Oral testosterone supplementation and chronic low-grade inflammation in elderly men: A 26-week randomized, placebo-controlled trial

Prevention and Rehabilitation Oral testosterone supplementation and chronic low-grade inflammation in elderly men: A 26-week randomized, placebo-controlled trial Hamid Reza Nakhai-Pour, MD, PhD,a,b Diederick E. Grobbee, MD, PhD,a Marielle H. Emmelot-Vonk, MD,a,b Michiel L. Bots, MD, PhD,a Harald J.J. Verhaar, MD, PhD,b and Yvonne T. van der Schouw, PhDa Utrecht, The Netherlands

Background To determine the effect of oral testosterone supplementation on systemic low-grade inflammation measured by high-sensitive C-reactive protein (hs-CRP) in aging men with low testosterone levels. Methods Two hundred thirty-seven men aged 60 to 80 years with a testosterone level of b13.7 nmol/L (below the 50th percentile of the population distribution) were recruited into a double-blind randomized placebo-controlled trial. Participants were randomized to either 4 capsules of 40 mg testosterone undecanoate (Andriol Testocaps, NV Organon, Oss, The Netherlands) or placebo daily for 26 weeks. Serum levels of hs-CRP were measured at baseline and at 26 weeks using a near-infrared particle immunoassay of the Synchron LX System (Beckman Coulter, Fullteron, CA). Results The median baseline hs-CRP level was 1.95 mg/L (0.30-6.43) in the testosterone group compared with 1.90 mg/L (0.40-5.91) in the placebo group. After 26 weeks of testosterone supplementation therapy, the 2 intervention groups were not statistically significantly different (median hs-CRP 2.20 vs 2.00 mg/L, interquartile range 0.40-6.54 vs 0.50-5.70, P = .36). In subgroup analysis, neither baseline testosterone level, nor age, nor baseline CRP-level modified the effect of testosterone supplementation on CRP levels. Conclusion

Oral testosterone undecanoate supplementation, in dosage of 160 mg daily for 26 weeks, does not increase hs-CRP levels in elderly men. (Am Heart J 2007;154:1228.e1-1228.e7.)

Aging is associated with a decrease in serum testosterone levels.1 At the same time, aging is accompanied by a pro-inflammatory state expressed by increasing levels of inflammatory cytokines.2 Inflammation plays a crucial role in atherogenesis,3 disability,4 and mortality.5 The ageassociated decline in sex hormones may affect the development of mild pro-inflammatory state.6 There is evidence suggesting that sex hormones may play a role in the regulation of inflammatory responses. For example, estrogen-based orally administered post-

From the aJulius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands, and bDepartment of Geriatrics, University Medical Center Utrecht, Utrecht, The Netherlands. Clinical trial registration number [ISRCTN23688581]. The study was financially supported by grant 014-91-063 from the Netherlands Organization for Health Research and Development. Submitted April 4, 2007; accepted September 9, 2007. Reprint requests: Yvonne T. van der Schouw, PhD, Julius Center for Health Sciences and Primary Care, Room STR 6.131, PO Box 85500, Utrecht Medical Center, 3508 GA Utrecht, The Netherlands. E-mail: [email protected] 0002-8703/$ - see front matter © 2007, Mosby, Inc. All rights reserved. doi:10.1016/j.ahj.2007.09.001

menopausal hormone therapy (HT) increases CRP levels in postmenopausal women.7-9 C-Reactive protein (CRP), which is a prototypic acute phase reactant and probably an important defense protein during inflammation, also seems to be partly regulated by androgens in women.10 In elderly men, evidence from a randomized clinical trial demonstrated no such effect after 13 weeks of testosterone supplementation with dihydrotestosterone.11 Also, short-term treatment with an aromatase inhibitor in elderly hypogonadal men, which increases testosterone levels, did not affect CRP levels.12 On the other hand, 3-week testosterone treatment before intracoronary stenting resulted in a significant suppression in highsensitive CRP (hs-CRP) and interleukin-6 (IL-6) levels after the stent implantation.13 Given the controversial findings regarding the sex hormones effect in chronic low-grade inflammation and because of increased interest in the potential for testosterone supplementation in elderly men, the possibility of regulatory effect of testosterone on circulating CRP is of importance. We determined the effect of oral testosterone supplementation on CRP levels in elderly men with moderately low testosterone levels.

