Imaging Mass Spectrometry (IMS) for drug discovery and development survey: Results on methods, applications and regulatory compliance.

Imaging mass spectrometry (IMS) is increasingly used for drug discovery and development to understand target enagement, tissue distribution, drug toxicity, and disease mechanisms, etc. However, this is still a relatively new technique that requires further development validation before it will be an acceptable technique to support regulated development of new drugs. Thus, best practices will need to be established to build more confidence and gain wider acceptance by the scientific community, pharmaceutical industry, and regulatory authorities. The Imaging Mass Spectrometry Society (IMSS) and the Japan Association for Imaging Mass Spectrometry (JAIMS) have conducted a thorough survey to gather information on the current state of IMS and to identify key issues. The survey was sent to researchers or managers in the position who are currently using IMS techniques in support of their drug discovery and development efforts and/or who plan to use such tools as best practices are established. The survey probes questions related to details regarding technical aspects of IMS, which includes data acquisition, data analysis and quantitation, data integrity, reporting, applications, and regulatory concerns. This international survey was conducted online through the Survey Monkey (https://www.surveymonkey.com) in both English and Japanese from September 14 through September 30, 2020.

Cell tracking of chromium-labeled mesenchymal stem cells using laser ablation inductively coupled plasma imaging mass spectrometry.

RATIONALE: Mesenchymal stem cells (MSCs) are widely used in regenerative medicine research. Evaluating the biodistribution of MSCs is important for determining whether the cells have reached the target tissue, and the time that the stem cells reside in each area is required to estimate the duration of efficacy. METHODS: A laser ablation inductively coupled plasma imaging mass spectrometry (LAICP-IMS) method was developed for highly sensitive and quantitative surface analysis of metal elements for solid samples. We evaluated the usefulness of a cell-tracking system with LAICP-IMS to investigate the biodistribution of mouse mesenchymal stem cells (mMSCs) labeled with the natural composition of chromium (Cr) in mice. To prepare the dosing solution, mMSCs were incubated with both Na2 CrO4 and fluorescent labeling solutions. The concentration of the cells was adjusted by vehicle solution at 2.0 to 2.5 × 107 cells/mL, and the dosing suspension of mMSCs was administered by intramuscular or intravenous injection to the mice. RESULTS: Thigh muscle sections after intramuscular injection of chromium- and fluorescence-labeled mMSCs were analyzed by LAICP-IMS and fluorescence microscopy, respectively. 52 Cr mass spectrometry and fluorescence signals were detected in the same thigh muscle sections after administration of mMSCs. A half-body section was also analyzed by LAICP-IMS. 52 Cr signals were mainly detected in the lungs. CONCLUSIONS: The 52 Cr signals were observed in sections through the thigh muscle and half body after intramuscular and intravenous administration, respectively, of Cr-labeled mMSCs to mice. Our results suggest that LAICP-IMS is a sensitive and useful technique to evaluate biodistribution in cell therapy research.

Applications of MALDI mass spectrometry imaging for pharmacokinetic studies during drug development.

The concentration and distribution of a drug or its metabolites in tissues are key factors for understanding drug efficacy or toxicity. Conventional pharmacokinetic studies show that the plasma concentration of a drug is often unrelated to the intra-tissue concentration. Moreover, it is difficult to predict the distribution of a drug in tissues, particularly those with complex structures, even though the overall tissue concentration is measured by using homogenizing procedures. Mass spectrometry imaging (MSI) enables visualization of the spatial distribution and quantities of drugs in tissue sections without labeling, which can significantly impact on the development of new drugs and translational research. Recent advances in instrument technology and the knowledge accumulated to date could further improve the sensitivity, spatial resolution, and reproducibility of MSI. Here we present current applications of matrix-assisted laser desorption/ionization (MALDI)-MSI in pharmacokinetic imaging (PK-imaging) studies, give an overview of MALDI-MSI procedures, highlight the importance of internal standards, and give details of quantitative approaches. We also point out the need for standardizing MALDI-MSI techniques. PK-imaging using standardized MALDI-MSI methods, independent of instrument or technician expertise, is expected to contribute to acquiring reliable data in drug development and translational research in the future.

