Population Pharmacokinetics of MYL-1402O, a Proposed Biosimilar to Bevacizumab and Reference Product (Avastin®) in Patients with Non-squamous Non-small Cell Lung Cancer.

BACKGROUND AND OBJECTIVES: MYL-1402O is a bevacizumab (Avastin®) biosimilar. Pharmacokinetic and safety similarity of MYL-1402O and reference Avastin® authorized in the European Union (EU-Avastin®) and the US (US-Avastin®) was demonstrated in healthy subjects (phase I, NCT02469987). The key objectives of this study were to establish a population pharmacokinetic (PopPK) model on pooled data from the phase I and phase III clinical studies to assess pharmacokinetic linearity of MYL-1402O and Avastin® across dose ranges, to assess the pharmacokinetic similarity of MYL-1402O and Avastin® in patients with non-squamous non-small cell lung cancer (nsNSCLC), and to explore potential covariates to account for systematic sources of variability in bevacizumab exposure. METHODS: Efficacy and safety of MYL-1402O compared with EU-Avastin® was investigated in a multicenter, double-blind, randomized, parallel-group study in patients with stage IV nsNSCLC (phase III, NCT04633564). PopPK models were developed using a nonlinear mixed effects approach (NONMEM® 7.3.0). RESULTS: The pharmacokinetics of Avastin® and MYL-1402O were adequately described with a two-compartment linear model. Fourteen covariates were found to be statistically significant predictors of bevacizumab pharmacokinectics. The impact of each covariate on area under the concentration-time curve, half-life, and maximum plasma concentration was modest, and ranges were similar between the treatment groups, MYL-1402O and EU-Avastin®, in patients with nsNSCLC. The pharmacokinectics of bevacizumab appeared to be linear. CONCLUSIONS: PopPK analysis revealed no significant differences between pharmacokinetics of MYL-1402O and Avastin® in patients with nsNSCLC. The developed PopPK model was considered robust, as it adequately described bevacizumab pharmacokinetics in healthy participants and nsNSCLC patients.

Phase III double-blind study comparing the efficacy and safety of proposed biosimilar MYL-1402O and reference bevacizumab in stage IV non-small-cell lung cancer.

PURPOSE: This phase III study compared the efficacy and safety of proposed biosimilar MYL-1402O with reference bevacizumab (BEV), as first-line treatment for patients with stage IV non-squamous non-small-cell lung cancer. PATIENTS AND METHODS: Patients were randomly assigned (1:1) to receive MYL-1402O or bevacizumab with carboplatin-paclitaxel up to 18 weeks (6 cycles), followed by up to 24 weeks (8 cycles) of bevacizumab monotherapy. The primary objective was comparison of overall response rate (ORR), based on independently reviewed best tumor responses as assessed during the first 18 weeks. ORR was analyzed per US Food and Drug Administration (ratio of ORR) and European Medicines Agency (difference in ORRs) requirements for equivalence evaluation. Secondary end points included progression-free survival, disease control rate, duration of response, overall survival, safety, and immunogenicity over a period of 42 weeks, and pharmacokinetics (up to 18 weeks). RESULTS: A total of 671 patients were included in the intent-to-treat population. The ratio of ORR was 0.96 [confidence interval (CI) 0.83, 1.12] and the difference in ORR was -1.6 (CI -9.0, 5.9) between treatment arms; CIs were within the predefined equivalence margins. Overall, the incidence of treatment-emergent adverse events and serious adverse events was comparable. Treatment-emergent anti-drug antibody (ADA) positivity was transient, with no notable differences between treatment arms (6.5% versus 4.8% ADA positivity rate in MYL-1402O versus BEV, respectively). The incidence of neutralizing antibody post-baseline was lower in the MYL-1402O arm (0.6%) compared to the bevacizumab arm (2.5%). CONCLUSIONS: MYL-1402O is therapeutically equivalent to bevacizumab, based on the ORR analyses, with comparable secondary endpoints. TRIAL REGISTRY INFORMATION: EU Clinical Trials Register, Registration # EudraCT no. 2015-005141-32https://www.clinicaltrialsregister.eu/ctr-search/search?query=2015-005141-32. PLAIN LANGUAGE SUMMARY: Previous studies established bioequivalence of the proposed bevacizumab biosimilar MYL-1402O to reference bevacizumab. In this randomized, double-blind, phase III trial, MYL-1402O (n = 337) demonstrated comparable efficacy to bevacizumab (n = 334) in treating advanced non-squamous non-small-cell lung cancer per Food and Drug Administration and European Medicines Agency requirements for equivalence; the ratio of objective response rate (ORR) was 0.96 [90% confidence interval (CI) 0.83, 1.12] and the difference in ORR (MYL-1402O:bevacizumab) was -1.6 (95% CI -9.0, 5.9). Median progression-free survival at 42 weeks was comparable: 7.6 (7.0, 9.5) with MYL-1402O versus 9.0 (7.2, 9.7) months (p = 0.0906) with bevacizumab, by independent review. Treatment-emergent adverse events leading to death (2.4% vs 1.5%), serious adverse events (17.6% vs 16.7%), and antidrug antibodies (6.5% vs 4.8%), were comparable in the MYL-1402O vs bevacizumab arms, respectively. The incidence of neutralizing antibody post-baseline was lower with MYL-1402O (0.6%) than with bevacizumab (2.5%). These findings confirm therapeutic equivalence of MYL-1402O to bevacizumab, providing opportunities for improving access to bevacizumab.

