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Testosterone Replacement Therapy in Older Male Subjective Memory

Complainers: Double-Blind Randomized Crossover Placebo-Controlled Clinical

Trial of Physiological Assessment and Sa...

Article  in  CNS & neurological disorders drug targets · April 2015

DOI: 10.2174/1871527314666150429112112 · Source: PubMed

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Testosterone Replacement Therapy in Older Male Subjective Memory Complainers: Double-Blind Randomized Crossover Placebo-Controlled Clinical Trial of Physiological Assessment and Safety

Prita R. Asih1,2,3,§, Eka J. Wahjoepramono1,2,§, Vilia Aniwiyanti2, Linda K. Wijaya1, Karl de Ruyck1,3, Kevin Taddei1,3, Stephanie J. Fuller3, Hamid Sohrabi1,3, Satvinder S. Dhaliwal4, Giuseppe Verdile3,5, Malcolm Carruthers3 and Ralph N. Martins*,1,3

1Centre of Excellence for Alzheimer’s Disease Research and Care, School of Medical Sciences, Faculty of Computing, Health, and Science, Edith Cowan University, Joondalup, WA, Australia 2Medical Faculty, Pelita Harapan University, Neuroscience Centre, Siloam Hospitals, Lippo Karawaci, Tangerang, Indonesia 3Sir James McCusker Alzheimer’s Disease Research Unit, School of Psychiatry and Clinical Neurosciences, University of Western Australia, Hollywood Private Hospital, Nedlands, WA, Australia 4School of Public Health, Curtin University of Technology, Bentley, WA, Australia 5School of Biomedical Sciences, Curtin University of Technology, Bentley, WA, Australia

Abstract: Testosterone replacement therapy (TRT) has been investigated in older men as a preventative treatment against Alzheimer’s disease and dementia. However, previous studies have been contradictory. We assessed TRT physiological effects in 44 older men (aged 61 ± 7.7 years) with subjective memory complaints using a double blind, randomized, cross-over, placebo-controlled study. Participants were randomized into 2 groups, one group received transdermal testosterone (50 mg) daily for 24 weeks, followed by a 4 week wash-out period, then 24 weeks of placebo; the other group received the reverse treatment. Blood evaluation revealed significant increases in total testosterone, free (calculated) testosterone, dihydrotestosterone, and a decrease in luteinizing hormone levels (p<0.001) following TRT. Although there were significant increases in red blood cell counts, hemoglobin and prostate specific antigen levels following TRT, they remained within normal ranges. No significant differences in plasma amyloid beta, estradiol, sex hormone binding globulin, insulin levels, body fat percentage, or body mass index were detected. This is the first carefully controlled study that has investigated the influence of TRT in Indonesian men on blood biomarkers linked to dementia risk. Our study suggests TRT is safe and well-tolerated in this Indonesian cohort, yet longitudinal studies with larger cohorts are needed to assess TRT further, and to establish whether TRT reduces dementia risk.

Keywords: Aging, Alzheimer’s disease, dementia, testosterone, luteinizing hormone, plasma.

1. INTRODUCTION

Hormonal changes due to the dysregulation of the hypothalamic pituitary gonadal (HPG) axis during aging have been associated with cognitive impairment, the risk of developing dementia and the pathogenesis of Alzheimer’s disease (AD). Longitudinal studies indicate that men normally experience a gradual loss of testosterone as they age [1-3], and neuropathological evidence suggests that testosterone levels in the brain also decrease with age [4]. However, men with neuropathological diagnoses of AD or mild cognitive impairment (MCI) have lower testosterone

*Address correspondence to this author at the Edith Cowan University, Suite 22, Hollywood Medical Centre, 85 Monash Avenue, Nedlands, WA 6009, Australia; Tel: (61 8) 93474200; Fax: 93474299; E-mail: r.martins@ecu.edu.au §Both authors contributed equally to this work.

levels compared to healthy men of a similar age [4]. The gradual decline in testosterone can manifest itself in many ways, including sexual dysfunction, diminished physical stamina, and loss of muscle and bone mass. Low testosterone has also been associated with systolic hypertension, the development of abdominal visceral fat mass, insulin resistance, high levels of cholesterol, LDL and triglycerides, low HDL levels, as well as impaired quality of life due to symptoms such as depression and cognitive impairment [5-8]. The age-related decline in testosterone levels usually starts in the fifth decade [9], with men aged 70 and older having the lowest levels. Estimates for the prevalence of men with low testosterone levels range from 1 in 200 [10], 1 in 20 [11], to 1 in 4 [12], depending on the cut-off point, the population age, ethnicity and health factors. Men with testosterone levels significantly lower than normal may benefit from carefully monitored testosterone replacement therapy (TRT).

Ralph N. Martins

2 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 4 Asih et al.

In vivo and in vitro studies have demonstrated that testosterone may be beneficial in the prevention and treatment of AD, at least in part by reducing brain beta amyloid (Aβ) levels. Testosterone treatment in castrated guinea pigs has been found to reduce cerebral and CSF Aβ levels [13]. Testosterone has also been shown to decrease Aβ levels in primary neuronal cultures [14]. In clinical studies, Gandy et al. [15] recorded an elevation in plasma Aβ levels in men being treated with testosterone suppression drugs for prostate cancer, and low testosterone levels have been associated with increased plasma Aβ levels in men [16]. Furthermore, testosterone supplementation in men with AD or MCI has been shown to improve spatial memory and cognition [17, 18]. Although there have been some inconsistencies in study results, most studies suggest that TRT may prevent or delay AD [19]. Some other studies have linked testosterone treatment to adverse outcomes such as increased risks of atherosclerosis, gynecomastia and in particular, prostate cancer [20-22]. However, the prostate cancer link has been strongly contested. Testosterone is now believed not to increase the risk when only raised to high physiological levels, even in people already at high risk of developing prostate cancer [23, 24]. TRT that has resulted in unfavourable outcomes has usually involved people deliberately trying to achieve supra-physiological levels to improve athletic performance, for example [25]. Such results do not apply to patients trying to maintain testosterone at physiological levels. Almost universally, a testosterone-deficient state in older men is permanent, and as a result, TRT may be for life. Therefore, testosterone treatments need to be carefully designed and extensively tested in properly controlled clinical trials. Many TRT studies have already been undertaken [17-19]. However, there is a lack of suitable longitudinal clinical studies of aging men that have investigated comprehensively the long-term effects of testosterone treatment, including effects on the cardiovascular and immune systems, body metabolism, and the brain, and no such studies have been carried out in an Indonesian population. In this double-blind, randomized, cross-over, placebo-controlled study, we have investigated the effect of testosterone treatment in a group of elderly male subjective memory complainers (SMC), who had testosterone levels at or below what is considered to be a low normal level. The blood biomarkers measured include total testosterone and its derivative dihydrotestosterone (DHT), SHBG, estradiol, luteinizing hormone (LH) and insulin. Free testosterone levels were calculated, lipid profiles were obtained, plasma Aβ levels were measured, and general hematology measures were also monitored. Potential unwanted side-effects of the treatment were monitored: we assessed markers of cardiovascular disease, and monitored prostate specific antigen (PSA) levels to assess prostate cancer risk, as well as any signs of gynecomastia.

