toxicokinetic evaluation and pharmacokinetic.pptx

Toxicokinetic evaluation in preclinical studies & Saturation kinetics Importance and applications of toxicokinetic Studies

1. Toxicokinetic evaluation in preclinical studies & Saturation kinetics Importance and applications of toxicokinetic Studies NAME :Nittal vekaria Roll no: 48 K.B.I.P.E.R

2. Introduction • Toxicokinetics (TK) is defined by The International Conference on Harmonization (ICH) as ‘the generation of pharmacokinetic data to design, conduct & Interpretation of drug safety evaluation studies , and it as an integral component in the (guideline S3A-S3B ) conduct of non-clinical toxicity studies • Toxicokinetics deals with absorption, distribution, biotransformation and excretion of chemicals • In these studies a minimum of two animal species are employed, as per regulation of FDA. Rodents(rat and mice) Non-rodents(dog)

3. Need of toxicokinetic studies • Generation of kinetic data for systemic exposure and toxicity assessment of the drug. • Safety/risk ratio. • Important in drug development stages especially in preclinical stage. • Useful for the setting safe dose level in clinical phases. • TK evaluation is useful in selection of dose, alternative dosing route , evaluation of toxicological mechanism

4. Objectives Primary objective • To describe the systemic exposure achieved in animals and its relationship to dose level and the time course of the toxicity study. Secondary objectives • To relate the exposure achieved in toxicity studies to toxicological findings and contribute to the assessment of human safety clinically . • To provide information which, in conjunction with the toxicity findings contributes to the design of subsequent non-clinical toxicity studies .

5. To support the choice of species and treatment regimen in nonclinical toxicity studies These objectives may be achieved by the derivation of one or more pharmacokinetic parameters from measurements made at appropriate time points during the course of the individual studies These measurements usually consist of plasma (or whole blood or serum) concentration for the parent compound and or metabolite and should be selected on a case by case basis Following parameters are usually measured - maximum plasma concentration(C) - Area under Curve (AUC) of plasma concentration(exposure) versus time - Time to reach maximum plasma concentration ( Tmax )

6. TOXICOKINETIC STUDY GOALS A). Pretoxicology study • recommend selection of species • route selection • develop data for dose selection B). Toxicology study – provide estimate of internal dose – determine effect of age and multiple exposure

7. The toxicology profile include: 1. Acute/short-term toxicology studies (7-28 days or 1 month duration) a) acute oral toxicity b) Acute dermal toxicity c) Acute inhalation d) Skin sensitization 2. Sub-chronic toxicology studies (3 or 6 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

8. 3.chronic\long term toxicology studies (12 months duration) a)Chronic oral toxicity b) Chronic dermal c) Chronic inhalation d) Skin sensitization 4.Long term test, studies also including a)Carcinogenicity studies 18 to 24 months b) Mutagenicity studies or genotoxicity

9. GENERAL PRINCIPLES OF TOXICOKINETICS TO BE CONSIDERED 1. Quantification of exposure 2. justification of time points of sampling 3. Setting of dose levels Low dose levels Intermediate dose levels High dose levels 4. Extent of exposure assessment in toxicity studies 5. Complicating factors in exposure interpretation 6. Route of administration. 7. Determination of metabolites 8. Statistical evaluation of data. 9. Analytical methods 10.Reporting

10. 1. Quantification and extent of exposure • The exposure might be represented by plasma concentrations or the AUCs of parent compound and sometimes by tissue concentrations . • Helps in the interpretation of similarities and differences in toxicity across species, dose groups and sexes. • To achieve relevant exposure at various dose levels in the animal toxicity studies. • Species differences in the pharmacodynamics of the substance should also be taken into consideration because sometimes it may have other effects. • This information may allow better interspecies comparisons than simple dose/body weight (or surface area) comparisons.

11. 2. Sampling point • The time points for collecting body fluids should be frequent. • No more than 10% of circulating volume can be taken. • Sample size is typically 0.25–0.50 ml/ day in rodents and upto 1ml /day in non rodents. • Sampling times vary based on the presence of pharmacokinetic data , but are often taken 0.5, 1.0, 2.0, 4.0, 8.0, 12.0 and 24.0 hr post-dose with only the parent drug generally being measured

12. 3. Dose level toxicity testing • The setting of dose levels in toxicity studies is largely governed by the toxicology findings and the pharmacodynamic responses of the test species. 1. Low dose levels - At the low dose, preferably a no-toxic-effect dose level , the exposure in the animals of any toxicity study should ideally equal or just exceed the maximum expected inpatients. 2. Intermediate dose levels - It must be between the high and low dose level. 3. High dose levels - The high dose levels in toxicity studies will normally be determined by toxicological consideration.

