Association between CYP2C19 genotype and proguanil oxidative polymorphism

AIMS: To examine the relationship between proguanil metabolism and the number of mutations in CYP2C19 by comparing the CYP2C19 genotype and proguanil phenotype of 10 subjects. METHODS: Partial clearance and urinary metabolic ratio data were obtained from a previous study of 10 subjects [5]. Analysis of CYP2C19 genotypes was performed using PCR amplification followed by restriction endonuclease digestion of genomic DNA from a blood sample. RESULTS: The intrinsic partial clearance of PG to CG ranged from 0.41-10.11 h-1, and was related to the number of functional CYP2C19 alleles present. Genotypic PMs had metabolic ratios > 13, while genotypic heterozygote EMs had metabolic ratios < 9. CONCLUSIONS: Proguanil may be a suitable phenotyping probe for the CYP2C19 genetic polymorphism, however the exact antimode of the urinary metabolic ratio chosen to separate poor and extensive metabolisers needs further investigation.     

Role of CYP3A4 in human hepatic diltiazem N-demethylation: inhibition of CYP3A4 activity by oxidized diltiazem metabolites

The antihypertensive agent diltiazem (DTZ) impairs hepatic drug metabolism by inhibition of cytochrome P450 (CYP). The accumulation of DTZ metabolites in serum occurs during prolonged therapy and leads to decreased DTZ elimination. Thus, DTZ metabolites may contribute to CYP inhibition. This study assessed the role of human CYPs in microsomal DTZ oxidation and the capacity of DTZ metabolites to inhibit specific CYP activities. DTZ N-demethylation varied 10-fold in microsomal fractions from 17 livers (0.33-3.31 nmol/mg of protein/min). DTZ oxidation was correlated with testosterone 6beta-hydroxylation (r = 0.82) and, to a lesser extent, tolbutamide hydroxylation (r = 0.59) but not with activities mediated by CYP1A2 or CYP2E1. CYP3A4 in lymphoblastoid cell microsomes catalyzed DTZ N-demethylation but CYP2C8 and CYP2C9 were also active (approximately 20% and 10% of the activity supported by CYP3A4); seven other CYPs produced little or no N-desmethyl DTZ from DTZ. The CYP3A4 inhibitors ketoconazole and troleandomycin decreased microsomal DTZ oxidation, but inhibitors or substrates of CYP2C, CYP2D and CYP2E1 produced no inhibition. Some inhibition was produced by alpha-naphthoflavone, a chemical that inhibits CYP1As and also interacts with CYP3A4. In further experiments, the capacities of DTZ and three metabolites to modulate human CYP 1A2, 2E1, 2C9 and 3A4 activities were evaluated in vitro. DTZ and its N-desmethyl and N,N-didesmethyl metabolites selectively inhibited CYP3A4 activity, whereas O-desmethyl DTZ was not inhibitory. The IC50 value of DTZ against CYP3A4-mediated testosterone 6beta-hydroxylation (substrate concentration, 50 microM) was 120 microM. The N-desmethyl (IC50 = 11 microM) and N,N-didesmethyl (IC50 = 0.6 microM) metabolites were 11 and 200 times, respectively, more potent. From kinetic studies, N-desmethyl DTZ and N,N-didesmethyl DTZ were potent competitive inhibitors of CYP3A4 (Ki = approximately 2 and 0.1 microM, respectively). CYP3A4 inhibition was enhanced when DTZ and N-desmethyl DTZ underwent biotransformation in NADPH-supplemented hepatic microsomes in vitro, supporting the contention that inhibitory metabolites may be generated in situ. These findings suggest that N-demethylated metabolites of DTZ may contribute to CYP3A4 inhibition in vivo, especially under conditions in which N-desmethyl DTZ accumulates, such as during prolonged DTZ therapy.     

