Medicilon Pharmacokinetic Service

2016-06-05

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Medicilon’s preclinical research department has professional knowledge in drug metabolism and pharmacokinetics research, and provides high-quality data and fast turnaround to support various drug development, preclinical research and clinical research. Medicilon’s pre-clinical research services cover all aspects: protocol design, in vivo research, sample analysis, professional data analysis, IACUC review and application data preparation.
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Pharmacokinetic experiment Pharmacokinetic experiment

The role of pharmacokinetics in the research and development of new drugs

Preclinical pharmacokinetic research is to reveal the dynamic changes of drugs in the body through animal research methods in vivo and in vitro, obtain basic pharmacokinetic parameters of drugs, and clarify the processes and characteristics of drug absorption, distribution, metabolism and excretion.

Main application areas:

①In the evaluation of pharmacodynamics and toxicology, the concentration data of drugs or active metabolites and their related pharmacokinetic parameters are the basis for producing, determining or elucidating the effect or toxicity of drugs, and can be used as the drug’s effect on target organs (pharmacodynamics or Toxicity) basis;
②In the study of pharmaceutical preparations, the results of preclinical pharmacokinetic studies are an important basis for evaluating the characteristics and quality of pharmaceutical preparations;
③In clinical research, the results of pre-clinical pharmacokinetic research can provide relevant reference information for designing and optimizing clinical research dosing regimens.

Pharmacokinetic research content:

Dissociation constant determination
In vitro ADME determination
Active metabolite analysis
Dissociation constant screening
Metabolic stability screening
CYP inhibition/PAMPA/Caco-2
Drug protein binding rate
Metabolic Site Screening
Active metabolite screening
Plasma concentration-time curve
Absorption, distribution, metabolism, excretion process
Plasma protein binding
Biotransformation (type of transformation, transformation pathway and metabolic enzymes)
Effect on the activity of drug metabolizing enzymes (cytochrome P450)
The metabolic pathway, structure and metabolic enzymes of the main metabolites

Pre-clinical pharmacokinetic studies are of great value for designing and optimizing clinical research. For example, it provides important reference information for the design of clinical research programs such as dosage, time interval and route of administration.

Pharmacokinetic experiment procedure

To conduct non-clinical pharmacokinetic studies, the following basic principles should be followed:
(1) The purpose of the test is clear
(2) Reasonable test design
(3) Reliable analysis method
(4) The obtained parameters are comprehensive and meet the evaluation requirements
(5) Comprehensive analysis and evaluation of test results
(6) Specific analysis of specific issues