2. MATERIALS AND METHODS

2.1. Participants

This study was undertaken at the Siloam Hospital in Lippo Karawaci, Tangerang, Indonesia. Human ethics

approval was obtained from the Independent Human Ethics Committee, Faculty of Medicine, University of Indonesia, as well as from Edith Cowan University, Western Australia, prior to study commencement. The participants were recruited from areas surrounding Jakarta and Tangerang, Indonesia, including patients from Siloam Hospital, Lippo Karawaci, Indonesia and the nursing homes in those areas. The following eligibility criteria were used: the male participants needed to 1) be 50 years of age or older, and have 2) a memory complaint, 3) testosterone levels between 300-600 ng/dL (~10.4 - 20.8 nmol/L), 4) normal levels of PSA, 5) blood pressure within normal limits, between 120/80 mmHg to 90/60 mmHg 6) no signs of diabetes mellitus, 7) normal liver and kidney enzyme function, and 8) they should not have suffered from major head injury. The diagnosis of subjective memory loss (complaint of memory loss but not meeting the criteria for dementia) was reached after a full clinical evaluation including a review of medical and drug history (for example, whether a participant had had any medication such as donepezil or rivastigmine) and a physical examination. Participants were partly selected based on their Mini Mental State Examination (MMSE) assessment: a score of 24 or above determined eligibility. The final decision for inclusion was determined jointly by a neurologist, a neuropsychologist, and the researchers in charge of the project. All participants in this study had at least 6 years of education. Written informed consent was obtained from all participants. Fifty male subjective memory complainers met the eligibility criteria, and 44 of these completed the study. They were recruited and assigned to one of the two parallel groups; placebo (n=22) or T (n=22) treatment from 2nd September 2006 to 20th October 2007. Calculation of the sample size based on the types of superiority design (randomized crossover trial) and measures of continuous outcomes. For allocation of the participants, a blocked randomization 1:1 ratio was used to ensure each arm of the study was balanced. Block randomization was by a random numbers-table prepared by an investigator with no clinical involvement in the trial. After the research nurse had obtained the patient’s consent, she telephoned a contact who was independent of the recruitment process for allocation consignment. The study design was such that participants received testosterone treatment or placebo for 24 weeks, followed by a wash-out period of 4 weeks. Following this step, the participant groups were “crossed over”, such that the placebo group received testosterone and vice versa for a further 24 weeks. For all participants, 11 clinic visits to Siloam Hospital, Lippo Karawaci (Indonesia) were required during the study period. The participants and the investigators were blinded to treatment assignment until study completion (Fig. 1).

2.2. Procedures

During the first (baseline) visit, participant blood pressure, height, weight, body fat percentage and body mass index (BMI) were measured and blood samples taken. Blood (± 45 ml) was collected using serum separating tubes (SST, that include a gel and blood clot activator), EDTA collection tubes, and heparin collection tubes. Blood in SST collection

Testosterone Replacement Therapy in Older Men CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 4 3

Fig. (1). Time course of the study, showing the weeks that blood samples were taken. Every 4 weeks for 0-28 weeks, and every 8 weeks of the 24-week cross-over period, blood samples were taken. Analysis of blood samples provided testosterone, dihydrotestos-terone (DHT), sex hormone-binding globulin (SHBG), luteinizing hormone (LH), estradiol, prostate-specific antigen (PSA), insulin and amyloid-β (Aβ) levels, and provided blood lipid profiles.

tubes was centrifuged at 2500 rpm for 10 min to separate the serum after clotting. This serum was removed, divided into 1 ml aliquots to which 10 µl (100 µg/ml) of protein inhibitor (aprotinin – Sigma) was added. Blood samples in EDTA and heparin collection tubes were centrifuged at 1000 rpm for 10 min at RT, to separate the plasma, leucocytes, and red blood cells (RBC). Plasma was removed and centrifuged further at 2500 rpm for 10 min to harvest platelets. The resulting supernatant (plasma) was aliquoted (~ 1 to 5 ml lots) and 10 µl protein inhibitor (aprotinin – Sigma) was added to each ml of plasma. These blood samples were frozen, then transported to Perth and used for serum testosterone levels, levels of LH and sex hormone binding globulin (SHBG), DHT (dihydrotestosterone), estradiol, cholesterol, insulin, glucose, PSA, plasma Aβ levels and to obtain lipid profiles. For the testosterone treatment group (n=22), testosterone (50 mg in the form of Andromen® 5% FORTE cream obtained from Lawley Pharmaceuticals, Perth, Western Australia) was applied daily to the scrotum, thus using a transdermal route (topically), for 24 weeks. For the placebo group (n=22) the same amount of the cream base [dl-α tocopherol acetate (vitamin E), without the testosterone] was applied with the same frequency and in the same manner. At each of the six remaining clinic visits i.e. at 4, 8, 12, 16, 20 and 24 weeks during the first treatment period, blood was collected as described for the baseline measurement. During the 4 week washout period, the participants were given an equivalent amount of cream base lacking testosterone and vitamin E, to be applied to the scrotum. The placebo and testosterone treatment groups of the study were then crossed-over for the second 24-week treatment period. During this period, the participants were examined every eight weeks (one bleed at the start of the period (end of washout), followed by bleeds at weeks 8, 16 and 24 of the cross-over period); and blood samples were analysed as described previously.

2.3. Blood Biomarker Measurement

Serum samples (n=535) were analysed for total testosterone (TT) and DHT using an isotope dilution LC-MS/MS method at the Anzac Research Institute, NSW. Other serum samples were analysed for LH, estradiol, PSA,

SHBG and insulin in PathWest Laboratory, Perth, WA. Lipid profiles were obtained and albumin levels measured using heparin-treated plasma samples. Serum-free testosterone (calculated free testosterone: CFT) was calculated using TT, SHBG and albumin levels following a standard formula [26]. Plasma Aβ40 and Aβ42 levels were measured using an ELISA kit [27]. Changes of all of these blood biomarker measurements are the primary outcomes of the study, compared to baseline.

2.4. Statistical Analysis

Statistical analysis was performed using the Statistical Package of Social Sciences (SPSS version 21, SPSS Inc, Chicago, USA). The mixed model ANOVA analysis consisted of fixed effects for treatment (to compare testosterone with placebo treatment), treatment period (testosterone or placebo), and sequence (testosterone period first or placebo period first), irrespective of the baseline. Carry-over effects were tested from differences from baseline in the first period (week 0) to the end of the washout period (week 24-28), which is the baseline time-point for the second period, based on APOEε4 genotype, on each arm of the study as well as on the entire data set. When directly comparing two groups based on the presence of APOEε4 genotype, two tailed independent t-tests were performed. All analyses were two-tailed and the alpha level was set at 0.05.