Imipramine demethylation in vivo: impact of CYP1A2, CYP2C19, and CYP3A4

Nephron loss leads to increased production of reactive oxygen intermediates. We measured the effect of carvedilol, a beta-blocking drug with radical scavenging properties, on renal function, glomerulosclerosis, antioxidant enzyme status and in vivo hydrogen peroxide (H2O2) production in rats with chronic renal failure caused by 5/6 nephrectomy (remnant kidney) and compared results to data obtained with propranolol, a beta-blocking drug without scavenging characteristics. Carvedilol and propranolol were administered during 11 weeks following reduction of nephron number. Kidneys were examined using enzymatic and histological techniques. Both carvedilol and propranolol decreased systolic blood pressure. Compared to propranolol, carvedilol offered some additional beneficial effects on renal function, particularly with regard to glomerulosclerosis. Lipid peroxidation, evaluated by malonaldehyde and 4-hydroxynonenal concentration in cortex homogenates, was decreased in carvedilol-treated rats only. Superior beneficial effect of carvedilol treatment is not linked to a significant up-regulation of the activities of the remnant kidney antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutase) or to a decreased in vivo H2O2 production.     

Simvastatin but not pravastatin is very susceptible to interaction with the CYP3A4 inhibitor itraconazole

MTG8 (HGMW-approved symbol CBFA2T1) was originally identified as one of the loci involved in the t(8;21)(q22;q22) of acute myeloid leukemia. We characterize two human MTG8-related genes, MTGR1 and MTGR2 (HGMW-approved symbols CBFA2T2 and CBFA2T3). The former is duplicated in mouse, one locus possibly being a retroposon. Multiple MTG8-related sequences are found in several vertebrate species, from fish to mammals, albeit not in a urodele. MTGR2 maps to 16q24 and, like MTG8 and MTGR1, is close to one of three loci encoding a syntrophin (dystrophin-associated proteins). Moreover, an alternative MTGR1 promoter/5' exon is contained within the alpha1-syntrophin locus. Thus, the two classes of genes may define novel paralogous groups. MTGR1 is expressed mainly in brain, while MTGR2 is expressed in the thymus and possibly in monocytes. Like MTG8, MTGR1 is transcribed into a number of isoforms due to alternative splicing of different 5' exons onto a common splice acceptor site. Comparison of the three predicted human MTG8-related polypeptides to their Drosophila counterpart (nervy) highlights four separate regions of sequence conservation that may correspond to distinct domains. The most NH2-terminal of these is proportionately more conserved among the human polypeptides, presumably due to specific structural/functional constraints.     

Association of cytomegalovirus genotype with graft rejection after liver transplantation

BACKGROUND: The envelope glycoprotein gB of human cytomegalovirus (CMV) occurs as one of four main genotypes. Some previous studies have proposed a relationship of CMV gB genotype to the frequency of symptomatic infection and to clinical outcomes in both transplant and human immunodeficiency virus-infected populations. Our aim was to define the distribution of CMV gB genotypes and the impact on acute cellular rejection and graft/patient survival after orthotopic liver transplantation (OLT). METHODS: Between October 1988 and December 1996, 325 patients underwent cyclosporine-based OLT at our center. CMV infection was surveyed prospectively and defined as viral isolation from blood or urine; 53 (16%) patients had detectable CMV. Isolates were genotyped by polymerase chain reaction amplification and restriction digest analysis. RESULTS: The distribution of CMV genotypes was: gB1, 19 (36%) patients; gB2, 15 (28%) patients; gB3, 13 (24%) patients; and gB4, 4 (8%) patients. Two patients (4%) had mixed infection (1 + 3, 1 + 4). Age, preOLT diagnosis, use of ganciclovir prophylaxis, basal immunosuppression, mean number of HLA donor/recipient mismatches, and United Network of Organ Sharing status were comparable among patients with different genotypes. Patients with gBl had a significantly higher mean number of acute rejection episodes (1.52+0.30 vs. 0.67+0.22; P=0.027). However, there was no difference in rejection severity, including OKT3 usage or FK506 conversion, or development of chronic rejection among patients with different genotypes. The gB genotype did not affect the development of symptomatic or tissue-invasive CMV disease, detected in 15 patients. Actuarial rates of patient (odds ratio [OR] 3.0; confidence interval [CI] 1.49-6.0) and graft (OR 2.57; CI 1.25-5.22) survival were significantly diminished in the group with CMV infection versus those without CMV (P<0.0001 for both), but there was no association with CMV genotype. CONCLUSIONS: (1) Patients with CMV infection had significantly reduced patient and graft survival rates at 1 and 5 years after OLT as compared with OLT recipients without CMV infection. (2) CMV genotype gB1 was associated with a higher mean number of acute rejection episodes.     

