Safety pharmacology tier 2

safety oharnacology Tier 2 - Renal and GI studies

1. Safety pharmacology Tier 2 Ragini Anil Dani 2 nd semester PJLCP

2. Tier 2 Safety P’cological Studies Also called as Supplemental pharmacological studies. Used to access potential adverse pharmacodynamic effect organ system function not addressed by core battery studies. It includes Renal/Urinary system Gastrointestinal System Other Organ system Skeletal system Immune and endocrine functions

3. Renal studies in safety pharmacology The kidney is a complex excretory organ Playing role in Fluid and electrolyte balance Control of blood pressure Removal of waste products Drug disposition Endocrine function

4. 30-40% drugs invented can cause renal injury Still the safety P’cological studies related to renal system in preclinical system is small Renal studies in safety pharmacology

5. Methods to assess drug effects on Renal Function It should cover Excretory function Hemodynamic function Endocrine aspects In-vivo models are frequently used.

6. Models Employed In-Vivo Mammalian models In-Vivo Non-Mammalian models In-Vitro Models In- Silico Models Glomerular Function Tubular Function Hemodynamic Function

7. In-Vivo Mammalian Models Rats, Dogs, Pigs are commonly used Conscious, Freely moving animals Analysis of urine and plasma are done. Urinary bladder catherterization and metabolism cage are used

8. Metabolism cage

9. a. Glomerular Function Clearance measurement of endogenous and exogenous small molecules (urea, creatinine, 2-MPT, inulin, cystatin C, iohexol , or iodixanol ) analysis of plasma and urine samples The noninvasive clearance (NIC)-kidney device that when mounted on the back of laboratory animals enables the transcutaneous measurement of the elimination kinetics of the fluorescent renal marker FITC- sinistrin This allows the measurement of the clearance of FITC- sinistrin from the plasma in real time without the need for any blood Sampling

10. b. Tubular Function allows the identification of the functional status of particular nephron segments includes visual assessment of urine (color, clarity), volume, specificgravity or osmolality, pH, quantitative or semiquantitative protein, and glucose content. Dipstick test strips assess other parameters, such as ketones, bilirubin, urobilinogen , hemoglobin, etc

11. c. Hemodynamic Function Direct renal blood flow measurment requires the placement of a flow probe around the renal artery conducted in large animal species such as dogs ,( mini-)pigs, and nonhuman primates usually coupled with systemic blood pressure monitoring, using a pressure catheter placed into an artery Another hemodynamic endpoint is the renal vascular resistance (RVR), calculatedas the ratio between RBF and mean arterial pressure (MAP) RVR can be increased in case of renal dysfunction or in case of systemic hypertension

12. 2. Indirect renal blood flow measurement para- aminohippuric acid(PAH ) clearance test all PAH passing through the kidneys appears in the urine PAH clearance is directly proportional to the rate of plasma flow through the kidneys If the hematocrit is known, the total renal blood flow can be easily calculated from the eRPF value

13. In Vivo Non-mammalian Models The zebrafish (Danio rerio ) larva has gained increasing interest over the last decade as an alternative to mammalian in vivo models. The zebrafish kidney is genetically and morphologically close to that of mammals measurements of FITC-inulin intensity in the caudal artery and excreted FITC-inulin validated using gentamicin and high salt loading high-throughput screening, visual transparency, low cost, and genetic manipulation

14.   The kidney is highly complex, composed of a filter unit and a tubular segment, together containing over 20 different cell types . Nephrotoxicants cause injury by selectively injuring specific cell types or by nonselectively injuring multiple cell types within the kidney, depending on their mechanism of action Assessment of the potential nephrotoxic effects of pharmaceutical compounds on different components of the kidney therefore requires the use of different model systems . in vitro model that recapitulates the in vivo response of renal cells to nephrotoxicants requires appropriate expression of the transporters and receptors that interact with the drug of interest . In addition to detecting expression of transporters at the transcript and protein level by quantitative PCR and immunohistochemistry, the function of transporters and endocytic receptors can be investigated by assessing the effect of fluorescent substrates and transport inhibitors In Vitro Models

15. In Silico Models The SAPHIR project (a Systems Approach for Physiological Integration of Renal , cardiac, and respiratory functions), initiated in 2008 targeting the short- and long-term regulation of blood pressure, body fluids, and homeostasis of the major solutes for renal and urinary disorders , the predictivity was not very high

