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Transdermal Testosterone Application: Pharmacokinetics and Effects on Pubertal Status, Short-Term Growth, and Bone Turnover

Department of Paediatrics (A.M.), Royal Aberdeen Children’s Hospital, Aberdeen AB25 2ZG, Scotland, United Kingdom; Department of Clinical Biochemistry (H.M., A.M.W.), Glasgow Royal Infirmary, Glasgow G4 0SF, Scotland, United Kingdom; and Bone and Endocrine Research Group, Department of Child Health (S.F.A.), Royal Hospital for Sick Children, Glasgow G3 8SJ, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Dr. S. F. Ahmed, Department of Child Health, Royal Hospital for Sick Children, Yorkhill, Glasgow G3 8SJ, Scotland, United Kingdom. E-mail: gcl328{at}clinmed.gla.ac.uk.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
The aim of the study was to assess the effect of transdermal testosterone on free testosterone concentrations in saliva and on short-term growth and bone turnover in boys with growth or pubertal delay.

A prospective, randomized, crossover study was conducted over 26 wk with 4 wk of run-in, 8 wk of treatment I (8 or 12 h), 4 wk of washout, 8 wk of treatment II (8 or 12 h), and 4 wk of final washout.

The main outcome measures were salivary testosterone profiles during the different study periods; weekly change in lower leg length (LLL) as measured by knemometry, i.e. LLL velocity; absolute and percentage change in bone alkaline phosphatase (bALP) levels; and deoxypyridinoline cross-links measured in urine.

Eight boys who took part in the study had a median age of 13.5 yr (range, 12.4–14.9 yr), testicular volume of 3 ml (range, 2–6 ml), height SD score of -2.4 (range, -1.44 to -3.35), and bone age delay of 2 yr (range, 1–3.2 yr).

Median salivary testosterone during 8- and 12-h treatments [179 pg/ml (range, 7–3579 pg/ml) and 150 pg/ml (range, 12–3472 pg/ml) (not significant)] was significantly higher than during the run-in and washout blocks (P < 0.0001) [9 pg/ml (range, <7 to 122 pg/ml) and 13 pg/ml (range, <7 to 285 pg/ml) (not significant)]. LLL velocity in the treatment blocks (median, 0.64 mm/wk; range, 0.1–1.08 mm/wk) was significantly higher than during the run-in and washout periods (median, 0.48 mm/wk; range, -0.06 to 0.92 mm/wk) (P < 0.001). The main rise in bALP occurred during the first treatment block with a median percentage change in bALP of 44.2% (range, -4 to 87%) and a smaller percentage change in bALP at the end of the second treatment block of 9.8% (range, -4 to 55%). The increases in bALP were not significantly different between the 8- and 12-h treatment periods, and there was no significant decline during the washout periods.

Overnight transdermal testosterone application, as Virormone (5 mg), may be a potentially acceptable method of induction of puberty and stimulates short-term growth and bone turnover.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
DELAYED GROWTH AND puberty are the commonest reasons for boys presenting to an endocrine clinic. They often cause great distress and can be successfully treated with androgenic steroids that induce a spurt of growth and development until endogenous puberty advances sufficiently for the growth spurt to be sustained without supplementation (1). Lifelong androgen replacement is also required in testicular failure that may be primary or secondary to pathological gonadotropin insufficiency.