Inhibitory anti-CYP3A4 peptide antibody: mapping of inhibitory epitope and specificity toward other CYP3A isoforms

The ultrastructures of colonies of two stable UV-induced morphological mutants and their parental strain of Candida albicans grown on glucose-containing solid medium were investigated by scanning electron microscopy. The structures and ultrastructures of these three types of colonies were determined not only in terms of the proportions of blastospores, hyphae and pseudohyphae, but also with regard to the mode of budding of blastospores and the positions of these particular cell types within the colonies. Hyphae with an atypical appearance and branching characters were observed both in regular-wrinkled and in irregular-wrinkled mutant colonies. Smooth colonies of the parental strain and the mutants exhibited the same hyphal network within the agar, suggesting that micro-environmental factors in the agar overcame the effects of these mutations.     

Trans-species gene transfer for analysis of glucocorticoid-inducible transcriptional activation of transiently expressed human CYP3A4 and rabbit CYP3A6 in primary cultures of adult rat and rabbit hepatocytes

BACKGROUND: The sensitive detection of circulating tumor cells and micrometastases may have important therapeutic and prognostic implications. METHODS: The molecular detection of occult tumor cells can be accomplished by amplifying tumor specific abnormalities present in the DNA or mRNA of malignant cells with the polymerase chain reaction (PCR). This approach has been used mainly for hemato-lymphoid malignancies. The other main PCR strategy for the detection of minimal residual disease (MRD) involves amplification of tissue-specific mRNA. This method was applied for the detection of occult disease in solid tumors. RESULTS: PCR was shown to be superior to conventional techniques in detecting circulating tumor cells and micrometastases allowing the identification of 1 tumor cell diluted with 10(6)-10(7) normal cells. The central question of whether PCR positivity reliably predicts relapse remains unanswered for many tumor types. Serial analysis of a large number of samples is needed and currently undertaken in many institutions. CONCLUSIONS: PCR is a highly sensitive method for the detection of circulating tumor cells and micrometastases in solid and hematopoietic malignancies. If PCR positivity is found to be a reliable tool, this will likely have a major impact on the treatment of many cancers. Patients could be selected for systemic therapy at an earlier stage when the metastatic tumor burden is low. PCR may improve the preoperative staging of patients with epithelial malignancies and therefore help avoid unnecessary radical procedures. Furthermore, this test may be useful in monitoring the effectiveness of adjuvant therapy, the intensity and duration of which is tailored to the individual patient. The impact of this PCR based approach on clinical oncology is likely to be profound.     

Modification of clinical presentation of prostate tumors by a novel genetic variant in CYP3A4

BACKGROUND: Pathways involved in androgen metabolism have been implicated in the etiology of prostate cancer. The goal of this study was to evaluate the effect of CYP3A4, a gene associated with the oxidative deactivation of testosterone, on the clinical presentation of prostate cancers. METHODS: A polymerase chain reaction-based approach was used to identify sequence variants of the human CYP3A4 gene. To ascertain whether allelic variants of the CYP3A4 gene were associated with tumor stage and grade and age of the patient at diagnosis, we determined CYP3A4 genotypes in 230 Caucasian men with incident prostate cancer. RESULTS: We identified a novel genetic variant (CYP3A4-V) that has an altered 5' regulatory element, containing an A to G mutation, upstream of the CYP3A4 gene. We then compared clinical characteristics of prostate cancers in men who did and did not carry this variant. The presence of the CYP3A4-V allele was associated with a higher tumor-lymph node-metastasis (TNM) stage and Gleason grade. The association between CYP3A4 genotype and tumor stage was most pronounced in men diagnosed at a relatively old age who reported no family history of prostate cancer. In this group, 46% of men with stage T3/T4 tumors carried CYP3A4-V, whereas only 5% of individuals with stage T1 tumors carried CYP3A4-V (adjusted odds ratio = 9.45; 95% confidence interval = 2.54-35.17; chi2(1) = 12.28; two-sided P<.001). CONCLUSIONS: We determined that a single base change in the 5' flanking region of the CYP3A4 gene was associated with higher clinical stage and grade in men with prostate tumors. Our results suggest that mutations in the CYP3A4 gene may influence prostate carcinogenesis.     

