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1.  Effects of different doses of testosterone on gonadotropins, 25-hydroxyvitamin D3, and blood lipids in healthy men 
To study the effect and time profile of different doses of testosterone enanthate on the blood lipid profile and gonadotropins.
Experimental design
Twenty-five healthy male volunteers aged 27–43 years were given 500 mg, 250 mg, and 125 mg of testosterone enanthate as single intramuscular doses of Testoviron® Depot. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), blood lipid profile (total cholesterol, plasma [p-] low-density lipoprotein, p-high-density lipoprotein [HDL], p-apolipoprotein A1 [ApoA1], p-apolipoprotein B, p-triglycerides, p-lipoprotein(a), serum [s-] testosterone, and 25-hydroxyvitamin D3) were analyzed prior to, and 4 and 14 days after dosing. Testosterone and epitestosterone in urine (testosterone/epitestosterone ratio) were analyzed prior to each dose after a washout period of 6–8 weeks.
Results and discussion
All doses investigated suppressed the LH and FSH concentrations in serum. LH remained suppressed 6 weeks after the 500 mg dose. These results indicate that testosterone has a more profound endocrine effect on the hypothalamic–pituitary–gonadal axis than was previously thought. There was no alteration in 25-hydroxyvitamin D3 levels after testosterone administration compared to baseline levels. The 250 and 500 mg doses induced decreased concentrations of ApoA1 and HDL, whereas the lowest dose (125 mg) did not have any effect on the lipid profile.
The single doses of testosterone produced a dose-dependent increase in serum testosterone concentrations together with suppression of s-LH and s-FSH. Alterations in ApoA1 and HDL were observed after the two highest single doses. It is possible that long-time abuse of anabolic androgenic steroids will lead to alteration in vitamin D status. Knowledge and understanding of the side effects of anabolic androgenic steroids are important to the treatment and care of abusers of testosterone.
PMCID: PMC4266340  PMID: 25525405
anabolic androgenic steroids; testosterone; gonadotropins; vitamin D; blood lipids; abuse
2.  Prenatal expression of thioredoxin reductase 1 (TRXR1) and microsomal glutathione transferase 1 (MGST1) in humans 
FEBS Open Bio  2014;4:886-891.
•The expression of the enzymes TRXR1 and MGST1 during fetal life are investigated for the first time.•The expression of TXRX1 is 7 times higher in fetuses than in adults.•The expression of MGST1 is higher in adults than in fetuses.•Hepatic TRXR1 and MGST 1 are co-expressed, suggesting common regulatory mechanisms.•The effect of the demethylating agent 5-AZA on TRXR1 expression is reported.
Thioredoxin reductase 1 (TRXR1) and microsomal glutathione transferase 1 (MGST1) are important redox and detoxifying enzymes in adult life. The aim of this study was to investigate the expression of these enzymes during fetal life. In addition, the role of gene methylation was studied since this might play an important role in the on-and-off switch of gene expression between fetal and adult life.
To this end, the expression of the TRXR1-encoding gene TXNRD1 and the MGST1-encoding gene MGST1 was studied in fetal tissues. The mean mRNA expression of TXNRD1 in fetal livers were seven times higher compared to the mean expression in adult livers (p < 0.001). Of the six studied splice variants of TXNRD1, four had a significantly higher expression in the fetal livers as compared to adult livers. The mean expression of MGST1 was twofold higher in adult compared to fetal liver tissue (p = 0.01). For MGST1 the alternative first exon 1B was the predominant splice variant in both fetal and adult liver samples. The highest mRNA expression of both TXNRD1 and MGST1 was found in fetal adrenals, whereas expression was lower in fetal liver, lungs and kidneys. There was a significant correlation between the hepatic expression of TXNRD1 and MGST1. Treatment with the demethylating agent 5-AZA resulted in decreased levels of TXNRD1 in human liver HepG2 cells but did not affect the expression of MGST1.
In conclusion, the expression of TXNRD1 is higher in fetuses than in adults and might be of importance during fetal life. Hepatic TXNRD1 and MGST1 are co-expressed in both fetuses and adults suggesting common regulatory mechanisms.