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1228.e2 Nakhai-Pour et al

Figure 1

Participant flow diagram.

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Methods A randomized, double-blind, placebo-controlled trial was conducted. Details of the study design and procedures have been published previously.14 In brief, we recruited 237 men with serum testosterone levels b13.7 nmol/L and aged 60 to 80 years. They were randomized to either 4 capsules of 40 mg testosterone undecanoate (TU) or placebo daily for 26 weeks. Primary end points were cognitive function, functional mobility, and quality of life. Secondary end points were body composition, aortic stiffness, cardiovascular risk factors (lipid levels, hs-CRP, glucose, and insulin), and bone mineral density. Effects on prostate, liver, and hematological parameters were studied with respect to safety. The Institutional Review Board of the University Medical Center Utrecht approved the study protocol and all participants provided written informed consent.

Participants We performed power calculations only for the 2 primary end points: the 15 Words Test for cognitive function and the Health Assessment Questionnaire for performance in activities of daily living. The planned number of subjects was 240 in total, 120 in each intervention arm. Participants were recruited by direct mailing to 8020 randomly selected men between 60 and 80 years of age whose addresses were obtained from the municipal register of the city of Utrecht, the Netherlands. Inclusion criteria included a testosterone level below the 50th percentile of the study population based on testosterone distribution. The 50th percentile cut-off level of testosterone was determined to be 13.7 nmol/L after screening of 50 candidates. Complete details about the recruitment procedures, participant selection, and retention have been published already.14 In short, after the primary recruitment, 684 men were screened using medical history, laboratory testing, and digital rectal examination. Finally, 237 men were eligible for entry into the study and agreed to participate. The flow of study participants' recruitment and enrolment is shown in Figure 1.

Intervention After completion of the baseline measurements, subjects were randomized to 4 capsules of 40 mg TU (Andriol Testocaps, NV Organon, Oss, The Netherlands) or placebo daily for 26 weeks. Capsules were taken before the onset of breakfast and dinner. Adherence was monitored by pill counting at each study visit. After finalization of the study, serum testosterone concentrations were assessed in the final visit using blood samples as an extra check on compliance.

Laboratory measurements Fasting blood samples were obtained between 8:00 and 11:00 AM to minimize diurnal variation. Serum levels of highsensitive CRP were measured using a near-infrared particle immunoassay of the Synchron LX System (Synchron LX, Beckman Coulter, Fullteron, CA). All laboratory measurements were done at the SHO laboratory, Velp, the Netherlands. The analytical sensitivity of the assay we used for measuring hs-CRP is 0.02 mg/dL (0.2 mg/L), and the lowest concentration that can be measured with an interassay coefficient of variation of 20% is estimated to be ≤0.018 (≤0.18 mg/L). High-sensitive

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Table I. Subject characteristics at baseline according to randomization group Testosterone (n = 113) Age (y) Testosterone (nmol/L) SHBG (nmol/L) Albumin (g/L) FT (nmol/L) BT (nmol/L) Body mass index (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Current smoker Alcohol user Cardiovascular disease ⁎ Hypertension Hyperlipidemia Antihypertensive medication Antilipid medication hs-CRP (mg/L)y

67.1 ± 5.1 11.0 ± 1.9 33.1 ± 10.6 44.1 ± 2.3 0.22 ± 0.04 5.3 ± 1.1 27.2 ± 3.4 152.0 ± 20.3 88.4 ± 11.5 17.5% 82.5% 48.6% 83.3% 41.7% 36.7% 22.5% 1.95 (0.30-6.43)

Placebo (n = 110) 67.3 ± 5.0 10.5 ± 1.8 33.0 ± 10.1 43.9 ± 2.3 0.21 ± 0.05 5.0 ± 1.2 27.3 ± 4.0 150.8 ± 23.0 86.3 ± 11.7 12.8% 76.9% 51.4% 76.9% 35.0% 41.0% 21.4% 1.90 (0.40-5.91)

FT, Free testosterone; BT, bioavailable testosterone. Values are shown as mean ± SD, percentage, or median (IQR). * Includes myocardial infarction, angina, hypertension, or stroke. y Median (IQR).

