Recommend NCE for development

Fig. 6.16. Role of Discovery MPK

than drugs in the human body, may be duplicates of naturally occurring substances, and are susceptible to many degradative processes. Proteins are quite large molecules with specificity of their amino acid sequence, disulfide bridges, tertiary structures (carbohydrates), three-dimensional conformation, isoforms of the same molecule, and other properties. These many structural features necessitate more testing in number, variety, and sophistication to ensure the integrity of the molecule especially in the manufacturing process and complicate measurement in the MPK studies.

In the past, drug discovery focused on finding the most potent lead compounds at a particular target. However, many compounds failed in development due to poor ADME properties. At the discovery stage nowadays, MPK is used via high-throughput screening to find lead candidates that have "drugability" properties or are "drug-like" to increase the chance of success in the development (Fig. 6.16) [5]. The studies are usually non-GLP compliant. The importance of identifying the physicochemical and the molecular components that dictate pharmacokinetics has been emphasized. The early understanding of the pharmacokinetic-chemical structure-activity relationship (PK-SAR), along with the pharmacological-chemical structure-activity relationship (PD-SAR), will increase the chance of success in finding a good drug candidate. The role of MPK at discovery is to predict if the drug will have acceptable pharmacokinetic properties in man; for example, it is bioavailable after oral administration, it is not extensively metabolized, the target tissue can be reached, pharmacological activity is achievable with blood concentrations that are attainable with reasonable doses. The figure at the right panel of Fig. 6.16 describes the sequence that is generally followed in the selection of lead candidates and characterization of a lead candidate's PK properties. A target is first identified in the disease process, followed by the screening and identification of analogues that modulate the target, which are called "hits." Once a considerable number of hits have been identified, two to four representative hits that show a promising pharmacological profile are selected as lead candidates. At this point, rank ordering takes place, and usually the one lead candidate that shows the most promise is chosen for optimization and assigned as a new chemical entity (NCE), with the others reserved as backups should the lead candidate fail. After this stage, the lead candidate goes through extensive profiling for ADME in parallel with drug safety studies and reconfirmation of pharmacological proof of concept including in vivo efficacy in animal disease model(s).

If a compound fails during the drug development process, it is vital that the reasons for failure are clearly understood as early as possible. Understanding the reasons would help in optimizing the appropriate PK/PD or metabolism properties that would enable the next series of compounds to be successful. The schema in Fig. 6.17 shows the sequential algorithm to analyze the various reasons of failures of drugs due to phar-macokinetic reasons, such as low bioavailability, short half-life, high or variable metabolism, high first-pass effect, excessive drug interaction potential, or poor tissues penetration. All these can lead to low and transient exposure of the drug, and thus the failure or lack of efficacy could be due to PK reasons. However, if the drug has favorable PK properties and still does not produce the desired effect, then the reason of failure is due to the lack of appropriate pharmacodynamics, such as poor affinity to target receptors or inappropriate target.

The aim of preclinical MPK studies depends on the stage of drug development the compound is at. Eleven possible

Fig. 6.17. Failure of NCEs

r GLP bioanalytical methods development c Analysis of drug and metabolites r PK profiling in two species c Usually toxicology species - rodent and non rodent o Safety margin assessment r Assessment of PK linearity o Prediction of dose related adverse events or lack of efficacy r Protein binding and erythrocyte-plasma distribution o Drug interaction potential c Determining the matrix for bioanalysis

c Target organ concentrations to assess distribution and toxicity Fig. 6.18. Preclinical DMPK to Support Development-1

