Tivantinib

The effect of proton pump inhibitors on the CYP2C19 enzyme activity evaluated by the pantoprazole-13C breath test in GERD patients: clinical relevance for personalized medicine

Keywords: pantoprazole breath test, GERD, CYP2C19 enzyme activity

Abstract

Patients with gastroesophageal reflux disease (GERD) are routinely prescribed one of the six FDA approved proton pump inhibitors (PPI). All of these PPI are inhibitors of CYP2C19 enzyme to varying degrees. The phenotype pantoprazole-13C breath test (Ptz-BT) was used to identify patients who are poor metabolizers (PM) and the extent of phenoconversion of CYP2C19 enzyme activity caused by four PPI (omeprazole, esomprazole pantoprazole and rabeprazole) in 54 newly diagnosed GERD patients prior to initiating randomly selected PPI therapy and 30 d after PPI therapy.

The phenoconversion after 30 d of PPI therapy in GERD patients was statistically significant (p =0.001) with omeprazole/esomeprazole (n = 27) strong CYP2C19 inhibitors, while there was no change in CYP2C19 enzyme activity (p = 0.8) with pantoprazole/ rabeprazole (n = 27), weak CYP2C19 inhibitors. The concommitant use of omeprazole/esomeprazole, therefore, could have critical clinical relevance in individualizing medications metabolized primarily by CYP2C19 such as PPI, clopidogrel, phenytoin, cyclophosphamide, thalidomide, citalopram, clonazepam, diazepam, proguanil, tivantinib etc. The rapid (30 min), in vivo, and non-invasive phenotype Ptz-BT can evaluate CYP2C19 enzyme activity. More importantly, it can identify GERD patients with low CYP2C19 enzyme activity (PM), caused by PPI or other concomitant medications, who would benefit from dose adjustments to maintain efficacy and avoid toxicity. The existing CYP2C19 genotype tests cannot predict the phenotype nor can it detect phenoconversion due to non genetic factors.

Introduction

Gastroesophageal reflux disease, or GERD, is a digestive disorder that affects the lower esophageal sphincter (LES), the ring of muscle between the esophagus and stomach. The range of GERD prevalence estimates was 18.1%–27.8% in North America, 8.8%–25.9% in Europe, 2.5%–7.8% in East Asia, 8.7%–33.1% in the Middle East, 11.6% in Australia and 23.0% in South America [1]. Antacids—Tums, Rolaids, Maalox; H2 Blockers— Tagamet, Pepcid, Zantac; and proton pump inhibitors (PPI)—Nexium, Prilosec, Prevacid, Dexilent, Aciphex, Protonix are commonly prescribed to treat the disease.

Human CYP2C19 enzyme is critical in the metabolism of PPI [2]. The effects of genetic poly- morphisms result in a 5- to 20-fold variability in the clearance of drugs metabolized by CYP2C19 among individuals and different ethnic groups pri- marily due to effects of genetic polymorphisms [2, 3], but also as a result of nongenetic factors, such as drug interactions [2, 4, 5], age [6], pregnancy [7], and disease state [2, 8] CYP2C19 is a clinically relevant drug-metabolizing enzyme for which genotyping and phenotyping information has the potential to improve drug safety and efficacy [9–11]. At least 27 variant alleles for CYP2C19 have been identified, with the most extensively described being CYP2C19*2, CYP2C19*3, and CYP2C19*1712.

The FDA has placed pharmacogenomic biomark- ers labels for several drugs metabolized by CYP2C19 enzyme to identify poor metabolizers (PM) and ultra rapid metabolizers (UM) to adjust the dosage or alter the drug. However, there is not a single diagnostic test in the market that can definitively identify these patients that need dose or drug alterations since the genetic test grossly underestimates the number of PM. There are mainly two classes of drugs on the FDA list for CYP2C19 pharmacogenomic biomarkers—cardiology (antiplatelet therapy Clopidogrel, Prasugrel and Tica- grelor) and gastroenterology (PPI) (table 1).

The aim of the study was to utilize the Ptz-BT to identify patients who are phenotype PM prior to PPI therapy and those who would undergo phenoconver- sion with PPI therapy to poor metabolizer (PM) status with either strong CYP2C19 inhibitors—omeprazole and esomeprazole or weak CYP2C19 inhibitors— pantoprazole and rabeprazole. Patients who are PM with inherent low CYP2C19 enzyme activity or caused due to phenoconversion at steady state after initiating PPI therapy would have elevated levels of the drugs omeprazole and esomeprazole and would benefit from a lower maintenance dose to reduce long-term toxicity while still retaining efficacy.