(1) Basic requirements

Test drugs
Pharmacokinetic studies for the purpose of revealing the pharmacokinetic characteristics of new drugs must ensure that the test drugs have stable quality.
Therefore, the basic requirements for pharmacokinetic research test drugs are: stable quality and consistent with the test drugs used in pharmacodynamics or toxicology research.
Number of test animals and tested animals
Preclinical studies of new drugs generally use adult and healthy animals. Commonly used are dogs, mice, rats, rabbits and guinea pigs.
The preferred animal should be consistent with pharmacodynamics or toxicology studies;
Kinetic studies should take multiple samples from the same animal, and try to avoid research methods that combine samples;
Innovative drugs should use two or more animals, one of which is a rodent and the other is a non-rodent;
For other types of drugs, one animal can be selected for experimentation;
Rabbits and other herbivorous animals should not be used for oral administration;
Try to experiment while awake.
When determining the required number of test animals, it can be calculated based on the limit of not less than 5 data at each sampling point of the blood drug concentration-time curve.
It is best to take multiple samples from the same animal. If the data of multiple animals form a blood concentration-time curve, the number of animals should be increased accordingly.
It is recommended that the test animals be divided into males and females. If there are obvious gender differences in pharmacokinetics, the number of animals should be increased in order to understand the gender differences in the pharmacokinetics of the test substance.
For single-sex medication, the gender consistent with clinical medication can be selected.
As with usual pharmacological experiments, experimental animals should be kept in the laboratory for 3 to 5 days before experimentation.
Route of administration and dosage
The route and method of administration used in the study of pharmacokinetics should be as consistent as possible with clinical medication.
Pharmacokinetic studies should have at least three dose groups, one of which should be equivalent to the effective dose of the pharmacodynamic test.
High doses are generally close to the maximum tolerated dose,
The medium and small doses are selected according to the upper and lower limits of the effective dose of the animal to understand whether the kinetic process of the drug in the body has nonlinear kinetic characteristics (the AUC of the three doses is linear with the dose).
For non-linear pharmacokinetics, the effect of dose on pharmacokinetics should also be studied.
When determining the dose, try to avoid arbitrarily increasing the dose in order to adapt to the sensitivity of the detection method.
Sampling time point arrangement
Blood drug concentration-time data is the core of pharmacokinetic research. Its accuracy and reliability depend on the analysis and detection technology on the one hand, and on the correct experimental design on the other hand. Among them, the reasonableness of sampling points has the most significant influence.
The design of sampling points should take into account the absorption phase, the distribution phase and the elimination phase.
According to the characteristics of the research sample, the sampling points can usually be arranged from 9 to 13 points.
Generally, at least 2 to 3 sampling points are required in the absorption phase. For drugs administered extravascularly with fast absorption, the first point should be avoided as Cmax;
At least 3 sampling points are required near Cmax;
4-6 sampling points are needed to eliminate the phase.
The entire sampling time should last for at least 3 to 5 half-lives, or until the blood concentration is 1/10 to 1/20 of Cmax.
Data processing of drug-time curve
According to the blood drug concentration-time data, a suitable compartment model or non-compartment model method can be used for data processing to calculate the pharmacokinetic parameters.
The basic pharmacokinetic parameters usually required for the pharmacokinetic study of new drugs are: t1/2, V, AUC and CL for intravenous administration;
Ka, Cmax, tmax, t1/2 and AUC for extravascular administration.
For water-soluble drugs, absolute bioavailability for extravascular administration should also be provided
For sustained and controlled-release preparations, the data should be based on the blood concentration-time data of the complete dosing interval when multiple dosing is in steady state,
Provide parameters such as steady-state peak time tmax, steady-state peak concentration, steady-state trough concentration, AUCss, volatility (DF) and steady-state average plasma concentration, etc.
And compare the absorption degree, DF and whether there is any difference with the generic drug or common preparation, and investigate whether the test preparation has the characteristics of slow and controlled release.

(2) Research content

Research on drug absorption
In new drug research, the absorption of drugs in extravascular preparations is mainly evaluated by whole animal pharmacokinetic tests or human bioavailability tests, but in vitro organ experiments can also help to investigate the influence of dosage form factors on drug absorption. For prescription screening.
Commonly used methods for studying drug absorption in the gastrointestinal tract include in vivo ileal perfusion, eversion intestinal sac method and Caco-2 cell model.
At present, intestinal epithelial cell culture technology has been increasingly used in the study of drug absorption mechanisms, and the most used is the Caco-2 cell model.
Research on the distribution of drugs
Through the tissue distribution research of new drugs, the distribution law, accumulation situation, main accumulation organs or tissues and accumulation degree of test drugs in experimental animals can be obtained.
Generally, mice or rats are used for tissue distribution research, and at least 3 dose groups should be set.
After administration, samples were taken at one time point each in the absorption phase, the distribution phase and the elimination phase.
There should be at least 5 animal counts at each time point.
The tested samples include important tissues such as heart, liver, spleen, lung, kidney, brain, stomach, intestine, uterus or testis and muscle;
By measuring the drug concentration in these tissues, we can understand the main tissues and organs where the drug is distributed in the body.
Especially the distribution characteristics of effect target organs and toxic target organs. (The site or tissue where the drug takes effect, such as a tumor)
In the experiment, attention must be paid to the representativeness and consistency of sampling.
If the concentration of the drug in a certain tissue is high, the elimination of the drug in the tissue should be further studied.
When the isotope measurement technology is selected for the detection of drugs, the tissue distribution test of isotope markers should be provided as much as possible after the administration of the whole autoradiographic images of different phases.