Correlation between P450 CYP1A1 inducibility, MspI genotype and lung cancer incidence

The aim of this study was to verify a possible correlation between CYP1A1 induction, MspI genotype and lung cancer incidence. A case-control study was performed on 48 lung cancer patients and 81 healthy subjects to test the existence of a correlation, within a European population. The hyperinducible group exhibited a significantly higher risk of lung cancer (odds ratio = 3.41; P = 0.036), especially for adenocarcinoma (odds ratio = 5.29; P = 0.033). In contrast with the situation observed in Asian populations, the frequency of the M2 allele did not differ significantly in the total lung cancer population (7.82%) and the group of healthy subjects (10.71%). The median inducibility value was slightly higher among cancer patients with one or two M2 alleles than among patients homozygous for the wild-type allele (P = 0.09). However, the percentage of individuals possessing at least one mutated allele was not significantly higher among hyperinducible patients (37.5%) than among non-hyperinducible patients (16.0%). No significant correlation could be found between M2 allele and lung cancer or between M2 allele and CYP1A1 inducibility; the only positive correlation found was between CYP1A1 hyperinducibility and lung cancer incidence. Our observations do not support the view that the presence of the M2 allele at the MspI site of the CYP1A1 gene constitutes a significant lung cancer risk in Caucasians.     

A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: expression of CYP3A4, CYP2A6, and CYP2E1

Heterologous expression of unmodified recombinant human cytochrome P450 enzymes (P450s) in Escherichia coli has proved to be extremely difficult. To date, high-level expression has only been achieved after altering the 5'-end of the native cDNA, resulting in amino acid changes within the P450 protein chain. We have devised a strategy whereby unmodified P450s can be expressed to high levels in E. coli, by making NH2-terminal translational fusions to bacterial leader sequences. Using this approach, we initially tested two leader sequences, pelB and ompA, fused to CYP3A4. These were compared with an expression construct producing a conventional NH2-terminally modified CYP3A4 (17alpha-3A4). Both leader constructs produced spectrally active, functional protein. Furthermore, the ompA-3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with 17alpha-3A4. We then tested the ompA leader with CYP2A6 and CYP2E1, again comparing with the conventional (17alpha-) approach. As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression than the 17alpha-construct. The ompA fusion strategy thus appears to represent a significant advance for the expression of P450s in E. coli, circumventing the previous need for individual optimization of P450 sequences for expression.     

The CYP3A4 inhibitor intraconazole does not affect the pharmacokinetics of a new calcium-sensitizing drug levosimendan

Itraconazole is a potent inhibitor of CYP3A4 isoenzyme and it can cause clinically significant interactions with some other drugs. Levosimendan is a new calcium-sensitizing drug intended for congestive heart failure. We aimed to study possible interactions of itraconazole with levosimendan in healthy volunteers. Twelve healthy male volunteers were included into a randomized, double-blind, two-phase crossover study. A wash-out period of 4 weeks was held between the phases. The subjects were given orally itraconazole 200 mg or placebo daily for 5 days. On the fifth day, they received a single oral dose of 2 mg of levosimendan. Levosimendan plasma concentrations were determined up to 12 hours and ECG, heart rate, and blood pressure followed-up to 8 hours after intake of levosimendan. Itraconazole had no significant effects on the pharmacokinetic parameters of levosimendan. Neither were there any differences in heart rate, PQ-, QTc- or QRS intervals between the placebo and itraconazole phases. The systolic blood pressure was decreased slightly more (p < 0.05) during the itraconazole phase than during the placebo phase. In conclusion, because the potent CYP3A4 inhibitor itraconazole had no significant pharmacokinetic interaction with levosimendan, interactions with CYP3A4 inhibitor, and oral levosimendan are unlikely.     