The top 200 FDA-approved drugs most often prescribed in the USA are primarily metabolized by CYP450 enzymes— CYP3A family contributed to 37% of the drugs, followed by CYP2C9 (17%), CYP2D6 (15%), CYP2C19 (10%), CYP1A2 (9%) and CYP2C8 (6%). Drug-induced phenoconversion of CYP2C19 metabolizer status is common with the use of certain commonly prescribed PPI [4, 5]. These CYP2C19 inhibitors can readily convert an extensive metabolizer (EM) or intermediate metabolizer (IM) phenotype to a poor metabolizer (PM) phenotype and thus alter drug clearance and response.

In the current study we evaluated the CYP2C19 enzyme activity with the phenotype Ptz-BT before and after 30 d of PPI therapy in GERD patients to identify patients who were CYP2C19 phenotype PM. These patients would benefit from dose reductions on their initial and maintenance dose of PPI therapy after steady state is reached. Patients were randomized to one of the two arms of the study each with two PPI. One arm was omeprazole (40 mg QD) and esomeprazole (20 mg QD), and the other was pantoprazole (20 mg QD) and rabeprazole (20 mg QD). We examined the genotype- phenotype discordance both at day 0 prior to initiating PPI therapy and at day 31 after PPI therapy in both arms of the study.

Methods

Study subjects
The study was approved by the IRB at the Crimean State Medical University in Simferopol, Russia (formerly Ukraine). A total of 64 GERD patients—male (19) and female (45)—were recruited at the outpatient clinic of the Crimean State Medical University. All study subjects signed a written informed consent before participation. Ten patients were excluded from the study for not complying with the inclusion/exclusion criteria for the study. Fifty-four patients were eligible for data analysis on the study (table 2).

Inclusion criteria

GERD patients age 16–70 years; willing to sign informed consent form; willing to give consent for drawing blood samples and/or mouth swabs for genotype; willing to perform overnight fasting of 8 h and 24 h alcohol abstention prior to Ptz-BT; and willing to perform two visits for the study.

Exclusion criteria

Prior adverse events from taking pantoprazole, sodium bicarbonate, esomeprazole, omeprazole, rabeprazole; infrared spectrometer (Photal Electronics, Tokyo, Japan) equipped with interference filters that are wavelength-selective for the absorbance of 13CO2 and 12CO2. Enrichment of 13CO2 in expired air was measured as the delta over baseline at 30 min (DOB30) after (±)-[13C]-pantoprazole administration relative to pre-dose (baseline) as described below [5, 13] kidney parameters exceeding 2.5 times the normal range.

Study design

This was an open label, 2-visit pantoprazole-13C-breath test (Ptz-BT) study recruiting GERD patients not on any PPI therapy. A blood sample was collected at the first visit for genotype (CYP2C19*2 and *3 alleles). On the same visit eligible GERD patients were administered a single aqueous 100 mg oral dose of (±)-pantoprazole, sodium salt: sesquihydrate (4-O-methyl-13C), 98%; CLM-7831-CTM lot number C-7831-RR-G1; Cambridge Isotope Laboratories, Inc., Andover, MA, with 2.1 g sodium bicarbonate to prevent degradation by stomach acid after a minimum 8 h fast and 24 h alcohol abstention. Breath samples were collected using 300 ml breath collection bags (Otsuka Pharmaceuticals Inc., Tokyo, Japan) at baseline pre ingestion and at 30 min post ingestion of (±)-[13C]-pantoprazole. The study patients were the randomized to one of the two arms of PPI—Arm 1 Omeprazole 40 mg QD or Esomeprazole 20 mg QD and Arm 2 Pantoprazole 20 mg QD or Rabeprazole 20 mg QD.
On their second and final visit on Day 31 patients were administered the Ptz-BT with the last PPI tablet taken 12 h prior to the breath test.

CYP2C19 genotype

The PCR was performed using SleepexR CYP2C19 ACE Genotyping a multiplex-PCR System for detecting CYP2C19 genotypes. SleepexR provide nucleic acid isolation, PCR amplification of target DNA using DPOTM primers (DPO-dual Priming Oligonucleotide technology), ScreenTapeR system for DNA detection. Isolation Kit used for nucleic acid isolation: NucleoSpin Blood (Man.MIN, Cat. No 740951). Genotyping of CYP2C19 alleles *2 (rs4244285) and *3 (rs4986893) was accomplished by using ScreenTapeR system.

Quantitation of 13CO2

The concentrations of 13CO2 and 12CO2 in exhaled breath samples were determined using the POCone where DOB was expressed as change per mille (‰).

