IN VIVO PK SCREENING - 2

üUnderstanding of in vitro-in vivo correlation and frontloading PK/PD is crucial for the compound selection even at an early stage. 

ü Obviously, an excellent PD data can save a poor PK as long as the PD effect is notable. 

In Vivo ADME studies are mostly custom-designed.  Often these studies are performed in multiple species by administering the test compounds by variety of routes (includes oral, intravenous, dermal, intraperitoneal, inhalation to name a few).  These in-life studies provide information for:

·        mass balance

·        metabolite isolation and characterization

·        dosing frequency

·        routes of administration

·        exposure

·        interspecies comparisons

·        PK/TK

·        tissue distribution studies to determine clearance routes and rates

·        tissue half-life

·        Potential sites of toxicity after systemic exposure.  

The basic assumption in all pre-clinical ADME studies is that the administered NCEs and dose levels are not toxic and do not cause any harm to the welfare of the animals.

In general rats of similar age and weight are chosen for the experiments.  Experimental procedures are kept strictly in accordance with the related ethics regulations.  In many studies, all the subjects are kept in an environmentally controlled breeding room (temperature maintained at about 25 ± 2 oC and with a 12 h light / 12 h dark cycle) for at least once week before starting the experiments and fed with standard laboratory food and water.  Prior to each experiment, the rats are fasted for 12 h with free access to water.

RANK ORDER OF COMPOUND EXPOSURE:  A small cohort of animals (selected number), is administered compound orally (p.o.) or by intraperitoneal injection (i.p.) at a single dose (5 - 50 mg/kg). Blood samples are collected at 20 and 120 minutes. The plasma samples are analyzed which provides a snapshot of compound exposure as the area under the curve (AUC(20-120 min)), providing a rank order of estimated AUC values to prioritize compounds for further investigation. 

Based on the concentration vs. time data, pharmacokinetic parameters such as body clearance, volume of distribution and bioavailability can be calculated, information which is used to refine in vivo experiments with the same series of drugs in the development.

MATERIAL BALANCE STUDY:

• Groups of male and female rats at a single dose level (equal number of male and female rats are used)
• Daily collection of urine and feces for 7 days
• Collect blood and major internal organs, and retain carcass (corpse) upon necropsy
• Total radioactivity level in each sample is assayed and the recovered dose is compared with the administered dose. The percentage of distribution of the administered dose in urine, feces, and each major internal organ and the half-time for excretion are calculated.

A necropsy is a surgical examination of a dead body, most commonly a dead animal, in order to learn why the animal died. A more common word for necropsy is autopsy. Either way, it's the dissection of a corpse performed to learn something about the cause of death or about a particular disease.

PLASMA LEVEL AND BIO-AVAILABLITY STUDY:

The objective of this study is to examine the pharmacokinetic profiles of the chemicals under test. These studies can be conducted by using unlabeled chemicals provided that appropriate analytical procedures (e.g., HPLC) are available.

·         Groups of 5 male and 5 female rats per route of dosing

·         One group is dosed intravenously and the second group is dosed by an extra-vascular route (e.g., oral, percutaneous)

·         The dosages for the 2 groups need not be identical

·         Blood is sampled at frequent intervals (e.g., 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hr), and plasma levels of the chemical are assayed.

·         The plasma- concentration-time data are analyzed to obtain the pharmacokinetic profile, including the area under the curve (AUC). Comparison of the AUCs after extra-vascular and intravenous dosing, after correcting for the dosage, provides a measure of bioavailability.  Alternatively, if a radioactive chemical is used, comparison of the administered doses excreted in urine can be used to calculate the approximate bioavailability of the orally dosed chemical.

IN VIVO PHARMACOKINETIC STUDY (PLASMA PHARMACOKINETICS):

Depending up on the study, rats were randomly assigned for pharmacokinetic investigation.  Test compound is administered by i.v. injection via the lateral tail vein and orally at the dose of for example, 10 and 20 mg/kg, respectively. At the time points of 0 (pre-dose), 5, 15, 30, 45, 60, 90, 120, 240 and 480 min post injection / administration, blood samples (0.5 mL) are collected in heparinized (add heparin to (blood or a container about to be filled with blood) to prevent it from coagulating) tubes from the orbital vein, and then centrifuged to obtain plasma. The plasma is stored at −70 °C prior to analysis by HPLC.

METABOLISM STUDY IN RAT URINE AND FECES:

Similar to above study, but here feces and urine samples are separately collected at 24 h after administration of test compound.  All the samples were stored at -70 ^oC prior to analysis.

TISSUE DISTRIBUTION STUDIES:

For tissue distribution study, number of rats is first divided into several groups randomly and test compound is administered intravenously through the tail vein at a particular dose, say 20 mg/kg.  After injection, the rats are sacrificed at 0.5, 1.0, 2.0, 4.0 and 8.0 h following administration and the tissue specimens including lung, liver, heart, spleen, stomach, small intestine, brain, thymus, muscle, fat and kidney are collected. Tissue samples are then rinsed in saline and blotted dry with filter paper, and then weighed for wet weight and homogenized in ice-cold physiological saline solution (500 mg/mL). The obtained tissue homogenates are stored at −70 °C until analysis performed.

ALTERNATIVE ROUTES OF ADMINISTRATION:

Alternative routes of administration, typically intraperitoneal (i.p.) or subcutaneous (s.c.), may be used depending on the targeted type of treatment and indication or to avoid first-pass metabolism in the liver occurring after absorption from the gut wall when oral dosing is used.

REPEATED DOSE STUDY:

Other typical experimental set-ups include a repeated dose study, where the drug is administered once a day for up to two weeks. This experimental design can be used to find out not only the drug pharmacokinetics but also pharmacokinetic changes in the drug, for example, in a situation where the drug metabolism is either induced or saturated.

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When the bioanalysis has been completed and the results processed to gain plasma concentrations, plasma concentration vs. time data is then input into a software program for the calculation of PK parameters.

Pharmacokinetic parameters calculated include:

The area under the plasma concentration–time curve (AUC) is first calculated.

C_max: After the drug administration, the highest concentration obtained is considered the peak plasma concentration.

T_max:  Time take for peak plasma concentration is T_max

C₀: Concentration at time zero

V_d: Volume of distribution.  The apparent volume to which the mass of compound is distributed in the body at any given time.

CL: Clearance.  This value describes the tendency of the NCE to disappear from plasma.

T_1/2: Half-life. Time taken for the plasma concentration of an NCE to halve.

BA: Bioavailability.  This is the fraction of the NCE that reaches the systemic circulation unchanged.

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WHAT IS A GOOD PK PROFILE OF A PRECLINICAL CANDIDATE THAT IS BEING CONSIDERED FOR TAKING INTO DEVELOPMENT?

Ideally, preclinical candidate should,

    have acceptable solubility for development; 

    be completely absorbed, preferably via passive absorption; 

    have high bioavailability (e.g., F>50%) for oral drug; 

    have a low plasma clearance CL (e.g, <30% blood flow), long half‐life (t1/2) (e.g., >6 hrs), and  acceptable distribution volume; 

    have a linear kinetics, i.e., exposure proportional to dose and a clear PK/PD correlation; 

    be eliminated by several pathways, i.e., renal excretion and hepatic metabolism, also  metabolised preferably by more than one enzyme for de‐risking DDI; 

    have a simple metabolite profile, with no reactive metabolite; 

    have no obvious CYP and major transporters like P‐gp inhibition or induction or low DDI potential;  and 

    have a sufficient or at least acceptable safety margin (safety margin >10x, depending  on different  therapeutic targets).