility indices Published data (e.g. transit times) variable and technique not widely used Non invasive but costly; size of capsule may limit size of animals used Bravo ® capsule SmartPill ® trace from SmartPill ® website; pH figure from Sagawa K et al, 2009. J Pharm Sci ,98:2494-2500; Bravo capsule image from BBC website

16. Current methods/technologies: X-ray Imaging Number of imaging techniques available including X-ray (shown above ), X-ray methods can be used on restrained, non anaesthetised animals ( fasted / non fasted) Can be performed in small and large animals Animals administered a contrast agent (e.g. barium meal) Image sequences are obtained at selected time points from restrained animals Multiple endpoints - measure of gastric emptying, intestinal transit and motility Non-invasive

17. Assessment of Gastric Secretion In Vitro Models Pharmacological studies on acid secretion can be performed on mucosal cell preparation from stomach mucosa obtained by enzyme dispersion and separated by centrifugation to separate parietal cells (H+ secreting cells) and histamine secreting cells, ECL ( Enterochromaffin like cells)cells . Whole-organ study in totally isolated vascularized perfused rat stomach was described by Kleveland to measure H+ concentration and pepsin secretion in the gastric effluent. This technique allows for drug infusion through the vasculature and direct measurement of gastric secretion using minimal amount of substance to construct a dose-response curve.

18. In Vivo Models Gastric secretion can be assessed in anesthetized or conscious animals. In anesthetized animals, a simple, acute model involves ligating the pylorus about 1 h after administration of the test compound. A few hours later, the stomach is removed, its contents centrifuged, and the supernatant analyzed for pH or other components. This technique does not permit the evaluation of chronic treatment and may be biased by the anesthesia.

19. Gastric fistula models have been extensively used in small animals (rats, guinea pigs, rabbits) and dogs for decades. Briefly, a cannula is placed into the body of the stomach, sutured and exteriorized so that gastric secretions can be collected in conscious, restrained animals. Changes in the content of gastric juice can be evaluated as well as the mechanisms of control involved in the secretion.

20. Models of Gastrointestinal Absorption Invitro : Everted small intestinal sac technique: T his method involves isolating a small segment of the intestine of a laboratory animal such as rat, inverting the intestine and filling the sac with a small volume of drug free buffer solution . Both the segments are tied off and the sac is immersed in an erlenmeyer flask containing a large volume of buffer solution that contains the drug . The flask and its contents are then oxygenated and the whole preparation is maintained at 37°C and shaken mildly . At predetermined time intervals, the sac is removed and the concentration of drug in the serosal fluid is determined/assayed for drug content.

21. Fig. Everted sac technique

22. CELL CUTURE TECHNIQUES Cell culture is the complex process by which cells are grown under controlled conditions, generally outside their natural environment. In this technique, differentiated cells of the intestine, originating from CaCo2 cells ( cells of carcinoma of colon) are placed on synthetic polycarbonate membrane previously treated with an appropriate material such as collagen which on incubation aids reproduction of cells while not retarding drug permeation characteristics. These models are based on the assumption that passage of drugs across the intestinal epithelium is the main barrier for drugs to reach the circulation.

23. Human intestinal cell lines are generally divided into four different groups: Type I : These cells differentiate spontaneously under normal culture conditions and hence are polarized (i.e. apical and basolateral surface), form domes, have tight junctions and brush border (eg.Caco-2 cells). Type II : These cells differentiate into enterocytes- type cells only under specific culture conditions e.g. HT29 in presence of glucose, HT29 clone can differentiate into mucus cells. Type III : These cells form domes but do not express any biochemical or morphological markers of differentiated cells. e.g. T84, SW116 and Col115 cell lines. Type IV : These cells do not differentiate. e.g. HCA7 and SE480 cell lines.

24. Fig. CaCo-2 cell monolayer to study drug absorption

25. CaCo-2 is the most widely used cell line and CaCo-2 are a human colon carcinoma cell line. The CaCo2(colon cancer cells) cell line is a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells. Solution of drug is placed on this layer of cultured cells and the system is placed on this layer of cultured cells and the system is placed in a bath of buffer solution. The drug that reaches the latter compartment is sampled and analysed periodically.

26. Invivo method: a. Direct method The drug levels in blood or urine is determined as a function of time. For this, a suitable sensitive reproducible analytical procedure should be developed to determine the drug in the biological fluid. In this method, blank urine or blood sample is taken from the test animal before the experiment. The test dosage form is administered to the animal and at appropriate intervals of time the blood or urine sample are collected and assayed for the drug content. From the data, we can determine the rate and extent of drug absorption.

27. b. Indirect method When the measurement of drug concentration in blood or urine is difficult or not possible, but a sensitive method is available to test the activity, then absorption studies can be done by this indirect method. In this method, pharmacological response of the drug is related to the amount of drug in the body. The response is determined after the administration of a test dosage form, LD 50 appears to be dependent on the rate of the absorption of drug.

28. ASSESSMENT OF EMESIS

29. Biomarkers of Gastrointestinal Injury The 13 C sucrose breath test (SBT) is utilized to report the status of health of the small intestinal villous. The test is based on the use of a selectively 13 C-labeled sucrose that enables a quantitative assessment of the absorptive capacity of the small intestine after ingestion of the stable isotope substrate, with an interval of collection of expired 13 CO 2 of 90 min. The cumulative percentage of the administered dose expired in a 90-min period is a marker of small intestinal damage and/or absorptive capacity. This level gives a quantitative indication of the status of small intestinal health, with a lower level indicating more impaired function. The SBT can be used in both animal models and in cancer patients to follow time courses of gut damage and repair with different drugs.

30. CITRULLINE

31. GASTRIN The primary physiological function of gastrin is the stimulation of gastric acid secretion from parietal cells. This is achieved via the activation of CCK2R( Cholecytokinin ) by gastrin on neighbouring ECL cells, resulting in histamine release which acts as a paracrine mediator stimulating acid release from parietal cells

32. Calprotectin and lactoferrin are proteins which are released when leukocytes become activated in response to tissue injury and are sensitive markers of intestinal inflammation . Calprotectin constitutes over half of the soluble cytosol proteins in neutrophil granulocytes and plays a central role in neutrophil defense, and its fecal level correlates well with the numbers of neutrophils infiltrating the intestinal mucosa and the overall severity of GI inflammation. High levels of fecal calprotectin can differentiate between patients with inflammatory or neoplastic bowel diseases and those with irritable bowel syndrome. Calprotectin and Lactoferrin

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