PMCID: PMC4215115  PMID: 25379386
TRXR1, thioredoxin reductase 1; MGST1, microsomal glutathione transferase 1; 5-AZA, 5-AZA-2-deoxyxytidine; Thioredoxin reductase 1; Microsomal glutathione transferase 1; Fetus; Liver; Splice variants; DNA methylation
3.  Detection of morphine-3-sulfate and morphine-6-sulfate in human urine and plasma, and formation in liver cytosol 
Morphine is still the mainstay in treatment of severe pain and is metabolized in the liver mainly by glucuronidation, partly to the pharmacologically active morphine-6-glucuronide (M6G). The sulfation pathway has attracted much less attention but may also form active metabolites. The aim of the present study was to study two sulfate metabolites of morphine in humans. Urine and plasma from newborns, adult heroin addicts, and terminal cancer patients was analyzed for the presence of morphine-3-sulfate (M3S) and morphine-6-sulfate (M6S) by a new liquid chromatography – tandem mass spectrometry (LC-MS/MS) method. In addition, morphine sulfation was studied in vitro in human liver cytosol preparations. M3S was present in urine and plasma from all study groups although at lower concentrations than morphine-3-glucuronide (M3G). The plasma M3S/M3G ratio was 30 times higher in newborns than in adults indicating that the relative sulfation is more important at early stage of life. M6S was measurable in only one plasma sample from a newborn patient, and in one of the urine sample from the drug testing group. The incubation of morphine with liver cytosol extracts resulted in approximately equal rate of formation of both M3S and M6S. In conclusion, sulfation of morphine is catalyzed in human liver but this minor metabolic pathway probably lacks clinical significance. The M6S metabolite is formed at a low rate, making it undetectable in most individuals.
PMCID: PMC4186450  PMID: 25505615
LC-MS/MS; morphine; morphine-3-sulfate; morphine-6-sulfate; plasma; urine
4.  PDE7B is involved in nandrolone decanoate hydrolysis in liver cytosol and its transcription is up-regulated by androgens in HepG2 
Most androgenic drugs are available as esters for a prolonged depot action. However, the enzymes involved in the hydrolysis of the esters have not been identified. There is one study indicating that PDE7B may be involved in the activation of testosterone enanthate. The aims are to identify the cellular compartments where the hydrolysis of testosterone enanthate and nandrolone decanoate occurs, and to investigate the involvement of PDE7B in the activation. We also determined if testosterone and nandrolone affect the expression of the PDE7B gene. The hydrolysis studies were performed in isolated human liver cytosolic and microsomal preparations with and without specific PDE7B inhibitor. The gene expression was studied in human hepatoma cells (HepG2) exposed to testosterone and nandrolone. We show that PDE7B serves as a catalyst of the hydrolysis of testosterone enanthate and nandrolone decanoate in liver cytosol. The gene expression of PDE7B was significantly induced 3- and 5- fold after 2 h exposure to 1 μM testosterone enanthate and nandrolone decanoate, respectively. These results show that PDE7B is involved in the activation of esterified nandrolone and testosterone and that the gene expression of PDE7B is induced by supra-physiological concentrations of androgenic drugs.
PMCID: PMC4038775  PMID: 24910615
phosphodiesterase 7B; nandrolone decanoate; testosterone; androgens; doping
5.  CYP2C8 and CYP2C9 mRNA expression profile in the human fetus 
CYP2C8 and CYP2C9 are involved in the inactivation of several non-steroidal anti-inflammatory drugs, including ibuprofen. CYP2C9 is the major form in human liver whereas CYP2C8 has been proposed to be the main CYP2C enzyme in fetal liver. The protein expression of CYP2C9 in the first trimester is low, only about 1% of the adult values, whereas the mRNA levels of CYP2C8/9 have not been determined at the fetal stage. In this study the mRNA expression levels of CYP2C8 and CYP2C9 were determined in 20 adult and 60 fetal liver tissue specimens. The expression profiles in fetal kidneys (n = 43), adrenals (n = 46), and lungs (n = 37) were also determined. Moreover the activity against ibuprofen hydroxylation was determined in fetus and adult liver microsomes. Adult liver samples expressed 140 and 400 times higher levels of CYP2C8 and CYP2C9 mRNA, respectively, as compared to fetal liver samples. Consistent with this, the hydroxylation of ibuprofen was 40 times higher in the adult liver microsomes. Hepatic CYP2C8 mRNA was three times more abundant than CYP2C9 mRNA in the fetus. Moreover, CYP2C8 were consistently expressed in all fetal tissues investigated, whereas CYP2C9 gene expression was confined to the liver in fetuses. Our results indicate that CYP2C8 plays a more important physiological role than CYP2C9 in the first trimester.
PMCID: PMC3971157  PMID: 24723938
CYP2C8; CYP2C9; fetus; drug metabolism; ibuprofen
6.  Ontogeny, distribution and potential roles of 5-hydroxymethylcytosine in human liver function 
Genome Biology  2013;14(8):R83.
Interindividual differences in liver functions such as protein synthesis, lipid and carbohydrate metabolism and drug metabolism are influenced by epigenetic factors. The role of the epigenetic machinery in such processes has, however, been barely investigated. 5-hydroxymethylcytosine (5hmC) is a recently re-discovered epigenetic DNA modification that plays an important role in the control of gene expression.
In this study, we investigate 5hmC occurrence and genomic distribution in 8 fetal and 7 adult human liver samples in relation to ontogeny and function. LC-MS analysis shows that in the adult liver samples 5hmC comprises up to 1% of the total cytosine content, whereas in all fetal livers it is below 0.125%. Immunohistostaining of liver sections with a polyclonal anti-5hmC antibody shows that 5hmC is detected in most of the hepatocytes. Genome-wide mapping of the distribution of 5hmC in human liver samples by next-generation sequencing shows significant differences between fetal and adult livers. In adult livers, 5hmC occupancy is overrepresented in genes involved in active catabolic and metabolic processes, whereas 5hmC elements which are found in genes exclusively in fetal livers and disappear in the adult state, are more specific to pathways for differentiation and development.
