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Institute of Reproductive Medicine, University Clinics, Muenster D-48149, Germany
Address all correspondence and requests for reprints to: Eberhard Nieschlag, Institute of Reproductive Medicine of the University, Muenster D-48149, Germany. E-mail: eberhard.nieschlag{at}ukmuenster.de.
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Abstract |
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Objective: The objective of the study was to assess the safety of TU regarding metabolic and pharmacogenetic confounders.
Design: This was a longitudinal one-arm open observation trial. A minimum of five individual assessments was a prerequisite. Putative modulators of safety parameters entering regression models were nadir and/or delta total testosterone concentrations, body mass index, androgen receptor (AR) gene CAG repeat length, and age.
Setting: The study was conducted at an andrological outpatient clinic.
Patients: Patients included 66 hypogonadal men (mean age 38 ± 9.9 yr).
Main Outcome Measures: A total of 515 data time points each related to prostate, erythropoiesis, lipoproteins, and circulation during 118 treatment-years with 1000 mg TU at 10- to 14-wk intervals.
Results: Testosterone substitution resulted in significant decrements of serum levels of low-density lipoprotein-cholesterol, resting diastolic and systolic blood pressure, and heart rate. Erythropoiesis was stimulated and concentrations of high-density lipoproteincholesterol increased. Parameters remained stable after four injections. No adverse effects regarding the prostate were observed. Significantly increased hematocrit greater than 50% was predicted by enhanced androgen action (shorter AR CAG repeats per higher testosterone levels). However, insufficient androgen action (longer AR CAG repeats per lower testosterone levels) caused pathological safety parameters (high blood pressure, adverse lipid profiles). In addition, a body mass index 30 kg/m2 or greater represents a clinically relevant factor for the occurrence of all pathological safety parameters. Risk calculations for obese patients and nonlinear pharmacogenetic models to tailor androgen substitution are presented.
Conclusions: Testosterone substitution with im TU is generally well tolerated. Modifications of androgen action are due to both AR CAG repeats and testosterone levels. Adverse observations are mostly seen in obese patients.
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Introduction |
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These preparations effectively establish, restore, and maintain T-dependent functions in men. Diagnostic procedures identifying the underlying cause of hypogonadism are a prerequisite for the start of therapy. T substitution therapy has to be accompanied by standardized procedures of surveillance to avoid adverse effects (1, 4).
Recently metaanalyses revealed that erythropoiesis, lipid constellations, and the prostate may be especially susceptible to undesired changes during T substitution (5, 6). However, these studies comprise data involving older injectable T preparations, producing unfavorable supraphysiologic serum levels. These are no longer seen with the new injectable TU, which may result in fewer or different side effects. We have long-term experience with this preparation in nonhuman primates as well as substitution therapy of hypogonadal men (7, 8, 9, 10, 11), which has also been evaluated by other groups (12, 13).
In addition, it has been previously shown that metabolic aspects such as body mass index (BMI) or age in general may influence the effect of T on safety parameters (14). The pharmacogenetic influence of the CAG repeat polymorphism of the androgen receptor (AR) gene on these parameters might also play a long-term role concerning safety aspects. It has been previously demonstrated that the effects of other T preparations, such as short-acting im injected esters, transdermal and oral preparations are modulated by this polymorphism (15, 16, 17, 18). These pharmacogenetic reports did not specifically evaluate the effects of BMI on the outcome of T substitution.
Therefore, we investigated the side effects of T substitution by im applied TU in 66 hypogonadal men during 118 treatment-years, and it is the first time that long-term aspects of safety in combination with both metabolic features and pharmacogenetics are considered.
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Patients and Methods |
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illustrates the process of patient selection: all patients currently receiving im TU were identified from our patient database. These men presented for examination and possible treatment of hypogonadism-related symptoms. Such symptoms were fatigue, loss of libido, depressiveness, change in body composition/weight, decreased physical performance, decrease in aggressive behavior, disability to cope, decreased work performance, and lack of androgenization. For inclusion, at least one of these symptoms had to be accompanied by low total T levels (<12 nmol/liter).