A phase I, open-label, single-dose micro tracer mass balance study of 14C-labeled ASP7991 in healthy Japanese male subjects using accelerator mass spectrometry.

ASP7991 is a calcimimetic that acts on the calcium-sensing receptor on parathyroid cell membranes and suppresses parathyroid hormone (PTH) secretion in the treatment of secondary hyperparathyroidism. The mass balance and metabolite profile of [14C]ASP7991 were investigated in six healthy male subjects after a single oral dose of [14C]ASP7991 [1 mg, 18.5 kBq (500 nCi)] in solution. [14C] radioactivity in plasma, urine and feces was analyzed using Accelerator mass spectrometry. ASP7991 was rapidly absorbed, metabolized and excreted. Mean recovery of [14C] radioactivity in urine and feces was 30.08% and 49.31%, respectively, and mean total recovery of [14C] radioactivity was 79.39%. The majority of [14C] radioactivity in urine and feces was excreted within the first 72 h following administration. Seven metabolites were detected in plasma, urine and feces samples, and their structures were determined by mass spectrometry. The main metabolic pathways of ASP7991 in humans were predicted to be N-dealkylation, followed by N-acetylation and taurine conjugation to a carboxylic acid moiety. Our findings show that a mass balance study using micro radioactivity doses is suitable for elucidating the pharmacokinetics of the absorption, metabolism and excretion of administered drugs.

Evaluation of the Utility of Chimeric Mice with Humanized Livers for the Characterization and Profiling of the Metabolites of a Selective Inhibitor (YM543) of the Sodium-Glucose Cotransporter 2.

PURPOSE: YM543 is a novel selective inhibitor of the sodium-glucose cotransporter 2. The objectives of the current study were to evaluate the utility of mice with humanized livers to predict human drug metabolites using YM543 as a case example. METHODS: Metabolites of YM543 generated in humans and experimental animals including chimeric mice with humanized liver, PXB mice, were analyzed via liquid chromatography-mass spectrometry, liquid chromatography-radiometric detector or nuclear magnetic resonance spectrometer. RESULTS: After oral administration of YM543, metabolites M1-M5 were detected in human plasma and urine. M2-M4 were detected in at least one species while M1 was not generated by experimental animals or in vitro systems. In the metabolite profiling in PXB mice, M1 was detected in both plasma and urine samples. CONCLUSIONS: Metabolite profile of YM543 in PXB mice and humans was closely resemble. and the human specific metabolite was detected in the model mice. The human specific metabolite, M1, was difficult to know in advance to clinical study. The ability to predict the human metabolite profile including presence of human specific metabolites using PXB mice will likely facilitate development of new drug candidates for human use.

Investigation of Metabolite Profile of YM758, a Novel If Channel Inhibitor.

BACKGROUND: YM758 monophosphate is a novel If channel inhibitor that has an inhibitory action for If current and shows a strong and specific activity, selectively lowering the heart rate and decreasing oxygen consumption by heart muscle. OBJECTIVES: The objectives of the current study were to investigate the in vivo metabolic profiles of YM758 in mice, rats, rabbits, dogs, and monkeys and to elucidate the structures of YM758 metabolites. METHODS: Biological samples were analyzed by liquid chromatography hyphenated with a radiometric detection system and liquid chromatography coupled with a mass spectrometer to clarify their metabolic patterns. To elucidate their structures, metabolites were isolated and analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. RESULTS: Our results from in vivo metabolic profiling in humans and animals indicated there is no significant species difference in the metabolism of YM758, and the metabolic pathways of YM758 are considered to be oxidation, hydration, and demethylation followed by sulfate or glucuronide conjugation.

Murine Cyp3a knockout chimeric mice with humanized liver: prediction of the metabolic profile of nefazodone in humans.