13. 4. Extent of exposure •Systemic exposure should be estimated in an appropriate number of animals and dose groups to provide a basis for risk assessment . •Concomitant toxicokinetics may be performed either in all or are presentative proportion of the animals used in the main study or in special satellite groups. •Both male and female animals are utilized in the main study it is normal to estimate exposure in animals of both sexes unless some justification can be made for not so doing. •Toxicokinetic data is not mandatory for studies of different duration if the dosing regimen is essentially unchanged.

14. 5. Ratifying factors on study to be considered • Species and sex differences had their effect on toxicokinetics . • There are other factors to be considered in this study is protein binding tissue uptake receptor properties metabolic profile • Systemic exposure may be decreased by protein binding and tissue uptake. •Due to the metabolism there will be formation of pharmacological active metabolites, the toxic metabolites and antigenic biotechnology products metabolites.

15. 6. Route of administration • For instance orally administered drugs bioavailability time is more than other routes. •If the drug is intended to administer through oral route then oral toxicity should be checked. •If any drug administering route is already established and new route of administration is going to establish it is necessary to ascertain whether changing the route will significantly reduce the safety margin. •In this case focusing on local toxicity is essential . • The alternative route of administration like inhalation, topical and parenteral delivery should be based on pharmacokinetic properties of a substance

16. 7. Metabolite determination • Many of the cases systemic exposure and toxic effect consider on the basis of parent drug concentration. •If it is a 'pro-drug' and the delivered metabolite is acknowledged to be the primary active entity. • Measurement of metabolite concentrations in plasma or other body fluids is especially important in the conduct of toxicokinetics . • If metabolites are >5% then determined . • Identification done by nuclear magnetic spectroscopy, mass spectroscopy.

17. 8. Statistical evaluation of data • The data should be evaluated statistically which allows assessment of the exposure. • Toxicokinetic values are normally calculated as mean . • Consideration should be given to the calculation of mean or median values . • If data transformation (e.g. logarithmic) is performed, a rationale should be provided.

18. H. Analytical methods • Regulatory authorities expects that analytical methods used to determine plasma concentrations of pharmaceuticals are of adequate sensitivity and precision • For evaluation validated analytical methods used and conforms to Good Laboratory Practice (GLP) • Analytical methods used in such studies include gas chromatography, HPLC, LC, LC–MS, LC-MS-MS, and capillary electrophoresis. • Results are then analysed using a set curve-prediction package

19. i ) REPORTING • An outline of the analytical method should be reported or referenced • In addition , a rationale for the choice of the matrix analysed and the analyte measured should be given • The positioning of the report within the application will depend upon whether the data are specific to any toxicity study or are supportive of all toxicity testing.

20. 1. Toxicokinetic studies in Preclinicalstage: a. Safety assessment b. Single dose and rising dose studies c. Repeated-dose toxicity studies d. Genotoxicity studies e. Reproduction toxicity studies i . Studies of fertility ii. In pregnant and lactating animals f. Carcinogenicity studies 2. Toxicokinetic studies in clinical phases: a. Approaches to decrease the animal usage in toxicokinetic b. Dried blood spot technology c. Alternative approaches to animal models d. Physiologically based pharmacokinetic (PBPK) modeling

21. Toxicokinetic studies in Preclinical stage: 1. Safety assessment • Generally safety of a molecule can be performed in in-vivo systems. • Core studies in safety pharmacology comprise in vivo CNS,Cardiovascular and respiratory assessments. • Toxicokinetic assesment is not specifically included in the guideline • But it is very useful for the researchers to assess the systemic exposure of the molecule and its effect on it

22. 2. Single dose and rising dose studies • Results from single-dose kinetic studies may help in the choice of formulation and in the prediction of rate and duration of exposure during a dosing interval • These studies are often performed in a very early phase of drug development before a bioanalytical method has been developed (usually performed in rodents) • Plasma samples may be taken in such studies and stored for later analysis.

23. 3. Repeated-dose toxicity studies • To give support for phase 1 studies this study is carried out for four weeks in both rodents as well as non-rodents. • Help to support dose-selection for subsequent studies • The treatment regimen and species should be selected when ever possible with regard to pharmacodynamic and pharmacokinetic principles. • Performing further repeated dose studies in both rodent and nonrodents up to 6-12 months enable estimation of drug and its metabolite(s), kinetic parameter assessment as well as long term clinical exposure assessment . • Another point to be considered is a few drugs shows tolerance when it is administered repeatedly.

24. 4. Genotoxicity studies • Two in vitro studies and one in vivo study is essential to support development of drug. • In vivo investigations usually use a rodent micronucleus (bone marrow or peripheral erythrocytes) test or chromosome aberration (bone marrow cells) test

25. 5. Reproduction toxicity studies : • Reproduction toxicity measurements are taken in studies of fertility (rat) , embryo- foetal development (rat and rabbit) and pre-or post-natal development (rat) • The study suggest the need to adjust the choice of species ,study design and dosing schedule Studies of fertility • Assessment of fertility toxicity has very important, because most of the drugs used in fertility conditions so has to strengthen at that time. • Usually this can be done in rats.