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No patient discontinued the treatment before receiving four injections. Further screening procedures excluded those men having yet received less than four injections, those with injections given intermediately at other centers, and those with missing data. Finally, 66 men were eligible for evaluation (Fig. 1
). These 66 hypogonadal men received a total of 383 injections of 1000 mg TU in 10- to 14-wk intervals. Eligibility for evaluation was given after assessment of baseline values, a minimum of four injections, and presentation to assess data before a putative further (minimum fifth) injection, accumulating to 515 data time points spanning 118 treatment-years. Thirty-five men had primary, 27 secondary, and four late-onset hypogonadism as previously defined (4, 19). None of these patients has been reported in manuscripts concerning T substitution and the modulation of androgen effects by the CAG repeat polymorphism (citations 16 and 17 refer to patients who received other preparations). Data of three of the 66 patients who are mentioned in this report have been referred to in an interim report about the efficacy of im TU in general, dating back to 2002 (9). Data of all other 63 patients have not been evaluated in previous publications.
Biochemical analyses
All venous blood samples were obtained between 0800 and 1200 h after a 30-min rest before the first or next injection of TU. Serum or plasma were separated at 800 g. Samples were immediately stored at –20 C. Serum T levels were measured by a commercial ELISA kit (DRG Instruments GmbH, Marburg, Germany), and mean interassay coefficient of variation (CV) was less than 5%. Concentrations of SHBG were determined by highly specific time-resolved fluoroimmunoassays (Autodelfia, Freiburg, Germany). Mean intraassay CVs were less than 5% and mean interassay CVs less than 10%. Levels of free T were calculated from levels of SHBG and total serum T according to previously published calculations (20). PSA was determined with highly specific time-resolved fluoroimmunoassay (Autodelfia), with a normal upper limit of reference range of less than 4 ng/ml. Mean intra- and interassay CVs were less than 2 and 5%, respectively. Sampling was performed before prostate palpation and transrectal ultrasonography. Red blood count was performed on a SE 9500 system (Sysmex Europe, Hamburg, Germany). A Hitachi 917 autoanalyzer (Hitachi/ Roche Diagnostics, Mannheim, Germany) was used for the quantification of serum concentrations of lipoproteins with enzymatic tests. Interassay CV was less than 5%.
Determination of AR CAG repeats
DNA was isolated from EDTA blood samples using the Nucleon kit (Amersham Life Science, Freiburg, Germany) and analysis of the AR gene microsatellite residues was performed as previously published (16).
Measurement of prostate size
Transrectal ultrasonography was performed as previously described using a high-resolution transrectal ultrasonography probe (7.5 MHz for sagittal and transversal scans with an ultrasound scanner type 2002 ADI; B-K Medical, Gentofte, Denmark) (16, 21).
Resting blood pressure and heart rate
Blood pressure and resting pulse were measured by trained physicians using a standardized oscillometric device (M5 Professional; Omron Medical Technics, Mannheim, Germany) with a cuff size appropriate to individual body habitus. Measurements were taken after the subject had rested in sitting position for at least 5 min in a quiet room.
Medication
In its injectable form, TU is a new preparation and has recently been made available (2004 in Germany). The medication used before market introduction was identical in preparation and content of TU and was provided by Bayer Schering Pharma, (Berlin, Germany; former Schering AG, Berlin, Germany): 1000 mg of TU (since 2004: Nebido) are dissolved in castor oil.
To achieve a fast steady-state after initiation of therapy, the second injection has to be given after 6–10 wk. Thereafter the dosing interval can be prolonged, reaching 10–14 wk. In general, patients were injected according to the following regimen: wk 0
wk 6wk 18wk 30further injections, starting wk 40–44 on an individual basis titrated according to nadir T levels at wk 30.
Statistics
All variables were checked for normal distribution by the Kolmogorov-Smirnov one-sample test for goodness of fit and log transformed if necessary. All data are presented as mean ± SD. In case of percent values (hematocrit), arcsin transformation was performed. Because most relevant changes of T treatment are likely to occur within the first year of treatment (6, 22), we compared baseline and T steady-state conditions before the fifth injection of TU (wk 40–44) by t tests for matched pairs. In case of significant differences, analysis of covariance (ANCOVA) models for repeated measurements including covariates of interest were assessed (BMI, age, 
-serum T concentrations, AR CAG repeats). Baseline correlations were calculated as Spearman rank correlations using nontransformed variables.
