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Bioavailability of testosterone enanthate dependent on genetic variation in the phosphodiesterase 7B but not on the uridine 50 -diphospho-glucuronosyltransferase (UGT2B17) gene
Lena Ekstro¨ma , Jenny J. Schulzea , Chantal Guillemetteb , Alain Belangerb and Anders Ranea
The major enzyme responsible for testosterone glucuronidation is uridine 50 -diphospho-glucuronosyltransferase (UGT2B17) [5,6]. A gene deletion polymorphism of UGT2B17 [7] was shown by us to be concordant with the rate of urinary testosterone excretion [5]. We were subsequently able to show that testosterone doping test results are highly dependent on the UGT2B17 deletion genotype as investigated in healthy volunteers [8,9].
Individuals homozygous for the UGT2B17 deletion polymorphism (del/del) have a markedly compromised testosterone glucuronide excretion capacity, both physiologically [5] and after testosterone administration [8]. Therefore, it is conceivable to assume that testosterone concentration in the circulation would be higher in patients devoid of the enzyme (del/del) compared with those with the UGT2B17 enzyme (del/ins, ins/ins), after testosterone administration.
Here, we have investigated the serum levels of testosterone and seven of its metabolites in 51 patients before and after 2 days of a single intramuscular dose of testosterone enanthate. Interestingly, a large interindividual variation and a bimodal distribution of the serum androgen levels were observed on day 2 indicating a genetic influence on the bioavailability. Affymetrix (Mercury Park, UK) analysis identified the phosphodiesterase (PDE7B) gene as potentially causative. Genotyping of selected single nucleotide polymorphisms (SNPs) in the PDE7B gene disclosed the PDE7B as a determinant of testosterone serum levels. Our data are further supported by studies of ester cleavage of testosterone enanthate in human liver homogenates.
Results
Serum steroid profile of exogenous testosterone
The serum levels of testosterone and the metabolites such as dihydrotestosterone, 5a-androstane-3b,17b-diol, ADT, Etio-G, 3a-Adiol-17G, 3a-Adiol-3G and ADT-G on days 0 and 2 are shown in Table 1. On day 2, a significant increase in the serum concentrations of all androgens was observed. For most of the metabolites analysed an approximately 100% increase was observed, whereas for testosterone and 3a-Adiol-17G a 200% increase was observed. The concentrations of all metabolites were positively-associated with testosterone level both on days 0 and 2.
There was a large interindividual variation in serum testosterone level 2 days after testosterone administration. The level was associated with body mass on day 2 (R2 = 0.16, P = 0.003). When the increase (nanogram/ millilitre) was corrected for body weight, the distribution of natural logarithms of serum testosterone level showed a multimodal (bi-modal or tri-modal) pattern (Fig. 1), suggesting a genetic contribution to the interindividual variation in serum testosterone concentration.
Genetic analysis
The results from the Affymetrix analysis of five individuals each with low or high-rise in testosterone concentration yielded approximately 300 hits (Supplementary Table 1, Supplemental digital content 1 http:// links.lww.com/FPC/A244). Of particular interest, 12 of the SNPs were localized in the PDE7B gene. Two of them were randomly chosen (rs7774640 and rs4896187) for further analysis, and Taqman reactions (Applied Biosystems) were used to genotype all participants (N = 51). The PDE7B genotypes were in Hardy–Weinberg equilibrium. The two PDE7B polymorphisms investigated showed the same genotype distribution in all the individuals indicating that they are in linkage disequilibrium. Haplotype analysis indicated that all the intron 1 SNPs identified from the Affymetrix analysis were in linkage disequilibrium with each other (data not shown). Therefore, only the analysis of the rs7774640, an A>G substitution, is presented here.
Individuals homozygous for the PDE7B G allele displayed significantly lower total testosterone concentrations on day 2 (13.5 ng/ml ± standard deviation 8.0) than individuals with one or two A alleles (19.3 ng/ml ± standard deviation 6.7) (R2 = 0.15, P = 0.0053) (Fig. 2a). Individuals homozygous for the G allele showed a significantly smaller increase (2.5-fold increase) in serum testosterone levels compared with carriers of the A allele (3.9-fold increase) (R2 = 0.22, P = 0.0006). As expected, the baseline testosterone levels were not dependent on the PDE7B genotype.
Inhibition studies
The esterase activity was determined in human liver homogenates by monitoring the release of testosterone from its enanthate salt. A peak in testosterone level was observed after 2 min (data not shown). To assess if PDE7B exhibits testosterone enanthate hydrolysis activity, inhibition studies using BRL 50481 (Sigma–Aldrich, St. Louis, Missouri, USA), a specific PDE7B/7A inhibitor, were carried out. BRL50481 was found to inhibit the testosterone formation in human liver homogenates in a concentration-dependent manner indicating that PDE7B is involved in the hydrolysis of the testosterone enanthate ester (Fig. 3).
