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Leuschner, M.; Cromarty, A.D. Phenotyping Cocktails Developed. Encyclopedia. Available online: https://encyclopedia.pub/entry/46809 (accessed on 27 July 2024).
Leuschner M, Cromarty AD. Phenotyping Cocktails Developed. Encyclopedia. Available at: https://encyclopedia.pub/entry/46809. Accessed July 27, 2024.
Leuschner, Machel, Allan Duncan Cromarty. "Phenotyping Cocktails Developed" Encyclopedia, https://encyclopedia.pub/entry/46809 (accessed July 27, 2024).
Leuschner, M., & Cromarty, A.D. (2023, July 14). Phenotyping Cocktails Developed. In Encyclopedia. https://encyclopedia.pub/entry/46809
Leuschner, Machel and Allan Duncan Cromarty. "Phenotyping Cocktails Developed." Encyclopedia. Web. 14 July, 2023.
Phenotyping Cocktails Developed
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This entry is adapted from the peer-reviewed paper 10.3390/jpm13071098

Interethnic and interindividual variability in in vivo cytochrome P450 (CYP450)-dependent metabolism and altered drug absorption via expressed transport channels such as P-glycoprotein (P-gp) contribute to the adverse drug reactions, drug–drug interaction and therapeutic failure seen in clinical practice. A cost-effective phenotyping approach could be advantageous in providing real-time information on in vivo phenotypes to assist clinicians with individualized drug therapy, especially in resource-constrained countries such as South Africa. 

phenotyping cocktail African genetic diversity personalized medicine CYP450 P-gp

1. Selectivity of Probe Drugs for Metabolizing Enzymes or Drug Transporters

The first main problem of current probes suggested by the FDA for phenotyping is the fact that no probe drug is completely selective for a single metabolizing enzyme or transporter. Nonetheless, the contribution of a specific pharmacokinetic pathway to the disposition of the probe drug should be primary and in addition must be indicative of changes in the phenotype when subject to an inducer or inhibitor [1]. For example, caffeine, a fully validated probe for CYP1A2, is also metabolized by CYP2E1, N-acetyl-transferase 2 (NAT2) and xanthine oxidase (XO) enzymes, but since CYP1A2 is the dominant metabolic pathway [2], most cocktails use the metabolic ratio of paraxanthine to caffeine plasma concentration [3][4][5][6][7][8] as a CYP1A2 phenotype identifier. Alternatively, provided the phenotyping measurement is carefully chosen, all metabolites of caffeine could be quantified to assess NAT2 and XO activity simultaneously, as in the Cooperstown [9] and Quebec [10] cocktails. Similarly, the metabolism of omeprazole to its hydroxylated metabolite and sulfone metabolite has been used to simultaneously assess CYP2C19 and CYP3A4 metabolism, respectively, in a recent cocktail [11]. Tolbutamide is an almost exclusive probe for CYP2C9, but the proposed phenotyping measurement of 24 h plasma concentration would restrict its usefulness in routine phenotyping. Metoprolol has been studied as a selective probe for CYP2D6 metabolism, but correlation with other CYP2D6 probes could not be established in an African population from Tanzania carrying a population-specific CYP2D6*17 allele [12], raising questions about its usefulness as a probe. This discordance between genotype and observed phenotype with altered substrate specificity in African populations has been shown in a number of studies [13][14][15]. These findings confirm the need for further research on different population groups before routine phenotyping can be implemented in clinical practice.
Phenotyping drug transporter activity may also provide a useful metric to assess and predict drug absorption or excretion (depending on the location of the drug transporter protein) in vivo [16]. The role of transporters in drug–drug interactions and the clinical safety and efficacy of drugs has been the focus of the International Transport Consortium since 2010 [17]. In a review by Ma et al., evaluating four P-gp probes, none met all the proposed validation criteria for an ideal probe drug [18]. Both digoxin and fexofenadine have overlapping substrate specificities with other transporters and their correlation with other P-gp probes was not established; in addition, digoxin has a narrow therapeutic window, limiting its usefulness as a probe in patient populations. Despite the fact that no ideal P-gp probe exist, fexofenadine is safe and has been used in phenotyping drug cocktail studies [6][19] and pharmacokinetic studies [20][21][22]. Understanding the pharmacokinetic processes influenced by xenobiotic exposure, the site of exposure and the expression and distribution of metabolizing enzymes and transporters at that site is imperative for assigning phenotype and making clinical decisions based on that assessment.
The chosen probe drugs should clearly elucidate the in vivo pharmacokinetic phenotype under investigation, and overlapping substrate specificities between P-gp and CYP3A4 in particular should be considered. A higher expression of CYP3A4 in enterocytes will significantly influence the first pass bioavailability of CYP3A4 substrates and therefore if the objective is to phenotype hepatic CYP3A4 activity, probe substrates should be administered by the intravenous route [23]. Changes in substrate selectivity for metabolizing enzymes and transporters when administered at lower subtherapeutic doses must be considered with the validation of low-dose cocktails. In most cases, a lower substrate dose will increase drug selectivity; however, even validated cocktails have to be re-evaluated when the dosages are lowered to ensure the applicability of the phenotype assessments [23]. An important factor to consider is dose-dependent plasma protein binding, as a result of the saturation of the available binding sites, influencing the fraction of unbound drug in systemic circulation as explained by Macheras and Rosen [24]. Micro dose strategies with phenotyping cocktails, containing dosages 100-fold lower than the normal dosages, have been proposed, but the authors stress that linear pharmacokinetics between normal and micro doses are required for the correct prediction of enzyme or transport activity. This is due to the fact that protein binding may be dose-dependent and both decreased bioavailability or the non-saturation of compartments during drug distribution may lead to non-linear pharmacokinetics. Furthermore, very precise and sensitive quantitation methods are required [25].