To assess long-term effects of safety, all data points except baseline values (n = 449) were analyzed in terms of passing respective thresholds into the pathological range. Such a condition was defined as yes or no and entered in binomial regression models involving the above-named covariates. Instead of -T levels, nadir T concentrations were chosen because this analysis applied to single time points. Because time under treatment was variable and a pathological condition could occur more than once in a single patient, duration of substitution was introduced as inverse weight to stratify for possible confounding effects.
Because BMI turned out to be a major confounder of treatment and safety, the relative risk was calculated for obese vs. nonobese men in 
2 tests.
The association between T levels in combination with AR CAG repeats and safety parameters may not be linear, as previous publications suggest (16). To this end, a nonlinear model to describe pharmacogenetic effects on hematocrit was created, involving data points of persons with a BMI less than 30 kg/m2 (n = 316) only because BMI is an additional factor of influence (see Fig. 3 and Table 3). A three-dimensional model to these data points was calculated, assuming a log-linear association of T concentrations with androgen effects (19, 23), a quasilog-linear binding of T to its receptor within the concentration range observed in humans (reviewed in Ref. 24) and a linear effect of the AR polymorphism on the transcription of androgen target genes (25). Thus, the underlying formula is based on these principles: Androgen effect = Log (T) x [m – (n x log (T) x CAGn)] + k.
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Computations were performed using the statistical software package SPSS (Chicago, IL; release 14.0) and GraphPad Prism (San Diego, CA; release 3.2).
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Results |
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. Table 2 displays major changes of safety parameters and the significant effectors of these changes after four injections of TU 1000 mg im at the time point before the fifth injection, i.e. after 40–44 wk of T substitution. Table 3 exhibits the frequency of pathological observations during the whole treatment period of 118 patient-years as well as predisposing factors. Figure 2 displays total serum T concentrations before and during therapy (also see Table 1). Figure 3 displays the crude relative risk of obese men vs. nonobese men to be diagnosed with safety parameters within the pathological range during T substitution. Figure 4 represents a nonlinear model of pharmacogenetics of T treatment in regard to hematocrit as a safety parameter and allows individual application of threshold levels for nadir T levels under substitution therapy.
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Discussion |
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In agreement with other long-term surveillance approaches that involve transdermal T preparations (22, 31), we observed major changes within the first year of treatment (Table 2). After reaching a plateau of androgenization, hypogonadal men seem to maintain a stable level of androgen-related effects; correspondingly, nadir total T levels are perpetuated within the normal range when hypogonadal men are regularly treated with injectable TU (Table 1).
Prostate safety is an issue of major concern among physicians treating hypogonadal men with T substitution. This study demonstrates that PSA levels are not necessarily affected to a clinically relevant degree, if at all, by external application of T. Nevertheless, to find a significant treatment effect on prostate volume without effect on serum PSA may seem rather unexpected. However, prostate volumes did not change to a major degree (Table 2). In addition, T treatment may not necessarily significantly alter PSA levels, as has been demonstrated by the largest metaanalysis of placebo-controlled trials regarding T substitution (6). Another large study involving transdermal application of T gel reported a significant increase in PSA levels. Nevertheless, these changes were subtle (baseline: 0.85 ± 0.06 ng/ml, 1.11 ± 0.08 ng/ml at month 6) and remained well within the normal range (22).
Such findings may be explained by low intraprostatic changes of hormone concentrations during T substitution (32).
Prostate size of hypogonadal men being substituted with TU im increases to values within the normal range, corroborating earlier findings with other T preparations (33). This effect is most visible within the first year of treatment and is more pronounced in older men and those with short AR CAG repeats and/or larger changes of T levels (Table 2). We have previously demonstrated such effects in a cohort of 131 men being treated with various androgen preparations (16).