3. RESULTS

Forty-four of the initial 50 male participants completed both treatment periods. The other 6 men did not complete the treatment due to personal reasons (n=5), and moving interstate (n=1), as shown in the participant flowchart (Fig. 2). After categorizing the men according to APOE ε4 status [32 APOE ε4- (72%) and 12 APOE ε4+ (27%)], the characteristics of the 44 SMC elderly men were as reported in Table 1. There were no significant differences between ε4+ and ε4- participants in terms of age or education. The treatment suppliers were provided with treatments labeled “A” and “B” to give to the participants, which eventually were revealed to be A = testosterone treatment, and B = placebo after the end of all treatment. For ease of understanding, testosterone and placebo treatment periods are referred to as “T” and “P” respectively, in the figures/tables of this paper. Overall, at week 0, there were no significant differences between the APOε4+ and ε4- subjects in any of the blood biomarkers measured. There were also no significant sequence effects, period effects or carry-over effects for any variable following the wash-out period (week 28) of either treatment sequence. As a result, all further analyses were carried out after pooling all testosterone treatment results and all placebo results, regardless of original order of treatment (TàP or PàT). This improved statistical power, and allowed us to focus on comparing placebo with testosterone treatments.

3.1. Hormones

From the graphs in Fig. (3) and Table 2 it is clear that testosterone levels were significantly higher following

4 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 4 Asih et al.

testosterone compared to placebo treatment (F=41.471, p<0.001). At four weeks post-testosterone treatment (end of washout period), testosterone was back at baseline levels, which indicated no carry-over effects. The results for DHT and calculated free testosterone levels were similar to total testosterone level results, with 4-fold and 2-fold increases respectively (F=151.793, p<0.001; F=37.875, p<0.001 Table 2 and Fig. 3b). LH levels significantly decreased following testosterone treatment (F=43.466, p<0.001, Table 1), yet rose back to pre-treatment levels by the end of the washout period in the T → P treatment group (Fig. 3c). In contrast, estradiol levels did not show significant changes following testosterone treatment (F=4.677, p=0.37). No significant changes in insulin levels were observed (F=1.32, p=0.261). Table 1. Average age and education level of each of the two

groups of participants at screening, after categorizing according to APOE ε4 status.

Group n Age Mean (SD)

Years of Education Mean (SD)

Treatment TàP 22 59.2 (7.18) 14.0 (2.84)

APOE ε4- 14 59 (5.82) 13.8 (3.31)

APOE ε4+ 8 59.6 (9.58) 14.2 (1.91)

Treatment PàT 22 62.9 (8.22) 13.8 (3.39)

APOE ε4- 18 62.7 (7.31) 13.5 (3.62)

APOE ε4+ 4 63.8 (13) 15 (2) T = testosterone, P = placebo. Results shown as means (SD).

3.2 .Plasma Aβ in Response to Testosterone Treatment

Overall, there were no significant changes in plasma Aβ levels, both Aβ40 and Aβ42 (F= 2.273, p=0.136; F=0.47, p=0.498 as shown in Table 2). Furthermore, based on the individual data (data not shown), most participants had no plasma Aβ40 and Aβ42 changes in response to testosterone treatment (24 plasma Aβ40 non-responsive (54.5%) and 29 plasma Aβ42 non-responsive participants (65.9%)). Whereas the rest of the participants had either a 10%-30% increase or decrease of their plasma Aβ compared to the baseline.

3.3. Lipid Profiles, Hematology and Cardiovascular Risk

There were no significant differences observed in total cholesterol (F=3.31, p=0.081), HDL (F=3.623, p=0.065), or LDL (F=0.159, p=0.693) levels following treatment with either testosterone or placebo (Table 1). Cholesterol/HDL and LDL/HDL ratios were also calculated to monitor atherosclerosis risk, and both did not show any significant changes following testosterone treatment (F=1.14, p=0.294; F=2.418, p=0.132, respectively). In contrast, RBC counts and hemoglobin levels both increased following testosterone treatment (F=43.317, p<0.001; F=41.634, p<0.001, Table 2).

3.4. High PSA Levels and Gynecomastia as Potential Side-Effects of TRT

Significant increases in PSA levels were observed following testosterone treatment compared to placebo

Fig. (2). Flow diagram of the study, includes detailed information on the excluded participants.

Assessed%for%eligibility%(n=65)%

Enrolment)

Excluded%(n=15)%•  Not%mee<ng%

inclusion%criteria%=%10%

•  Refused%to%par<cipate%=%5%Randomiza<on%

Alloca<on%to%1st%arm%treatment%(T!P)%(n=25)% Alloca-on) Alloca<on%to%2nd%arm%treatment%

(P!T)%(n=25)%

Lost%to%followGup%(n=3)%(withdraw)%

Follow0up) Lost%to%followGup%(n=3)%(withdraw)%

Analysis)Sta<s<cal%analysis%(n=22)%%

Sta<s<cal%analysis%(n=22)%%

Testosterone Replacement Therapy in Older Men CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 4 5

(F=6.634, p=0.014). The risk of developing gynecomastia was estimated by measuring the estradiol/testosterone ratio, and testosterone treatment caused no change to this ratio (F=0.31, p=0.974 Table 2).

3.5. Physical Measures

There were no significant differences following testosterone and placebo treatments in body fat percentage (F=0.088, p=0.77) and body mass index (BMI) (F=0.51, p=0.481 Table 2).

4. DISCUSSION

This study investigated the effect of topical transdermal testosterone treatment in a group of Indonesian male subjective memory complainers (SMC). A selection of blood biomarkers was investigated, including plasma Aβ. In addition, the study monitored possible risks of testosterone treatment such as the potentially increased risks of developing prostate cancer, cardiovascular disease, and gynecomastia. All participants in this 12 month double-blind, randomized, and placebo-controlled trial underwent both

Fig. (3). Individual graphs of the blood biomarkers that demonstrated significant changes following testosterone treatment. T = testosterone, P = placebo.

(a)# (b)#

# #(c)# (d)#

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Treatment"T!P" Treatment"P!T"

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Treatment(T!P( Treatment(P!T(

Dihydrotestosterone((DHT)(levels(

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Prostate(Specific(An0gen((PSA)(levels(

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Red$Blood$Cell$(RBC)$levels$

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0$$$$3.5$$$$$$$$$4$$$$$$$$$$4.5$$$$$$$$$5$$$$$$$$$$5.5$$$$$$$$$6$$$$$$$$$6.5$$

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6 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 4 Asih et al.

24 weeks of testosterone and 24 weeks of placebo treatments. Thus all participants served as their own “controls”, and the study reduced the problems of patient-to-patient variability. Another strength of the study came from the fact that both potential orders of treatment were tested – testosterone followed by placebo, as well as the reverse. From the participant point of view, the role was the same all year –all stages required a cream to be applied daily.