types of preclinical studies or research questions are enumerated on these two figures (Figs. 6.18 and 6.19). As much information as possible should be obtained in animals and the laboratory to design the optimal human studies, to screen for the best drug candidates to move forward into humans, and especially to discontinue a molecule as early as possible to create more efficient and cost-effective product development. During development, traditional preclinical pharmacokinetic studies in animals, including toxicokinetics, will be carried out to support filing of an investigational New Drug (iND) application. Also, in vitro studies, such as isolated hepato-cytes or purified enzymes, might be used to assess the metabolic clearance of the lead candidate. When the drug enters the clinic, preclinical pharmacokinetics is then used to answer specific questions (e.g., does the compound show a drug interaction?). Hence, preclinical studies help in selecting the first dose in man, selection of the correct dosing regimen, and appropriate interpretation of toxicological studies. They also r Mass balance in the toxicology species c Metabolite identification and primary routes of elimination r In vitro and in vivo metabolic profiling c Identification of major active or toxic metabolites or intermediates o Explanation of toxicological differences between species r Identification of major metabolizing enzymes c Identification of isozymes involved in formation of metabolites and potential for drug-drug interaction r Enzyme inhibition and induction o Potential effect of drug candidate on other drugs r Formulation support studies c Optimizing drug substance r PK/PD correlation o Rational drug development

Fig. 6.19. Preclinical DMPK to Support Development -2

provide input in helping bridge historical toxicological data to new formulations of drug candidates. Preclinical MPK studies are GLP compliant.

The term toxicokinetics refers to the kinetics when compounds are administered to animal models at doses in the range of those used in toxicity studies, while pharmacokinet-ics refers to the kinetics of compounds given to humans or animal models at lower (i.e., pharmacological) doses (Fig. 6.20). Every compound that is identified as a potential lead candidate will undergo a battery of safety/toxicity screens prior to being considered as a NCE. Typical studies include genotox-icity, carcinogenicity, mutagenicity, ion channel safety (hERG potassium channel), reproductive toxicity, and target organ toxicity. The toxicokinetic support for these toxicology studies may help to determine the concentrations that cause toxicity, choose appropriate species for research questions, examine species variability, evaluate exposure-toxicity relationships, assess the safety margin, and define the therapeutic window.

Safety margin of a compound can be expressed as the ratio of drug exposure (Cmax or AUC) at NOAEL doses in the most sensitive animal species to the corresponding parameter in human at a particular dose (Fig. 6.21). Allometric scaling can be applied to estimate human exposure if this is used for first-in-man dose selection. Modeling and simulation technologies can be used to generate the exposure if particular doses or dose regimens have not been tested in humans. Assumptions on PK linearity and others may be required. Because the safety margin is assessed across different species, the total drug concentrations should be converted to unbound fraction, if there is significant species dependency on protein binding. On the same plot, exposures that produce side effects either benign or serious, such as hERG interaction (toxicokinetic data), and exposures that produce the desired effects (pharmacological data) can also be presented. This kind of plot k Objectives o Systemic exposure following single & multiple dosing (safety margin of parent & metabolites); o Dose Proportionality c Sex related differences in exposure

- Types of toxicokinetic studies c Single dose ranging toxicokinetic studies o Four-week, 13-week, 26-week toxicokinetic studies in "two species" c Two year carcinogenicity studies in rats & mice at 3 doses

- Role of Toxicokinetics c Selecting appropriate animal species for toxicity study

> E.g., same metabolites as human & sufficiently high c Assisting dose selection

> E.g., dose non-proportional c Comparing findings across species

> E.g., species-specific toxicity c Comparison of findings from toxicity studies employing different routes c Relating exposure to toxicity findings

> E.g., differentiate lack of toxicity from lack of exposure c Assessing if plasma concentrations change over time course of dosing

Fig. 6.20. Toxicokinetics to Support Development

Based on preclinical (pharmacology / toxicology) information and projected human exposure using simulation

Fig. 6.21. Safety Margin provides an integrated view on margin of safety and a means of dose finding based on animal pharmacology both in vivo and in vitro, toxicokinetic data, and human PK.