26. In pregnant and lactating animals • There is a regulatory expectation for toxicokinetic data in pregnant animals, although no specific guidance is given. • Data from non-pregnant animals is useful to set dose levels • The limitation of exposure is usually governed by maternal toxicity • Toxicokinetics may involve exposure assessment of dams,embryos , foetuses or newborn at specified days. • Secretion in milk may be assessed to define its role in the exposure of newborns . • In some situations, additional studies may be necessary or appropriate in order to study embryo/ foetal transfer and secretion in milk.

27. 6. CARCINOGENECITY STUDIES • Sometimes drugs are used for longtime for curing purposes, this may lead to the toxicity or carcinogenicity. • So lifetime studies in the rodent are needed to support the long-term clinical use of pharmaceuticals and non-rodents can also be used. • Dose selection is usually determined as the maximum tolerated dose (MTD) , which is a 25-fold AUC ratio (rodent to human),or by dose-limiting pharmacodynamic effects, saturation of absorption, or a maximum feasible dose. • Monitoring should occur occcasinally i.e more than six months is not necessary. However, pharmaceutical companies use various strategies for such monitoring times (e.g. Weeks 1, 13, 26 and 52, Weeks 1and 26, or Weeks 26 and 52). • It should be noted that, owing to high variability in plasma concentration, toxico kinetic data from aged rats (above one year old) are not useful for estimating exposure. • Sampling times depend on available kinetic data but can range from full profile (up to 24 h) to limited time-points which are earlier stated.

28. Protocol for toxicokinetic validation Rationale selection of animal Preliminary study Method of Evaluation Main study Chronic Study Statistical Analysis Results and Reporting

29. IMPORTANCE AND APPLICTIONS OF TOXICOKINETICS • Generation of kinetic data for systemic exposure and toxicity assessment of the drug • Simply it means if the safety/risk ratio is balanced or safety is more then it will be used as good therapeutic agent • Important in drug development stages especially in preclinical stage. • TK evaluation is useful for the setting safe dose level in clinical phases.

30. • TK evaluation is useful in selection of dose dosing form alternative dosing route evaluation of toxicological mechanism. • TK studies also used to reduces the animal number. • TK evaluation is very important in drug development phase in both regulatory and scientific perspective

31. • TK data are practically used for the purpose of drug discovery such as lead-optimization and candidate-selection. Toxicokinetic data is important to know the toxic response to that of drug exposure obtained in drug development stages. It is used to set safe dose for clinical use of new drugs. Useful in the understanding of differences in responses or sensitivity between individual animals, genders, species or life stages , and supporting the extrapolation of findings in experimental animals to humans. Even though toxicokinetic evaluation is only a small part of the process of understanding the fate of a drug , it has a vital part in drug development a role that proceeds to advance.

32. Saturation kinetics

33. INTRODUCTION • kinetics -Branch of science concerned with measuring and studying the rates of reactions. • First order elimination kinetics - a constant proportion ( eg. A percentage) of drug is eliminated per unit time First order kinetics is a concentration-dependent process (i.e. the higher the concentration, the faster the clearance) • Zero order elimination kinetics : a constant amount ( eg. so many milligrams) of drug is eliminated per unit time zero order elimination rate is independent of concentration.

34. • Michaelis-Menten kinetics describes enzymatic reactions where a maximum rate of reaction is reached when drug concentration achieves 100% enzyme saturation. • Non-linear elimination kinetics is the term which describes drug clearance by Michaelis Menten processes, where a drug at low concentration is cleared by first-order kinetics and at high concentrations by zero order kinetics ( eg. phenytoin or ethanol).

35. SATURATION KINETICS • Also called as Nonlinear Pharmacokinetics • For a few drugs it is observed that the elimination of the drug appears to be zero order at high concentrations and first order at low concentrations. • That is 'concentration' or 'dose' dependent kinetics are observed. • At higher doses, which produce higher plasma concentrations, zero order kinetics are observed, whereas at lower doses the kinetics are linear or first order. • This occurs especially with drugs which are extensively metabolized. A typical characteristic of enzymatic reactions and active transport is a limitation on the capacity of the process.

36. DETECTION OF NON –LINEARITY INPHARMACOKINETICS There are several tests to detect non –linearity in pharmacokinetics but the simplest ones are: 1) First test :- Determination of steady state plasma concentration at different doses. 2) Second test :- Determination of some important pharmacokinetic parameters such as fraction bioavailability, elimination half life or total systemic clearance at different doses of drug. Any change in these parameters is indicative to non-linearity which are usually constant.

