Drug Metabolism and Pharmacokinetics – an overview

The journey from molecular target and early drug lead to the clinic is an arduous one with many hurdles to cross prior to developing a successful clinical candidate. The high rate of attrition of drug molecules has forced drug researchers to pay greater attention to drug metabolism and pharmacokinetics (DMPK) of lead molecules at even the earliest stages of drug discovery. Throughout the development of a successful molecule the researcher must bear in mind three important questions: will enough drug reach the target (pharmacokinetics)? What form will it arrive in (metabolism)? And what will it do when it gets there (pharmacodynamics)? These are the main questions that the DMPK scientist attempts to answer.

Physico-chemical properties

Selection of drug leads in modern drug discovery is a balancing act between desirable properties i.e. oral absorption, metabolic stability and between those properties that are less desirable i.e. toxicity and rapid clearance. Many of these properties can be predicted based on physico-chemical properties, and of particular use to the DMPK scientist is knowledge of the lipophilicity of the drug leads under investigation. Lipophilicity (LogD) is a measure of the partition of the molecule between an aqueous and a lipid environment. In order for a molecule to cross a biological membrane it passes from an aqueous environment through a lipid bilayer and into the aqueous cytostol. Thus, lipophilicity will give an indication of the permeability of a drug molecule. Additionally a measure of lipophilicity can, to a certain degree, indicate the route of clearance. For example, low lipophilic molecules tend to be renally cleared and highly lipophilic molecules are more likely to undergo metabolic clearance.

Solubility is another important parameter in DMPK. Highly insoluble compounds are unlikely to progress very far in the drug discovery process as the poorly soluble compounds are more difficult to test. These difficulties are due to either the compound precipitating from solution and/or the selected diluents adversely affecting enzymatic action in a metabolic assay. Conversely, those compounds that are very easily dissolved in an aqueous media will by their very nature struggle to cross biological membranes and hence the chances of such compounds reaching their designated target will decrease. Despite all this, drug leads that lean towards either extreme of solubility or lipophilicity may still progress in the drug development process depending on additional DMPK findings. Thus, the physico-chemical properties of a drug lead can enable scientists to prioritise leads based on known metabolic and pharmacokinetic properties associated with those physico-chemical properties1. Solubility and lipophilicity can also give an indication of many pharmacokinetic properties including gut absorption, brain permeability, toxicity and renal clearance. However, knowledge of solubility and lipophilicity alone is insufficient to enable selection of drug leads. In order to further characterise drug leads in vitro and in vivo studies must be performed.

Oral absorption

Let us assume the drug lead(s) of interest have crossed the first barriers to progress; that they can be formulated in a suitable diluent and have shown by virtue of their lipophilicity the potential to cross biological membranes. This in itself may not be enough for a drug lead to progress to pre-clinical testing. Other factors contributing to oral absorption i.e. stability of drug in low pH and resistance to proteolytic enzymes must be considered. Two such examples are aspirin and insulin. Aspirin is a readily recognised drug, associated with pain relief. The problem with aspirin is lack of solubility. Soluble aspirin is prepared as the sodium salt form to increase solubility. In the acidic environment of the gut the solubility decreases due to the equilibration shifting to the acidic form and hence some of the drug precipitates from solution and remains in the stomach.

Insulin is currently not available to diabetics as an oral administration due to it being a polypeptide and hence sensitive to both the extremes of pH in the gut and the proteolytic enzymes present there. Recent research has attempted to develop oral formulations of insulin2 and thus alleviate the problem for diabetics of regular IV administration.

Metabolism

In reality the body wants to remove/detoxify any substance that cannot be otherwise utilised to serve the needs of the body. In a nutshell that is what metabolism entails. The modification of a xenobiotic to aid removal from the body. This removal process is carried out predominately by the liver. The liver has a host of enzymes that are extremely effective at this work.

Drug metabolism is characterised into two phases. Phase 1 metabolism consists of reactions such as oxidation, reduction and hydrolysis and is primarily carried out by cytochrome P450s (CYPs) and flavin containing monooxygenases. The purpose of these phase 1 reactions is either to decrease the lipophilicity sufficiently in order to facilitate renal clearance or preparative for phase 2 reactions which add a water soluble endogenous molecule to reduce lipophilicity.

CYPs are the most important enzymes involved in drug metabolism3 and as such are among the most studied and best characterised enzyme families. These enzymes are studied at all stages of drug development. Screening libraries of compounds for CYP inhibition is now one of the earliest assays performed within DMPK. A CYP inhibitor can disrupt normal cellular metabolism and lead to alterations in physiological processes4.

CYPs are also studied by way of microsomal and hepatic fractions (these fractions will contain other enzymes but the CYPs tend to predominate) which contain phase 1 and phase 1 and 2 enzymes respectively.

Recombinant CYP enzymes can be used to determine exactly which CYP enzyme is causing the metabolic effect. This is what is known as reaction phenotyping. Reaction phenotyping quantifies the amount different enzymes contribute to the metabolism of a compound and is useful in pre-clinical studies to understand metabolism in a variety of tissues by correlating it with enzyme expression in those tissues.

Patients taking one particular drug are often advised/prevented from taking another treatment if the same enzyme contributes to the metabolism of both drugs. This can lead to what is commonly referred to as drug drug interactions, whereby the reduced metabolism of one of the drugs leads to an unwanted effect.

Further complications arise due to differential expression of particular enzymes, or slightly different forms of the same enzyme (polymorphism)5 not only across species but differential expression of CYPs can be observed with genetic factors such as age, sex, ethnicity and environmental factors such as smoking or illness for example. Due to these factors CYPs metabolism studies continue throughout the drug developmental cycle.