CRP levels N10 mg/L can be taken as evidence of active inflammatory processes (eg, trauma, infection); therefore, subjects with hs-CRP N10 mg/L at baseline or at the end of the study were excluded from the analysis (n = 37). The levels of total testosterone and sex hormone–binding globulin (SHBG) were measured with a solid-phase, competitive, chemiluminescent enzyme immunoassay (IMMULITE 2000, Diagnostic Products Corporation, Los Angeles, CA) at baseline and at the end of the study. The intra- and interassay coefficients of variation were 7.2% and 8.2% for testosterone, and 2.5% and 5.2% for SHBG, respectively. Free and bioavailable testosterone (FT and BT, respectively) were calculated from testosterone, SHBG, and albumin using the method described by Vermeulen et al.15

Data analysis We used the t test to evaluate differences in means and the χ2 statistic to evaluate differences in proportions. As the distributions of hs-CRP were skewed, differences in medians between treatment groups in the final visit were tested by using the Wilcoxon signed rank test. All analyses were based on a modified intention-to-treat approach (ie, the intention-totreat group consists of all subjects, including those who withdrew from blinded medication, who received at least one dose of study drug, and who had at least one postbaseline assessment of the outcome variable). In addition, a per-protocol analysis was performed in the subjects who complied with the study protocol for 26 weeks. Next, we studied whether men changed from low- to higher risk categories according to cut-off levels used in clinical practice; we also analyzed hs-CRP as a categorical variable using χ2 statistics. Furthermore, prespecified subgroup analysis was performed according to baseline testosterone level (the lowest tertile vs the

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Table II. Effect of treatment on CRP level in overall subjects (intention-to-treat and per-protocol analyses) Change in CRP between final and baseline visit (mg/L) Testosterone Intention-to-treat analysis Unadjusted Adjusted for smoking Adjusted for alcohol Adjusted for smoking and alcohol Per-protocol analysis Unadjusted Adjusted for smoking Adjusted for alcohol Adjusted for smoking and alcohol

Placebo

Median CRP at final visit (mg/L) Difference in change (95% CI)

Testosterone

Placebo

P value

0.12 0.12 0.12 0.12

(2.1) (2.1) (2.1) (2.1)

0.06 0.06 0.06 0.06

(1.9) (1.9) (1.9) (1.9)

0.06 0.07 0.08 0.09

−0.50 −0.49 −0.48 −0.48

to 0.62 to 0.64 to 0.64 to 0.66

2.20 (0.40-6.54)

2.00 (0.50-5.70)

.36

0.10 0.10 0.10 0.10

(2.16) (2.16) (2.16) (2.16)

0.06 0.06 0.06 0.06

(1.9) (1.9) (1.9) (1.9)

0.04 0.06 0.06 0.07

−0.55 −0.54 −0.54 −0.53

to 0.63 to 0.65 to 0.65 to 0.67

2.20 (0.42-6.54)

2.00 (0.50-5.70)

.35

Table III. Difference in mean CRP change in selected subgroups (intention-to-treat and per-protocol analyses) Δ CRP in the testosterone group Intention-to-treat analysis 1st tertile of testosterone 2nd and 3rd tertile of testosterone Age bMedian ≥Median hs-CRP bMedian ≥Median Per-protocol analysis 1st tertile of testosterone 2nd and 3rd tertile of testosterone Age bMedian ≥Median hs-CRP bMedian ≥Median

Δ CRP in the placebo group

Mean difference CRP between groups

95% CI

0.62 (2.3) −0.10 (2.0)

0.43 (1.5) −0.15 (2.0)

0.19 0.05

−0.75 to 1.13 −0.65 to 0.74

0.21 (2.2) 0.01 (2.0)

0.01 (2.3) 0.10 (1.5)

0.21 −0.09

−0.70 to 1.11 −0.78 to 0.60

0.81 (1.5) 0.46 (2.4)

0.34 (0.7) 0.22 (2.5)

0.47 −0.24

0.01 to 0.93 −1.20 to 0.72

0.60 (2.31) 0.12 (2.07)

0.39 (1.54) 0.12 (2.08)

0.22 −0.00

−0.77 to 1.20 −0.74 to 0.74

0.18 (2.25) 0.01 (2.08)

0.01 (2.31) 0.11 (1.47)

0.17 −0.10

−0.75 to 1.09 −0.85 to 0.65

0.88 (1.51) 0.23 (2.59)

0.35 (0.73) 0.52 (2.40)

0.53 −0.29

0.04 to 1.02 −1.29 to 0.72

2 highest tertiles), age (bmedian vs ≥median), and baseline level of CRP (bmedian vs ≥median). Level of statistical significance was set at P b .05. All analyses were performed with SPSS statistical software package, version 12 (SPSS Inc, Chicago, IL).