Functional analysis of phenylalanine residues in the active site of cytochrome P450 2C9.

The two published crystal structures of cytochrome P450 2C9, complexed with ( S)-warfarin or flurbiprofen, implicate a cluster of three active site phenylalanine residues (F100, F114, F476) in ligand binding. However, these three residues appear to interact differently with these two ligands based on the static crystal structures. To elucidate the importance of CYP2C9's active site phenylalanines on substrate binding, orientation, and catalytic turnover, a series of leucine and tryptophan mutants were constructed and their interactions with ( S)-warfarin and ( S)-flurbiprofen examined. The F100-->L mutation had minor effects on substrate binding and metabolism of each substrate. In contrast, the F114L and F476L mutants exhibited substantially reduced ( S)-warfarin metabolism and altered hydroxy metabolite profiles but only modestly decreased nonsteroidal antiinflammatory drug (NSAID) turnover while maintaining product regioselectivity. The F114-->W and F476-->W mutations also had opposing effects on ( S)-warfarin versus NSAID turnover. Notably, the F476W mutant increased the efficiency of ( S)-warfarin metabolism 5-fold, yet decreased the efficiency of ( S)-flurbiprofen turnover 20-fold. (1)H NMR T 1 relaxation studies suggested a slightly closer positioning of ( S)-warfarin to the heme in the F476W mutant relative to the wild-type enzyme, and stoichiometry studies indicated enhanced coupling of reducing equivalents to product formation for ( S)-warfarin, again in contrast to effects observed with ( S)-flurbiprofen. These data demonstrate that F114 and F476, but not F100, influence ( S)-warfarin's catalytic orientation. Differential interactions of F476 mutants with the two substrates suggest that their catalytically productive binding modes are not superimposable.

Substrate proton to heme distances in CYP2C9 allelic variants and alterations by the heterotropic activator, dapsone.

CYP2C9 polymorphisms result in reduced enzyme catalytic activity and greater activation by effector molecules as compared to wild-type protein, with the mechanism(s) for these changes in activity not fully elucidated. Through T(1) NMR and spectral binding analyses, mechanism(s) for these differences in behavior of the variant proteins (CYP2C9.2, CYP2C9.3, and CYP2C9.5) as compared to CYP2C9.1 were assessed. Neither altered binding affinity nor substrate (flurbiprofen) proton to heme-iron distances differed substantially among the four enzymes. Co-incubation with dapsone resulted in reduced substrate proton to heme-iron distances for all enzymes, providing at least a partial mechanism for the activation of CYP2C9 variants by dapsone. In summary, neither altered binding affinity nor substrate orientation appear to be major factors in the reduced catalytic activity noted in the CYP2C9 variants, but dapsone co-incubation caused similar changes in substrate proton to heme-iron distances suggesting at least partial common mechanisms in the activation of the CYP2C9 forms.

Influence of fluorescent probe size and cytochrome b5 on drug-drug interactions in CYP2C9.

7-Methoxy-4-trifluoromethylcoumarin (MFC) has been used extensively in high-throughput screens for the identification of potential CYP2C9 interactions. More recently, additional probes from Invitrogen have been used. Vivid 2C9 Green is the largest of the probes and has had limited prior characterization. The new series of probes differ significantly from MFC and were examined for their ability to identify interactions with 19 CYP2C9 substrates/inhibitors. The inhibition profiles depend largely on the physical differences between the fluorescent probe substrates. Cytochrome b5 (cyt b5) was also investigated for the ability to alter the inhibition profile of a given compound. The stoichiometric addition of cyt b5 caused an increase in V max of MFC and Vivid 2C9 Green 4.4 and 1.7 times, respectively. Furthermore, cyt b5 imposes a steric component to the active site as the inhibition profiles were significantly affected in incubations with MFC. The addition of cyt b5 had limited impact on the inhibition profiles generated with Vivid 2C9 Green. The K(m) of Vivid 2C9 Green increased from 1.2+/-0.2 micro M to 4.8+/-0.3 micro Mas a result of cyt b5 addition. These results illustrate that multiple substrate probes may be necessary for screening drug-drug interaction in CYP2C9 and that cyt b5 effects can impart steric restraints on the CYP2C9 active site.