Our findings suggest that 5-hydroxymethylcytosine plays an important role in the development and function of the human liver and might be an important determinant for development of liver diseases as well as of the interindividual differences in drug metabolism and toxicity.
PMCID: PMC4054829  PMID: 23958281
7.  Androgens and doping tests: genetic variation and pit-falls 
The large variation in disposition known for most drugs is also true for anabolic androgenic steroids. Genetic factors are probably the single most important cause of this variation. Further, there are reasons to believe that there is a corresponding variation in efficacy of doping agents. Doped individuals employ a large variety of doping strategies in respect of choice of substance, dose, dose interval, duration of treatment and use of other drugs for enforcement of effects or correction of side effects.
Metabolic steps up-stream and down-stream of testosterone are genetically variable and contribute substantially to the variation in disposition of testosterone, the most common doping agent in sports and in society. Large inter- and intra-ethnic variation in testosterone glucuronidation and excretion is described as well as the pit-falls in evaluation of testosterone doping test results. The hydrolysis and bioactivation of testosterone enanthate is also genetically variable yielding a 2–3 fold variation in excretion rate and serum concentration, thereby implicating a substantial variation in ‘efficacy’ of testosterone.
Given this situation it is logical to adopt the new findings in the doping control programme. The population based cut-off level for the testosterone : epitestosterone ratio should be replaced by a Bayesian interpretation of consecutive tests in the same individual. When combined with the above genetic information the sensitivity of the test is considerably improved. The combination of the three approaches should reduce the rate of falsely negative or positive results and the number of expensive follow-up tests, stipulated by the World Anti-Doping Agency.
PMCID: PMC3394124  PMID: 22506612
androgens; doping; genetic variation; testosterone; UGT2B17
8.  Non-Steroidal Anti-Inflammatory Drugs Do Not Influence the Urinary Testosterone/Epitestosterone Glucuronide Ratio 
The UDP Glucuronosyl Transferase (UGT) enzymes are important in the pharmacokinetics, and conjugation, of a variety of drugs including non-steroidal anti-inflammatory drugs (NSAIDs) as well as anabolic androgenic steroids (AAS). Testosterone glucuronidation capacity is strongly associated with a deletion polymorphism in the UGT2B17 gene. As the use of high doses of NSAIDs has been observed in athletes there is a risk for a drug–drug interaction that may influence the doping tests for AAS. In vitro studies show inhibitory potential on UGT2B7, 2B15, and 2B17 enzymes by NSAIDs. The aim of this study was to investigate if concomitant use of NSAIDs and a single dose of testosterone enanthate would affect the excretion rate of testosterone and epitestosterone glucuronide (TG and EG) as well as the T/E ratio, thereby affecting the outcome of the testosterone doping test. The study was designed as an open, randomized, cross-over study with subjects being their own control. The 23 male healthy volunteers, with either two, one or no allele (ins/ins, ins/del, or del/del) of the UGT2B17 gene, received the maximum recommended dose of NSAID (Ibuprofen or Diclofenac) for 6 days. On day three, 500 mg of testosterone enanthate was administered. Spot urine samples were collected for 17 days. After a wash-out period of 4 months the volunteers received 500 mg testosterone enanthate only, with subsequent spot urine collection for 14 days. The glucuronides of testosterone and epitestosterone were quantified. NSAIDs did not affect the excretion of TG or EG before the administration of testosterone. The concomitant use of NSAIDs and testosterone slightly increased the TG excretion while the EG excretion was less suppressed compared to testosterone use only. The effects of the NSAIDs on the TG and EG excretion did not differ between the UGT2B17 genotype groups. In conclusion, the outcome of testosterone doping tests does not seem to be affected by the use of NSAIDs.
