A quote from Rudolph Buchheim, an experimental pharmacologist who lived from 1820 to 1879, summarizes the nature and importance of studies in drug metabolism, including the pharmacokinetics and pharmacology, which deal with the amount of the drug and its action in the body, as well as, and particularly, the critical nature of knowing the metabolites of any drug formed by its transformation:
“In order to understand the actions of drugs it is an absolute necessity to have knowledge of the transformations they undergo in the body…we must not judge drugs according to the form and amount administered, but rather according to the form and amount which actually is eliciting the action.”
One of the more intriguing and important aspects of drug metabolism studies is stereoselective metabolism, or enantio-selective, or chiral-selective metabolism. While chirality was formerly an academic area, and thought to be a property of certain natural chemicals, this was discovered to be the effect of the artificial bounds set by the capabilities of separation chemistry techniques at that time.
Now, however, chirality is of great importance in the process of drug development. In fact, the majority of new molecules with active pharmacology being sold in this century are enantiomers and not the achiral or racemic mixtures so common throughout the waning years of the last.
The study of stereo-selective metabolism is thus vital in understanding pharmacokinetics, pharmacodynamics, drug safety and biochemical analysis. It also affects drug regulation, intellectual property rights, business ethics and practices, and drug development.
It is true that studies on the effect of selective enantiomers on drug adsorption, distribution and elimination are only being initiated or has been shown to be negligible. However, it does influence drug metabolism in almost all cases. Drugs are subject to enantiomer-dependent changes in their metabolism, whether they act as the product or the substrate, and this holds good over the action of many enzyme systems, as shown in the following figure:
- The transformation of the chiral compound (S)-warfarin to the chiral compound (S)-7- or (S)-6-hydroxywarfarin;
- The transformation of the chiral compounds 1,4-dihydropyridine calcium antagonists to their achiral pyridine analogs
- The transformation of chiral compounds such as warfarin through keto-reduction to diastereomer compounds, in this case two pairs of diastereomer alcohols shown below:
- Chiral inversions, whether or not mediated by enzymes, which are, for instance, seen with 2-arylpropionic acid NSAIDS
Potential Interactions Between Drugs
When racemic drugs are used on a patient, the fact is that multiple drugs are being administered. Thus when a ‘second’ drug is then added, drug-drug interactions become an important issue, along with pharmacology and drug safety. This is because one of the drugs may induce or inhibit the metabolism of another. This is why chirality must be considered when drug metabolites are being studied.
When warfarin and cimetidine are prescribed together, the clearance of the R form of warfarin is inhibited; warfarin with sulfinpyrazone reduces S-warfarin clearance; and similar racemic mixtures affect both drug pharmacodynamics and safety.
- For instance, only one enantiomer in a racemic mixture may have pharmacologic efficacy while the other is inert in terms of biologic effect
- Each enantiomer in a racemic mixture may have varying activity on the body and thus each should be treated as a different drug and developed separately
- The enantiomers may act on the same pharmacological target but with opposing effects
- Both enantiomers may have the same type of pharmacological effect but only one causes adverse reactions
Stereo-selective chemistry received a boost with the development of separating columns to help separate enantiomers from a racemic mixture. In fact, some say that it is of no value to evolve pharmacokinetic models or to derive relationships between plasma concentration and effect based on the total drug concentration when the drug administered is in racemic form.
Bio-Analytical Chemistry Approaches6
Similar to other branches of analytical quantitative chemistry, bioanalytical chemistry should focus on achieving precise and reproducible effects, while not requiring special sample preparation. The methods should use mobile phases or pH which are sample-friendly.
On the other hand, chiral separation methods do need particular care to develop and validate them. The method of choice is to separate enantiomers on a chiral stationary phase such as immobilized protein or a polysaccharide phase, though indirect methods are available in which derivatives of the enantiomers are created using a chiral reagent to yield diastereoisomers, which are capable of separating on traditional separation columns.
Stereoselective Metabolism in Action
Using single enantiomers as candidates for drug development rather than racemic mixtures is expected to lead to reduction in drug-drug interactions. Yet the possibility of achiral-to-chiral, chiral-to-chiral, or chiral-to-diastereoisomer transformations remains.
The following instance describes a hypothetical case study though based upon real data:
A drug candidate is presented, a small molecule, which is pharmacologically effective in a mouse model, and has safety and pharmacokinetic limits within acceptable limits in its preclinical studies. When the metabolites were identified and characterized in mouse models, the result showed that keto-reduction to the alcohol was the transformation of greatest significance, giving an alcohol like that in the above example, but with a chiral center:
This shows that stereoselective metabolism should be studied with respect to its transformation to the alcohol. The points to consider include:
- How does the compound’s metabolism relate to that in other species and in humans as observed in preclinical in vitro studies?