Chimeric mice with humanized livers (PXB mice) are used to investigate the metabolism and pharmacokinetics of drugs in humans. However, residual murine enzymatic activities derived from the liver and the presence of mouse small intestinal metabolism can hamper the prediction of human drug metabolism. Recently murine Cytochrome P450 3a gene knockout chimeric mice with humanized livers (Cyp3a KO CM) were developed. To evaluate the prediction of drug metabolism, nefazodone (NEF) was administered orally at 10 mg/kg to the following mouse strains: Cyp3a KO CM, murine Cyp3a gene knockout (Cyp3a KO), PXB and severe combined immunodeficiency (SCID) mice. Liquid chromatography-mass spectrometry was used for metabolic profiling of plasma, urine and bile. The prediction of human metabolite levels such as hydroxy nefazodone (OH-NEF), triazoledione form (TD), m-chlorophenylpiperazine and dealkyl metabolites in Cyp3a KO CM was superior to that in Cyp3a KO, PXB or SCID mice. Further, clinical exposure levels of NEF, OH-NEF and TD were reproduced in Cyp3a KO CM. In contrast, NEF was rapidly metabolized to TD in both PXB and SCID mice but not in Cyp3a KO mice, suggesting that murine CYP3A is involved in the elimination of NEF in these mice. These findings demonstrate that the metabolic profile of NEF in Cyp3a KO CM differs qualitatively and quantitatively from that in PXB mice due to the higher metabolic rate of NEF and its metabolites via murine CYP3A. Therefore Cyp3a KO CM might be useful in predicting the metabolic profiles of drug candidates in humans.

Human mass balance, metabolite profile and identification of metabolic enzymes of [¹4C]ASP015K, a novel oral janus kinase inhibitor.

1. The human mass balance of (14)C-labelled ASP015K ([(14)C]ASP015K), an orally bioavailable Janus kinase (JAK) inhibitor, was characterized in six healthy male subjects after a single oral dose of [(14)C]ASP015K (100 mg, 3.7 MBq) in solution. [(14)C]ASP015K was rapidly absorbed with tmax of 1.6 and 1.8 h for ASP015K and total radioactivity in plasma, respectively. Mean recovery in urine and feces amounted to 36.8% and 56.6% of the administered dose, respectively. The main components of radioactivity in plasma and urine were ASP015K and M2 (5'-O-sulfo ASP015K). In feces, ASP015K and M4 (7-N-methyl ASP015K) were the main components. 2. In vitro study of ASP015K metabolism showed that the major isozyme contributing to the formation of M2 was human sulfotransferase (SULT) 2A1 and of M4 was nicotinamide N-methyltransferase (NNMT). 3. The in vitro intrinsic clearance (CLint_in vitro) of M4 formation from ASP015K in human liver cytosol (HLC) was 11-fold higher than that of M2. The competitive inhibitory effect of nicotinamide on M4 formation in the human liver was considered the reason for high CLint_in vitro of M4 formation, while each metabolic pathway made a near equal contribution to the in vivo elimination of ASP015K. ASP015K was cleared by multiple mechanisms.

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice.

Identification and characterization of metabolites of ASP015K, a novel oral Janus kinase inhibitor, in rats, chimeric mice with humanized liver, and humans.

1. Here, we elucidated the structure of metabolites of novel oral Janus kinase inhibitor ASP015K in rats and humans and evaluated the predictability of human metabolites using chimeric mice with humanized liver (PXB mice). 2. Rat biological samples collected after oral dosing of (14)C-labelled ASP015K were examined using a liquid chromatography-radiometric detector and mass spectrometer (LC-RAD/MS). The molecular weight of metabolites in human and the liver chimeric mouse biological samples collected after oral dosing of non-labelled ASP015K was also investigated via LC-MS. Metabolites were also isolated from rat bile samples and analyzed using nuclear magnetic resonance. 3. Metabolic pathways of ASP015K in rats and humans were found to be glucuronide conjugation, methyl conjugation, sulfate conjugation, glutathione conjugation, hydroxylation of the adamantane ring and N-oxidation of the 1H-pyrrolo[2,3-b]pyridine ring. The main metabolite of ASP015K in rats was the glucuronide conjugate, while the main metabolite in humans was the sulfate conjugate. Given that human metabolites were produced by human hepatocytes in chimeric mice with humanized liver, this human model mouse was believed to be useful in predicting the human metabolic profile of various drug candidates.