4.1. Hormones

When measuring testosterone levels, the assay for total plasma testosterone levels is the test of choice as it is one of the most widely available as well as the gold standard of the testosterone assays. Total testosterone levels were also used to monitor the efficacy of testosterone treatment. Total plasma testosterone represents testosterone that is tightly bound to SHBG (44%) as well as that weakly associated with albumin (54%), with the remaining 2% free (and able to pass the blood brain barrier) [28]. With aging, total testosterone levels decrease on average, approximately 110 ng/dL (~3.8 nmol/L) per decade [29]. An early morning plasma testosterone level of less than 300 ng/dL (~ 10.4 nmol/L) indicates that a patient should be considered testosterone-deficient [30]. However, the threshold of testosterone levels for various symptoms can vary widely,

and individual variation also occurs. The total testosterone levels of the study participants were slightly above the borderline for testosterone-deficient men, as they ranged between 10-20 nmol/L. This study aimed to raise serum testosterone levels to that seen in the mid-normal range, which for these Indonesian elderly men is 20-35 nmol/L, based on the laboratory reference range using immunoassays. Since day-to-day variations in testosterone levels may be considerable, blood samples were taken at 4 week intervals in weeks 4-24, and at 8 week intervals in weeks 28-52 and averages for treatment periods were calculated. From the results (Fig. 3a), it is clear testosterone levels were significantly higher during testosterone treatments, irrespective of treatment order (TàP or PàT). However, individual increases in testosterone levels varied somewhat (data not shown). This may have been due to the possibility that the amounts of testosterone cream applied may have varied from one individual to another, although the testosterone cream application instructions had been well demonstrated and documented on the T/P tubes. In addition, androgen receptor polymorphisms (CAG repeats) conferring different individual sensitivity to testosterone may also affect testosterone treatment efficiency. The calculated free testosterone result showed a similar outcome.

Table 2. Combined blood biomarker results (n=44) from the two treatment groups.

Blood Biomarkers Baseline Testosterone Placebo p Value

Testosterone (nmol/L)a 17.4 27.5 ± 1.22 16.6 ± 1.22 *** p<0.001

DHT (nmol/L)a 1.87 8.96 ± 1.66 1.73 ± 0.42 *** p<0.001

Calculated free testosteronea 363 662 ± 36.4 362 ± 36.4 *** p<0.001

Estradiol (pmol/L) 82.3 89.1 ± 4.28 78.6 ± 4.28 p= 0.37

Estradiol/ Testosterone 0.005 0.003 ± 0.05 0.004 ± 0.05 p= 0.974

LH (U/L)a 4.83 2.77 ± 0.26 5.17 ± 0.26 *** p<0.001

Abeta 40 (pg/mL) 137 137 ± 2.94 143 ± 2.94 p= 0.136

Abeta 42 (pg/mL) 31.6 31.2 ± 0.69 31.8 ± 0.67 p= 0.498

Abeta 42 : Abeta 40 0.22 0.21 ± 0.01 0.18 ± 0.02 P=0.084

Total cholesterol (mmol/L) 4.77 4.65 ± 0.087 4.8 ± 0.087 p= 0.081

HDL (mmol/L) 1.17 1.12 ± 0.02 1.17 ± 0.02 p= 0.065

LDL (mmol/L) 3.01 2.98 ± 0.076 3.01 ± 0.076 p= 0.693

Total cholesterol/HDL 4.09 4.25 ± 0.08 4.16 ± 0.08 p= 0.294

LDL / HDL 2.58 2.72 ± 0.07 2.61 ± 0.07 p= 0.132

Insulin (mU/L) 7.29 7.42 ± 0.32 7.87 ± 0.32 p= 0.261

PSA (µg/L) a 1.32 1.41 ± 0.07 1.19 ± 0.07 ** p= 0.014

Red blood cells (x106/µL)a 4.88 5.2 ± 0.04 4.81 ± 0.04 *** p<0.001

Hemoglobin (g/dL)a 14.4 15.2 ± 0.11 14.2 ± 0.11 *** p<0.001

SHBG (nmol/L) 36.0 34.5 ± 0.56 34.3 ± 0.56 p= 0.843

% body fat 22.5 25.4 ± 0.29 25.4 ± 0.29 p= 0.77

Body mass index 25.5 22.8 ± 0.4 22.5 ± 0.4 p= 0.481 Data are expressed as mean and SEM of the average of readings from the blood samples taken over the 6 months from both treatment groups (TàP and PàT). Baseline levels are the week 0 (starting baseline) and week 28 (end of washout period) readings. aLevels on these measures were significantly different post-testosterone vs post-placebo.

Testosterone Replacement Therapy in Older Men CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 4 7

Testosterone’s mechanisms of action on target organs may be mediated directly via testosterone or indirectly via a major metabolic product, dihydrotestosterone (DHT). The testosterone cream resulted in elevated serum DHT levels, with no carry-over effect, most likely due to high scrotum levels of 5-a reductase. Much of the testosterone must have been converted to DHT, which can bind to androgen receptors. The results agree with those of Cunningham et al. [31] and other studies [32] where it has also been found that DHT is elevated following testosterone treatment using a transdermal cream. Another metabolic product of testosterone is estradiol. Testosterone’s conversion to estradiol via cytochrome P450 aromatase in men, unlike women, helps to maintain or modulate levels of serum estradiol as men age [9, 33, 34]. Thus, in the presence of aromatase, testosterone can exert its effects on cell metabolism by influencing estrogen receptors as well as androgen receptors [35]. Aging has been linked to enhanced peripheral aromatization of androgens as well as increased estradiol levels. Higher estradiol levels promote increased adiposity or increased body fat, as well as an increase in SHBG levels [30]. In our patients, there was a small (non-significant) rise in estradiol levels observed during testosterone treatment (Table 2). The lack of change in estradiol levels may partly explain why there were no significant changes observed in body fat or SHBG levels. Estradiol levels were still in the mid- to lower-normal range, which is 10-82 pg/mL in fertile Asian men [36]. It was clear that testosterone treatment had a significant inhibitory effect on LH levels in plasma (Table 2; Fig. 3c), and that once the participants stopped TRT, LH levels rose back to baseline levels. This indicated no carry-over or long-term effects, and that the normal feedback loops and enzymatic conversions of testosterone were occurring. In men, both testosterone and DHT exert their effects on LH mainly at the hypothalamic level, by decreasing the frequency of LH pulses, whilst estradiol reduces the amplitude of LH at the pituitary level [37]. Among testosterone-deficient men, LH has been shown to be suppressed down to levels in the eugonadal range following injectable and implantable testosterone [38, 39].

4.2. Effect of Testosterone Treatment on Plasma Aβ Levels

Ideally, CSF Aβ40 or Aβ42 levels would be investigated to determine testosterone effects and future risk of AD, as CSF levels of these biomarkers are considered much more useful than plasma Aβ levels when assessing AD risk [40, 41]. However, CSF sampling is not likely to be adopted for routine population screening, mostly due to cost and discomfort to patients, thus we have concentrated on blood screening. In this study, there were no significant changes in plasma Aβ40 or Aβ42 levels following testosterone treatment. Many factors influence the measurement as well as the levels of Aβ40 or Aβ42 levels. For example: most of the Aβ in plasma is bound to triglyceride-rich lipoproteins, soluble low-density lipoprotein receptor-related protein-1 and possibly albumin, leaving little free Aβ [42-44]. Levels of albumin and lipoproteins and the amounts of Aβ bound to