MPK plays a central role in discovery and preclinical screening phases to identify the ideal physicochemical (PC), bioavailability, biopharmaceutical, pharmacokinetic, and pharmacodynamic characteristics among the candidate compounds. Figures 6.22 and 6.23 present 12 representative possible problems with the 4 ADME areas, including PC characteristics and their physiologic relevance to product selection in the product development process. Absorption issues revolve mostly around the compound's bioavailability, especially its variability. Distribution examines both protein binding in the blood and tissue effects, which can impact both efficacy and toxicity. Metabolism involves a compound's degradation or activation, including metabolites and their effect on drug interactions, efficacy, and toxicity. Elimination focuses on half-life and dosing impacts. By applying PC profiling, preclinical PK and metabolite screening, and safety evaluations early, it minimizes the probability of candidate failures in clinical development due to poor solubility and stability, lack of high permeability, absorption from the gastrointestinal tract, inadequate PK characteristics, short duration of action, metabolite(s), covalent binding, cofactor depletion, and so on.

A key question in the design of first-in-man studies is how to select an appropriate starting dose: too high a dose may lead to severe adverse events (AEs), and too low a dose may require many dose escalation steps before pharmacological evidence of activity is observed. Safety margin assessment


ADME Issues

Physiological relevance c Low oral bioavailability (<20%)

p Large food/beverage effect

> Larger variability

Distribution c Poor target tissue penetration o High plasma protein binding

<~: High tissue binding

> Lack of efficacy

> Displacement potential

> Site-specific toxicological concerns

Fig. 6.22. ADME Factors as Development Issues -1

ADME Issues

Physiological relevance


Elimination r Autoinduction c Enzyme induction o Enzyme inhibition (including the mechanism based inhibition)

c Multiple active metabolites Reactive / toxic metabolites c: Short persistence in plasma o Long half-life

> Loss of efficacy

> Loss of efficacy for concurrent medication

> Toxicity of concurrent medication

> Hard to predict drug response

- Toxicity

> Inconvenient multiple dose regimen

> Excessive accumulation

Fig. 6.23. ADME Factors as Development Issues -2

- Css (steady state concentrations)

O IC50 or EC50 or MIC = Cssav c EC50 in animals, when scaled across species, has shown remarkable correlation with negligible slope for either pharmacodynamics or toxicity, thus in vitro EC50 or values from animal studies can be used

- Tau can be based on several parameters but usually QD is dosing interval of Choice or 24 hours

- CL needs to be scaled across species. Common methods are:

v Allometric scaling ^ Campbell method o In vitro metabolic prediction of CL

Fig. 6.24. First Dose in Man: Allometric Scaling -1

based on the ratio of exposure at NOAEL dose in animals and human exposure at a particular dose estimated according to allometric interspecies scaling may be a useful guide. Figures

6.24 and 6.25 enumerate key principles in determining the first dose in man [11, 12].

Interspecies scaling of PK data to predict human PK is based on similarities in physiology and anatomy among species. Allometric scaling can be conducted using the following relationship: CL = Wtb, where the total clearance is scaled based on the body weights of various species. Similarly, volume of distribution can also be scaled, which is generally proportional to the body weight. Generally, the exponent, b, has a value of 0.75 for clearance and 1 for volume of distribution.

The second method that was used was the Campbell method where scaling method uses the body weight and the maximum life span. The projected dose can be calculated according to the equation Dose = CL • Css • tau, where tau is the dosing interval (24 hours). Because the pharmacological effects have been shown to be similar across species, the in vitro IC50 can be used for the target concentration for the efficacious dose, while the concentration at the NOAEL (no adverse effect level) in toxicity studies can be used to predict the maximum tolerated dose in humans.

Fig. 6.25. First Dose in Man: Allometric Scaling -2

" Single Dose Studies - Objectives:

c Safety and tolerability in healthy subjects o PK and its metabolites in plasma o Urinary excretion of the drug and its metabolites c PD markers of safety & efficacy o Initial insights in to putative human metabolites o Initial PKPD modeling o Can include effect of gender or food in these studies

■< Multiple Dose Study - Objectives o Safety and tolerability in healthy subjects o Steady-state PK parameters such as fluctuation

& accumulation ratio o Refinement of the PK, PD and PK-PD relationship k FIM studies: Oncology & HIV conducted in patient populations.