16. A survey conducted in the top 15 pharmaceutical companies

17. Gastrointestinal Safety Pharmacology 2-3% discontinuation of drug project Related ADRs and AEs are not life threatening but hampers quality of life for patients 18% of total ADRs Overuse of NSAIDs in US is a reason behind >1,00,000 hospitalizations and 17,000 deaths in a year (2003) Hence there is a need for improved and more extensive GI Screening

18. Methods to Assess Drug Effects on Gastrointestinal Function Assessment of Gastric Emptying and Intestinal Motility Assessment of Gastric Secretion Models of Nausea and Emesis Models of Gastrointestinal Absorption

19. Assessment of Gastric Emptying and Intestinal Motility In Vitro Models subcellular, cellular, tissue, or whole organ to study the pharmacological effects and mechanism of action of drugs on GIT Stomachs or isolated segments of GIT from small laboratory animals can be suspended in organ baths to investigate the mechanisms underlying a novel drug target and to validate physiological or pharmacological responses . Typically muscular strips or whole segments from the GIT are suspended in organ baths containing a suitable nutrient solution held at 37 C and gassed with carbogen study the enteric nerves is done by measuring changes in motility caused by electrical field stimulation (EFS)

20. In Silico Organ Modeling Computational fluid dynamic (CFD) techniques ( Ferrua and Singh 2010 ) predict the dynamic effects of luminalcontents on GI motility, luminal content mixing, and propulsion This model showed a complex and highly three-dimensional flow profile during gastric contraction

21. In Vivo Models I n rodents by administering meals containing markers such as phenol red, barium sulfate (BaSO4 ), or charcoal, subsequent to the administration of the test compound. The test meals can be used either as an indicator of liquid (phenol red) or solid transport (charcoal , BaSO4 ). For gastric emptying assessment, the stomach is weighed,after definite time as its weight directly correlates with its content. Any difference in the gastric weight between treatment groups indicates altered gastric emptying.

22. For intestinal motility assessment, the intestines are removed (usually from duodenum to ileum) and the length of intestine filled with charcoal or BaSO4 is measured and compared to the full gut length by visual inspection. The percentage of intestinal length filled by the test meal is proportional to the intestinal transit. Any difference between treatment groups indicates an alteration in the intestinal motility (increase or decrease ). With phenol red, the intestinal transit is evaluated by measuring the spectral absorbance in specific subparts of the gut.

23. Assessment of Gastric Secretion In Vitro Models performed on mucosal cell preparation from stomach mucosa obtained by enzyme dispersion and separated by centrifugation to separate parietal cells (H+ secreting cells ) Measures the H+ concentration and pepsin secretion in the gastric effluent

24. In Vivo Models Rats fasted for 24 hrs prior to pyloric ligation.  Randomly divided into 5 groups of 3 animals each.                                      Group I    : Control vehicle                                       Group II   : Standard drug (Omeprazole)                                       Group III  : ‘x’ concentration of test drug                                       Group IV  : ‘2x’ concentration of test drug                                       Group V  : ‘ 4x’ concentration of test drug Drugs administered once for 2 days and 30 mins prior to ligation Rats anesthetized with ether. Pyloric ligation procedure done. Rats  placed in separate cages and allowed to recover. 19 hrs after pyloric ligation, animals sacrificed by decapitation. Pyloric ligation method

25. Models of Nausea and Emesis In Vivo Models assessed in conscious animal models Ferrets and dogs are typically considered to be the best species for the evaluation of emesis, dogs being usually more sensitive to emetic stimuli compared to humans Rats and mice are not able to vomit, but can display some behavioral reactions that can represent nausea, such as gasping or pica Emesis evaluation in dogs, ferrets, or nonhuman primates usually consists of visual recordings of retching and vomiting events, and possibly premonitory signs such as licking, during a defined period following compound administration The number of events and the latency are the most frequent parameters reported.

26. Models of Gastrointestinal Absorption In Vitro Models Cell culture-based permeability screening models are used for the rapid assessmentof intestinal permeability of drug candidates The human monolayer of colon adenocarcinoma cells (Caco-2) model used to predict the intestinal absorption of orally administered drugs or measurement of flux of a marker molecule or active ion transport across epithelial cells . NSAID-induced mucosal damage has been demonstrated in gastric mucosa

27. In Vivo Models In situ absorption models have been used in the anesthetized rat, with intraluminal administration of the test drug and collection of inflow and outflow of an isolated segment of the gut In parallel, arterial blood is sampled and the disappearance rate of the substance is evaluated

28. THANK YOU!