PMCID: PMC3655282  PMID: 23720652
NSAID; testosterone; epitestosterone; diclofenac; ibuprofen; UGT2B17; T/E ratio
9.  Implication of Human UGT2B7, 2B15, and 2B17 in 19-Norandrosterone Metabolism 
Nandrolone (19-nortestosterone) is an anabolic androgenic steroid commonly abused for doping purposes. Nandrolone is mainly metabolized in the liver into 19-norandrosterone prior to glucuronidation and excretion through urine over an extended period of time. Several UGTs (i.e., UGT2B7, UGT2B15, and UGT2B17) are thought to be the major enzymes responsible for conjugation of androgens in human. An in vitro study using recombinant enzymes expressed in insect cells showed that UGT1A4 and UGT2B7 are the two main enzymes responsible of 19-norandrosterone glucuronidation. However, the identity of the enzyme involved in nandrolone metabolism in vivo together with their relative contribution and regulation remain unknown. Inhibition assays using human liver microsomes (HLM) incubated with 19-norandrosterone and selective inhibitors confirmed that UGT2B7 and UGT2B15 are involved in 19-norandrosterone glucuronidation, since the presence of the specific UGT2B7 and UGT2B15 inhibitors gemfibrozil and valproic acid inhibited the 19-norandrosterone glucuronidation by 35 and 45%, respectively. HLM were genotyped for UGT2B15 D85Y, UGT2B7 H268Y, and the UGT2B17 deletion polymorphism. The glucuronidation activity on 19-norandrosterone was significantly higher in UGT2B15 DD than in the other UGT2B15 genotypes (p < 0.05). Moreover, human liver cancer HepG2 cells were exposed to androgens to determine if the transcriptional activity of the genes of interest was affected. Only UGT2B7 mRNA expression was significantly increased (1.8-folds) after incubation with nandrolone decanoate. These results show that the UGT2B7 and UGT2B15 are involved in 19-norandrosterone glucuronidation and that the UGT2B15 polymorphism (D85Y) is the only UGT genetic variation that influences the glucuronidation activity. This could partly explain the inter-individual variation in 19-norandrosterone excretion.
PMCID: PMC3693077  PMID: 23805127
anabolic androgenic steroid; nandrolone; 19-norandrosterone; UGT2B7; UGT2B15
10.  SULT2A1 Gene Copy Number Variation is Associated with Urinary Excretion Rate of Steroid Sulfates 
Human cytosolic sulfotransferases (SULT) 2A1 is the main enzyme involved in the sulfate conjugation of dehydroepiandrosterone, a weak androgen, and the main androgen precursor, whereas estrogens are mainly conjugated by SULT1A1. Here we have identified a copy number variation (CNV) polymorphism in the SULT2A1 gene in a Swedish population including healthy men (N = 30). Moreover, the CNV of SULT1A1 and SULT2A1 was further characterized in relation to urinary levels of androgen sulfate metabolites before and after an intramuscular dose of 500 mg testosterone enanthate. Individuals expressing two or more CNVs excrete 80 and 40% higher levels of DHEAS (p = 0.02) and androsteroneS (p = 0.01), respectively as compared to individuals with one gene copy. The mean area under the urine concentration time-curve from time 0 (prior to the administration of 500 mg testosterone) to 15 days post dose values were 80% higher for DHEAS (p = 0.046) and testosteroneS (p = 0.019) in individuals with two and three SULT2A1 gene copies as compared to individuals with one gene copy. The SULT1A1 CNV on the other hand did not affect the sulfation activity toward the androgens. In conclusion our results indicate that functional CNV polymorphisms in SULT2A1 and SULT1A1 are common in a Swedish population and that SULT2A1 CNV is associated with the urinary concentrations of androgen sulfate metabolites.
PMCID: PMC3709130  PMID: 23874324
SULT2A1; SULT1A1; copy number variation; DHEAS; androgens; testosterone
11.  Single dose testosterone increases total cholesterol levels and induces the expression of HMG CoA Reductase 
Cholesterol is mainly synthesised in liver and the rate-limiting step is the reduction of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) to mevalonate, a reaction catalysed by HMG-CoA reductase (HMGCR). There is a comprehensive body of evidence documenting that anabolic-androgenic steroids are associated with deleterious alterations of lipid profile. In this study we investigated whether a single dose of testosterone enanthate affects the cholesterol biosynthesis and the expression of HMGCR.
39 healthy male volunteers were given 500 mg testosterone enanthate as single intramuscular dose of Testoviron®--Depot. The total cholesterol levels prior to and two days after testosterone administration were analysed. Protein expression of HMGCR in whole blood was investigated by Western blotting. In order to study whether testosterone regulates the mRNA expression of HMGCR, in vitro studies were performed in a human liver cell-line (HepG2).
The total cholesterol level was significantly increased 15% two days after the testosterone injection (p = 0.007). This is the first time a perturbation in the lipoprotein profile is observed after only a single dose of testosterone. Moreover, the HMGCR mRNA and protein expression was induced by testosterone in vitro and in vivo, respectively.
Here we provide a molecular explanation how anabolic androgenic steroids may impact on the cholesterol homeostasis, i.e. via an increase of the HMGCR expression. Increasing knowledge and understanding of AAS induced side-effects is important in order to find measures for treatment and care of these abusers.
PMCID: PMC3342114  PMID: 22433938
Testosterone; Cholesterol; HMG CoA reductase
12.  Pharmacogenetic Warfarin Dose Refinements Remain Significantly Influenced by Genetic Factors after One Week of Therapy 
Thrombosis and Haemostasis  2011;107(2):232-240.
By guiding initial warfarin dose, pharmacogenetic (PGx) algorithms may improve the safety of warfarin initiation. However, once INR response is known, the contribution of PGx to dose refinements is uncertain. This study sought to develop and validate clinical and PGx dosing algorithms for warfarin dose refinement on days 6–11 after therapy initiation.