Differential activation of CYP2C9 variants by dapsone.

Studies have shown that CYP2C9.1 mediated metabolism of flurbiprofen or naproxen is activated by co-incubation with dapsone. However, dapsone activation has not been examined in the known variant forms of CYP2C9. Six concentrations of flurbiprofen (2-300microM) or naproxen (10-1800 microM) were co-incubated with six concentrations of dapsone (0-100 microM) and with reconstituted, purified CYP2C9.1, CYP2C9.2 (R144C), CYP2C9.3 (I359L), or CYP2C9.5 (D360E), in order to assess degrees of activation. Dapsone increased the efficiency (V(m)/K(m)) of flurbiprofen 4'-hydroxylation by CYP2C9.1, CYP2C9.2, CYP2C9.3, and CYP2C9.5 by 8-, 31-, 47-, and 22-fold, respectively. In similar experiments using the substrate naproxen, dapsone increased the efficiency of naproxen demethylation 7-, 15-, 13-, and 22-fold, in CYP2C9.1, CYP2C9.2, CYP2C9.3, and CYP2C9.5, respectively. Also, dapsone normalized naproxen's kinetic profile from biphasic (CYP2C9.1 and CYP2C9.2) or linear (CYP2C9.3 and CYP2C9.5) to hyperbolic for all variant forms. Thus, amino acid substitutions of CYP2C9 variants affect the degree of dapsone activation in a genotype-dependent fashion. Furthermore, the degree of effect noted across variants appeared to be dependent on the substrate studied.

CYP2C9 genotype-dependent effects on in vitro drug-drug interactions: switching of benzbromarone effect from inhibition to activation in the CYP2C9.3 variant.

The CYP2C9.3 variant exhibits marked decreases in substrate turnover compared with the wild-type enzyme, but little is known regarding the effect this variant form may have on the occurrence of drug-drug interactions. To examine this possibility, the effect of the potent CYP2C9 inhibitor, benzbromarone, was studied with regard to CYP2C9.1- and CYP2C9.3-mediated flurbiprofen metabolism to evaluate whether the variant enzyme exhibits differential inhibition kinetics. Although benzbromarone inhibited CYP2C9.1 activity as expected, CYP2C9.3-mediated flurbiprofen 4'-hydroxylation was activated in the presence of benzbromarone. T1 relaxation studies revealed little change in distances of flurbiprofen protons from the heme iron of either CYP2C9.1 or CYP2C9.3 in the presence of benzbromarone compared with flurbiprofen alone. Spectral binding studies were also performed to investigate whether benzbromarone affected substrate binding, with the addition of benzbromarone having little effect on flurbiprofen-binding affinity in both CYP2C9.1 and CYP2C9.3. Docking studies with the 2C9.1 structure crystallized with a closed active site identified multiple but overlapping subsites with sufficient space for benzbromarone binding in the enzyme when flurbiprofen was positioned closest to the heme. If the closed conformation of 2C9.3 is structurally similar to 2C9.1, as expected for the conservative I359L mutation, then the dynamics of benzbromarone binding may account for the switching of drug interaction effects. In conclusion, the I359L amino acid substitution found in CYP2C9.3 not only reduces metabolism compared with CYP2C9.1 but can also dramatically alter inhibitor effects, suggesting that differential degrees of drug inhibition interactions may occur in individuals with this variant form of CYP2C9.

Modeling kinetic data from in vitro drug metabolism enzyme experiments.

Modeling of in vitro enzyme kinetic data derived from drug metabolism experiments can greatly facilitate the drug development process because estimation of kinetic parameters can facilitate decision making regarding whether to continue development of a compound. From this information, predictions can be made regarding the "metabolic stability" of a compound and even the in vivo intrinsic clearance of the drug. Many drugs exhibit typical Michaelis-Menten-type kinetics in vitro that result in a hyperbolic kinetic profile from which Km and Vm can be readily estimated. However, it is increasingly being recognized that many drug compounds exhibit "atypical" enzyme kinetics in vitro, requiring use of more complex kinetic models for data fitting and parameter estimation. These atypical kinetic profiles may include sigmoidal kinetics (autoactivation), biphasic kinetics, substrate inhibition kinetics, and heterotropic cooperativity (activation). This article briefly summarizes the types of equations necessary to adequately model both typical and atypical kinetic profiles in order to facilitate correct estimation of the relevant kinetic parameters.

Effector-mediated alteration of substrate orientation in cytochrome P450 2C9.

