In vivo cocktail pathway phenotyping (ICPP) is a relatively new approach that facilitates the simultaneous, but independent, assessment of the activity of multiple metabolic pathways. ICPP is ideally suited for the assessment of the magnitude of in vivo metabolic drug–drug interactions (mDDIs) involving cytochrome P450 (CYP) enzymes (Snyder et al., 2014). Indeed, ICPP is routinely used to assess the mDDI potential of new drug candidates (NDC) during drug development. However, there are a number of potential limitations to this approach, such as the capacity for interactions between probe drugs, increased risk of adverse effects caused by probe drugs, and increased analytical complexity (Tanaka et al., 2003). As such, the use of validated drug cocktails and study protocols is essential.

Frye et al. (1997) the “Pittsburgh cocktail” was reported as the first validated in vivo cocktail for the assessment of CYP enzyme activities. Today, validated drug cocktails include the “Cooperstown” (Streetman et al., 2000a,b), “Karolinska” (Christensen et al., 2003), and “Inje” (Ryu et al., 2007) cocktails. Each cocktail has advantages and disadvantages, and in some cases they have been modified to expand their capacity (Chainuvati et al., 2003; Snyder et al., 2014) or address limitations. The four probe drugs in the Cooperstown cocktail; caffeine (CYP1A2), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and intravenous midazolam (CYP3A), are generally considered the core ‘probes’ of the CYP cocktail approach. These drugs do not interact with each other and are not associated with significant adverse effects. However, the intravenous administration of midazolam (probe for CYP3A) precludes the assessment of gastrointestinal CYP3A enzyme activity. Furthermore, this cocktail does not contain a probe drug for CYP2C9, which accounts for approximately 18% of the CYP protein in human liver (Miners and Birkett, 1998). These limitations are partially ameliorated by using warfarin as a probe for CYP2C9 in the “Cooperstown 5+1” cocktail (Chainuvati et al., 2003). The Karolinska 5-drug cocktail uses caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), debrisoquine (CYP2D6), and quinine (CYP3A) as probe drugs. However, the simultaneous administration of the cocktail drugs causes a significant increase of metabolic ratio of debrisoquine. Therefore, this approach requires the separate oral intake of debrisoquine, with additional logistical complexity. The “Inje” cocktail comprises caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6) and oral midazolam (CYP3A). These probe drugs do not interact with each other and are not associated with significant adverse effects. Furthermore, the use of these drugs as probes facilitates the assessment of the effects of mDDIs on all major drug metabolizing CYP enzymes including both hepatic and gastrointestinal CYP3A.

Historically ICPP based mDDI studies have only assessed the impact of interactions following multiple (3 to 5) doses of the postulated ‘perpetrator,’ where an interaction perpetrator is a compound that has the potential to alter the metabolic clearance of other drugs. As such, the emphasis in terms of validation of these studies has been ensuring that there are no interactions between probe substrates. Increasingly, however, it is recognized that the mechanism of mDDIs both in terms of inhibition (i.e., mechanism based versus reversible) and activation (i.e., induction of enzyme expression versus enhanced substrate binding) of CYP activity can have important clinical implications. While multiple dose mDDI studies are useful in describing the effect of steady state dosing of the postulated perpetrator on CYP activity, they provide limited insights regarding the mechanism of mDDIs. Studies assessing the comparative impact of single and multiple doses of the postulated perpetrator, providing information both on the clinical impact and mechanism of mDDIs, have not been routinely performed and appropriate validated dosing protocols to facilitate such studies remain to be defined.

Modafinil, a drug able to both induce and inhibit CYP activities in vitro, particularly CYP3A (Robertson et al., 2000), is an ideal candidate to assess the comparative impact of single dose versus steady state dosing on CYP activity using an ICPP mDDI study design. We sought to develop an optimized ICPP mDDI study protocol to evaluate the potential magnitude and clinical relevance of mDDIs perpetrated by modafinil using a physiologically based pharmacokinetic (PBPK) modeling approach.

This study reports a validated dosing protocol for the administration of Inje cocktail probes that facilitates the assessment of the magnitude of potential mDDIs following a single perpetrator dose and steady state perpetrator dosing. Consistent with the estimated half-life of modafinil in healthy volunteers (∼15 h; Robertson and Hellriegel, 2003), simulated steady-state concentrations were obtained with 5 days of dosing. Repeatable simulated mean trough concentrations (defined as within 5%) were observed from Day 5 onwards. The minimal duration of perpetrator dosing considered sufficient to facilitate assessment of potential induction and mechanism-based inactivation of CYP enzymes e.g., time-dependent changes in CYP activity is 7 days (FDA, 2012). Simulations demonstrate that this dosing duration was also sufficient to ensure steady state modafinil concentrations.

In order to assess changes in CYP activity caused by the administration of the postulated mDDI perpetrator (single or steady state dosing), ICPP based mDDI studies require administration of probes prior to perpetrator administration to establish baseline CYP activities (Day 0). Simulations demonstrate that when assessing the impact of a single dose of the postulated mDDI perpetrator, repeat probe dosing on Day 1 (i.e., the Day 0, 1 and 7 protocol) is suitable for evaluation of CYP 2C9 (losartan), 2C19 (omeprazole) and 3A4 (midazolam) activities since no residual probe is present and AUC and C_Max values are equivalent to the first dose. However, this protocol is not suitable for assessment of CYP 1A2 (caffeine) or 2D6 (dextromethorphan) activities due to the presence of significant residual probe in the systemic circulation at the time of the second dose (24 h), leading to an overestimation of AUC and C_Max for these probes (Table 3). Use of this protocol will bias activity assessment toward inhibitory mDDIs involving CYP 1A2 and 2D6 increasing the risk of either detecting an inhibitory interaction (false positive) or failing to detect induction (false negative).