Materials and Methods
An international sample of 2,022 patients at 13 medical centers on 3 continents provided clinical, INR, and genetic data at treatment days 6–11 to predict therapeutic warfarin dose. Independent derivation and retrospective validation samples were composed by randomly dividing the population (80%/20%). Prior warfarin doses were weighted by their expected effect on S-warfarin concentrations using an exponential-decay pharmacokinetic model. The INR divided by that “effective” dose constituted a treatment response index.
Treatment response index, age, amiodarone, body surface area, warfarin indication, and target INR were associated with dose in the derivation sample. A clinical algorithm based on these factors was remarkably accurate: in the retrospective validation cohort its R2 was 61.2% and median absolute error (MAE) was 5.0 mg/week. Accuracy and safety was confirmed in a prospective cohort (N=43). CYP2C9 variants and VKORC1-1639 G→A were significant dose predictors in both the derivation and validation samples. In the retrospective validation cohort, the PGx algorithm had: R2= 69.1% (P<0.05 vs. clinical algorithm), MAE= 4.7 mg/week.
A pharmacogenetic warfarin dose-refinement algorithm based on clinical, INR, and genetic factors can explain at least 69.1% of therapeutic warfarin dose variability after about one week of therapy.
PMCID: PMC3292349  PMID: 22186998
warfarin; VKORC1; CYP2C9; pharmacogenetic
13.  Pharmacogenetics of Anticoagulants 
Warfarin, acenocoumarol, and phenprocoumon are among the major anticoagulant drugs worldwide. Because of their low therapeutic index and serious adverse reactions (ADRs), their wide use, and their varying kinetics and pharmacogenetic dependence, it is of great importance to explore further possibilities to forecast the dose beyond conventional INR measurements. Here, we describe particulars of the relative pharmacogenetic influence on the kinetics of these agents, the population distribution of genetics risk groups, and novel data on clinical features with influence on dose requirement and ADR risk. The usefulness of genetic information prior to and soon after start of therapy is also discussed. The current renewed focus on these issues is caused not only because of new genetic knowledge and genotyping facilities but also because of the high rate of serious ADRs. Application of these measures in the care of patients with anticoagulant therapy is important awaiting new therapeutic principles to be introduced, which may take long time still.
PMCID: PMC2958670  PMID: 20981234
14.  A Genome-Wide Association Study Confirms VKORC1, CYP2C9, and CYP4F2 as Principal Genetic Determinants of Warfarin Dose 
PLoS Genetics  2009;5(3):e1000433.
We report the first genome-wide association study (GWAS) whose sample size (1,053 Swedish subjects) is sufficiently powered to detect genome-wide significance (p<1.5×10−7) for polymorphisms that modestly alter therapeutic warfarin dose. The anticoagulant drug warfarin is widely prescribed for reducing the risk of stroke, thrombosis, pulmonary embolism, and coronary malfunction. However, Caucasians vary widely (20-fold) in the dose needed for therapeutic anticoagulation, and hence prescribed doses may be too low (risking serious illness) or too high (risking severe bleeding). Prior work established that ∼30% of the dose variance is explained by single nucleotide polymorphisms (SNPs) in the warfarin drug target VKORC1 and another ∼12% by two non-synonymous SNPs (*2, *3) in the cytochrome P450 warfarin-metabolizing gene CYP2C9. We initially tested each of 325,997 GWAS SNPs for association with warfarin dose by univariate regression and found the strongest statistical signals (p<10−78) at SNPs clustering near VKORC1 and the second lowest p-values (p<10−31) emanating from CYP2C9. No other SNPs approached genome-wide significance. To enhance detection of weaker effects, we conducted multiple regression adjusting for known influences on warfarin dose (VKORC1, CYP2C9, age, gender) and identified a single SNP (rs2108622) with genome-wide significance (p = 8.3×10−10) that alters protein coding of the CYP4F2 gene. We confirmed this result in 588 additional Swedish patients (p<0.0029) and, during our investigation, a second group provided independent confirmation from a scan of warfarin-metabolizing genes. We also thoroughly investigated copy number variations, haplotypes, and imputed SNPs, but found no additional highly significant warfarin associations. We present power analysis of our GWAS that is generalizable to other studies, and conclude we had 80% power to detect genome-wide significance for common causative variants or markers explaining at least 1.5% of dose variance. These GWAS results provide further impetus for conducting large-scale trials assessing patient benefit from genotype-based forecasting of warfarin dose.