Monthly im preparations of testosterone esters, such as Sustanon, are the most commonly used regimen for treatment. The dose regimen usually ranges between 50 and 100 mg monthly for 3–12 months, but even with low doses, supraphysiological levels of testosterone are unavoidable in the first 48–72 h after injection, followed by gradual waning of effect over the next 3–4 wk (2). The repeated im injections are painful and disliked by many young adolescents. Daily oral testosterone undecanoate (TU) is claimed to be an effective and well-accepted treatment for delayed puberty but produces a different drug profile to the depot preparation and is not used as commonly as the injections (3). There is at present no consensus regarding the optimal regimen for testosterone replacement therapy in children or adults. This issue is more important now, because new forms of androgen replacement, such as transdermal testosterone (TT) delivery as adhesive patches, are introduced in children as well as adults (4). TT has obvious advantages as a mode of testosterone delivery and has been shown to increase short-term growth in children with end-stage renal disease (5). There is a lack of information about the pharmacokinetics of testosterone preparations as well as comparison of the biological effects of different regimens used in boys with hypogonadism. Butler et al. (3) have shown variable serum testosterone concentrations in boys given TU, but these studies are difficult to perform. By using knemometry to look at short-term growth and markers of bone turnover, we have previously compared the effects of TU and im depot testosterone esters (6). Salivary testosterone (SalT) measurements provide a noninvasive method of assessing the free, biologically active fraction of circulating testosterone (7). In this study, we have used SalT measurements in the investigation of the pharmacokinetics of TT and related results to the effects on short-term growth and bone turnover.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

Boys requiring testosterone therapy for pubertal or growth delay were recruited from an endocrine clinic. Over the recruiting period, 12 boys were eligible to enter the study. Two of 12 decided against treatment, and two did not wish to take part due to the number of visits involved in the study. Exclusion criteria included the use of any other drugs known to affect growth. Recruitment was halted after the study drug was withdrawn from the United Kingdom for commercial reasons. The eight boys who took part in the study had a median age of 13.5 yr (range, 12.4–14.9 yr), testicular volume of 3 ml (range, 2–6 ml), height SD score of -2.4 (range, -1.44 to -3.35), and bone age (BA) delay of 2 yr (range, 1–3.2 yr). One of them suffered from type 1 diabetes mellitus, and another had cystic fibrosis. Routine biochemistry, including liver function tests and gonadotropin levels, were measured at the beginning and end of the study.

The study was approved by the local research ethics committee, and informed consent was obtained from all children and their parents. All of the children completed the study.

Design

The study was a prospective, randomized, crossover, open trial over 26 wk with 4 wk of run-in, 8 wk of treatment I (8 or 12 h), 4 wk of washout, 8 wk of treatment II (8 or 12 h), and 4 wk of final washout (Fig. 1). During the treatment blocks, TT [Virormone (5 mg); Ferring Pharmaceuticals, Copenhagen, Denmark] was applied over 8 (starting at 2400 h) or 12 h (starting at 2000 h) overnight to the skin of the back or buttocks.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Flowchart of study. Four weeks of run-in (Run In) followed by a randomization step into the first 8-wk treatment block, receiving 5 mg Virormone either 8 h nightly or 12 h nightly. The first treatment block is followed by another 4 wk of no-treatment (Wash Out I) and another 8 wk of the other testosterone timing. Finally, there is another 4-wk period of observation (Wash Out II). Knemometry () was performed every 4 wk throughout the study, and blood samples were collected at the beginning of each treatment and no-treatment block (*).

Twenty-four-hour salivary profiles were collected at the beginning and end of the run-in period and at the end of the two treatment blocks and the two washouts. Samples were collected at 2000 h on application of TT, and at 2400, 0800, 1200, and 1600 h. The boys were given instructions to rinse their mouth with water 10 min before collecting the samples and not to eat or brush their teeth in the preceding hour. Samples were stored at home in the freezer until the next visit and then stored in the lab at -20 C. Before analysis, samples were thawed and centrifuged to precipitate mucins, and the clear supernatant was used for measurement.

SalT was measured by RIA after solvent (diethyl ether) extraction. The assay uses an antibody raised in sheep against testosterone-3-carboxymethyloxime, which binds testosterone with high affinity (code no. 505) (8). The antibody is specific with the only major cross-reactant being dihydrotestosterone (12%). The final antibody titer is 1:700,000. An iodinated testosterone label is used, prepared from testosterone-3-carboxymethyloxime-histamine by the chloramine-T method. Separation of antibody bound from free testosterone is achieved by binding to donkey antisheep globulin chemically linked to a magnetic solid phase. All assay reagents are prepared in-house. The assay has a sensitivity of 7 pg/ml, and between-batch coefficients of variation over the working range of the assay is less than 15%.