Despite the challenges with the differential expression of similar enzymes this can sometimes be a potential area to exploit. Many therapeutic strategies attempt to exploit the natural or, in disease case, unnatural cellular metabolism. By utilising a particular group of over-expressed enzymes or simply exploiting normal physiological processes a drug lead could be made that is a specific substrate for a particular enzyme.

In vitro metabolism can be used to predict in vivo metabolism. Robust assays are used across the industry and the DMPK scientist can make use of the ability to separate cellular fractions armed with the knowledge of the properties of those fractions i.e. S9, microsomal, whole cell or even recombinant enzymes. Each fraction will exhibit different components of the cell and hence the in vitro metabolic pathway can be elucidated with the knowledge of the enzymatic make up of the particular cellular fraction. Studies in mouse or rat liver microsomes are often one of the earliest experiments to assess drug metabolism as data from these experiments is used to select compounds for further studies. One of the goals of in vitro work with cellular fractions is to calculate intrinsic clearance. This is often done in a variety of species and then the data is extrapolated to obtain a prediction of human clearance.

Detection and measurement of drugs and drug metabolites

Having discussed the challenges with metabolism and the approaches used to test and assess drug leads we now query how do we measure these drug and drug metabolite molecules?

Thankfully common techniques can be employed on a range of drug molecules enabling laboratories throughout the world to have a few different types of detection and quantification systems that will cover most drug leads. The workhorse of all DMPK laboratories is the mass spectrometer (MS); enabling identification of molecules based on molecular weight. Different types of MS can be used for specific functions. For example, the selectivity and specificity of the tandem MS/MS can be utilised for quantification of molecules from early discovery studies to clinical trial samples and is also more than able to provide an insight into metabolic products. Time of Flight (TOF) Mass Spectrometers come into their own when optimised for metabolite identification studies6 and are able to assign accurate mass and some TOF MS can easily distinguish between isomers. These techniques and their routine use can be further enhanced by coupling to an appropriate separation technique i.e. chromatography. This enables separation of the drug and metabolites which aids characterisation of the metabolites.

Additionally NMR (Nuclear Magnetic Resonance) spectroscopy is increasingly being used to provide structural metabolic information that the above techniques do not completely provide on their own. By determining the intra and inter molecular distance between known atoms the DMPK scientist can hypothesise the metabolic product structure i.e. MS may provide information that a hydroxylation has occurred but where on the molecule it has occurred may be confirmed by proton NMR.

Pharmacokinetics and pharmacodynamics

Pharmacodynamics (PD), which studies the effects the drug has on the body, is becoming more integrated within DMPK. Early clinical trials which once determined safety and tolerability now include PK and PD objectives. Pre-clinical studies also incorporate PD at an early stage. This makes sense as a drug lead could have all the desirable properties but not show efficacy, the sooner this is understood the better so the drug leads can be terminated.

In vitro and in silico studies can determine or estimate the binding affinity of a drug lead to a molecular target and hence give an indication of potency. This is undoubtedly extremely useful information but the potency in vitro may not necessarily correlate with what will be observed in the clinic and this can be related to a number of factors. One major fact being pharmacokinetics.

Pharmacokinetics (PK), is the study of how the body deals with the drug. It can be predicted from in vitro and in silico data but the drug lead(s) must be studied in a whole organism in order to properly define the pharmacokinetic parameters.

Prior to the first in vivo experimentation sufficient data is required to design a suitable study. A good study design will glean maximum information using an appropriate species. Most commonly used in initial in vivo studies are mouse and rat models hence the need for good mouse and or rat in veto data. Following the first PK experiments more in vitro studies tend to be carried out in order to understand what has been happening in vivo.

The first in vivo studies will determine a suitable dose to study and assess tolerability. Subsequent PK studies will look at PK parameters in more depth such as clearance (the volume of blood from which the drug is completely removed per unit time), AUC (drug exposure) and half-life (time taken for drug concentration to drop to 50%). Depending on the results of these experiments further studies would be conducted to assess in vivo metabolism and would eventually lead to long term toxicity studies.

Eventually a strong data package is required to be presented to the regulatory authorities showing a new drug candidate for unmet clinical need or an improvement on current therapy. Following this, well designed clinical trials will first focus on safety and tolerability. Often a first in man study will incorporate a single ascending dose with the subjects monitored closely for any adverse events. This first study will determine the dosage used in subsequent studies. Further trials will measure PK parameters in more detail and include pharmacological objectives. As the clinical trial progresses drug metabolism is studied in depth to ensure any significant metabolites observed in humans were present in pre-clinical toxicological species. If not the metabolite will be synthesised and subjected to the barrage of DMPK tests as if it were the parent compound.

In conclusion, DMPK has a central role to play in drug development. With increases in the sensitivity, accuracy and speed of analytical techniques and additional increases in the quantity and quality of the data, the DMPK scientist has a wealth of information upon which to select drug leads for progression. This puts DMPK in a unique position of having a decision making role in many of the stages from drug discovery to development; from concept to consumer.

References

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4. Pollack TM, McCoy C, Stead W. Clinically significant adverse events from a drug interaction between quetiapine and atazanavir-ritonavir in two patients. Pharmacotherapy. 2009 Nov;29(11):1386-91.
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6. Mortishire-Smith RJ, Castro-Perez JM, Yu K, Shockcor JP, Goshawk J, Hartshorn MJ, Hill A. Generic dealkylation: a tool for increasing the hit-rate of metabolite rationalization, and automatic customization of mass defect filters. Rapid Commun Mass Spectrom. 2009 Apr;23(7):939-48.