Results Between January 2004 and October 2004, we enrolled 237 men in the study; 120 were assigned to testosterone and 117 to placebo. There were 30 early withdrawals, 16 in the testosterone group and 14 in the placebo group. Of the subjects completing the study, N90% of the participants used at least 80% of their medication. The mean age of the participants was 67 ± 5 years and the serum testosterone concentration was 10.7 ± 1.9 nmol/L. There

were no material differences between the 2 groups at baseline. Baseline hs-CRP levels were similar in the intervention groups (Table I). The effects of 26 weeks of oral supplementation therapy on safety parameters and testosterone and SHBG levels have been reported elsewhere (EmmelotVonk et al, submitted for publication). In brief, total testosterone was unchanged from baseline in the testosterone group and increased slightly in the placebo group; the difference between the testosterone and placebo group at month 6 was −3.2 nmol/L (95% CI −4.2 to −2.2, P b .001). Sex hormone–binding globulin levels declined from baseline in the testosterone group but not in the placebo group (difference −10.1 nmol/L, 95% CI −11.7 to −8.5, P b .001). Also, the

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between-group difference for FT and BT was significant at month 6 (FT difference −0.03, 95% CI −0.05 to −0.00, P = .04; BT difference −0.69, 95% CI −1.24 to −0.13, P = .02, respectively). After 26 weeks of therapy, median hs-CRP was 2.20 vs 2.00 mg/L (interquartile range [IQR] 0.40-6.54 vs 0.50-5.70; P = .36) in the testosterone and the placebo group, respectively. The results of per-protocol analysis were similar to those of intent-to-treat analysis (Table II). Based on the intention-to-treat analysis, 32% of subjects in the testosterone treatment group were in the high-risk category (hs-CRP N3 mg/L) versus 26% in the placebo treatment group (P = .37). After 26 weeks of supplementation, this percentage was 29% in the testosterone treatment group versus 28% in the placebo treatment group (P = .86). Based on the per-protocol analysis in the baseline visit, 32% of subjects in the testosterone treatment group were in the high-risk category versus 28% in the placebo treatment group (P = .44). After 26 weeks of supplementation, this percentage was 30% in the testosterone treatment group versus 28% in the placebo treatment group (P = .86). Neither baseline testosterone level, nor age, nor baseline CRP level modified the effect of testosterone supplementation on CRP levels (Table III) (P values for interaction .70, .28, and .65, respectively).

Discussion The results of this double-blind randomized trial in 237 elderly men with moderately low testosterone levels indicate that 26 weeks of 160 mg oral TU supplementation has no effect on their serum hs-CRP levels. The results reported here need to be interpreted with a few issues in mind. It is possible that the testosterone dose that we used was too low to exert a beneficial effect. However, the dose of testosterone we used is the same as or even higher than the dose that is used in clinical practice to treat hypogonadal men. For this dose, a significant beneficial effect has been shown with respect to quality of life,16 sexual interest and behavior,17 prostate symptoms and sexual function,18 and lean body mass and fat mass19 by previous studies. The fact that the serum testosterone levels were not increased at the end of the study in the testosterone treatment group is a known effect of supplementation with oral TU and agrees with previous studies.20 However, we showed the same statistically significant biochemical (increased hematocrit) and physical effects (decreased fat mass and increased lean body mass) (Emmelot-Vonk et al, submitted for publication) as in the studies reporting an increase of the serum testosterone concentration with use of intramuscular or transdermal testosterone.

