In vivo Pharmacokinetics Services

Cyprotex’s vision is to accurately predict the human clinical outcome following exposure to a chemical or drug using high quality, robust in vitro methods combined with in silico technology. By using this approach we are committed to finding alternative methods to reduce animal testing.

Despite this vision, Cyprotex has to accept that in order to bring a drug to market it is a requirement by the regulatory authorities to perform some animal testing to ascertain exposure and safety information. Although Cyprotex does not perform testing on animals itself, it partners with Avastus Preclinical Services to offer a combined pharmacokinetics and bioanalysis service in order to meet the needs of its pharmaceutical-based clients. Our customers appreciate our ability to offer both pharmacokinetics and bioanalysis services through Cyprotex because it streamlines study management, reduces cost and provides faster turnaround times.

Pharmacokinetics (PK)Â Services offered by Avastus Preclinical Services

Wide variety of small rodent (rat, mice, others on request) strains available.
Multiple routes of administration including cannulated animals.
Many dosing routes have pre-approved IACUC protocols in place, improving turnaround time.
Extensive bioanalytical capabilities for analyzing many types of tissues (brain, tumor, muscle, liver, kidney, heart).
Formulation design available for discovery-stage dosing.
PK/PD models also available (xenograft, diabetes models, infection models, EAE, arthritis, inflammatory).

Protocol

In vivo Pharmacokinetics offered by Avastus Preclinical Services

Species	Rat, mouse, others (multiple strains available on request)
Dosing Routes Available	Oral, intravenous, buccal, ocular, sublingual, intraperitoneal, subcutaneous, topical
Dosing Options	Repeat, single, cassette, infusion, cannulated
Analysis Method	LC-MS/MS, ELISA

How to Interpret a Pharmacokinetic (PK) Profile

Administration can occur by many different routes (e.g., oral, intravenous, subcutaneous, intramuscular, topical, sublingual and ocular administration). Two of the most common routes (intravenous and oral administration) are described below.

Intravenous Administration

When a drug is administered intravenously, there is no absorption stage and the entire dose enters the circulatory system. The early stage rapid fall in plasma concentration is often followed by a slower more steady fall in concentration. These 2 stages relate to the distribution and elimination phases respectively. Elimination typically follows first order kinetics.

Figure 1
Example of a Plasma Concentration Time Profile following Intravenous Administration.

Common PK Parameters (Intravenous administration)

C₀: The C₀ is the initial plasma concentration following an intravenous dose. This is calculated by extrapolating the plot of log plasma concentration versus time back to zero time.

Elimination Half Life: The half life is the time taken for the drug plasma concentration to decrease by 50%. Compound elimination typically follows first order kinetics. The following equation can be used to estimate the elimination half life.

Where k = elimination rate constant (determined from the slope of the elimination phase of the log concentration time plot).

The half life is used to calculate how frequently a drug should be administered to maintain a therapeutic concentration.

Apparent Volume of Distribution: The apparent volume into which a drug distributes in the body at equilibrium is called the apparent volume of distribution and this term is used to quantify the distribution of a drug between plasma and the rest of the body after dosing.

The volume of distribution is dependent on the properties of the compound.
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For example,

drugs which are very highly bound to plasma proteins or highly hydrophilic have a low volume of distribution i.e., approaching blood volume of 3L /70kg man.
drugs which are neither bound in plasma nor tissue have a volume of distribution which varies between 16L (extracellular fluid volume) and 42L (total body water volume).
drugs which are retained in the tissues of the body have a high volume of distribution. For example, chloroquine has a volume of distribution of 15,000L/70Kg man, as it sequesters in body fat.

The following equation can be used to calculate the volume of distribution:

Volume of distribution is useful for estimating dose required to give a particular plasma concentration or estimating plasma concentration when a known amount of drug is in the body.

Area under the curve (AUC): AUC is the total area under the concentration time curve. The AUC is used to measure the level of exposure. AUC can be used in the calculation of clearance and bioavailability.

Total Clearance: The clearance of a drug is the volume of plasma from which the drug is completely removed per unit time. Total body clearance is the sum of all the different clearance processes occurring for a given drug (e.g., metabolic, renal and biliary are the predominant routes). Clearance is an important parameter as it indicates how rapidly the compound is being eliminated from the body. The following equation can be used to calculate the clearance after an intravenous bolus injection:

Oral Administration

Oral administration is the preferred route for the majority of intended drugs. However the drug has to pass through a number of challenges before it reaches the systemic circulation. These include absorption from the gastrointestinal tract, intestinal metabolism and first pass metabolism.

Initially, the rate of absorption is maximal as all the drug is at the site of absorption. However, as the drug is absorbed the rate of absorption decreases and the rate of elimination increases. t_max (the time of maximal plasma concentration) is reached when the rate of absorption equals the rate of elimination. The concentration at t_max is known as C_max (i.e., the maximal plasma concentration). The plasma concentration decreases when the rate of elimination exceeds the rate of absorption.

Figure 2
Example of a Plasma Concentration Time Profile following Oral Administration.

Common PK Parameters (Oral administration)

C_max: C_max is the maximal plasma concentration. The C_max is commonly used to relate to pharmacological effect.

t_max : t_max is the time at which the maximal concentration is reached.

Oral Bioavailability: Bioavailability is the fraction of the administered dose that reaches the systemic circulation unchanged. Bioavailability is 100% for intravenous injection. Oral bioavailability can be influenced by solubility, intestinal stability, drug efflux, intestinal metabolism and first pass metabolism. Oral bioavailability is calculated using the following equation:

Although it is possible to calculate the bioavailability at different oral and intravenous doses, this can lead to misleading results in certain circumstances and it is always advisable to calculate the bioavailability using an equal oral and intravenous dose.

Fraction Absorbed: Bioavailability is a function of absorption as well as first pass metabolism. In order to identify if low bioavailability is related to low absorption, it is important to calculate the fraction absorbed. The equation for calculation of the fraction absorbed relates bioavailability, clearance and liver blood flow as detailed below: