Anil S Modak

Stable isotope breath tests in clinical medicine: a review

Diagnostic (13)C-stable isotope probes are currently being expanded in their scope, to provide precise evaluations of the presence or absence of etiologically significant changes in metabolism due to a specific disease or the lack of a specific enzyme. The salient features of the (13)C-breath test are that they are non-invasive, non-radioactive, safe, simple, and effective. The simplicity of the (13)C-breath test makes it very applicable in a clinical setting: the physician can obtain valuable diagnostic information by distinguishing between two groups or populations on the basis of the recovery of (13)CO(2) from the ingested (13)C-substrate. The breath tests can also be used to monitor the progress of disease severity or to evaluate the efficacy of medications. This review concentrates on current research in the medical field dedicated to the metabolite (13)C-labelled carbon dioxide in exhaled air following ingestion of (13)C-labelled substrates.

Rapid identification of the hepatic cytochrome P450 2C19 activity using a novel and noninvasive [13C]pantoprazole breath test.

We tested the hypothesis that the stable isotope [13C]pantoprazole is O-demethylated by cytochrome P450 CYP2C19 and that the 13CO2 produced and exhaled in breath as a result can serve as a safe, rapid, and noninvasive phenotyping marker of CYP2C19 activity in vivo. Healthy volunteers who had been genotyped for the CYP2C19*2, CYP2C19*3, and CYP2C19*17 alleles were administered a single oral dose of [13C]pantoprazole sodium-sesquihydrate (100 mg) with 2.1 g of sodium bicarbonate. Exhaled 13CO2 and 12CO2 were measured by IR spectroscopy before (baseline) and 2.5 to 120 min after dosing. Ratios of 13CO2/12CO2 after [13C]pantoprazole relative to 13CO2/12CO2 at baseline were expressed as change over baseline (DOB). Maximal DOB, DOB15 to DOB120, and area under the DOB versus time curve (AUC0–120 and AUC0–∞) were significantly different among three genotype groups (CYP2C19*1/*1, n = 10; CYP2C19*1/*2 or CYP2C19*1/*3, n = 10; and CYP2C19*2/*2, n = 5) with predicted extensive metabolizers (EMs), intermediate metabolizers (IMs), and poor metabolizers (PMs) of CYP2C19, respectively (Kruskal-Wallis test, p < 0.01); linear regression analysis indicated a gene-dose effect relationship (r2 ranged between 0.236 and 0.522; all p < 0.05). These breath test indices were significantly lower in PMs than IMs (p < 0.05) or EMs (p < 0.01) of CYP2C19. [13C]Pantoprazole plasma exposure showed significant inverse correlation with breath test indices in the respective subjects (Pearson r = -0.74; p = 0.038). These feasibility data suggest that the [13C]pantoprazole breath test is a reliable, rapid, and noninvasive probe of CYP2C19 and seems to be a useful tool to optimize drug therapy metabolized by CYP2C19.

Stereoselective pharmacokinetics of stable isotope (+/-)-[13C]-pantoprazole: Implications for a rapid screening phenotype test of CYP2C19 activity.

AIMS We have previously shown that the (±)-[(13) C]-pantoprazole breath test is a promising noninvasive probe of CYP2C19 activity. As part of that trial, plasma, breath test indices and CYP2C19 (*2, *3, and *17) genotype were collected. Here, we examined whether [(13) C]-pantoprazole exhibits enantioselective pharmacokinetics and whether this enantioselectivity is correlated with indices of breath test. METHODS Plasma (-)- and (+)-[(13) C]-pantoprazole that were measured using a chiral HPLC were compared between CYP2C19 genotypes and correlated with breath test indices. RESULTS The AUC( 0-∞) of (+)-[(13) C]-pantoprazole in PM (*2/*2, n = 4) was 10.1- and 5.6-fold higher that EM (*1/*1or *17, n = 10) and IM (*1/*2or *3, n = 10) of CYP2C19, respectively (P < 0.001). The AUC( 0-∞) of (-)-[(13) C]-pantoprazole only significantly differed between PMs and EMs (1.98-fold; P = 0.05). The AUC( 0-∞) ratio of (+)-/(-)-[(13) C]-pantoprazole was 3.45, 0.77, and 0.67 in PM, IM, and EM genotypes, respectively. Breath test index, delta over baseline show significant correlation with AUC( 0-∞) of (+)-[(13) C]-pantoprazole (Pearsons r = 0.62; P < 0.001). CONCLUSIONS [(13) C]-pantoprazole exhibits enantioselective elimination. (+)-[(13) C]-pantoprazole is more dependent on CYP2C19 metabolic status and may serve as a more attractive probe of CYP2C19 activity than (-)-[(13) C]-pantoprazole or the racemic mixture.

Single time point diagnostic breath tests: a review

The metabolism of ingested xenobiotics is clinically significant to minimize risk and optimize therapeutic benefits. A majority of the drugs approved by the FDA are metabolized by phase I enzymes. Stable isotope-labeled xenobiotics can be used to provide rapid in vivo phenotype assessment of phase I enzymes. In this paper, we describe three simple, noninvasive phenotype breath tests using [13C]-dextromethorphan and [13C]-pantoprazole for assessing polymorphic CYP2D6 and CYP2C19 enzyme activity and [13C]-uracil to assess the enzyme activity of DPD, DPHD and β-ureidopropionase for identifying pyrimidine metabolic disorder. The results of the [13C]-dextromethorphan, [13C]-pantoprazole and [13C]-uracil breath test studies suggest that they have great potential for evaluating CYP2D6, CYP2C19 and DPD enzyme activities in a relatively short time with a single time point breath collection in a clinic or hospital setting. This would enable physicians to prescribe personalized therapy for each individual by selecting the ideal medication at the right dose for optimal efficacy of xenobiotics metabolized by these enzymes.

CYP2C19 Phenoconversion by Routinely Prescribed Proton Pump Inhibitors Omeprazole and Esomeprazole: Clinical Implications for Personalized Medicine

The phenotype pantoprazole-13C breath test (Ptz-BT) was used to evaluate the extent of phenoconversion of CYP2C19 enzyme activity caused by commonly prescribed proton pump inhibitors (PPI) omeprazole and esomprazole. The Ptz-BT was administered to 26 healthy volunteers and 8 stable cardiovascular patients twice at baseline and after 28 days of PPI therapy to evaluate reproducibility of the Ptz-BT and changes in CYP2C19 enzyme activity (phenoconversion) after PPI therapy. The average intrapatient interday variability in CYP2C19 phenotype (n = 31) determined by Ptz-BT was considerably low (coefficient of variation, 17%). Phenotype conversion resulted in 25 of 26 (96%) nonpoor metabolizer (non-PM) volunteers/patients as measured by the Ptz-BT at baseline and after PPI therapy. The incidence of PM status by phenotype following administration of omeprazole/esomeprazole (known inhibitors of CYP2C19) was 10-fold higher than those who are genetically PMs in the general population, which could have critical clinical implications for personalizing medications primarily metabolized by CYP2C19, such as clopidogrel, PPI, cyclophosphamide, thalidomide, citalopram, clonazepam, diazepam, phenytoin, etc. The Ptz-BT can rapidly (30 minutes) evaluate CYP2C19 phenotype and, more importantly, can identify patients with phenoconversion in CYP2C19 enzyme activity caused by nongenetic factors such as concomitant drugs.

Changes in CYP2C19 enzyme activity evaluated by the [13C]-pantoprazole breath test after co-administration of clopidogrel and proton pump inhibitors following percutaneous coronary intervention and correlation to platelet reactivity

Dual antiplatelet therapy (DAPT) with clopidogrel and aspirin is used for the prevention of cardiovascular events following percutaneous coronary intervention (PCI). These agents increase the risk of gastrointestinal bleeding. To prevent these events, proton pump inhibitors (PPI) are routinely prescribed. It has been reported that with the exception of pantoprazole and dexlanzoprazole, PPIs can impede conversion of clopidogrel by cytochrome P450 2C19 (CYP2C19) to its active metabolite, a critical step required for clopidogrel efficacy. Changes in CYP2C19 enzyme activity (phenotype) and its correlation with platelet reactivity following PPI therapy has not yet been fully described. In this study we attempted to determine if the [ (13)C]-pantoprazole breath test (Ptz-BT) can evaluate changes in CYP2C19 enzyme activity (phenoconversion) following the administration of PPI in coronary artery disease (CAD) patients treated with DAPT after PCI. Thirty (30) days after successful PCI with stent placement, 59 patients enrolled in the Evaluation of the Influence of Statins and Proton Pump Inhibitors on Clopidogrel Antiplatelet Effects (SPICE) trial (ClinicalTrials.gov Identifier: NCT00930670) were recruited to participate in this sub study. Patients were randomized to one of 4 antacid therapies (omeprazole, esomeprazole. pantoprazole or ranitidine). Subjects were administered the Ptz-BT and platelet function was evaluated by vasodilator-stimulated phosphoprotein (VASP) phosphorylation and light transmittance aggregometry before and 30 d after treatment with antacid therapy. Patients randomized to esomeprazole and omeprazole had greater high on-treatment platelet reactivity and lowering of CYP2C19 enzyme activity at Day 60 after 30 d of PPI therapy. Patients randomized to ranitidine and pantoprazole did not show any changes in platelet activity or CYP 2C19 enzyme activity. In patients treated with esomeprazole and omeprazole, changes in CYP2C19 enzyme activity (phenoconversion) correlated well with changes in platelet reactivity. Co-administration of omeprazole or esomeprazole in patients treated with clopidogrel results in lower CYP2C19 enzyme activity and increased platelet reactivity as measured by VASP phosphorylation test while patients given pantoprazole or ranitidine did not show any significant changes in CYP2C19 enzyme activity and platelet reactivity.

Breath tests with 13C substrates

Labeled (stable and radio) isotope compounds ((2)H, (3)H, (14)C, (13)C, (15)N) have been widely used as diagnostic probes in research laboratories for over 30 years in the fields of gastroenterology, hepatology, oncology, and nutrition, as well as pharmacokinetic studies in the development of drugs. (13)C stable isotope diagnostic probes are now being expanded in their scope, to provide precise evaluations of the presence or absence of etiologically significant changes in metabolism due to a specific disease or the lack of a specific enzyme. The concept exploits the use of the (13)C- label that is incorporated at the appropriate site into a selected substrate specifically designed for the targeted enzyme in a discrete metabolic pathway. The enzyme-substrate interaction results in the release of (13)CO(2) in the expired breath. The subsequent quantification of (13)CO(2) allows for the indirect determination of pharmacokinetics and the evaluation of enzyme activity. Breath tests, although non-invasive, have not been integrated routinely in clinical practice due to most of them requiring multiple breath sample collection over an extended time period. The use of area-under-the-curve (AUC) and percent-dose-recovery (PDR) parameters of breath tests to differentiate between controls and patients has been a huge barrier to implementing them into routine clinical practice due to time constraints on clinical staff. In order to get breath tests accepted in clinical practice as in vivo diagnostic tools, the tests need to be accurate with high sensitivity and specificity with a single time point breath collection post ingestion of a (13)C substrate. It is now incumbent on diagnostic test companies to invest capital for the development of promising single time point breath tests and getting regulatory board approval (FDA, EMEA), CPT codes and reimbursement. Following regulatory approvals, the breath tests would also need to be marketed aggressively by making physicians, patients, and insurance companies aware of the medical benefits to patients and lowering of healthcare costs. The diagnostic breath tests will enable physicians and patients to benefit from rapid, novel and non-invasive ways to detect enzyme deficiencies, to monitor the progress of disease severity or medication efficacy, to trace acquired and/or congenital metabolic defects, to study in vivo the pharmacokinetics of xenobiotics, and to optimize individually tailored treatment regimens for drugs with narrow therapeutic windows. The primary reason for publishing this special section on (13)C breath tests is to highlight some of the recent advances in the field of breath tests as well as to review the literature.

Barriers to overcome for transition of breath tests from research to routine clinical practice

Over the last decade noninvasive diagnostic phenotype [13C]-breath tests using suitably labeled 13C substrates as well as breath tests using endogenous/exogenous volatile organic compounds in breath have been extensively researched. Despite the potential benefits of these companion diagnostic tests and stand-alone diagnostic tests for patient/disease stratification and market segmentation to personalize medicine, the clinical and commercial development of these diagnostic tests will need to overcome a number of regulatory, financial and scientific hurdles prior to their acceptance into routine clinical practice.

L-[1-13C] phenylalanine breath test for monitoring hepatic function after living donor liver transplant surgery

Although metabolic response after partial hepatectomy has been well studied in animal models, there are few studies examining restoration of metabolic capacity after right hepatectomy in humans. The L-[1-(13)C]-phenylalanine breath test (PBT) is a simple non-invasive diagnostic tool which allows measurement of liver functional reserve. We investigated the PBT for monitoring hepatic function in living liver donors by measuring the metabolism of L-[1-(13)C]-phenylalanine ((13)C-Phe). We used (13)C-Phe administered orally and iv to adult living liver donor patients and measured exhaled (13)CO(2) to determine the extent of metabolic impairment and time course of its return. Patients given oral (13)C-Phe had approximately 70-90% reduction in (13)CO(2) production compared with baseline 2-3 days after surgery. Patients given iv (13)C-Phe had only 40-50% reduction in (13)CO(2) production and recovered their baseline (13)C-Phe metabolism much sooner than their oral (13)C-Phe metabolic capacity (P < 0.05). In some cases oral (13)C-Phe did not recover to baseline for as long as 56 days after surgery. Patients recovering (13)C-Phe metabolism had significantly higher (13)CO(2) recovery 60 min after ingestion by day 4 (0.97 versus 3.06, P = 0.033) and day 7 (1.50 versus 5.02, P = 0.031). We conclude that orally administered amino acids may not be well absorbed and/or metabolized in some subjects for weeks after partial hepatectomy whereas intravenously delivered substrates are much better oxidized by the regenerating liver. These findings may be due to impaired gut motility due to trauma to the gastrointestinal tract or portal venous flow that reduces delivery of oral agents after liver surgery. In early recovery phase for living liver donor patients, the iv PBT would be a better predictor of functional hepatic reserve.

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

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.