In vitro/in vivo investigations to examine the gender differences in the pharmacokinetics of novel oral Janus kinase (JAK) inhibitor ASP015K and sulfate metabolite M2 in rats.

1. Although marked gender differences have been reported for the exposure level of the sulfate metabolite M2 of ASP015K in rats, no such differences have been reported for the unchanged drug. To clarify the cause of these pharmacokinetic gender differences, we investigated the in vitro hepatic sulfation, glucuronidation, and cytochrome P450 (CYP) metabolism of ASP015K in rat liver cytosols or rat liver microsomes. Further, in vivo excretion and metabolic profiles were investigated using rat urine, bile, and feces post-ASP015K administration. 2. In vitro metabolism study using liver cytosols clearly suggested that the gender differences in the M2 exposure were mainly attributed to the female-predominant ASP015K metabolism mediated by sulfotransferase (SULT). Metabolic profiles in urine and bile from male rats suggested that the major elimination pathway of ASP015K is glucuronidation in rats. No remarkable gender differences in the in vitro glucuronidation were observed. 3. The contribution of the sulfation pathway to the clearance of ASP015K was markedly lower than that of the glucuronidation pathway in both male and female rats. These results might explain why gender differences were not marked for ASP015K exposure but were for M2.

Assessment of chimeric mice with humanized liver as a tool for predicting circulating human metabolites.

The ability to predict circulating human metabolites of a candidate drug before first-in-man studies are carried out would provide a clear advantage in drug development. A recent report demonstrated that while in vitro studies using human liver preparations reliably predict primary human metabolites in plasma, the predictability of secondary metabolites, formed by multiple reactions, was low, with total success rates of < or =65%. Here, we assess the use of chimeric mice with humanized liver as an animal model for the prediction of human metabolism in vivo. Metabolism studies with debrisoquine and (S)-warfarin demonstrated significantly higher concentrations of their primary human abundant metabolites in serum or plasma in chimeric mice than in control mice. Humanized chimeric mice were also capable of producing human-specific metabolites of several in-house compounds which were generated through more than one metabolism reaction. This model is closer to in vivo human physiology and therefore appears to have an advantage over in vitro systems in predicting complex metabolites in human plasma. However, prediction of human metabolites failed for other compounds which were highly metabolized in mice. Although requiring careful consideration of compound suitability, this model represents a potential tool for predicting human metabolites in combination with conventional in vitro systems.

Investigation of long-term retention of unchanged (-)-N-{2-[(R)-3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide, a novel "funny" If current channel inhibitor, and its metabolites in the eyeball and thoracic aorta of rats.

(-)-N-{2-[(R)-3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758), a novel "funny" If current channel inhibitor, was being developed as a treatment for stable angina and atrial fibrillation. After a single oral administration of (14)C-YM758, extensive accumulation and long-term retention of radioactivity were observed in the eyeballs of nonalbino rats and in the thoracic aorta of albino/nonalbino rats. Radioluminograms of the eyeballs of nonalbino rats indicated that the radioactivity was localized to the uveal tract, which suggests that the radioactivity may be positively charged and bound mainly to the melanins. Treatment with a mixture of 2 mol/l hydrochloric acid and methanol (5:95, v/v) allowed for the recovery of the major portion of radioactivity from the eyeball, which suggests reversible binding. The radioactive constituents in eyeballs consisted of the unchanged drug (YM758) and three metabolites [mainly 6,7-dimethoxy-2-[(3R)-piperidin-3-ylcarbonyl]-1,2,3,4-tetrahydroisoquinoline (YM-252124)]. Using the organic solvent mixture described above, almost all of the radioactivity was not collected from the thoracic aorta, and approximately 90% was recovered by treatment with elastase, which suggests that some metabolites covalently bind to the elastin fiber localized in the tunica media.

Interactive effect of paraoxonase-1 Q192R polymorphism and smoking history on the lung function decline in grain workers.