them are influenced by many factors, and thus measured Aβ levels may also be affected by such confounding factors [45]. Plasma Aβ is also rapidly broken down or removed by peripheral tissues, particularly the liver [46]. Recent clinical trials have shown that the diagnostic utility of plasma Aβ levels in relation to risk of AD have been limited due to the lack of correlation between plasma, CSF Aβ, and PET amyloid plaque measurement [47-49]. There are several reasons that can explain these findings. Firstly, Aβ in the plasma and blood can be found not only in the brain, but also in almost all peripheral cells that metabolize APP [50, 51], that travel through the blood-brain barrier penetration which is limited. Thus plasma Aβ might only be partially reflect altered APP metabolism in the brain. Secondly, plasma Aβ was found to be independent to that in the CSF of both dementia and healthy people [47]. Thirdly, like CSF, plasma Aβ has been found to have a circadian pattern that is inversely correlated with age but is unaffected by amyloid deposition [52]. Even though plasma Aβ is not a significant diagnostic AD biomarker, but it can be an important prognostic biomarker. Several studies have demonstrated that plasma levels of Aβ40 and Aβ42, when combined with a panel of other peripheral biomarkers, can help in the assessment of AD risk. However, Aβ changes are not substantial, and not all studies agree. Individual variation may be influenced by the confounding factors mentioned above [45], as well as genetic differences such as APOE allele status, stage of AD neuropathology (if present) and the many health conditions that can afflict the elderly as well as associated medications. Nevertheless, animal and cell culture studies support the concept that lower testosterone levels are associated with higher Aβ levels [13, 14]. Furthermore, in clinical studies, low levels of testosterone due to androgen blockade therapy (flutamide and leuprolide) have been associated with increased plasma Aβ levels in men [16], and serum total testosterone and SHBG have been found to correlate inversely with plasma Aβ40 levels in older men with memory loss or dementia [53]. The authors of these studies suggested that subclinical androgen deficiency enhances the expression of AD-related peptides. One study which did not find an inverse relationship between testosterone levels and Aβ levels is a study which found that testosterone treatment raised plasma Aβ40 levels, in an animal model of hypogonadism [13]. Studies have also not been conclusive concerning the relationship between pre-MCI plasma Aβ and the risk of AD or dementia. For example, individuals with plasma Aβ40 levels above the median baseline level for the group in one particular study had an increased risk of developing dementia [54]; however, another study found low plasma Aβ40 levels can predict incident AD in elderly men [55]. Yet another study found that plasma Aβ40 and Aβ42 levels increase with age and are strongly correlated with each other, and that plasma Aβ40 and Aβ42 levels are elevated in some patients before and during the early stages of AD but decline thereafter [56]. A recent review has found that low plasma Aβ42:Aβ40 ratios are the best predictors of AD and dementia development [57]. Interestingly, an increase in plasma Aβ oligomers, known to be important in AD pathogenesis, has been associated with decreased MMSE

8 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 4 Asih et al.

and AMT (Abbreviated Mental Test) scores [58]. In our study however, plasma Aβ42:Aβ40 ratios did not change though it should be recognized that a longer follow up on these subjects would be needed to reach a definitive conclusion. The lack of conclusion in all these studies may be partly attributable to small cohort sizes, as well as differences between the studies, for example in the ethnic origins of the populations studied, the ages of study participants, the follow-up periods used, plasma Aβ assays, and the cognitive and physical health status of participants at the start of the studies [59]. The standardization of procedures will be essential to improve study comparisons [59, 60]. Plasma Aβ peptides or oligomer levels may eventually be part of a blood biomarker panel used to determine AD risk or AD status [40].

4.3. Lipid Profiles and Hematology

Studies of the effects of testosterone on lipid metabolism have produced conflicting results. As in this study, some have found no significant effects on LDL levels, testosterone has also been found to result in a mild decrease in HDL levels [61-63]. However, some have demonstrated an inverse correlation between testosterone and LDL levels [64-67]. The significance of a small HDL level decrease is still unknown. It is known, however, that serum HDL levels are lower in men than in premenopausal women [68], and a key enzyme responsible for HDL clearance, hepatic endothelial triglyceride lipase, has been found to have greater activity in men, and to be stimulated by androgens and suppressed by estrogens [69]. Overall, the effects of testosterone on lipid profiles remain uncertain. Testosterone has long been known to stimulate red blood stem cell numbers [70]. Several TRT studies have reported significant increases in hemoglobin and hematocrit levels [29, 71-73], with greater increases in RBC in older men than in young men [71]. Our results also show that TRT enhances RBC and hemoglobin levels. However, it has been found that testosterone-treated men are nearly four times as likely to have hematocrits of >50% compared to placebo-treated men [74]. Therefore, TRT should not be administered to men with baseline RBC or hematocrits of 50% or greater without appropriate evaluation and treatment of erythrocytosis [75], since TRT may worsen pre-existing erythrocytosis. Although it had been postulated that testosterone stimulates erythropoiesis by modulating erythropoietin and stem cell proliferation, recent data suggests testosterone increases red cell mass by inhibiting hepcidin [71]. The normal hemoglobin range in men is 14-18 ng/dL [75], and although significant increases in hemoglobin were observed following TRT, the levels seen in our results were still in the safe healthy range.

4.4. PSA, Cardiovascular Disease, and Gynecomastia as Potential Risks of TRT

In the past, there have been major concerns regarding testosterone therapy promoting the growth of metastatic prostate cancer. However, it has now been established that there is no evidence for an increased risk of prostate cancer [24, 76]. The view now is that testosterone (at physiological

levels) does not promote prostate cancer, but can exacerbate existing cancer [77, 78] and should not be administered to men with already high PSA levels [75]. In fact, one study has found that an increased risk of prostate cancer was associated with low plasma testosterone [20]. Most studies have shown no significant increases in PSA or prostate volume following TRT in testosterone-deficient men [79]. Although PSA levels did increase following TRT in our study, levels still remained within the normal range of 0-4 µg/L (American Cancer Society). Similarly, a study by Khera et al. [80] showed a small increase in PSA levels following 12 months of testosterone treatment. According to the American Cancer Society, a PSA level of 4 - 10 µg/L indicates a 25% increased risk of prostate cancer, and a level over 10 µg/L increases the risk by 50% or more. A number of open-label trials have also reported that testosterone treatment results in very low rates of prostate cancer [81, 82]. Cross-sectional studies indicate an inverse relationship between cardiovascular disease (CVD) incidence and/or severity, and endogenous testosterone levels, irrespective of age, when considering men with low testosterone levels [83]. For example, in hypogonadal men, testosterone treatment increases coronary blood flow and reduces signs of myocardial ischemia in CVD [21, 84, 85]. However, testosterone treatment has been contra-indicated in some studies of people with hypertension, diabetes and hyperlipidemia, thus maintenance of testosterone levels within a normal range is recommended, and the monitoring of CVD indicators is advisable [84]. To this end, total cholesterol:HDL ratios and LDL:HDL ratios were monitored in our study. The total cholesterol:HDL ratio should be kept below 5:1, ideally below 3.5:1. In our study, no significant changes in total cholesterol/HDL ratios were found, and the ratios remained below 5:1. Also, no significant changes to HDL levels or LDL/HDL ratios were detected, which suggests that the testosterone treatment in this study did not increase CVD risk. An increased production or action of estradiol compared to testosterone will result in gynecomastia: benign enlargement of the male breast; a side-effect occasionally associated with TRT. In our studies, the ratio of testosterone to estradiol was calculated to monitor the risk of gynecomastia. No significant changes were observed, reflecting the fact that testosterone increases were greater than estradiol increases, and implying that gynecomastia risk was minimal. Studies with larger numbers of participants are needed to get a clearer picture of any differences in all of the blood parameters measured, particularly plasma Aβ40 and lipid profiles. Ideally, CSF Aβ40 and Aβ42 levels should be measured, as these reflect AD neuropathology more directly. However, sampling CSF is uncomfortable and costly. If plasma Aβ40 or Aβ42 levels are found to correlate with AD risk, then plasma would clearly be preferred for screening. Another possible problem is the reliance on daily TRT self-administration, some men may have forgotten to apply the cream daily, and/or the dosage may have been incorrect. However the blood testosterone levels suggest strongly that these problems did not occur.