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In addition, the duration of the intervention may have been too low, but given the existing evidence regarding the effect of various treatments on hs-CRP levels, a 6-month intervention has been shown to be long enough to observe any clinically important effect.21,22 The primary end points for which this study was powered were the 15 Words Test (for cognitive function) and the Health Assessment Questionnaire (for physical functioning). The usefulness of post hoc power analyses is questionable. When looking at our findings and their 95% CIs, our results are compatible with an increase of maximally 0.65 mg/L due to testosterone treatment. With an increase of this magnitude, men are still in the low-moderate risk category (1-3 mg/L) according to currently used cut-off levels.23 This is also true for the men who were at baseline in the lower half of the CRP distribution, even though in this group the subgroup analysis may suggest that testosterone might increase CRP levels stronger than in the men in the upper half of the CRP distribution. In randomized clinical trials, compliance is always a major concern. However, based on count of returned study medication, N90% of the subjects completing the study used at least 80% of their medication. Moreover, the per-protocol analysis did not suggest a more pronounced effect of testosterone. Although this trial could not disclose a relevant effect of testosterone supplementation on CRP levels, such an effect was plausible in view of other research findings. The higher incidence and prevalence of autoimmune diseases in androgen-deficient men suggest that testosterone may have an immunosuppressive role.24 In addition, the anti-inflammatory effect of testosterone may explain why hypogonadal men have a higher risk of metabolic syndrome and coronary artery disease.25,26 Data show that IL-6 is the main inducer of acute-phase CRP response.27 Some evidence of androgen regulation of the inflammatory response comes from observations that testosterone downregulates cytokine IL-6, a potent stimulator of inflammation.28 In vitro studies showed that testosterone downregulates the IL-6 gene through androgen receptors,29 although conflicting data are available regarding the effects of IL-6 on testosterone as well.30 Furthermore, it has been shown that the induction of hypogonadism in older men is followed by a significant increase in IL-6.31 The few existing epidemiologic studies have been inconsistent in showing a relationship between testosterone and inflammatory markers. In a cross-sectional study in 497 samples of men aged ≥65 years, no significant associations have been reported between testosterone and IL-6.32 Moreover, no association has been found between CRP and testosterone in a population-based cross-sectional study in 770 middle-aged white men.33 However, a significant negative association of FT levels with hs-CRP levels was noted among 1865 men with the metabolic syndrome.34

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Our findings are consistent, at least in part, with the findings of previous studies on testosterone supplementation. In one study, testosterone replacement in 27 hypogonadal men (crossover study) resulted in decreased levels of TNF-α and IL-1β without significant changes in IL-6 and CRP.35 Furthermore, it has been demonstrated that a wide dose range of testosterone administration in 61 eugonadal young-aged men for 20 weeks had no effect on CRP levels.36 In addition, a randomized, double-blind, placebo-controlled trial conducted in healthy middle-aged men treated with dihydrotestosterone for 13 weeks could not show any change in CRP levels.11 However, in 41 middle-aged men with stable angina, high dose of testosterone for 3 weeks after angiography suppressed hs-CRP levels.13 The current randomized trial is one of the largest studies of testosterone supplementation to date. The compliance was high and the dropout rate was low. In addition, we found in this study that testosterone supplementation during 26 weeks was safe and well tolerated, with no adverse effects on the prostate (Emmelot-Vonk et al, submitted for publication). It remains possible that testosterone supplementation may be of benefit to protect against inflammatory burden by aging during a longer period of use or at a higher dose. In conclusion, oral testosterone supplementation at levels of 160 daily does not increase CRP levels in elderly men with moderately low circulating testosterone levels. Trial medication was provided by Organon NV, Oss, The Netherlands. Organon NV neither controlled nor influenced the contents of the research or of this paper, nor played any part in the decision to submit this manuscript for publication.

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32. Maggio M, Basaria S, Ble A, et al. Correlation between testosterone and the inflammatory marker soluble interleukin-6 receptor in older men. J Clin Endocrinol Metab 2006;91:345-7. 33. Van Pottelbergh I, Braeckman L, De Bacquer D, et al. Differential contribution of testosterone and estradiol in the determination of cholesterol and lipoprotein profile in healthy middle-aged men. Atherosclerosis 2003;166:102. 34. Laaksonen DE, Niskanen L, Punnonen K, et al. Sex hormones, inflammation and the metabolic syndrome: a population-based study. Eur J Endocrinol 2003;149:601-8. 35. Malkin CJ, Pugh PJ, Jones RD, et al. The effect of testosterone replacement on endogenous inflammatory cytokines and lipid profiles in hypogonadal men. J Clin Endocrinol Metab 2004;89:3313-8. 36. Singh AB, Hsia S, Alaupovic P, et al. The effects of varying doses of T on insulin sensitivity, plasma lipids, apolipoproteins, and C-reactive protein in healthy young men. J Clin Endocrinol Metab 2002;87:136-43.