2. Tolerability of Drug Doses Used in Phenotyping Cocktails and Safety Profiles of Some Proposed Probes

Secondly, earlier cocktails contained probe drugs at therapeutic doses, contributing to possible side effects, especially considering drugs with narrow therapeutic indexes, such as tolbutamide, warfarin and digoxin. Any small variation in enzyme or transport activity could contribute greatly to the disposition of drugs with a narrow therapeutic index, causing severe adverse reactions. Possible side effects with therapeutic probe drug doses included hypotension with debrisoquin (CYP2D6 probe), hypoglycemia with tolbutamide [26] (CYP2C9 probe), bleeding risk with warfarin (CYP2C9 probe, requiring co-administration of vitamin K) and gastrointestinal side effects and sedation with mephenytoin (CYP2C19) [27]. The incidence of side effects has been largely eliminated since the introduction of low-dose phenotyping cocktails; however, they present pharmaceutical complications, because probe drugs are not commercially available at these low doses and have to be compounded from available dosage forms. More importantly, low-dose phenotyping cocktails require optimized, sensitive bioanalytical methods to detect low concentrations of metabolites in biological matrixes, especially when probe drugs and their metabolites, all with different physicochemical properties, are to be simultaneously quantified in a single run. An example of an ideal probe drug is flurbiprofen for phenotyping CYP2C9. It is almost exclusively metabolized by this enzyme, has a wide therapeutic window and is not dependent on urinary conjugation for excretion; therefore, it has a much better safety profile than tolbutamide and warfarin [28], justifying its incorporation into the Pittsburg cocktail [5].

3. Sample Collection Protocols and Corresponding Phenotyping Measurements Chosen for Phenotype Assessment

A third main challenge of current proposed phenotyping cocktails is the inconvenient and impractical sample collection protocols. Multiple time point venous plasma sampling or collective urine sampling would not be feasible in a routine clinical environment. Use of a single or limited time point sampling strategy to measure metabolic or transporter activity would be advantageous especially when coupled with probe drugs with short elimination half-lives to reduce the time patients have to spend at the clinic for observation. Studies comparing the systemic clearance (AUC) of probe drugs or the clearance ratio of probe drug to metabolite to limited AUC or single time point metabolic ratios are currently underway [7][29][30][31][32][33]. No consensus has yet been reached and results are conflicting. In validating their Basel phenotyping cocktail, Donzelli et al. correlated the AUC0–24 h ratios for probe versus metabolite to a number of single time point plasma metabolic ratios (including a 2 h single time point midazolam metabolic ratio (r2 of 0.959). Yang et al., on the other hand, found a 4 h limited sampling AUC for midazolam and a 4 h single time point concentration to best fit a two-compartmental population PK model, derived from 2122 observations from 152 healthy subjects, for the estimation of CYP3A4 metabolic activity [32]. A 5 h single time point plasma midazolam concentration [34] and limited sampling at 0.5, 2 and 6 h for midazolam [29] have also been suggested. Similarly, many single time point paraxanthine over caffeine metabolic ratios have been shown to correlate with the systemic clearance of caffeine, ranging from 2 h [6], 4 h [8] and 8 h [7] post oral dose. Care should be taken in choosing the phenotyping measurement to infer metabolic or transport activity in different patient populations. Chosen phenotyping measures should be validated; correlate with enzyme or transport activity and represent change clearly under induction or inhibition conditions; account for confounding factors such as glomerular filtration rate or urinary pH; and have low intra-individual variability [2][23]. Intraindividual variability is usually lower with plasma sampling rather than urinary sampling.

4. Pharmacokinetic, Pharmacodynamic and Bioanalytical Interaction between Probe Drugs in Simultaneous Assessment of Phenotype

An understanding of the PK and PD interaction between probe drugs used together in a cocktail approach is essential. Interactions at the target receptor sites (PD interactions) should also be considered; for example, using the antihypertensives losartan and debrisoquin together might cause hypotension. Each probe drug used in a proposed cocktail must be validated individually and then in combination to exclude interaction with other probe drugs. In the Basel cocktail, chlorzoxazone (a CYP2E1 probe) had to be excluded due to a significant interaction with CYP3A4, significantly increasing midazolam AUC0–24h when administered together [7]. To overcome this, Blakey et al. administered the midazolam intravenously to exclude this intestinal CYP3A4 interaction with chlorzoxazone [35]. Although separate intravenous dosing is feasible during drug interaction studies and during drug development, it would be difficult to implement in clinical practice. Chlorzoxazone also interacts with CYP1A2 and when administered together with caffeine caused a 16–20% decrease in caffeine metabolism in urine and plasma [36]. Simultaneous probe drug and metabolite quantitation using bioanalytical methods requires optimization due to different physicochemical properties to reduce competition for charge and to optimize individual extraction recovery, ionization efficiency and detection limits.

References

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