Fig. 6.26. First in Human Study: Single/Multiple Doses

Usually, single dose, first-in-man (FIM) studies are designed as placebo-controlled, double-blind, randomized, parallel-group studies involving several groups of 8-12 healthy volunteers (males and/or females) that receive escalating doses (Fig. 6.26). Initial doses are based on allometric scaling with at least 1/10 to 1/20 of the NOAEL dose. Dose escalation is usually based on various methods including Fibonacci series or PK/PD driven, where the concentration of the next dose is predicted based on concentration of the prior dose and compared with a target for effective or safe concentration based on animal data. The studies evaluate safety, tolerability, pharmacokinetics, and pharmacodynamics in the first-in-man studies. The stop dose can be based on the maximum tolerated dose or the stop dose criteria based on the exploratory IND guidance by the U.S. FDA [13].

Multiple doses studies are of similar design, but their duration is usually based on the pharmacokinetics of the drug, so that steady state may be achieved on the anticipated duration of responses for the pharmacodynamic marker. These studies usually have 3-4 dose groups and the dose escalation and regimen based on single-dose study. Although these studies are usually conducted in healthy subjects and they can be extended to patient population. FIM studies for oncology and HIV should be conducted in patient populations because of the toxicity of the drugs and to accelerate development of the compound for the potentially life-extending drugs.

The objectives of mass balance studies include recovery of radioactivity in administered dose, excretion routes (urine vs. feces) of radioactivity in administered dose, and metabolite profile of excreta. Mass balance studies are usually singlecenter, open-label, single-dose studies after oral administration of the intended route in 6-8 healthy male volunteers [14, 15]. 14C is the most common radiolabel used. The amount of radioactivity can not exceed 100 |Ci and is based on the dosimetery calculation, taking into account the 14C mass balance studies in two animal species and the animal quantitative whole-body autoradiography (QWBA) data.

In these studies, a series of samples of blood and excreta are collected to assess the distribution (in RBC and protein binding) and elimination of radioactivity after dose administration and to determine the PK. Plasma (blood), urine, and feces samples are collected for up to several days after dose administration, provided that discharge criteria have been met (i.e., all radioactivity is taken into account [>90%]). Metabolic profiling of plasma, feces, and urine is performed to determine the metabolic fate of the drug. The mean (±SD) 14C radioactivity in plasma and blood over time and the mean (±SD) plasma concentration over time profiles of M100240 and MDL 100173 (active metabolite) following oral

10 15 20 Time (hours)

Shah et al. Am J Therap 2003;10:356-362.

Fig. 6.27. Mass Balance Study k Objectives o For Innovator compounds BA/BE studies allow bridging the clinical data throughout drug development ° For Generic drugs BA/BE studies are pivotal data for approval

General Features c Two-way crossover design o "n" determined by reference treatment variability o Study population relatively homogeneous o PK sampling adequate to capture early and full exposure metrics

^ Cmax & Tmax: rate of abSorption

> AUC: extent of absorption o Analysis involves individual PK metric estimation o Bioequivalence criteria (Confidence Interval Methods)

> mean ratio (T/R) and associated 90% CI of AUC & Cmax are within 80% - 125%

> Sustained release formulation should include Cmin

Fig. 6.28. BA/BE Studies administration of 14C M100240 (25 mg/50 |Ci) to 6 healthy male subjects for a mass balance study are presented in Figure 6.27.

Bioavailability and bioequivalence studies measure how much of the drug gets into the body and how fast is the absorption (Fig. 6.28). The pharmacokinetic parameter, area under the curve (AUC), explains the extent of absorption, and the PK parameter, C , explains the rate of absorption.

Although Tmax can also explain the rate of absorption, this parameter is not used for determining bioequivalanece. The role of BA/BE studies in product development differs for innovator versus generic drugs. That is, they are pivotal for approval for generic drugs, but they can serve as bridging studies for new formulations of innovator drugs.