Statistical analysis

All statistical tests for lowering of CYP2C19 enzyme activity as measured by the DOB30 values (‰) at baseline prior to PPI therapy and after administration of PPI were evaluated using the two-tailed P values. A P value <0.05 was considered statistically significant. The DOB30 differences between various genotypes and phenotypes have been reported as mean ± SD. Results Genotype-phenotype discordance From previous studies [4, 5, 13–18] correlating CYP219 genotype and phenotype using pantoprazole metabolites in plasma, the DOB30 cutoff values for the Ptz-BT were set at <1.2‰ for PMs, 1.2–3.4‰ for intermediate metabolizers (IMs), ⩾3.5 to 7‰ for extensive metabolizers (EMs), and >7‰ for ultrarapid metabolizers (UMs).

In the omeprazole/esomeprazole arm by geno- type 19 of 27 patients enrolled were EMs and 8 of 27 patients were IMs. The genotype-phenotype discord- ance for genotype EMs was 26% at baseline prior to PPI therapy with 1 UM, 14 EMs, 3 IMs and 1 PM by phenotype (DOB30 = 4.7 ± 1.8‰). After 30 d of PPI therapy the genotype phenotype discordance was 63% with 7 EMs, 9 IMs and 1 PM (DOB30 = 2.9 ± 1.5‰).

The genotype-phenotype discordance for genotype IMs was 25% at baseline prior to PPI therapy with 6 IMs and 2 PMs by phenotype (DOB30 = 2.4 ± 0.9‰).After 30 d of PPI therapy the genotype-phenotype discordance was 50% with 4 IMs and 4 PMs (DOB30 = 1.4 ± 1.0‰) figure 1(A).
In the pantoprazole/rabeprazole arm by geno- type 12 of 27 patients enrolled were EMs and 15 of 27 patients were IMs. The genotype-phenotype discord- ance for genotype EMs (n = 12) was 58% at baseline prior to PPI therapy with 5 EMs, 5 IMs and 2 PM by phenotype (DOB30 = 3.1 ± 1.4‰). After 30 d of PPI therapy the genotype phenotype discordance was 58% with 4 EMs, 7 IMs and 1 PM (DOB30 = 3.1 ± 1.4‰).

The genotype-phenotype discordance for geno- type IMs (n = 15) was 36% at baseline prior to PPI therapy with 11 IMs and 4 PMs by phenotype (DOB30 = 1.7 ± 0.9‰). After 30 d of PPI therapy the genotype-phenotype discordance was 36% with 11 IMs and 4 PMs (DOB30 = 1.5 ± 0.9‰). The geno- type-phenotype discordance or the DOB30 (CYP2C19 phenotype) did not significantly alter before and after pantoprazole/rabeprazole therapy (figure 1(B)).

Phenoconversion

The DOB30 values, reflecting CYP2C19 enzyme activity of GERD patients (n = 27) on omeprazole/ esomeprazole therapy arm, were significantly lowered (phenoconverted) from before (DOB30 = 4.0 ± 1.9‰) and after (DOB30 = 2.4 ± 1.5‰) 30 d of therapy (p < 0.001), as shown in figure 2(A). However, DOB30 values of GERD patients (n = 27) on pantoprazole/ rabeprazole therapy arm exhibited no statistically significant change before (DOB30 = 2.3 ± 1.4‰) and after (DOB30 = 2.2 ± 1.4‰) 30 d of therapy (p = 0.80) figure 2(B). This clearly indicates the inhibition of CYP2C19 enzyme by potent inhibitors of CYP2C19 omeprazole/ esomeprazole leading to phenoconversion while pantoprazole/rabeprazole both weak inhibitors showed no phenoconversion. There would be no need for dose adjustments of PPI with pantoprazole/ rabeprazole unless they are PM by phenotype while patients on omeprazole/esomeprazole may require dose reductions depending on the extent of phenoconversion. Resolution of GERD Two of 27 patients in each of the omeprazole- esomeprazole group and pantoprazole-rabeprazole group did not have resolution of their GERD following 30 d of PPI therapy. Comparison with two prior studies In the Austrian study [4] carried out in 31 healthy volunteers the PPI—omeprazole/esomeprazole were primarily involved in inhibition of CYP2C19 enzyme activity to evaluate the rate of phenoconversion. Consequently, the phenoconversion rate (genotype- phenotype discordance) in healthy volunteers was substantially higher (table 3). In the Canadian study [5] with cardiovascular patients who underwent PCI surgery followed by PPI therapy in conjunction with dual antiplatelet ther- apy—Clopidogrel and aspirin, the Ptz-BT was capa- ble of identifying patients with lowered efficacy of the antiplatelet therapy Clopidogrel—a prodrug caused by drug–drug interaction between co-administered CY2C19 inhibitor PPI especially the more potent inhibitors omeprazole and esomperazole. The PPI in this study were primarily used to study the influence of CYP2C19 inhibition on the antiplatelet therapy and evaluate efficacy of Clopidogrel. Incontrast in the current study we carried out the Ptz- BT study in a completely different cohort of Ukrainian patients suffering from GERD, taking multiple co-med- ications, who needed PPI therapy to resolve gastrointes- tinal acidity. The PPI were primarily being used to treat an underlying disease—GERD for a pronounced and long-lasting reduction of gastric acid production with less of the dose being utilized for lowering the CYP2C19 enzyme activity (phenoconversion). We observed the lowest number of PM after PPI therapy with omeprazole/ esomeprazole compared to the other two studies [4, 5] due to lower CYP2C19 phenoconversion (table 4). PPI are the most common drugs used in a gastroen- terologist’s practice worldwide [23]. This class of acid- reduction agents has been available for commercial use for nearly 27 years, having displaced the use of hista- mine-2-receptor antagonists (H2RA) for patients with moderate to severe gastric acid-related diseases as well as for prophylaxis of upper gastrointestinal (GI) injury caused by NSAIDS [24]. The safety profile of PPI with very low toxicity has been recently challenged by stud- ies published on long-term potential adverse effects associated with use of PPI. The Food and Drug Admin- istration (FDA) has issued a number of broad-based product warnings, for all of the available PPI drugs available either with prescription or over-the-counter. The rise in gastric pH with the prolonged use of PPI at higher doses results in • alteration of absorption of essential vitamins and minerals • adversely impacting cardiovascular function [25] • decrease in bone density [26] • increase in dementia and Alzheimer’s disease especially in elderly patients [27, 28] • alteration of the pharmacogenomics resulting in drug interactions (clopidogrel) • increased risk of hypocalcemia, hypomagnesemia, Clostridium difficile infections and pneumonia [29, 30]. Patients often become dependent on PPI, using them for years rather than for the 8- to 12-week period usually recommended. Therefore, it is clinically impor- tant to use the lowest doses for the shortest duration to treat the underlying gastrointestinal disorder. With the FDA recommending the use of exist- ing genetic tests in the labels of drugs metabolized by CYP2C19 to identify PM, there is a clinical unmet need to develop a diagnostic test that would reliably iden- tify these PM patients. Unfortunately, the genetic tests fall woefully short in identifying all CYP2C19 pheno- type PM due to nongenetic reasons resulting in phe- noconversions. We have now clearly demonstrated in the current study and two past studies [4, 5] that the Ptz-BT meets that clinical unmet need to help physi- cians personalize cardiovascular, and GERD patient medications. The clinical utility of the non invasive, point of care Ptz-BT is to rapidly identify all GERD patients who are CYP2C19 phenotype PM that need lowered initial or maintenance PPI doses to avoid the long term use side effects and maintain efficacy. The current study sets up future directions for research into conducting clinical studies in GERD patients for personalizing initial and maintenance doses of PPI. The Ptz-BT is able to noninvasively and rapidly identify all PM both before and after PPI ther- apy, thereby providing the physician a tool for personal- izing medications metabolized by CYP2C19. Limitations Genotyping for CYP2C19*17 allele would have been useful to ascertain if it would lead to higher CYP2C19 phenotype since there were three GERD patients with DOB30 values over 6‰. These patients could have benefitted from higher initial doses of the PPI. In our study we did not personalize (adjust/lower) the maintenance dose for the GERD patients that had a significant change in CYP2C19 phenotype. Conclusions Predicting the phenotype for the CYP2C19 enzyme solely from the genetic test or in combination with concomitant medications in clinical practice for individualizing therapy is virtually impossible. This is mainly due to the genotype-phenotype discordance even prior to initiating PPI therapy in GERD patients. Individuals withthe same genotype do not have the same phenotype (large interindividual variability) due to nongenetic factors, such as age, diet, environment, liver disease, and, most importantly, drug–drug interactions. The CYP2C19 phenotype can be ascertained by invasive (plasma metabolites of drug), time consuming, laborious and expensive therapeutic drug monitoring (TDM) or via the Ptz-BT, a rapid, noninvasive, in vivo point of care test to evaluate CYP2C19 enzyme activity (phenotype) which presents the physician with a diagnostic tool to individualize drugs metabolized by CYP2C19 especially when they are taking commonly prescribed and widely used CYP2C19 inhibitors such as PPI and cimetidine. Patients with the same phenotype do not drop in CYP2C19 enzyme activity (phenotype) to the same extent with more potent CYP2C19 inhibitors omeprazole/esomeprazole. This information on CYP2C19 phenotype is critical for a physician and eliminates the prediction of phenotype. The Ptz-BT is capable of identifying PM prior to initiating PPI therapy and detecting the extent of phenoconversion after initiating PPI therapy when plasma steady state of drug is reached. The CYP2C19 phenotype can be utilized to personalize both the initial and maintenance doses of PPI especially for PM to maintain efficacy and eliminate long-term toxicity.