Author Summary
Recently, geneticists have begun assaying hundreds of thousands of genetic markers covering the entire human genome to systematically search for and identify genes that cause disease. We have extended this “genome-wide association study” (GWAS) method by assaying ∼326,000 markers in 1,053 Swedish patients in order to identify genes that alter response to the anticoagulant drug warfarin. Warfarin is widely prescribed to reduce blood clotting in order to protect high-risk patients from stroke, thrombosis, and heart attack. But patients vary widely (20-fold) in the warfarin dose needed for proper blood thinning, which means that initial doses in some patients are too high (risking severe bleeding) or too low (risking serious illness). Our GWAS detected two genes (VKORC1, CYP2C9) already known to cause ∼40% of the variability in warfarin dose and discovered a new gene (CYP4F2) contributing 1%–2% of the variability. Since our GWAS searched the entire genome, additional genes having a major influence on warfarin dose might not exist or be found in the near-term. Hence, clinical trials assessing patient benefit from individualized dose forecasting based on a patient's genetic makeup at VKORC1, CYP2C9 and possibly CYP4F2 could provide state-of-the-art clinical benchmarks for warfarin use during the foreseeable future.
PMCID: PMC2652833  PMID: 19300499
15.  Effect of renal impairment on the pharmacokinetics of bupropion and its metabolites 
What is already known about this subject
There is an ongoing debate regarding the effect of renal impairment on CYP related metabolic activities.
The possible effect of renal impairment on hepatic CYP2B6 activity, or on bupropion pharmacokinetics in renally impaired subjects without dialysis treatment has not yet been investigated.
What this study adds
Bupropion clearance was found to be significantly decreased in patients with renal impairment.
This study provides further evidence for interplay between the role of the kidney and liver in drug disposition, and opens novel lines of research with respect to the regulation of CYP2B6.
To investigate the effect of kidney disease on bupropion pharmacokinetics and on cytochrome P450 (CYP) 2B6 activity as measured by bupropion hydroxylation.
In an open parallel group study, 17 healthy, nonsmoking subjects and 10 patients with impaired kidney function received a single 150 mg oral dose of sustained release bupropion. Plasma concentrations of bupropion and its metabolites were measured for up to 72 h. Subjects were genotyped for the CYP2B6 SNPs 1459 C>T, 785 A>G and 516 G>T.
Bupropion AUC was 126% higher (P< 0.0001, 95% CI +72%, +180%), Cmax 86% higher (P = 0.001, 95% CI +40%, +131%), CL/F 63% lower (P = 0.001, 95% CI −29%, −96%), and t1/2 140% longer (P = 0.001, 95% CI +76%, +204%) in renally impaired patients. However, only minor changes were detected in the concentrations of the metabolites. In renally impaired subjects the hydroxybupropion : bupropion AUC ratio was decreased by 66% (P = < 0.0001, 95% CI −19%, −114%) and the hydrobupropion : bupropion AUC ratio by 69% (P = 0.001, 95% CI +8%, −146%) compared with controls.
The CL/F of bupropion was significantly lower in subjects with renal impairment. Because the principal metabolites of bupropion possess similar pharmacological activity to the parent compound, dosage recommendations for patients with renal impairment cannot be given. A direct effect of renal impairment on CYP2B6 activity could not be demonstrated by the present study design.
PMCID: PMC2000634  PMID: 17335546
bupropion; cytochrome P450; pharmacokinetics; renal failure
16.  Effects of the Antifungals Voriconazole and Fluconazole on the Pharmacokinetics of S-(+)- and R-(−)-Ibuprofen 
Our objective was to study the effects of the antifungals voriconazole and fluconazole on the pharmacokinetics of S-(+)- and R-(−)-ibuprofen. Twelve healthy male volunteers took a single oral dose of 400 mg racemic ibuprofen in a randomized order either alone, after ingestion of voriconazole at 400 mg twice daily on the first day and 200 mg twice daily on the second day, or after ingestion of fluconazole at 400 mg on the first day and 200 mg on the second day. Ibuprofen was ingested 1 h after administration of the last dose of voriconazole or fluconazole. Plasma concentrations of S-(+)- and R-(−)-ibuprofen were measured for up to 24 h. In the voriconazole phase, the mean area under the plasma concentration-time curve (AUC) of S-(+)-ibuprofen was 205% (P < 0.001) of the respective control value and the mean peak plasma concentration (Cmax) was 122% (P < 0.01) of the respective control value. The mean elimination half-life (t1/2) was prolonged from 2.4 to 3.2 h (P < 0.01) by voriconazole. In the fluconazole phase, the mean AUC of S-(+)-ibuprofen was 183% of the control value (P < 0.001) and its mean Cmax was 116% of the control value (P < 0.05). The mean t1/2 of S-(+)-ibuprofen was prolonged from 2.4 to 3.1 h (P < 0.05) by fluconazole. The geometric mean S-(+)-ibuprofen AUC ratios in the voriconazole and fluconazole phases were 2.01 (90% confidence interval [CI], 1.80 to 2.22) and 1.82 (90% CI, 1.72 to 1.91), respectively, i.e., above the bioequivalence acceptance upper limit of 1.25. Voriconazole and fluconazole had only weak effects on the pharmacokinetics of R-(−)-ibuprofen. In conclusion, voriconazole and fluconazole increased the levels of exposure to S-(+)-ibuprofen 2- and 1.8-fold, respectively. This was likely caused by inhibition of the cytochrome P450 2C9-mediated metabolism of S-(+)-ibuprofen. A reduction of the ibuprofen dosage should be considered when ibuprofen is coadministered with voriconazole or fluconazole, especially when the initial ibuprofen dose is high.