Erythropoiesis is stimulated by T supplementation (reviewed in Ref. 34). This effect is generally considered beneficial because anemia is frequently encountered in male hypogonadism and contributes to overall complaints of weakness and fatigue (22). However, overstimulation of erythropoiesis may cause an elevated hematocrit. Hematocrit values over 50–52% are related to increased blood viscosity, and there is evidence from larger neurological studies unrelated to T that elevated hematocrit can result in cerebral ischemia: erythrocyte aggregation may facilitate platelet activation and aggregation (35, 36, 37). Identifiable risk factors in a prospective study involving 1000 stroke patients were arterial hypertension, smoking, diabetes mellitus, hypercholesterolemia, and high hematocrit (
50%) (38). However, it remains unclear whether high hematocrit caused by T treatment has the same relevance as described in the stroke studies because T exerts direct counterregulatory effects on hemostasis (39). Although hematocrit was observed to be higher in a recent metaanalysis of T substitution, the incidence of cardiovascular events was not increased in comparison with placebo-treated men (6). It is also known that the T effect on hematocrit is dose as well as age related (40, 41). In agreement, we demonstrate that effects of injectable TU on hematocrit and erythropoiesis are augmented by high nadir T concentrations as well as advanced age (Tables 2 and 3). In addition, we are able to report that short AR CAG repeats and high BMI contributed to the elevation of hematocrit during T substitution. Table 2 displays general, significant changes in hemoglobin content and hematocrit after initiation of therapy, and Table 3 exhibits risk markers for elevation of these parameters above safety thresholds. Remarkably, men with a BMI 30 kg/m2 or greater and/or CAG repeats less than 20 seem to have a markedly increased risk of developing blood hyperviscosity (Table 2 and Fig. 3). We present a nonlinear pharmacogenetic model to estimate androgen effects on hematocrit (Fig. 4).
We also considered estradiol concentrations as pivotal in regard to obesity. Aromatase activity may be increased due to augmented fat tissue in obese men and may thus cause higher estradiol concentrations on T substitution therapy. Indeed, serum concentrations of estradiol under treatment were strongly related to BMI (see Results). Estradiol levels were used as additional independent variables for analyses in second-step approaches and replaced BMI as confounder of hemoglobin levels and hematocrit (Tables 2 and 3), suggesting an independent effect of this sex steroid. This finding supports a previous, similar observation (41). Preexisting lung disease may present an additional factor in regard to the increase of hematocrit but was not diagnosed in our patients. Cigarette smoking did not exhibit a significant effect on the red blood picture under T substitution.
In regard to lipid metabolism, the general effect induced by androgen substitution within our study is a shift toward favorable profiles with lower low-density lipoprotein (LDL)-cholesterol and higher high-density lipoprotein (HDL)-cholesterol, which seems to be especially the case in younger, slimmer men receiving higher doses of T (Table 2). These changes in LDL-cholesterol concentrations are in agreement with a recent metaanalysis (5). Alterations in HDL-cholesterol under T therapy are usually described as a slight decline. For example, a study conducted in younger hypogonadal men showed a decrease of HDL levels with T treatment (im and transdermal) relative to the untreated state (e.g. Refs. 5 , 42), whereas we report a significant increase, especially in younger, slimmer men. Such findings are in agreement with cross-sectional, noninterventional observations (reviewed in Ref. 43). It can be speculated that our findings are due to the longer observation period of therapy, which may induce favorable alterations in body composition. This aspect was not measured here but is known from previous observations in T-treated hypogonadal men (44, 45). In opposition to this general treatment effect within the total cohort (Table 2), we also observed some hypogonadal men with elevated LDL-cholesterol and decreased HDL-cholesterol during androgen substitution: their specific characteristics are detailed in Table 3 and are due to a higher BMI (also see Fig. 3). Thus, a higher body mass in hypogonadal men may attenuate or even reverse general effects of T treatment in regard to lipid metabolism.
An association between hypogonadism and arterial blood pressure in men has been suggested by a cross-sectional approach: inverse correlations between endogenous T levels and systolic as well as diastolic blood pressure were described in 356 healthy elderly men (46). Here we report a systematic approach to treatment effects of T substitution in men and demonstrate favorable effects because diastolic and systolic blood pressure as well as resting heart rate decreased significantly during treatment with TU. This new and important observation is especially notable in the case of younger, slimmer men with higher androgen activity due to the CAG repeat AR polymorphism and/or higher -T concentration under treatment (Table 2). However, and similar to the effects described in lipid metabolism, some patients exhibited safety parameters regarding blood pressure and heart rate above the normal range and thus of inverse nature to the general treatment effect (Table 3). Blood pressure values (systolic and/or diastolic) or resting heart rates were elevated in men with higher BMI, lower nadir T concentrations, and attenuated androgen action due to the AR polymorphism (Table 3).