Cytochrome P450 2C9 (CYP2C9)-mediated flurbiprofen 4'-hydroxylation is activated by the presence of dapsone resulting in reduction of the K(m) for flurbiprofen hydroxylation and an increase in V(m). Previous spectral binding studies have demonstrated that the binding of flurbiprofen with CYP2C9 is increased (decrease in K(S)) by the presence of dapsone. We hypothesized that the two compounds are simultaneously in the active site with the presence of dapsone causing flurbiprofen to be oriented more closely to the heme. T(1) relaxation rates determined by NMR were used to estimate the distances of protons on these compounds from the paramagnetic heme-iron center. Samples contained 0.014 microM CYP2C9 and 145 microM flurbiprofen in the presence and absence of 100 microM dapsone. Estimated distances of various flurbiprofen protons from the heme ranged from 4.2 to 4.5 A in the absence of dapsone and from 3.2 to 3.8 A in the presence of dapsone. The 4' proton of flurbiprofen, the site of metabolism, showed one of the greatest differences in distance from the heme in the presence of dapsone, 3.50 A, as compared to the absence of dapsone, 4.41 A. Dapsone protons were less affected, being 4.40 A from the heme in the absence of flurbiprofen and 4.00-4.01 A from the heme in the presence of flurbiprofen. Molecular modeling studies were also performed to corroborate the relative orientations of flurbiprofen and dapsone in the active site of CYP2C9. Shift of the 4' proton of flurbiprofen closer to the heme iron of CYP2C9 in the presence of dapsone may play a role in activation.

Activation of CYP2C9-mediated metabolism by a series of dapsone analogs: kinetics and structural requirements.

Cytochrome P450 2C9-mediated metabolism has been shown to be activated in the presence of the effector dapsone. However, it has yet to be established what effector structural features are necessary to activate CYP2C9 activity. To address this question, kinetic studies were conducted with nine analogs of dapsone containing various functional properties (three sulfone compounds, three carbonyl compounds, and three sulfonamide compounds), to examine the functional groups important for enzyme activation by the effector (dapsone). Results show that phenylsulfone (dapsone without the para-amino groups) activates flurbiprofen 4'-hydroxylation comparable to dapsone but inhibits naproxen demethylation. Meanwhile, p-tolylsulfone had little effect on flurbiprofen metabolism, but activated naproxen demethylation, albeit only at high concentrations. These substrate-dependent differences in effect suggest that naproxen has a different binding orientation compared with flurbiprofen. Perhaps most interesting is that replacement of only one amino group from dapsone with a nitro group (4-(4-nitrophenylsulfonyl)-aniline) resulted in substantial inhibition of flurbiprofen 4'-hydroxylation, suggesting that electronic effects may influence activation of this substrate. Other analogs either had minor or no effect on CYP2C9-mediated metabolism. Overall, it is apparent from these studies that a sulfone group in direct association with two benzene rings with para-electron-donating groups represents the most efficient activator of CYP2C9. However, the effects of these analogs appear to be concentration- and substrate-dependent, further complicating the prediction of these types of in vitro interactions.

Polymorphic variants (CYP2C9*3 and CYP2C9*5) and the F114L active site mutation of CYP2C9: effect on atypical kinetic metabolism profiles.

CYP2C9 wild-type protein has been shown to exhibit atypical kinetic profiles of metabolism that may affect in vitro-in vivo predictions made during the drug development process. Previous work suggests a substrate-dependent effect of polymorphic variants of CYP2C9 on the rate of metabolism; however, it is hypothesized that these active site amino acid changes will affect the kinetic profile of a drug's metabolism as well. To this end, the kinetic profiles of three model CYP2C9 substrates (flurbiprofen, naproxen, and piroxicam) were studied using purified CYP2C9*1 (wild-type) and variants involving active site amino acid changes, including the naturally occurring variants CYP2C9*3 (Leu359) and CYP2C9*5 (Glu360) and the man-made mutant CYP2C9 F114L. CYP2C9*1 (wild-type) metabolized each of the three compounds with a distinctive profile reflective of typical hyperbolic (flurbiprofen), biphasic (naproxen), and substrate inhibition (piroxicam) kinetics. CYP2C9*3 metabolism was again hyperbolic for flurbiprofen, of a linear form for naproxen (no saturation noted), and exhibited substrate inhibition with piroxicam. CYP2C9*5-mediated metabolism was hyperbolic for flurbiprofen and piroxicam but linear with respect to naproxen turnover. The F114L mutant exhibited a hyperbolic kinetic profile for flurbiprofen metabolism, a linear profile for naproxen metabolism, and a substrate inhibition kinetic profile for piroxicam metabolism. In all cases except F114L-mediated piroxicam metabolism, turnover decreased and the K(m) generally increased for each allelic variant compared with wild-type enzyme. It seems that the kinetic profile of CYP2C9-mediated metabolism is dependent on both substrate and the CYP2C9 allelic variant, thus having potential ramifications on drug disposition predictions made during the development process.