Polycyclic aromatic hydrocarbon-DNA adducts in human placenta and modulation by CYP1A1 induction and genotype

This study investigated the relationship in human placenta between polycyclic aromatic hydrocabon (PAH)-DNA adduct levels and two biomarkers of cytochrome P4501A1 (CYP1A1): gene induction evidenced by CYP1A1 mRNA, and a genetic polymorphism, the CYP1A1 MspI RFLP. CYP1A1 codes for an inducible enzyme system that catalyzes the bioactivation of PAHs. Prior research found a high correlation in human lung tissue between CYP1A1 activity and DNA damage from PAHs. The CYP1A1 Mspi RFLP has been linked in some studies to risk of lung cancer. The relationships in human placenta between DNA damage, CYP1A1 activity and genotype have not been well characterized and may be relevant to risks from transplacental PAH exposure. The study cohort consisted of 70 newborns from Krakow, Poland, a city with elevated air pollution, and 90 newborns from nearby Limanowa, an area with lower air pollution but greater indoor coal use. Contrary to results seen previously in lung tissue, CYP1A1 mRNA was not significantly correlated with PAH-DNA adduct levels in the placenta. Smoking (self-reported maternal and infant plasma cotinine) was significantly associated with CYP1A1 mRNA levels (P < 0.01), but not with PAH-DNA adduct levels. Placental PAH-DNA adduct levels were significantly higher in infants with the CYP1A1 MspI restriction site compared with infants without the restriction site (P < 0.01), implicating a genetic factor in inter-individual variation in DNA damage in human placenta. Further studies are needed to determine the relevance of this finding to risk of transplacental carcinogenesis.     

Inhibition of cytochrome P-450 3A (CYP3A) in human intestinal and liver microsomes: comparison of Ki values and impact of CYP3A5 expression

Seckel syndrome has been described as the prototype of the primordial bird-headed type of dwarfism. Since Seckel originally defined the disorder, less than 60 cases have been reported. In addition to the characteristic craniofacial dysmorphism and skeletal defects, abnormalities have been described in the cardiovascular, hematopoietic, endocrine, and central nervous systems. This pleiotropy has implied genetic heterogeneity and prompted reviews of previously reported cases of Seckel syndrome. As a result, the characteristic diagnostic features of Seckel syndrome have been highly debated. Although deletions in chromosome 2q have been described, to date, no genetic defect has been defined. We report three cases of Seckel-like syndrome in siblings from nonconsanguinous Caucasian parents. In addition to the typical Seckel phenotypic features, all three cases were characterized by severe hydrocephalus. We review the literature and propose that there is a spectrum of Seckel conditions that share some common key features, but also demonstrate a wide range of phenotypic features.     

CYP2C19 genotype and phenotype determined with omeprazole in patients with acid-related disorders with and without Helicobacter pylori infection

Mandatory exploration is the standard method for managing patients with gunshot wounds to the abdomen and back. This policy is associated with a high incidence of unnecessary laparotomies and significant morbidity. Reports from our center have shown that a policy of selective management, based on clinical findings, is safe in such patients. Patients with bullet trajectories that carry a high likelihood for intraabdominal organ injury may constitute a subgroup at particular risk. The need for routine or selective exploration in similar patients must be assessed. Therefore we decided to analyze patients with transpelvic gunshot wounds. The objective of the study was to examine if a policy of selective management of patients with transpelvic gunshot wounds is safe. This prospective study was conducted at an academic level I trauma center. We admitted 37 patients with transpelvic gunshot wounds over a 12-month period. All patients were managed according to a protocol that dictated laparotomy in the presence of significant clinical findings (peritoneal signs, hemodynamic instability, gross hematuria, rectal bleeding) and observation in the absence of the above. Additional diagnostic workup was performed only in appropriate cases rather than routinely. Nineteen (51.3%) patients were immediately operated on the basis of clinical findings. Sixteen of these laparotomies were therapeutic. Eighteen (48.6%) patients were initially observed. Subsequently, three of them underwent exploration for development of abdominal tenderness. All three laparotomies were nontherapeutic. The remaining 15 (40.5%) patients were successfully managed nonoperatively. There were no delays in diagnosis or missed injuries. Clinical examination had a sensitivity of 100% and specificity of 71.4% in detecting the need for laparotomy. A policy of selective management is thus safe, even for patients who suffer gunshot wounds with a high likelihood for intraabdominal organ injury. Clinical examination, supported by additional studies in appropriate cases, is the main method of selecting patients for operation or nonoperative treatment.     