Simulations demonstrate that when assessing the single dose effect of the postulated mDDI perpetrator, administration of probes 48 h after the initial dose (Day 0, 2 and 8 protocol) is suitable for evaluation of all CYP activities since essentially no residual probe is present and AUC and C_Max values are comparable between the first and second dose (Table 3). On this basis, the Day 0, 2 and 8 protocol was used for the assessment of modafinil as a perpetrator of mDDIs involving CYP and is recommended for ICPP mDDI studies performed to assess single dose and steady state dosing effects of the a postulated mDDI perpetrator.

The capacity of modafinil to inhibit and induce CYP activity has been demonstrated in vitro. Classical (i.e., [I]/K_i) extrapolation of in vitro data suggests that modafinil may induce expression of CYP 1A2 and 3A4 and inhibit CYP 2C19 and 3A4 activities in a reversible, competitive manner (Robertson et al., 2000). On the basis of these classically extrapolated in vitro data, modafinil is classified as a ‘moderate’ inducer of CYP 3A4 by the US FDA. Increasingly, however, it is accepted that there are several limitations (such as the appropriate selection of [I] (Rowland et al., 2006)) associated with the use of classical extrapolation of in vitro data when assessing mDDIs that preclude sensible assessment of the clinical relevance of novel interactions. Indeed, the substantial benefits of mechanistic (PBPK) extrapolation of mDDI assessments have been extensively reported and adopted (Sheiner and Steimer, 2000; Danhof et al., 2008; Rowland Yeo et al., 2010; Rowland et al., 2011) and this approach is recommended for the preclinical assessment of mDDIs for regulatory review (Zhao et al., 2011; FDA, 2012).

On the basis of the in vitro interaction profile (warfarin; CYP2C9; Robertson et al., 2002a), or potential for concomitant use with other vigilance (dexamphetamine, methylphenidate) or sedative (triazolam) agents (Wong et al., 1998b; Hellriegel et al., 2001, 2002; Robertson et al., 2002b), a limited number of in vivo studies assessing the mDDI potential of modafinil have been reported. Notably, these study ‘victim’ drugs, including warfarin, which was administered as a racemic mix or r- and s-enantiomers (metabolised by CYP 3A4 and 2C9, respectively) have been substrates for multiple metabolic pathways, and as such these studies do not readily facilitate the direct assessment of the effects of modafinil on individual CYP enzymes. Only one in vivo interaction study has reported the capacity of modafinil to induce drug metabolizing CYP (Moachon et al., 1996). This study used antipyrine, a pan-CYP substrate, with 7 days of modafinil dosing at 100 to 500 mg BD. The results of the study suggest that modafinil may be a weak general inducer of CYP at doses ≥400 mg/day (double the recommended daily dose), but no definitive conclusions were possible. The study design did not facilitate assessment of effects on individual CYP enzymes or account for concurrent inhibition of these enzymes.

In the current study, a clinically relevant increase in omeprazole exposure was predicted following both a single modafinil dose and dosing of modafinil to steady state. Notably, the apparent magnitude of the inhibitory interaction was attenuated with dosing of modafinil to steady state; omeprazole AUC ratios following a single modafinil dose and steady state modafinil dosing were 1.56 (1.52–1.59) and 1.38 (1.32–1.44), respectively. While omeprazole is primarily (88%) cleared by CYP2C19 catalyzed demethylation and hydroxylation, the remainder of omeprazole clearance occurs via CYP3A4 catalyzed sulfone formation. Following repeated dosing of modafinil, which induces CYP3A4 while inhibiting CYP2C19, the inhibitory effect on CYP2C19 is partially offset by induction of CYP3A4. The resulting change in fraction metabolized (f_m) for omeprazole is shown in Figure 7. These data highlight both the advantages of mechanistic extrapolation of mDDI data when assessing complex interactions involving victim drugs that are metabolized by multiple enzymes (Rowland Yeo et al., 2010), and the need to consider even minor metabolic pathways when considering the selection of probe substrates as induction of a minor metabolic pathway can result in a significant contribution to clearance when induced. Where multiple enzyme involvement results in the formation of unique metabolites for each enzyme, this potential confounder may be overcome by the consideration of an enzyme specific metabolic ratio. Simulations also predict that steady state dosing of modafinil may cause clinically relevant mDDIs resulting in reduced exposure to drugs metabolized by CYP3A4. This observation supports the current FDA classification of modafinil as a ‘moderate inducer’ of this enzyme (FDA, 2012).

These data support consideration of the risk of clinically relevant mDDIs when co-administered modafinil with drugs that are primarily cleared by CYP 2C19 and 3A4 catalyzed metabolic pathways. In particular, given the major role of CYP3A4 in the clearance and metabolic activation of a myriad of drugs from various therapeutic classes including the oral contraceptive pill and orally administered non-cytotoxic anticancer drugs (e.g., erlotinib, pazopanib and sunitinib; Rowland et al., 2016) potential induction of CYP3A4 by modafinil should be considered when evaluating new indications for this drug such as the management of chemotherapy induced fatigue and cognitive impairment in order to avoid a potential decrease in therapeutic response for the co-prescribed drug that may result in therapeutic failure.

Participated in research design: AgR, AM, MS, and AdR. Conducted experiments: AgR and AdR. Performed data analysis: AgR, AM and AdR. Wrote or contributed to the writing of the manuscript: AgR, AM, AH, MS, and AdR.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.