PURPOSE: This retrospective longitudinal study investigated the association between the Q192R polymorphism of the high-density lipoprotein-associated multifunctional antioxidant enzyme, paraoxonase-1 (PON1), and lung function decline, while taking into account smoking history. METHODS: The demographic, occupational, and respiratory symptom information and lung function variables were obtained from 216 male Saskatchewan grain workers. RESULTS: An interaction between the PON1 genotypes and smoking status was observed. Current smokers with the 192R allele had a lower forced expiratory volume in the first second (FEV(1)) and FEV(1) per forced vital capacity (FVC). The annual decline rate of FEV(1)/FVC in current smokers was greater among 192R allele carriers than noncarriers (0.58+/-0.05 vs. 0.35+/-0.04 %/yr, p<0.0001). A similar result was observed with FEV(1) (40.9+/-6.4 vs. -33.0+/-7.0 mL/yr, p=0.10). The annual decline rate of FVC was not influenced by the genotypes. CONCLUSIONS: These results strengthened the previous findings of our cross-sectional study, suggesting that the 192R allele may be a novel genetic risk factor for airway injury among current smokers.

Population estimation of the effects of cytochrome P450 2C9 and 2C19 polymorphisms on phenobarbital clearance in Japanese.

A nonlinear mixed-effect modeling (NONMEM) program was used to evaluate the effects of cytochrome P450 (CYP) 2C9 and CYP2C19 polymorphisms on the phenobarbital (PB) population clearance for Japanese epileptics. The pharmacokinetics of the 260 PB concentrations at a steady-state obtained from 79 patients was described with a one-compartment open pharmacokinetic model with first-order elimination. The covariates screened included the total body weight (BW), age, gender, PB daily dose, CYP2C9 and CYP2C19 genotypes, the coadministered antiepileptic drugs (AEDs), and complications. The final model of PB apparent clearance was as follows: CL = 0.23 x (BW/40)0.21 x 0.52CYP2C9*1/*3 x 0.68VPA x 0.85PHT x 0.85SMID x (1 + etaCL) where CL = the clearance of PB; CYP2C9*1/*3 = 1, otherwise 0; VPA = 1 if valproic acid is coadministered, otherwise 0; PHT = 1 if phenytoin is coadministered, otherwise 0; SMID = 1 if complications of severe or profound mental retardation with a significant behavior impairment are presented, otherwise 0; and etaCL = the independent random error distributed normally with the mean zero and variance equal to omegaP2. The total clearance of PB decreased by 48% in patients with CYP2C9*1/*3 genotype in comparison with those with CYP2C9*1/*1 genotype (P < 0.001). An effect of CYP2C19 polymorphisms was not detected. To our knowledge, this is the first report to demonstrate that the CYP2C9 genotype affects the PB metabolism in routine care, but the results should be further verified in other ethnic populations.

ABCB1 polymorphisms influence the response to antiepileptic drugs in Japanese epilepsy patients.

OBJECTIVES: The efflux transporter P-glycoprotein encoded by the ATP-binding cassette (ABC)B1 gene may play a role in drug-resistant epilepsy by limiting gastrointestinal absorption and brain access of antiepileptic drugs (AEDs). Our objective was to investigate the effect of ABCB1 polymorphisms on AED responsiveness and on the pharmacokinetics of carbamazepine (CBZ) in epileptic patients with the indication for CBZ therapy. METHODS: The ABCB1 T-129C, C1236T, G2677T/A and C3435T polymorphisms were genotyped in 210 Japanese epileptics who had been prescribed AEDs, including CBZ, for longer than 2 years. Haplotype and diplotype frequencies were estimated by expectation-maximization algorithm. Drug resistance was determined by the presence of seizures. Association of the polymorphisms with the risk of drug resistance was estimated by logistic regression analysis and the odds ratios (ORs) were adjusted for the clinical factors affecting the outcome of AED therapy. CBZ concentrations to the dose (C/D) ratios were compared among the ABCB1 polymorphisms. RESULTS: Drug-resistant patients were more likely to have the T allele (OR [95% confidence interval (CI)], 2.02 [1.14-3.58]) and the TT genotype at C3435T (OR [95% CI], 3.64 [1.16-11.39]), and the TT genotype at G2677T/A (OR vs the GG genotype [95% CI], 3.43 [1.01-11.72]). The frequency of the T-T-T haplotype at C1236T, G2677T/A and C3435T was significantly higher (OR [95% CI], 1.84 [1.03-3.30]), and the CC-GG-CC diplotype was lower (OR [95% CI], 0.09 [0.01-0.85]) in the drug-resistant patients than in the drug-responsive patients. None of the ABCB1 polymorphisms were observed to influence the C/D ratios of CBZ. CONCLUSION: We demonstrated that ABCB1 polymorphisms may influence the AED responsiveness without significant changes in the plasma concentrations of CBZ. Our findings were the inverse of previous results in European epileptics, thus the influence of ABCB1 polymorphisms on the AED responsiveness and/or the P-glycoprotein activity may vary among races.