37. Drugs that demonstrate saturation kinetics usually show the following characteristics: 1)Elimination of drug does not follow simple first-order kinetics-that is, elimination kinetics are nonlinear . 2)The elimination half-life changes as dose is increased . Usually, the elimination half-life increases with increased dose due to saturation of an enzyme system. However, the elimination half-life might decrease due to "self"-induction of liver biotransformation enzymes, as is observed for carbamazepine. 3)The area under the curve (AUC) is not proportional to the amount of bioavailable drug. 4)The saturation of capacity-limited processes may be affected by other drugs that require the same enzyme or carrier-mediated system ( ie , competition effects). 5)The composition and/or ratio of the metabolites of a drug maybe affected by a change in the dose.

38. CAUSES OF NON-LINEARITY 1.Drug absorption 2.Drug Distribution 3.Drug metabolism 4.Drug Excreation

39. DRUG ABSORPTION • When absorption is solubility or dissolution rate limited e.g. griseofulvin- at high concentration in intestine • When absorption involve carrier mediated transport system. e.g. absorption of the riboflavin, ascorbic acid, cyanocobalamine etc. • When presystemic gut wall or hepatic metabolism attains saturation. e.g. propranolol, hydralazine and verapamil • These parameters affected F, Ka , Cmax and AUC • A decrease in these parameters is observed in former two causes and an increase in latter cause.

40. DRUG DISTRIBUTION • Non-linearity in drug distribution of drugs administered at high doses may be due to Saturation of binding sites on plasma proteins. e.g. phenylbutazone and naproxen -Saturation of tissue binding sites. e.g. thiopental and fentanyl • In both cases there is increase in plasma drug concentration. • Increase in Vd only in saturation of binding sites on plasma proteins

41. DRUG METABOLISM Non-linearity occurs due to capacity limited metabolism small changes in dose administration large variations in plasma concentration at steady state - large inter-subject variability Two important causes of non-linearity in metabolism are- • Capacity limited metabolism due to enzyme and cofactor saturation. e.g. phenytoin, alcohol, theophylline • Enzyme induction- Decrease in plasma concentration e.g. carbamazepine

42. DRUG EXCREATION The two active processes in renal excretion of a drug that are saturable are- Active tubular secretion. e.g. penicillin G II. Active tubular reabsorption. e.g. water soluble vitamins and glucoseSaturation of carrier systems • After saturation of carrier system decrease in renal clearance incase of I & increase in II. Half life also increases. • Other reasons like forced diuresis, change in urine pH, nephrotoxicity & saturation of binding sites. • In case of biliary excretion non - linearity due to saturation -Tetracycline & Indomethacin

43. SATURABLE ENZYMATIC ELIMINATION PROCESSES MICHAELIS MENTEN ENZYME KINETICS • It is also called as Capacity-limited metabolism or Mixed order kinetics. E + D ED E + M • Enzymes usually react with the substrate to form enzyme substrate complexes; then the product is formed. The enzyme can go back to react with another substrate to form another molecule of the product.

44. MICHAELIS MENTEN EQUATION The kinetics of capacity limited or saturable processes is best described by Michaelis-Menten equation. • If Cp is the concentration of drug in the plasma • Vmax is the maximum elimination rate. • KM is the Michaelis constant that reflects the capacity of the enzyme system. • When the drug concentration Cp is large in relation to KM (Cp >>Km), saturation of the enzymes occurs and the value for KM is negligible.

45. • The rate of elimination proceeds at a fixed or constant rate equal to Vmax. Thus, elimination of drug becomes a zero-order process and Eq. becomes: • When the drug concentration Cp is small in relation to the KM, therate of drug elimination becomes a first-order process. • Where: k' is a first-order rate constant • When given in therapeutic doses, most drugs produce plasmadrug concentrations well below KM for all carrier-mediated enzyme systems affecting the pharmacokinetics of the drug. • Therefore, most drugs at normal therapeutic concentrations follow first order rate processes

46. • Only a few drugs, such as salicylate and phenytoin, tend to saturate the hepatic mixed-function oxidases at higher therapeutic doses. • With these drugs, elimination kinetics are first-order with very small doses, mixed order at higher doses, and may approach zero-order with very high therapeutic doses.

47. References Barton, H.A., et .al (2006), The Acquisition and Application of Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety Assessments, Critical Reviews in Toxicology, 36: -935. OECD Guideline for Testing of Chemicals: 428 "Skin Absorption: in Vitro Method" (Adopted 31 April 2004) Gingrich, J., Pu, Y., Ehrhardt, R., Karthikraj , R., Kannan, K., Veiga -Lopez, A., Brahmankar , D.M. and Jaiswal, S.B. (2009) Biopharmaceutics and Pharmacokinetics. 2nd Edition, Vallabh Prakashan , Delhi, 399-401