Testosterone Replacement Therapy in Older Men CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 4 9

Truly testosterone-deficient men who have no contraindications to TRT may benefit more from such treatment. Lower testosterone levels are associated with increased risks of osteoporosis, metabolic syndrome, type 2 diabetes mellitus and mortality. Nevertheless, it is still uncertain whether a reduced testosterone level causes ill-health or whether it is a marker of pre-existing condition or disease, as some illnesses can lower testosterone levels.

CONCLUSION

This study has shown that 6 months of transdermal TRT in Indonesian elderly men with SMC is well-tolerated, and results in a significant increase in testosterone and its metabolic products, which in turn suppress LH levels. The TRT-induced trend towards decreasing plasma Aβ40 levels warrants investigation using a larger cohort, to establish more conclusively the benefits of TRT in elderly men with SMC with low testosterone level.

TRIAL REGISTRATION

The Australian New Zealand Clinical Trials Registry (ANZCTR). Trial ID ACTRN12614000277640

LIST OF ABBREVIATIONS

Aβ = Beta Amyloid AD = Alzheimer’s Disease CFT = Calculated Free Testosterone DHT = Dihydrotestosterone HDL = High Density Lipoprotein LDL = Low Density Lipoprotein LH = Luteinizing Hormone MCI = Mild Cognitive Impairment P = Placebo PSA = Prostate Specific Antigen RBC = Red Blood Cells SMC = Subjective Memory Complainers SHBG = Sex Hormone Binding Globulin TRT = Testosterone Replacement Therapy T = Testosterone

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

We acknowledge the McCusker Alzheimer’s Disease Research Foundation, Perth Western Australia and Siloam Hospitals Lippo Village, Tangerang Indonesia for generously funding this study. We are indebted to the Indonesian participants and research volunteers for making this study possible.

REFERENCES

[1] Morley JE, Kaiser FE, Perry HM, 3rd, et al. Longitudinal changes in testosterone, luteinizing hormone, and follicle-stimulating hormone in healthy older men. Metabol Clin Exp 1997; 46(4): 410-3.

[2] Harman SM, Metter EJ, Tobin JD, Pearson J, Blackman MR. Baltimore Longitudinal Study of A. Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metabol 2001; 86(2): 724-31.

[3] Feldman HA, Longcope C, Derby CA, et al. Age trends in the level of serum testosterone and other hormones in middle-aged men: longitudinal results from the Massachusetts male aging study. J Clin Endocrinol Metabol 2002; 87(2): 589-98.

[4] Rosario ER, Chang L, Stanczyk FZ, Pike CJ. Age-related testosterone depletion and the development of Alzheimer disease. JAMA 2004; 292(12): 1431-2.

[5] Kenny AM, Prestwood KM, Gruman CA, Marcello KM, Raisz LG. Effects of transdermal testosterone on bone and muscle in older men with low bioavailable testosterone levels. J Gerontol Ser A Biol Sci Med Sci 2001; 56(5): M266-72.

[6] Moffat SD, Zonderman AB, Metter EJ, Blackman MR, Harman SM, Resnick SM. Longitudinal assessment of serum free testosterone concentration predicts memory performance and cognitive status in elderly men. J Clin Endocrinol Metabol 2002; 87(11): 5001-7.

[7] Schroeder ET, Zheng L, Ong MD, et al. Effects of androgen therapy on adipose tissue and metabolism in older men. J Clin Endocrinol Metabol 2004; 89(10): 4863-72.

[8] Wang C, Alexander G, Berman N, et al. Testosterone replacement therapy improves mood in hypogonadal men--a clinical research center study. J Clin Endocrinol Metabol 1996; 81(10): 3578-83.

[9] Gray A, Feldman HA, McKinlay JB, Longcope C. Age, disease, and changing sex hormone levels in middle-aged men: results of the Massachusetts Male Aging Study. J Clin Endocrinol Metabol 1991; 73(5): 1016-25.

[10] Morley JE. Andropause, testosterone therapy, and quality of life in aging men. Cleveland Clin J Med 2000; 67(12): 880-2.

[11] Araujo AB, Esche GR, Kupelian V, et al. Prevalence of symptomatic androgen deficiency in men. J Clin Endocrinol Metabol 2007; 92(11): 4241-7.

[12] Leifke E, Gorenoi V, Wichers C, Von Zur Muhlen A, Von Buren E, Brabant G. Age-related changes of serum sex hormones, insulin-like growth factor-1 and sex-hormone binding globulin levels in men: cross-sectional data from a healthy male cohort. Clin Endocrinol 2000; 53(6): 689-95.

[13] Wahjoepramono EJ, Wijaya LK, Taddei K, et al. Distinct effects of testosterone on plasma and cerebrospinal fluid amyloid-beta levels. J Alzheimers Dis 2008; 15(1): 129-37.

[14] Gouras GK, Xu H, Gross RS, et al. Testosterone reduces neuronal secretion of Alzheimer's beta-amyloid peptides. Proc Natl Acad Sci USA 2000; 97(3): 1202-5.

[15] Gandy S, Almeida OP, Fonte J, et al. Chemical andropause and amyloid-beta peptide. JAMA 2001; 285(17): 2195-6.

[16] Almeida OP, Waterreus A, Spry N, Flicker L, Martins RN. One year follow-up study of the association between chemical castration, sex hormones, beta-amyloid, memory and depression in men. Psychoneuroendocrinology 2004; 29(8): 1071-81.

[17] Tan RS, Pu SJ, Culberson JW. Role of androgens in mild cognitive impairment and possible interventions during andropause. Med Hypotheses 2003; 60(3): 448-52.

[18] Cherrier MM, Matsumoto AM, Amory JK, et al. Testosterone improves spatial memory in men with Alzheimer disease and mild cognitive impairment. Neurology 2005; 64(12): 2063-8.

[19] Traish AM, Miner MM, Morgentaler A, Zitzmann M. Testosterone deficiency. Am J Med 2011; 124(7): 578-87.

[20] Raynaud JP. Prostate cancer risk in testosterone-treated men. J Steroid Biochem Mol Biol 2006; 102(1-5): 261-6.