Tumor necrosis factor alpha and pulmonary function in Saskatchewan grain handlers.

OBJECTIVE: The objective of this study was to estimate the contribution of lifestyle (cigarettes) and tumor necrosis factor (TNF) alpha polymorphisms at position 308 of the tumor necrosis factor alpha gene promotor (TNF-308*1/*2) to pulmonary function among grain handlers. METHODS: Employed male grain handlers (157) provided occupational and respiratory symptom information, pulmonary function measurements, and DNA for genotyping. RESULTS: The genotypes of 101 were TNF-308*1/*1, 47 were *1/*2, and nine were *2/*2. Current smokers whose genotype was *2/*2 or *1/*2 had lower values compared with other combinations of genotype and smoking status. Among *1/*1 homozygotes, current smokers had better percent of predicted forced expiratory volume in 1 second (P = 0.04) mean values than nonsmokers and better percent of predicted forced vital capacity than exsmokers (P = 0.017) or nonsmokers (P = 0.008). CONCLUSIONS: These results indicate the complexity of determining which workers will develop acute and chronic adverse pulmonary conditions in response to exposure to grain dust and the toxins in cigarette smoke interacting with their genotype.

Phenotype-genotype analysis of CYP1A2 in Japanese patients receiving oral theophylline therapy.

OBJECTIVE: To clarify the association between the cytochrome P450 (CYP) 1A2 genotype with the CYP1A2 phenotype and to search for the CYP1A2*1K haplotype, which has been shown to decrease CYP1A2 inducibility and/or other functional polymorphisms in Japanese. METHODS: Two polymorphisms, CYP1A2*1C and CYP1A2*1F, were genotyped in 126 patients receiving oral slow-release theophylline (TP) therapy and in 224 healthy volunteers. The CYP1A2 phenotype was assessed by the plasma [1-methyluric acid (1U)+3-methylxanthine (3X)]/TP ratio in the patients. The volunteers were given 150 mg caffeine, and the urine [1X+1U+5-acetylamino-6-amino-3-methyluracil (AAMU)]/17U ratio was used for CYP1A2 phenotyping. CYP1A2 intron 1 and six exons (exon 2-exon 7) were sequenced in the patients whose (1U+3X)/TP ratios were below the mean-2SD of those of all patients, and intron 1 was also sequenced in an additional 20 healthy volunteers exhibiting putative low CYP1A2 activities. RESULTS: The individual (1U+3X)/TP ratios ranged from 0.007 to 0.21 (a 30-fold difference) in the patients, and the (1X+1U+AAMU)/17U ratios ranged from 1.6 to 112 (a 70-fold difference) in the healthy volunteers. The CYP1A2 activities were not significantly influenced by CYP1A2*1C or CYP1A2*1F. We found no functional polymorphisms by a sequencing analysis. CONCLUSION: These results suggest that the CYP1A2*1C and CYP1A2*1F genotypes are not crucial factors for the variability of CYP1A2 activity and that the CYP1A2*1K haplotype is either nil or only shows a very low frequency in Japanese.