Inhibition of CYP3A7 DHEA-S Oxidation by Lopinavir and Ritonavir: An Alternative Mechanism for Adrenal Impairment in HIV Antiretroviral-Treated Neonates

Abstract

Prophylactic antiretroviral therapy (ART) in HIV infected pregnant mothers and their newborns can dramatically reduce mother-to-child viral transmission and seroconversion in the neonate. The ritonavir-boosted lopinavir regimen, known as Kaletra, has been associated with premature birth and transient adrenal insufficiency in newborns, accompanied by increases in plasma dehydroepiandrosterone 3-sulfate (DHEA-S). In the fetus and neonates, cytochrome P450 CYP3A7 is responsible for the metabolism of DHEA-S into 16α-hydroxy DHEA-S, which plays a critical role in growth and development. In order to determine if CYP3A7 inhibition could lead to the adverse outcomes associated with Kaletra therapy, we conducted in vitro metabolic studies to determine the extent and mechanism of CYP3A7 inhibition by both ritonavir and lopinavir and the relative intrinsic clearance of lopinavir with and without ritonavir in both neonatal and adult human liver microsomes (HLMs). We identified ritonavir as a potent inhibitor of CYP3A7 oxidation of DHEA-S (IC 50 = 0.0514 μM), while lopinavir is a much weaker inhibitor (IC 50 = 5.88 μM). Furthermore, ritonavir is a time-dependent inhibitor of CYP3A7 with a K I of 0.392 μM and a k inact of 0.119 min –1 , illustrating the potential for CYP3A mediated drug–drug interactions with Kaletra. The clearance rate of lopinavir in neonatal HLMs was much slower and comparable to the rate observed in adult HLMs in the presence of ritonavir, suggesting that the addition of ritonavir in the cocktail therapy may not be necessary to maintain effective concentrations of lopinavir in neonates. Our results suggest that several of the observed adverse outcomes of Kaletra therapy may be due to the direct inhibition of CYP3A7 by ritonavir and that the necessity for the inclusion of this drug in the therapy may be obviated by the lower rate of lopinavir clearance in the neonatal liver. These results may lead to a reconsideration of the use of ritonavir in neonatal antiretroviral therapy.

Characterization of fluorescent probe substrates to develop an efficient high-throughput assay for neonatal hepatic CYP3A7 inhibition screening

“…4 C). In comparison, the values reported previously in Kandel et al 9 were 5.88 μM for lopinavir and 0.0514 μM for ritonavir.

Figure 4 CYP3A7 IC50 inhibition curve by ( A ) DHEA-S, ( B ) Lopinavir, and ( C ) Ritonavir assessed using DBF as a fluorescent marker.

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“…For DHEA-S, previous work reports the K M value of 16α-hydroxylation via CYP3A7 to be 5.4 µM 9 . The DHEA-S IC 50 value against CYP3A7 metabolism of DBF we obtained was 5.31 µM, corresponding closely to the K M for DHEA-S, assuming a strictly competitive model of inhibition (Fig.…”

“…It is common industrial practice to screen drugs against major hepatic CYP enzymes that are expressed in adults, but it is not routine or standard to test for the inhibitory effects of these drugs on CYP3A7, despite the continued off label use of drugs in the neonatal patient population. The lack of preclinical testing of drugs for CYP3A7 inhibition in a neonate-relevant model has led to downstream adverse drug reactions and toxic effects in clinical settings 9 , 19 , 32 – 35 . Adopting and applying a standard, simple assay to screen for CYP3A7 inhibition would significantly reduce the possibility of CYP3A7-mediated adverse drug reactions by identifying potent CYP3A7 inhibitors prior to drug administration, thereby allowing the clinician to select a safer alternative.…”

“…Drug dosing in neonates is rarely based on neonatal physiology and, as a result, dosing regimens in neonates have been formed on a trial-and-error basis, at times resulting in either subtherapeutic doses or adverse drug reactions (ADRs) from overdosing 4 , 7 , 8 . For instance, Kaletra, a ritonavir-boosted lopinavir regimen used in newborns as a prophylactic treatment for HIV infection, has been reported to cause serious health problems, including renal toxicity and collapse, central nervous system impairment, cardiac arrhythmia, hemolysis and transient adrenal insuffiency 9 – 13 . Intriguingly, inhibition of the hepatic CYP3A7 enzyme by the Kaletra pharmacoenhancer ritonavir may be a leading cause of the substantial increase in plasma level of the adrenal steroid hormone DHEA-S and associated adrenal insufficiency observed in the treated newborns 9 .…”

“…A significant factor leading to ADRs like those experienced from Kaletra is the difference in the hepatic metabolism profile of neonates versus adults 9 , 14 , 15 . Newborns and fetuses predominantly express cytochrome P450 CYP3A7, which accounts for ~ 50% of the total CYP content in their livers, but express negligible amounts of CYP3A4/5 16 .…”