Chlorotrifluoroethylene trimer and tetramer are inducers of the CYP4A subfamily

Male Wistar albino rats were treated for a 7 day period with equimolar doses of the trimer and tetramer oligomers of chlorotrifluoroethylene (CTFE), resulting in significant hepatomegaly for both compounds. In addition, both trimer and tetramer significantly induced the peroxisomal beta-oxidation of fatty acids as assessed by increases in palmitoyl-coenzyme A (CoA) oxidation, thus confirming these oligomers as peroxisome proliferators. Consistent with these conclusions, both trimer and tetramer increased the hydroxylation of lauric acid indicating that the CTFEs were inducers of the CYP4A subfamily, a conclusion further supported by substantial increases in the steady-state levels of the cognate CYP4A1 mRNA as determined by northern blotting. The liver appeared to be more susceptible to induction than the kidney and the CTFE tetramer was more potent than the trimer. These results are discussed with respect to both the differential hepatotoxicity, and biotransformation/disposition of the two polyhalogenated oligomers.     

Establishment of transgenic mice carrying human fetus-specific CYP3A7

OBJECTIVE: To determine the value of cytology in the follow-up of cervical cancer. STUDY DESIGN: The study group consisted of 230 patients with invasive cervical carcinoma who were followed for one to seven years. Forty-four patients developed recurrences or metastases. During this period, cytologic investigations involved 795 exfoliative smears from the cervix or vaginal vault, 10 fine needle aspirates and 5 fluids. RESULTS: Thirty-three patients had positive or inconclusive cervical or vault smears that were histologically proven to be recurrences, and the other 11 patients had clinically obvious recurrences that were not smeared. Cytology first alerted the clinicians to recurrence in eight patients. Of 25 cervical or vault smears reported as malignant, 24 (96%) were histologically confirmed, and 1 showed radiation change on biopsy. In all 22 cases of smears reported as inconclusive, a biopsy followed, and in 9 (41%) of these, recurrence was demonstrated histologically. Inability to distinguish radiation change from recurrent malignancy was the chief cause of inconclusive smears. Five fluids and seven fine needle aspirates were diagnosed as malignant, saving patients an invasive diagnostic procedure. CONCLUSION: Cytology is a useful, cost-effective, noninvasive and accurate investigation in the follow-up of cervical cancer.     

Effect of androgen administration during puberty on hepatic CYP2C11, CYP3A, and CYP2A1 expression in adult female rats

This biochemical and pharmacokinetic investigation was undertaken to evaluate the effects of androgen administration during puberty on sex-dependent cytochrome P450 (CYP or P450) enzyme expression in adult female rats. Hepatic testosterone 2alpha-hydroxylase activity and CYP2C11 and CYP3A protein levels were elevated in prepubertally ovariectomized rats injected subcutaneously with testosterone enanthate at 35-49 days of age and killed 41 days after discontinuation of treatment. In contrast, testosterone 6beta- and 7alpha-hydroxylase activities and CYP2A1 protein content were not affected. The increase in CYP2C11 and CYP3A was likely not due to circulating testosterone because plasma testosterone was undetectable. The calculated elimination half-life was 51 +/- 6 hr (mean +/- SE) after testosterone enanthate administration. By 80 days after treatment, CYP2C11 and CYP3A levels were no longer increased. To determine if CYP2C11 expression was responsive to a more periodic pattern of androgen release, ovariectomized rats were injected subcutaneously once or twice daily with unesterified testosterone (elimination half-life was 2.0 +/- 0.3 hr, mean +/- SE). Once- or twice-daily dosing (5 or 2.5 micromol/kg/injection, respectively) during days 35-49 of age did not increase the mean CYP2C11 expression in 90-day-old female rats, although testosterone 2alpha-hydroxylase activity and CYP2C11 protein content were elevated in three of the eight rats injected twice daily. Neither dosing regimen increased CYP3A or decreased CYP2A1 expression. In summary, the results indicate that treatment with testosterone enanthate during puberty resulted in a prolonged but reversible increase in hepatic expression of CYP2C11 and CYP3A.     