PMCID: PMC1479148  PMID: 16723553
17.  Cytochromes P450 and MDR1 mRNA expression along the human gastrointestinal tract 
The aim of this study was to quantify the mRNA expression of three cytochromes P450 (CYP) and P-glycoprotein (P-gp) in the human gastrointestinal (GI) tract.
Biopsies were obtained from gastric, duodenal, colonic and rectal mucosa during routine gastro-colonoscopy in 27 patients. The biopsies were snap-frozen in liquid nitrogen. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used for the quantitative analyses of mRNA expressed by the CYP2E1, CYP3A4 and CYP3A5 genes, and the MDR1 gene coding for P-gp protein. The mRNA expression of b-actin was used as an internal standard for comparisons between samples.
All CYP genes were expressed at all locations throughout the GI tract, although all showed substantial interindividual variation. CYP2E1 had the highest expression at all locations (P < 0.05 to P < 0.0001), except in the right colon. CYP3A4 and CYP3A5 had their highest mRNA expression in the duodenum (P < 0.001 and P < 0.000 001, respectively) and CYP2E1 in the stomach (P < 0.01). MDR1 mRNA concentrations increased along the GI tract with the highest expression being in the left colon (P < 0.000001).
Multiple sampling within the same individual enabled us to study the intraindividual variation in expression of CYP and MDR1 genes along the GI tract. We find that CYP2E1 mRNA expression is higher than that of the other CYPs. CYP3A expression is highest in the duodenum and that of MDR1 increases from stomach and duodenum to colon.
PMCID: PMC1884914  PMID: 15963094
Cytochrome P450; Drug metabolism; GI-tract
18.  Internet based clinical trial protocols – as applied to a study of warfarin pharmacogenetics 
To describe and evaluate the use of an Internet-based study protocol in a multicentre study of genetic risk factors in anticoagulant treatment.
A web-based study protocol, similar to existing anticoagulation medical record systems, was developed for entry of clinical data. It was also supplied with a separate interface for study monitoring. Measures were taken to assure the confidentiality of transferred data. In addition, software modifications were made to enable automated transfer of clinical data from an existing medical record system to the study database.
The system has been in use since March 2002, and at present 39 centres have included 909 patients with a dropout rate of 2.8%. The need for education of participating clinicians has been satisfactorily provided for by means of written instructions and telephone support.
Our study demonstrates the usability of Internet-based data acquisition techniques in a full-scale multicentre clinical trial. The main advantages of such a protocol are automated data validation and standardization, fast data transfer independent of geographical distance, user feedback, synchronization of protocol updates and automatic data formatting facilitating statistical analyses. Safety and accessibility are possibly cumbersome areas and should be addressed duly.
PMCID: PMC1884621  PMID: 15521895
data collection/methods; Internet; multicentre studies; warfarin
19.  The effect of the CYP1A2 *1F mutation on CYP1A2 inducibility in pregnant women 
To investigate the influence of the CYP1A2*1F mutation on CYP1A2 activity in smoking and nonsmoking pregnant women.
Pregnant women (n = 904) who served as control subjects in a case-control study of early fetal loss were investigated. They were phenotyped for CYP1A2 using dietary caffeine and the urinary ratio AFMU + 1X + 1 U/1,7 U. An assay for CYP1A2*1F using 5′-nuclease assay (Taqman) was developed to genotype the population.
The frequencies of *1 A and *1F alleles among Swedish women were 0.29 and 0.71, respectively. There was no statistically significant difference in CYP1A2 activity between the genotypes, although a trend towards enhanced activity was observed in *1F/*1F (log MRc 0.77) and *1F/*1 A (log MRc 0.82) genotypes compared with the *1 A/*1 A genotype (log MRc 0.71) (anovaP = 0.07). The mean difference between the *1 A homozygotes and the heterozygotes was 0.11 [95% confidence interval of the difference: (−0.21, −0.01)] and that between the *1 A and *1F homozygotes was 0.05 [95% confidence interval of the difference: (−0.13, 0.03)]. No significant effect (P = 0.22) of the *1F on CYP1A2 activity was observed in smokers, tested using an interaction term (smoking * genotype) in the anova model (*1F/*1F log MRc 0.79, *1F/*1 A log MRc 0.86, and *1 A/*1 A log MRc 0.73). In smokers, there was no difference in ratio between homozygotes for the *1 A and *1F alleles [mean difference −0.06; 95% confidence interval of the difference: −0.22, 0.11] or between *1 A/*1 A and *1 A/*1F genotypes [mean difference −0.13; 95% confidence interval of the difference: −0.29, 0.04].
The effect of the CYP1A2*1F mutation on CYP1A2 activity in smoking pregnant women could not be confirmed.