Discussion
The large interindividual variation in the increase of testosterone serum levels 2 days after a single dose observed by us may be ascribed to genetic variation in an esterase enzyme, as we suggested earlier [15]. Our hypothesis was supported by the Affymetrix hit at the PDE7B gene, which was subject to further investigation. Interestingly, the serum level of testosterone was associated with genetic variation in the PDE7B gene. Our findings were corroborated in experiments of human liver samples in which we observed an inhibition of the hydrolysis of testosterone enanthate by the PDE7B/7A specific inhibitor, BRL50485. To our knowledge this is the first time an enzyme involved in testosterone enanthate hydrolysis has been studied and identified. It is likely that other enzymes are also involved in the hydrolysis as there was a residual activity after inhibition of the specific PDE7B/7A inhibitor.
A multiple regression analysis showed that the body weight and PDE7B genotype together explained 36% of the variation observed in serum testosterone level 2 days after testosterone administration. Other factors are known to influence the release of depot drugs. Thus, it has been shown that the body fat may serve as a depot for injected steroids, resulting in a retarded release [16]. Moreover, exercise will increase the blood flow through the tissue near the site of injection and hence increase the release rate.
In addition to our findings of a large variation in testosterone levels in healthy volunteers after testosterone administration, Di Luigi et al. [22] observed interindividual variation in testosterone levels in hypogonadal volunteers after testosterone treatment 1 week after intramuscular administration of testosterone enanthate. Other investigations have also described large differences in serum concentration of testosterone among patients after androgen replacement therapy in hypogonadal men [23,24]. It is of great interest to identify the genetic reasons for the variation in bioavailability of testosterone after androgen replacement therapy. Any measures to compensate for the variation in bioavailability of testosterone and presumably also other esterified drugs such as neuroleptic depot preparations, will improve the outcome of treatment and diminish the risk of side-effects. The effect of repeated testosterone administrations as in chronic treatment or abuse of anabolic androgenic steroids remains to be studied. In addition to polymorphisms in the androgen receptor known to modulate the androgenic effects [25], we believe that genetic variation in the PDE7B gene also modulates the response to testosterone therapy or abuse.
Other testosterone preparations than testosterone enanthate are available, that is, undecanoate, cypionate and propionate. In addition, many other anabolic androgenic steroids are provided as esters, for example, nandrolone decanoate, drostanolone propionate, trenbolone enanthate, etc. It is not known which enzymes catalyze the hydrolysis activity on these esters, but it is possible that PDE7B, or other PDE family members, may be involved. Importantly, a large number of other drugs are esterified with enanthate, for example, antipsychotic drugs (haloperidol, perfenazine, zuklopentixol, etc) and antiasthmatic drugs (flutikason, beklametason). Dose requirement and injection interval of these drugs differ widely between patients, and it is possible that genetic variation in the PDE pathway may contribute to this interindividual variation. This has great interest in clinical medicine, in particular in therapeutics with
Lena Ekstro¨ma , Jenny J. Schulzea , Chantal Guillemetteb , Alain Belangerb and Anders Ranea
The major enzyme responsible for testosterone glucuronidation is uridine 50 -diphospho-glucuronosyltransferase (UGT2B17) [5,6]. A gene deletion polymorphism of UGT2B17 [7] was shown by us to be concordant with the rate of urinary testosterone excretion [5]. We were subsequently able to show that testosterone doping test results are highly dependent on the UGT2B17 deletion genotype as investigated in healthy volunteers [8,9].
Individuals homozygous for the UGT2B17 deletion polymorphism (del/del) have a markedly compromised testosterone glucuronide excretion capacity, both physiologically [5] and after testosterone administration [8]. Therefore, it is conceivable to assume that testosterone concentration in the circulation would be higher in patients devoid of the enzyme (del/del) compared with those with the UGT2B17 enzyme (del/ins, ins/ins), after testosterone administration.
Here, we have investigated the serum levels of testosterone and seven of its metabolites in 51 patients before and after 2 days of a single intramuscular dose of testosterone enanthate. Interestingly, a large interindividual variation and a bimodal distribution of the serum androgen levels were observed on day 2 indicating a genetic influence on the bioavailability. Affymetrix (Mercury Park, UK) analysis identified the phosphodiesterase (PDE7B) gene as potentially causative. Genotyping of selected single nucleotide polymorphisms (SNPs) in the PDE7B gene disclosed the PDE7B as a determinant of testosterone serum levels. Our data are further supported by studies of ester cleavage of testosterone enanthate in human liver homogenates.
Results
Serum steroid profile of exogenous testosterone
The serum levels of testosterone and the metabolites such as dihydrotestosterone, 5a-androstane-3b,17b-diol, ADT, Etio-G, 3a-Adiol-17G, 3a-Adiol-3G and ADT-G on days 0 and 2 are shown in Table 1. On day 2, a significant increase in the serum concentrations of all androgens was observed. For most of the metabolites analysed an approximately 100% increase was observed, whereas for testosterone and 3a-Adiol-17G a 200% increase was observed. The concentrations of all metabolites were positively-associated with testosterone level both on days 0 and 2.
There was a large interindividual variation in serum testosterone level 2 days after testosterone administration. The level was associated with body mass on day 2 (R2 = 0.16, P = 0.003). When the increase (nanogram/ millilitre) was corrected for body weight, the distribution of natural logarithms of serum testosterone level showed a multimodal (bi-modal or tri-modal) pattern (Fig. 1), suggesting a genetic contribution to the interindividual variation in serum testosterone concentration.