[21] English KM, Steeds RP, Jones TH, Diver MJ, Channer KS. Low-dose transdermal testosterone therapy improves angina threshold in men with chronic stable angina: A randomized, double-blind, placebo-controlled study. Circulation 2000; 102(16): 1906-11.

[22] Rhoden EL, Morgentaler A. Treatment of testosterone-induced gynecomastia with the aromatase inhibitor, anastrozole. Int J Impot Res 2004; 16(1): 95-7.

10 CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 4 Asih et al.

[23] Rinnab L, Gust K, Hautmann RE, Kufer R. Testosterone replacement therapy and prostate cancer. The current position 67 years after the Huggins myth. Der Urol Ausg A 2009; 48(5): 516-22.

[24] Feneley MR, Carruthers M. Is testosterone treatment good for the prostate? Study of safety during long-term treatment. J Sex Med 2012; 9(8): 2138-49.

[25] Aaronson AJ, Morrissey RP, Nguyen CT, Willix R, Schwarz ER. Update on the safety of testosterone therapy in cardiac disease. Expert Opin Drug Safety 2011; 10(5): 697-704.

[26] Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metabol 1999; 84(10): 3666-72.

[27] Mehta PD, Pirttila T, Mehta SP, Sersen EA, Aisen PS, Wisniewski HM. Plasma and cerebrospinal fluid levels of amyloid beta proteins 1-40 and 1-42 in Alzheimer disease. Arch Neurol 2000; 57(1): 100-5.

[28] Bates KA, Harvey AR, Carruthers M, Martins RN. Androgens, andropause and neurodegeneration: exploring the link between steroidogenesis, androgens and Alzheimer's disease. Cell Mol Life Sci 2005; 62(3): 281-92.

[29] Bachman E, Feng R, Travison T, et al. Testosterone suppresses hepcidin in men: a potential mechanism for testosterone-induced erythrocytosis. J Clin Endocrinol Metabol 2010; 95(10): 4743-7.

[30] Basaria S, Dobs AS. Hypogonadism and androgen replacement therapy in elderly men. Am J Med 2001; 110(7): 563-72.

[31] Cunningham GR, Cordero E, Thornby JI. Testosterone replacement with transdermal therapeutic systems. Physiological serum testosterone and elevated dihydrotestosterone levels. JAMA 1989; 261(17): 2525-30.

[32] Gooren LJ, Bunck MC. Androgen replacement therapy: present and future. Drugs 2004; 64(17): 1861-91.

[33] Burger HG, Dudley EC, Cui J, Dennerstein L, Hopper JL. A prospective longitudinal study of serum testosterone, dehydroepiandrosterone sulfate, and sex hormone-binding globulin levels through the menopause transition. J Clin Endocrinol Metabol 2000; 85(8): 2832-8.

[34] Cherrier MM, Matsumoto AM, Amory JK, et al. The role of aromatization in testosterone supplementation: effects on cognition in older men. Neurology 2005; 64(2): 290-6.

[35] Oettel M, Hubler D, Patchev V. Selected aspects of endocrine pharmacology of the aging male. Exp Gerontol 2003; 38(1-2): 189-98.

[36] Yamamoto M, Hibi H, Katsuno S, Miyake K. Serum estradiol levels in normal men and men with idiopathic infertility. Int J Urol 1995; 2(1): 44-6.

[37] Carruthers M. Testosterone deficiency syndrome: cellular and molecular mechanism of action. Curr Aging Sci 2013; 6(1): 115-24.

[38] Conway AJ, Boylan LM, Howe C, Ross G, Handelsman DJ. Randomized clinical trial of testosterone replacement therapy in hypogonadal men. Int J Androl 1988; 11(4): 247-64.

[39] Fennell C, Sartorius G, Ly LP, et al. Randomized cross-over clinical trial of injectable vs implantable depot testosterone for maintenance of testosterone replacement therapy in androgen deficient men. Clin Endocrinol 2010; 73(1): 102-9.

[40] Lista S, Faltraco F, Prvulovic D, Hampel H. Blood and plasma-based proteomic biomarker research in Alzheimer's disease. Progr Neurobiol 2013; 101-102: 1-17.

[41] Henriksen K, O'Bryant SE, Hampel H, et al. The future of blood-based biomarkers for Alzheimer's disease. Alzheimers Dement 2014; 10(1): 115-31.

[42] Biere AL, Ostaszewski B, Stimson ER, Hyman BT, Maggio JE, Selkoe DJ. Amyloid beta-peptide is transported on lipoproteins and albumin in human plasma. J Biol Chem 1996; 271(51): 32916-22.

[43] Mamo JC, Jian L, James AP, Flicker L, Esselmann H, Wiltfang J. Plasma lipoprotein beta-amyloid in subjects with Alzheimer's disease or mild cognitive impairment. Ann Clin Biochem 2008; 45(Pt 4): 395-403.

[44] Sagare A, Deane R, Bell RD, et al. Clearance of amyloid-beta by circulating lipoprotein receptors. Nat Med 2007; 13(9): 1029-31.

[45] Prvulovic D, Hampel H. Amyloid beta (Abeta) and phospho-tau (p-tau) as diagnostic biomarkers in Alzheimer's disease. Clin Chem Lab Med 2011; 49(3): 367-74.

[46] Hone E, Martins IJ, Fonte J, Martins RN. Apolipoprotein E influences amyloid-beta clearance from the murine periphery. J Alzheimers Dis 2003; 5(1): 1-8.

[47] Le Bastard N, Aerts L, Leurs J, Blomme W, De Deyn PP, Engelborghs S. No correlation between time-linked plasma and CSF Abeta levels. Neurochem Int 2009; 55(8): 820-5.

[48] Toledo JB, Shaw LM, Trojanowski JQ. Plasma amyloid beta measurements - a desired but elusive Alzheimer's disease biomarker. Alzheimers Res Ther 2013; 5(2): 8.

[49] Hansson O, Zetterberg H, Vanmechelen E, et al. Evaluation of plasma Abeta(40) and Abeta(42) as predictors of conversion to Alzheimer's disease in patients with mild cognitive impairment. Neurobiol Aging 2010; 31(3): 357-67.

[50] Roher AE, Esh CL, Kokjohn TA, et al. Amyloid beta peptides in human plasma and tissues and their significance for Alzheimer's disease. Alzheimers Dement 2009; 5(1): 18-29.

[51] Selkoe DJ, Podlisny MB, Joachim CL, et al. Beta-amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues. Proc Natl Acad Sci USA 1988; 85(19): 7341-5.

[52] Huang Y, Potter R, Sigurdson W, et al. beta-amyloid dynamics in human plasma. Arch Neurol 2012; 69(12): 1591-7.

[53] Gillett MJ, Martins RN, Clarnette RM, Chubb SA, Bruce DG, Yeap BB. Relationship between testosterone, sex hormone binding globulin and plasma amyloid beta peptide 40 in older men with subjective memory loss or dementia. J Alzheimers Dis 2003; 5(4): 267-9.

[54] Hansson O, Stomrud E, Vanmechelen E, et al. Evaluation of plasma Abeta as predictor of Alzheimer's disease in older individuals without dementia: a population-based study. J Alzheimers Dis 2012; 28(1): 231-8.