In summary, a marked tendency to augment the effects of T substitution (in case of hematocrit) or inverse to the favorable androgenic response (in case of lipid metabolism and circulation) can be observed in obese hypogonadal men. The crude relative risk for hypogonadal men with a BMI 30 kg/m2 or greater to be diagnosed with adverse safety parameters during treatment with TU is quite relevant (Fig. 3). In principle, this finding reflects the generally increased cardiovascular risk of obese men and may not be directly related to T substitution (47).
Because the effect of BMI on cardiovascular risk might not be related to serum concentrations of total T, this might still be the case for free T because BMI reduces SHBG. However, overall levels of free T rather followed concentrations of total T and results of analyses did not change (see Results and Tables 2 and 3). Thus, observation of factors that can be considered adverse in regard to cardiovascular risk (high LDL-cholesterol, low HDL-cholesterol) were not related to total or free T but high BMI as such (Table 3). Rather, the general treatment trend was reduction of these parameters (Table 2) and related to higher T concentrations and lower BMI. In addition, insufficient T concentrations seemed to contribute to the observation of high blood pressure (Table 3). Free T levels tended to be higher in obese men (see Results). Thus, it is, from a clinically intuitive point, unlikely that free T itself contributed to the generation of unfavorable observations (Table 3). These observations are most likely due to other factors inherent to obesity (47). However, a statistical dissociation of obesity from levels of free T is not possible within this setting. Hence, we cannot exclude that in some obese men free T levels might have contributed to adverse effects, which may have been facilitated via lower SHBG and hence high levels of free T.
In addition, the relevance of the AR CAG repeat polymorphism in pharmacogenetics of T treatment seems to be of clinical significance, as previously indicated (16, 17). The length of the repeat tract (normal range 9–37) is inversely associated with the transcriptional and physiological activity induced by T in vitro (25), in mouse models (48), and in humans (15, 16, 17, 49 , metaanalysis in Ref. 50). The modulatory effect on androgen-dependent gene transcription is linear, given a fixed concentration of T over a range from 0 to 200 repeats as seen in in vitro studies (51), and is probably mediated by a differential affinity of coactivator proteins to the encoded polyglutamine stretch of the actual AR protein, such as ARA24 and p160 (26, 27). Because these proteins are ubiquitously but nevertheless nonuniformly expressed, the modulatory effect of the CAG repeat chain on AR target genes is most likely not only dependent on androgenic saturation and AR expression but also varies from tissue to tissue.
However, androgen effects seem to follow a nonlinear pattern, when nadir total T levels under substitution therapy with TU and AR CAG repeat length are simultaneously taken into consideration: we present a nonlinear model derived from general hormone kinetics to describe the effects of T treatment on hematocrit (see Statistics and Fig. 4; also see review in Ref. 52). Quite in agreement with previous clinical findings (50), an AR CAG repeat length greater than 25 seems to require T levels quite above the normal range and may cause a picture of hypogonadism in the presence of normal T levels.
According to the number of AR CAG repeats, the nadir T level (respectively dosing interval of TU im) may be individually modulated to achieve beneficial effects (Table 2) yet simultaneously avoid an elevated hematocrit. According to our data and a recent metaanalysis (6), this safety parameter may deserve even greater recognition in comparison with prostate issues when treating hypogonadal men.
In conclusion, this long-term observation demonstrates that the use of the novel modality of T substitution by injectable, long-acting TU is generally well tolerated. Special attendance during T treatment in general is required in obese men because adverse effects are more prevalent in this subgroup. Pharmacogenetics exerted by the AR will be a future option to tailor individual T dosage.
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Acknowledgments |
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Footnotes |
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Disclosures: Both authors received lecture fees of less than $10,000 in regard to this topic. Both authors received consulting fees of less than $10,000 in regard to this topic.
First Published Online July 17, 2007
Abbreviations: ANCOVA, Analysis of covariance; AR, androgen receptor; BMI, body mass index; CV, coefficient of variation; HDL, high-density lipoprotein; LDL, low-density lipoprotein; PSA, prostate-specific antigen; T, testosterone; TU, T undecanoate.
Received March 20, 2007.
Accepted July 9, 2007.
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