Neither dapsone hydroxylation nor cortisol 6beta-hydroxylation detects the inhibition of CYP3A4 by HIV-1 protease inhibitors

OBJECTIVE: This study examined the use of dapsone N-hydroxylation and cortisol 6beta-hydroxylation, well accepted in vivo probes of cytochrome P4503A4 (CYP3A4) activity, on defining the effect of three HIV protease inhibitors on CYP3A4 activity. METHODS: Subjects from University Hospital Infectious Disease Clinic about to be started on indinavir, and subjects from two clinical studies, one using ritonavir and the other using amprenavir, were recruited to participate in the study. Subjects received dapsone 100 mg p.o. followed by an 8-h urine collection for dapsone, dapsone N-hydroxylamine, cortisol, and 6beta-hydroxycortisol concentrations before HIV protease inhibitor administration, and 3 4 weeks into receiving HIV protease inhibitors. RESULTS: None of the HIV protease inhibitors demonstrated statistically significant alterations in dapsone recovery ratio and 6beta-hydroxycortisol/cortisol ratio. In fact, with ritonavir, the dapsone recovery ratio tended to increase rather than decrease, suggesting induction. These negative results were found despite evidence of CYP3A4 inhibition by these three HIV protease inhibitors via published drug-drug interactions with drugs that are substrates for CYP3A4. CONCLUSIONS: These in vivo assays used to probe CYP3A4 activity are suboptimal, most likely because of the presence of extrahepatic sites of metabolism for both dapsone and cortisol, and multiple CYP isozymes involved in dapsone N-hydroxylation.     

Use of omeprazole as a probe drug for CYP2C19 phenotype in Swedish Caucasians: comparison with S-mephenytoin hydroxylation phenotype and CYP2C19 genotype

A single oral dose of omeprazole (20 mg) was given orally to 160 healthy Caucasian Swedish subjects and tested as a probe for CYP2C19. The study was nonrandomized and included seven subjects previously classified as poor metabolizers (PM) of S-mephenytoin. The ratio between the plasma concentrations of omeprazole and hydroxyomeprazole (metabolic ratio; MR) was determined by HPLC in a blood sample drawn 3 h after drug intake. In 17 subjects the test was repeated and the MRs of omeprazole on the two occasions were correlated (rs = 0.85; p < 0.0001). There was a significant correlation between the MR of omeprazole and the S/R mephenytoin ratio among 141 subjects, in whom both ratios were determined (rs = 0.63, p < 0.001). All seven PMs of S-mephenytoin had higher MRs of omeprazole (7.1-23.8) than extensive metabolizers (EM) (0.1-4.9). All 160 subjects and another 15 Caucasian Swedish PMs previously phenotyped with mephenytoin were analysed with respect to the presence of the CYP2C19m1 allele by PCR amplification of the intron 4/exon 5 junction followed by Sma I digestion. EMs heterozygous for the CYP2C19m1 gene had MRs of omeprazole and S/R ratios of mephenytoin that were higher than those of subjects who were homozygous for the wild-type allele (p = 0.0001). Nineteen of the 22 PMs were homozygous for the CYP2C19m1 gene. Three were heterozygous for this allele. Thus, 41 of the 44 alleles (93%) of PMs were defective CYP2C19m1. One of the remaining three PM alleles was subsequently found to contain the CYP2C19m2 mutation, which has earlier been shown to be associated with the PM phenotype in Oriental populations. In conclusion, the phenotype determined by omeprazole correlated with that of mephenytoin, and was in good agreement with the genotype.