Genetic analysis
The results from the Affymetrix analysis of five individuals each with low or high-rise in testosterone concentration yielded approximately 300 hits (Supplementary Table 1, Supplemental digital content 1 http:// links.lww.com/FPC/A244). Of particular interest, 12 of the SNPs were localized in the PDE7B gene. Two of them were randomly chosen (rs7774640 and rs4896187) for further analysis, and Taqman reactions (Applied Biosystems) were used to genotype all participants (N = 51). The PDE7B genotypes were in Hardy–Weinberg equilibrium. The two PDE7B polymorphisms investigated showed the same genotype distribution in all the individuals indicating that they are in linkage disequilibrium. Haplotype analysis indicated that all the intron 1 SNPs identified from the Affymetrix analysis were in linkage disequilibrium with each other (data not shown). Therefore, only the analysis of the rs7774640, an A>G substitution, is presented here.
Individuals homozygous for the PDE7B G allele displayed significantly lower total testosterone concentrations on day 2 (13.5 ng/ml ± standard deviation 8.0) than individuals with one or two A alleles (19.3 ng/ml ± standard deviation 6.7) (R2 = 0.15, P = 0.0053) (Fig. 2a). Individuals homozygous for the G allele showed a significantly smaller increase (2.5-fold increase) in serum testosterone levels compared with carriers of the A allele (3.9-fold increase) (R2 = 0.22, P = 0.0006). As expected, the baseline testosterone levels were not dependent on the PDE7B genotype.
Inhibition studies
The esterase activity was determined in human liver homogenates by monitoring the release of testosterone from its enanthate salt. A peak in testosterone level was observed after 2 min (data not shown). To assess if PDE7B exhibits testosterone enanthate hydrolysis activity, inhibition studies using BRL 50481 (Sigma–Aldrich, St. Louis, Missouri, USA), a specific PDE7B/7A inhibitor, were carried out. BRL50481 was found to inhibit the testosterone formation in human liver homogenates in a concentration-dependent manner indicating that PDE7B is involved in the hydrolysis of the testosterone enanthate ester (Fig. 3).
Discussion
The large interindividual variation in the increase of testosterone serum levels 2 days after a single dose observed by us may be ascribed to genetic variation in an esterase enzyme, as we suggested earlier [15]. Our hypothesis was supported by the Affymetrix hit at the PDE7B gene, which was subject to further investigation. Interestingly, the serum level of testosterone was associated with genetic variation in the PDE7B gene. Our findings were corroborated in experiments of human liver samples in which we observed an inhibition of the hydrolysis of testosterone enanthate by the PDE7B/7A specific inhibitor, BRL50485. To our knowledge this is the first time an enzyme involved in testosterone enanthate hydrolysis has been studied and identified. It is likely that other enzymes are also involved in the hydrolysis as there was a residual activity after inhibition of the specific PDE7B/7A inhibitor.
A multiple regression analysis showed that the body weight and PDE7B genotype together explained 36% of the variation observed in serum testosterone level 2 days after testosterone administration. Other factors are known to influence the release of depot drugs. Thus, it has been shown that the body fat may serve as a depot for injected steroids, resulting in a retarded release [16]. Moreover, exercise will increase the blood flow through the tissue near the site of injection and hence increase the release rate.
In addition to our findings of a large variation in testosterone levels in healthy volunteers after testosterone administration, Di Luigi et al. [22] observed interindividual variation in testosterone levels in hypogonadal volunteers after testosterone treatment 1 week after intramuscular administration of testosterone enanthate. Other investigations have also described large differences in serum concentration of testosterone among patients after androgen replacement therapy in hypogonadal men [23,24]. It is of great interest to identify the genetic reasons for the variation in bioavailability of testosterone after androgen replacement therapy. Any measures to compensate for the variation in bioavailability of testosterone and presumably also other esterified drugs such as neuroleptic depot preparations, will improve the outcome of treatment and diminish the risk of side-effects. The effect of repeated testosterone administrations as in chronic treatment or abuse of anabolic androgenic steroids remains to be studied. In addition to polymorphisms in the androgen receptor known to modulate the androgenic effects [25], we believe that genetic variation in the PDE7B gene also modulates the response to testosterone therapy or abuse.
Other testosterone preparations than testosterone enanthate are available, that is, undecanoate, cypionate and propionate. In addition, many other anabolic androgenic steroids are provided as esters, for example, nandrolone decanoate, drostanolone propionate, trenbolone enanthate, etc. It is not known which enzymes catalyze the hydrolysis activity on these esters, but it is possible that PDE7B, or other PDE family members, may be involved. Importantly, a large number of other drugs are esterified with enanthate, for example, antipsychotic drugs (haloperidol, perfenazine, zuklopentixol, etc) and antiasthmatic drugs (flutikason, beklametason). Dose requirement and injection interval of these drugs differ widely between patients, and it is possible that genetic variation in the PDE pathway may contribute to this interindividual variation. This has great interest in clinical medicine, in particular in therapeutics with