[55] Sundelof J, Giedraitis V, Irizarry MC, et al. Plasma beta amyloid and the risk of Alzheimer disease and dementia in elderly men: a prospective, population-based cohort study. Arch Neurol 2008; 65(2): 256-63.

[56] Mayeux R, Honig LS, Tang MX, et al. Plasma A[beta]40 and A[beta]42 and Alzheimer's disease: relation to age, mortality, and risk. Neurology 2003; 61(9): 1185-90.

[57] Koyama A, Okereke OI, Yang T, Blacker D, Selkoe DJ, Grodstein F. Plasma amyloid-beta as a predictor of dementia and cognitive decline: a systematic review and meta-analysis. Arch Neurol 2012; 69(7): 824-31.

[58] Zhou L, Chan KH, Chu LW, et al. Plasma amyloid-beta oligomers level is a biomarker for Alzheimer's disease diagnosis. Biochem Biophys Res Commun 2012; 423(4): 697-702.

[59] Gupta VB, Sundaram R, Martins RN. Multiplex biomarkers in blood. Alzheimers Res Ther 2013; 5(3): 31.

[60] Rosen C, Hansson O, Blennow K, Zetterberg H. Fluid biomarkers in Alzheimer's disease - current concepts. Molecular neurodegeneration. 2013; 8: 20.

[61] Bhasin S, Storer TW, Berman N, et al. Testosterone replacement increases fat-free mass and muscle size in hypogonadal men. J Clin Endocrinol Metabol 1997; 82(2): 407-13.

[62] Friedl KE, Hannan CJ, Jr., Jones RE, Plymate SR. High-density lipoprotein cholesterol is not decreased if an aromatizable androgen is administered. Metabol Clin Exp 1990; 39(1): 69-74.

[63] Thompson PD, Cullinane EM, Sady SP, et al. Contrasting effects of testosterone and stanozolol on serum lipoprotein levels. JAMA 1989; 261(8): 1165-8.

[64] Howell SJ, Radford JA, Adams JE, Smets EM, Warburton R, Shalet SM. Randomized placebo-controlled trial of testosterone replacement in men with mild Leydig cell insufficiency following cytotoxic chemotherapy. Clin Endocrinol 2001; 55(3): 315-24.

[65] Ly LP, Jimenez M, Zhuang TN, Celermajer DS, Conway AJ, Handelsman DJ. A double-blind, placebo-controlled, randomized clinical trial of transdermal dihydrotestosterone gel on muscular strength, mobility, and quality of life in older men with partial androgen deficiency. J Clin Endocrinol Metabol 2001; 86(9): 4078-88.

[66] Saad F, Gooren L, Haider A, Yassin A. An exploratory study of the effects of 12 month administration of the novel long-acting testosterone undecanoate on measures of sexual function and the metabolic syndrome. Arch Androl 2007; 53(6): 353-7.

[67] Saad F, Gooren LJ, Haider A, Yassin A. A dose-response study of testosterone on sexual dysfunction and features of the metabolic

Testosterone Replacement Therapy in Older Men CNS & Neurological Disorders - Drug Targets, 2015, Vol. 14, No. 4 11 syndrome using testosterone gel and parenteral testosterone undecanoate. J Androl 2008; 29(1): 102-5.

[68] Heiss G, Johnson NJ, Reiland S, Davis CE, Tyroler HA. The epidemiology of plasma high-density lipoprotein cholesterol levels. The Lipid Research Clinics Program Prevalence Study. Summary. Circulation 1980; 62(4 Pt 2): IV116-36.

[69] Glueck CJ, Gartside P, Fallat RW, Mendoza S. Effect of sex hormones on protamine inactivated and resistant postheparin plasma lipases. Metabol Clin Exp 1976; 25(6): 625-32.

[70] Shahidi NT. Androgens and erythropoiesis. N Engl J Med 1973; 289(2): 72-80.

[71] Coviello AD, Kaplan B, Lakshman KM, Chen T, Singh AB, Bhasin S. Effects of graded doses of testosterone on erythropoiesis in healthy young and older men. J Clin Endocrinol Metabol 2008; 93(3): 914-9.

[72] Hajjar RR, Kaiser FE, Morley JE. Outcomes of long-term testosterone replacement in older hypogonadal males: a retrospective analysis. J Clin Endocrinol Metabol 1997; 82(11): 3793-6.

[73] Sih R, Morley JE, Kaiser FE, Perry HM, 3rd, Patrick P, Ross C. Testosterone replacement in older hypogonadal men: a 12-month randomized controlled trial. J Clin Endocrinol Metabol 1997; 82(6): 1661-7.

[74] Calof OM, Singh AB, Lee ML, et al. Adverse events associated with testosterone replacement in middle-aged and older men: a meta-analysis of randomized, placebo-controlled trials. J Gerontol Ser A Biol Sci Med Sci 2005; 60(11): 1451-7.

[75] Bhasin S, Cunningham GR, Hayes FJ, et al. Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metabol 2010; 95(6): 2536-59.

[76] Jackson G. Late onset hypogonadism in males - think of it - act on it. Int J Clin Pract 2012; 66(2): 115-6.

[77] Bhasin S, Singh AB, Mac RP, Carter B, Lee MI, Cunningham GR. Managing the risks of prostate disease during testosterone replacement therapy in older men: recommendations for a standardized monitoring plan. J Androl 2003; 24(3): 299-311.

[78] Fowler JE, Jr., Whitmore WF, Jr. The response of metastatic adenocarcinoma of the prostate to exogenous testosterone. J Urol 1981; 126(3): 372-5.

[79] Behre HM, Bohmeyer J, Nieschlag E. Prostate volume in testosterone-treated and untreated hypogonadal men in comparison to age-matched normal controls. Clin Endocrinol 1994; 40(3): 341-9.

[80] Khera M, Bhattacharya RK, Blick G, Kushner H, Nguyen D, Miner MM. Changes in prostate specific antigen in hypogonadal men after 12 months of testosterone replacement therapy: support for the prostate saturation theory. J Urol 2011; 186(3): 1005-11.

[81] Agarwal PK, Oefelein MG. Testosterone replacement therapy after primary treatment for prostate cancer. J Urol 2005; 173(2): 533-6.

[82] Kaufman JM, Graydon RJ. Androgen replacement after curative radical prostatectomy for prostate cancer in hypogonadal men. J Urol 2004; 172(3): 920-2.

[83] Wu FC, von Eckardstein A. Androgens and coronary artery disease. Endocr Rev 2003; 24(2): 183-217.

[84] Xu L, Freeman G, Cowling BJ, Schooling CM. Testosterone therapy and cardiovascular events among men: a systematic review and meta-analysis of placebo-controlled randomized trials. BMC Med 2013; 11: 108.

[85] Webb CM, McNeill JG, Hayward CS, de Zeigler D, Collins P. Effects of testosterone on coronary vasomotor regulation in men with coronary heart disease. Circulation 1999; 100(16): 1690-6.

Received: August 11, 2014 Revised: January 9, 2015 Accepted: January 20, 2015

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