PMCID: PMC1874450  PMID: 12445029
CYP1A2 activity; CYP1A2*1F; polymorphism; pregnancy; smoking
20.  Dose requirement and prolactin elevation of antipsychotics in male and female patients with schizophrenia or related psychoses 
The aim of this study was to investigate the prolactin (PRL) secretion and the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis in relation to gender and side-effects and dose of antipsychotic drugs during long-term treatment.
Forty-seven patients (21 men and 26 women), diagnosed with schizophrenia or related psychoses according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria and treated with different classical antipsychotics, were studied. Prolactin, GH and IGF-I were measured, as well as the serum concentration of the antipsychotics. In addition, body mass index (BMI) was calculated.
The median daily, as well as the median body weight, adjusted daily dose of antipsychotic drugs was twofold higher in male compared with female patients. Antipsychotic-induced hyperprolactinaemia was more frequent and occurred at a lower daily dose of antipsychotics in women. Irrespective of sex, more than half of the patients had elevated BMI. Two patients had a slight increment in IGF-I levels, whereas the GH concentration, as assessed on a single occasion, was normal in all patients.
Patients on long-term antipsychotic therapy, with doses adjusted according to therapeutic efficiency, exhibited hyperprolactinaemia and elevated BMI, but no obvious influence on the GH-IGF-I axis. Furthermore, it appeared that the males required twice the dose of antipsychotic compared with females.
PMCID: PMC2014456  PMID: 11318766
antipsychotic agents; body mass index (BMI); hyperprolactinaemia; insulin-like growth factor I (IGF-I); sex differences
21.  Survey of unlicensed and off label drug use in paediatric wards in European countries 
BMJ : British Medical Journal  2000;320(7227):79-82.
To determine the extent of use of unlicensed and off label drugs in children in hospital in five European countries.
Prospective study of drugs administered to children in general paediatric medical wards over four weeks.
Children’s wards in five hospitals (one each in the United Kingdom, Sweden, Germany, Italy, and the Netherlands).
Children aged 4 days to 16 years admitted to general paediatric medical wards.
Main outcome measure
Proportion of drugs that were used in an unlicensed or off label manner.
2262 drug prescriptions were administered to 624 children in the five hospitals. Almost half of all drug prescriptions (1036; 46%) were either unlicensed or off label. Of these 1036, 872 were off label and 164 were unlicensed. Over half of the patients (421; 67%) received an unlicensed or off label drug prescription.
Use of off label or unlicensed drugs to treat children is widespread. This problem is likely to affect children throughout Europe and requires European action.
Key messagesMany drugs are not tested in children, which means that they are not specifically licensed for use in childrenLicensed drugs are often prescribed outside the terms of the product license (off label) in relation to age, indication, dose of frequency, route of administration, or formulationOver two thirds (67%) of 624 children admitted to wards in five European hospitals received drugs prescribed in an unlicensed or off label manner39% of the 2262 drug prescriptions given to children were off labelThe problem of off label and unlicensed drug prescribing in children is a European problem that requires European action
PMCID: PMC27251  PMID: 10625257
22.  Dietary caffeine as a probe agent for assessment of cytochrome P4501A2 activity in random urine samples 
To validate the use of randomly collected urine samples for assessment of cytochrome P4501A2 (CYP1A2) activity based on dietary caffeine (caffeine metabolic ratio, MRcaff), and to relate the MRcaff to caffeine intake and smoking habits in a larger group of individuals.
Nineteen healthy volunteers were included in the validation study. Caffeine (100 mg) was ingested and a urine sample was collected after 6 h. Within the following week a random urine sample was collected in the individuals without a preceding test dose of caffeine. Urine samples were analysed for caffeine and its metabolites by h.p.l.c. and the (AFMU+1U+1X)/1,7U metabolic ratio was used to reflect CYP1A2 activity. In an extended investigation of 522 healthy pregnant women the MRcaff was related to intake of caffeine from various sources, and to smoking.
The results from the random and standardised sampling methods correlate with each other (correlation coefficient of MRcaff was 0.91). The MRcaff as assessed by the random sampling method in a larger population was not affected by source or amount of caffeine ingested. Significantly higher MRcaff was found in smokers compared to non-smokers. In the large group of individuals the random sampling method was possible to use in 80% of the cases. In the residual 20% one or several of the metabolite concentrations were too low or unmeasurable.
Our study demonstrates that the random urine caffeine phenotyping method is possible to use in as many as 80% of the individuals when based on dietary caffeine. Our approach should prove applicable in most countries with widely spread caffeine consumption. The method is useful in larger studies of drug metabolising enzyme activities and minimises the time consumption and costs.
PMCID: PMC2014237  PMID: 10233204
dietary caffeine; CYP1A2 activity; phenotyping; validation; metabolic ratio
23.  Endocrine and adrenergic pharmacological intervention in diseasesof the prostate 
PMCID: PMC1873965  PMID: 9578179
benign prostatic hyperplasia; BPH; prostate cancer; 5α-reductase inhibition; α-adrenoceptor blockade

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