The bioequivalence of the test formulation (test) versus reference formulation (ref) is assessed by examining the logarithmically transformed PK parameters (AUC and Cmax) using an analysis of variance (ANOVA) model with subject as random effect and treatment regimen as factors. Point estimate and 90% confidence interval are calculated for the geometric mean ratio of the test to reference. If the 90% confidence interval for the geometric mean ratio falls within (0.8, 1.25), then the formulations are considered to be bioequivalent. For sustained-release formulations, the calculations will include the minimum concentrations as well. Other design issues include sample size (n), which is dependent on the treatment variability (more variability means more patients). Study populations are usually quite homogeneous to assist in reducing variability and permitting smaller sample sizes. PK sampling needs to cover early and later metrics for full exposure. Therapeutic equivalence is determined when, instead of phar-macokinetic parameters, clinical or safety end points are used in the calculations.

In the previous sections, most of the discussion of PK properties was limited to the behavior of the drug after oral administration with immediate release formulation, which is usually the most desired route of administration. However, as can be seen in the marketplace, there are various other routes of administration for a drug. Figures 6.29 and 6.30 review three alternative formulations, transdermal, extended release, and inhalation, including key features (advantages and design issues) and a few product examples. For these other formulations, the pharmacokinetics can be used to bridge the information of an existing formulation to develop a new formulation (e.g., extended-release formulation). The extended-release formulation may be desired for a drug with probably a short half-life requiring multiple administrations


Key Features



c Evaluate release profile of drug from patch.

o Conduct skin metabolism & penetration studies.

c Compare BA/BE with respect to either IV or oral formulation.

c Can bypass first pass effect thus altering metabolic profile.

Progestagel Estrogen Nicoderm Duragesic

Extended Release

c Eliminate multiple doses & increase compliance. c Site specific absorption & metabolism issues.

c Drugs with short-half-life & poor absorption good candidates for extended release formulations. o Consistent & predictable performance without dose-dumping, in vitro /in vivo formulation correlation. o Effect of food can be substantial.






Fig. 6.29. Alternative Formulation Evaluation -1


Key Features



o Examine lung deposition, & gamma scintigraphy

SPECT imaging, & charcoal block studies. o Correlate particle size of formulation with site of absorption.

o Consider differences in DPI and MDI, solution and suspension MDI. c Account for extra variability. o Evaluate both oral and inhaled bioavailability; Ideal for drug to have low systemic exposure with low oral bioavailability, high protein binding to avoid systemic side effects. c Conduct lung metabolism studies o Long lung retention times & conduct binding studies in lung or evaluate lung residence time. o Inhaled drugs for systemic use should have low. lung metabolism and no long-term toxicity in lungs.




Fig. 6.3G. Alternative Formulation Evaluation -2

during the day. By improving the formulation, a more convenient and compliance-friendly once-daily or even less frequent formulation (e.g., bisphosphonates for osteoporosis) may be developed.

In order to avoid side effects of the drugs or inordinate first-pass metabolism, drugs may be administered to the target organ directly, such as in the case of inhaled drugs or ophthalmics. The use of inhaled corticosteroids in treatment and management of asthma have significantly reduced the systemic side effects, such as cortisol suppression observed after oral administration. Drugs with poor and variable oral bioavailability due to the first-pass effect can be challenging (e.g., selegiline). However the proposed use of selegiline in a transdermal patch can not only reduce the variability but also has led to the investigations for possible use of the drug in new indications such as Alzheimer disease [16].

Objectives of dose proportionality include dose-exposure relationship, changes in ADME in relation to dose, and accumulation of multiple doses. Dose proportionality of a dose-dependent PK metric implies that the surrogate measure divided by dose (e.g., dose-normalized AUC) is independent of dose (Fig. 6.31). An analysis of variance (ANOVA) can be performed on the log-transformed, dose-normalized surrogates (i.e., AUC and Cmax), using dose as a fixed effect rather than a continuous variable. If a crossover design is applied, then a repeated measures ANOVA would be used. If the F-test for the treatment effect is not statistically significant, one r Objectives o Dose-exposure relationship O Changes in ADME in relation to dose C Accumulation if multiple doses r General features o X-way crossover designs (single or multiple dose)

o Statistic analysis

> ANOVA with dose-normalized AUC or Cmax

> Power model

20 r

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