- Will minimal method development or sample preparation be sufficient to develop an early bioanalytical method?
- Is it possible to refine the method to an optimal technique suitable for validation?
- Does it undergo stereoselective metabolism, of either the R or the S form, to the alcohol?
- Do the in vivo results mirror the in vitro findings?
- What enzyme mediates this transformation?
- Does the stereoselectivity affect the drug development process in terms of drug regulation, intellectual property rights, or business development?
The drug was tested by in vitro experiments in many different species which showed that the drug was preferentially metabolized via keto-reduction to the S-enantiomer of the metabolite, to a level of over 90 percent in human, canine and rat models, and over 80 percent in mouse models. This was significantly absent in monkey models which would mean this species should not be chosen for studies on the toxicology of this drug. This was followed by pharmacokinetic experiments in the mouse, rat and dog subjects, and corresponding results were obtained. The preference for the S enantiomer and the same ratios were found to persist.
The keto-reduced form was present in plasma at concentrations several times higher than the original parent drug, which needed study as to the disproportion between the parent and the metabolite. The S-enantiomer of the keto-reduced metabolite also possessed effective activity against the target.
Looking from the viewpoint of bioanalytical testing, the earliest tests used an immobilized protein column which achieved excellent separation of the enantiomers of the metabolite formed by keto-reduction, but this took over 60 minutes of run time.
The resulting peaks were not optimal in shape and thus their sensitivity was also limited. The technique was not robust and failed to perform properly with repeated injections of biological samples. The next round was performed using another chiral chemistry with separation columns that reduced the run time and enhanced the sensitivity, allowing cost-effective monitoring of the metabolite in clinical studies.
This case study, though hypothetical, shows why drug development is dependent upon the possibility of stereoselective metabolism and how it affects multiple areas such as pharmacokinetics and pharmacodynamics, toxicology and bioanalytical chemistry, and business development opportunities, besides drug regulation and intellectual property.
EAG Experience with Stereoselective Metabolism
EAG has been dealing with ADME (adsorption, distribution, metabolism, and excretion) studies for over 30 years in preclinical animal species so as to promote the process of drug development.
The directors of each study are responsible to create and supervise the in-life stages of testing in partnership with AAALAC-accredited site laboratories, all of which are tested and qualified by EAG. The EAG metabolism scientists have immense expertise when it comes to preparing the dose, determining the mass balance, preparing samples, extractions, profiling metabolites of drugs and identifying unknown metabolites. Other services offered include quantitative autoradiography of the whole body.
In particular, EAG offers the following facilities:
- Scientists who have published reports in drug metabolism with good credibility
- ADME studies which are either radiolabeled or not, and in preclinical animal or human clinical trials
- Preparation of samples and development of extraction methods
- Development of chromatographic methods
- Characterization of chemicals by radiochromatographic testing
- Identification using LC-MS/MS, including the use of high-resolution mass spectrography
- Identifying potential metabolic pathways
- Radiolabeled or stable-labeled, or non-radiolabeled synthesis of the drug and its metabolites
- Conti and M. H. Bickel, Drug Metab. Rev., 1977, 6, 1–50.
- V. Campo, L. Bernardes and I. Carvalho, Curr. Drug Metab., 2009, 10, 188–205.
- D . Brocks, Biopharm. Drug Dispos., 2006, 27, 387–406.
- Chirality in Drug Design and Development, ed. I. Reddy and R. Mehvar, CRC Press, New York, 2004.
- A . Hutt, Metab. Drug Interact., 2007, 22, 79–112.
- Adapted from Campo, et al. (ref. 2, above)
- J. Schmidt, A. Nouraldeen, L. Moran, L. Li and A. Wilson, Enantio-selective and Species-Dependent Carbonyl Reductase Metabolism of LX6171, 12th Annual Conference on Drug Metabolism and Applied Pharmacokinetics, 2009.
- L. Li, W. Heydorn, J. Kramer, A. Nouraldeen, J. Schmidt, J. Jiang, L. Moran and A. Wilson, Metabolism Mediated CYP2B Induction by LX6171 (3′-chlorobiphenyl-4-yl)-1-(pyrimidine-2-yl) piperidin-4-yl methanone in the Rat, International Society for the Study of Xenobiotics (ISSX) Annual Meeting, 2009.
- J. Schmidt, “Metabolite Profiling,” in A.G.E. Wilson (ed.), New Horizons in Predictive Drug Metabolism and Pharmacokinetics, RSC Publishing, Cambridge, UK, 2015.
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