Targeting Drug Delivery System (TDDS) is currently the most concerned research direction in the research of new drug formulations. The distribution characteristics of these drugs in vivo and their influencing factors are the focus of research.
The main indicators for quantitatively evaluating the distribution characteristics of targeted preparations in vivo are:
Targeting index (drug targeting index, DTI)
Selectivity index (drug selectivity index, DSI)
Targeting efficiency (drug targeting efficiency, DTE)
Relative targeting efficiency (relative targeting efficiency, RTE)

3. Binding study of plasma protein:
Under normal circumstances, only free drugs can diffuse to tissues through lipid membranes, be filtered by renal tubules, or be metabolized by the liver. Therefore, the binding of drugs and proteins will significantly affect the kinetic process of drug distribution and elimination, and reduce the drug at the target site. The strength of the action.
For drugs with a protein binding rate higher than 90%, an in vitro drug competition binding test should be carried out, that is, a drug with a high protein binding rate that may be used clinically is selected to investigate the effect on the protein binding rate of the drug under study.
In the study of plasma protein binding of new drugs, the main purpose is to determine the plasma protein binding rate.
Various methods can be used to study the binding test of drugs and plasma proteins, such as balanced dialysis, ultrafiltration, ultracentrifugation, and gel filtration.
According to the physical and chemical properties of the drug and the test conditions, a method can be selected to conduct at least 3 concentrations (including effective concentration) of plasma protein binding test, and each concentration shall be repeated at least three times to understand whether the plasma protein binding rate of the drug has a concentration Dependence.

4. Drug metabolism research:
For innovative drugs, it is necessary to understand the biotransformation of the drug in the body, including the type of transformation, the main transformation pathway and the metabolic enzymes that may be involved.
For new prodrugs, in addition to studying their metabolic pathways and the structure of the main active metabolites, systematic pharmacokinetic studies on the original drug and active metabolites are also required.
For drugs that are mainly metabolically eliminated in the body (prototype drug excretion<50%), biotransformation research can be divided into two stages:
Chromatographic methods or radionuclide labeling methods can be used to analyze and separate the possible metabolites before the clinic, and their structure can be preliminarily inferred by methods such as chromatography-mass spectrometry.
If the Phase II clinical study suggests that it has a development prospect in terms of effectiveness and safety, further research and clarification of the possible metabolic pathways, structures and metabolic enzymes of the main metabolites should be carried out.
If there are multiple signs suggesting that there may be a metabolite with strong activity, research on the active metabolite should be carried out to determine the necessity of conducting a metabolite kinetic test.
The induction or inhibition of drugs on drug metabolizing enzymes, especially cytochrome P450 isoenzymes, should be observed.
In the pre-clinical stage, the substrate method can be used to observe the inhibitory effect on animal and human liver microsomal P450 enzymes, and compare species differences.
When conducting research on drug-induced enzyme induction, observe the changes in liver P450 enzyme activity after multiple administrations of whole animals or hepatocyte P450 enzyme activity after repeated drug action to understand whether the drug has potential metabolic interactions .

5. Research on excretion of drugs:

The purpose of excretion research is to determine the excretion route, excretion rate and excretion volume of each excretion route.
The drug excretion test is generally performed in mice or rats.
Put the animal into a metabolic cage, and collect all urine or feces samples at a certain time interval after administration, until the collected drug has been exhausted.
Determine the drug concentration and calculate the rate and amount of drug excretion through this route.
There are at least 5 animal test data at each time point, and urine and feces samples should be collected before administration, and with reference to the results of the pre-test, the time point for collecting samples after administration should be designed, including the drug’s excretion from urine or feces , Excretion peak and the whole process of excretion basically ending.
During bile excretion, the rats are generally used for bile duct drainage under ether anesthesia. After the animals are awake, the drug is administered, and the bile is collected at appropriate intervals for drug determination.
Record the speed of drug excretion from feces, urine, and bile and total excretion (percentage of total administration), and provide data on material balance.

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