Anthropometric measurements

Lower leg length (LLL) was measured at each of eight visits at 4-wk intervals with a knemometer. The random zero method (9) was used during the measurements. In this method, the baseline reading is changed before each measurement, and each estimation is subsequently calculated by subtracting the actual recording from the allocated baseline value. On each occasion, four readings were obtained, the most deviant was discarded, and the median of the remaining three was taken as the true reading.

All of the measurements were carried out by the same operator (AM) in the afternoon. The median technical error (1 SD from the mean of a set of triplicate measurements) was 0.17 mm. Measurements were available from seven of the boys, because one of them was unable to bend his leg sufficiently. Height was measured with a Holtain stadiometer (Holtain, Crymych, Dyfed, UK), and pubertal status was assessed by the same observer at the start and end of each treatment block. BA (radius-ulna-short bones) assessment at the start and end of the study was performed by the TW2 method (10).

Bone markers

Blood and urine samples were collected at the start and end of each block at approximately the same time in the afternoon. All samples were analyzed in duplicate, and samples from each patient were analyzed in a single run to minimize analytical variation. Bone alkaline phosphatase (bALP) was measured in plasma by ELISA (Alkphase-B; Metra Biosystems, Inc., Mountain View, CA). The sensitivity of the assay was 0.7 U/liter, and within-run and between-runs coefficients of variation were less than 5% and less than 8%, respectively. Deoxypyridinoline cross-links (DPD) were measured in urine by ELISA (Pyrilink-D; Metra Biosystems). Assay sensitivity was 1.1 nmol/liter, and within-run and between-runs coefficients of variation were less than 6% and less than 11%, respectively. The results were expressed in relation to creatinine measured on the same urine sample as nanomoles of DPD per millimoles of creatin.

Statistical analyses

For the knemometry data, LLL velocity (LLLV) was calculated for each time point by subtracting the LLL at that time point from that measured at the previous time point, and dividing by the time interval (in weeks) between the two measurements. LLLV was expressed as millimeters per week. bALP was expressed as absolute values as well as percentage change in bALP (%bALP). The data were expressed as medians and ranges and analyzed using nonparametric tests. Comparison between groups was performed using the Mann-Whitney U test. Data were analyzed using SPSS software, version 9.0.0 (SPSS, Inc., Chicago, IL), and Microsoft Excel 97 SR-2 (Microsoft Corporation, Redmond, WA).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
User acceptability

Children in this study did not report any significant side effects from the use of the transdermal preparation. Skin irritation was minimal and did not require any active treatment when it occurred. Occasionally, the patch would become detached at night from sweating, but this was usually overcome by placing the patch on the buttocks under the underwear. No abnormalities in the liver function tests were detected at the beginning and end of the study (detailed results not shown).

Anthropometric parameters

Clinical and anthropometric details of the patients at the beginning and end of the study can be seen in Table 1. There was evidence of physical and biochemical pubertal advance over the period of the study in the boys (Table 1). In addition, there was a reduction in BA delay from a median of 2.0 to 1.75 yr [not significant (NS)].

SalT profiles during the run-In and washout reflect endogenous production of testosterone, and there was evidence of circadian variation (Fig. 2). Testosterone peaked in the early hours of the morning with a median value at 0800 h of 84 pmol/liter (range, <25 to 721). SalT levels were detectable only at this time of the day in most children with Tanner stage 1. The levels declined throughout the day with lowest levels found at 2400 h (median, 25 pmol/liter; range, <25 to 898 pmol/liter). Higher SalT levels were noted at the end of the study as puberty advanced, but this did not achieve statistical significance.

View larger version (32K): [in this window] [in a new window]   FIG. 2. Saliva testosterone profiles in all boys performed at the beginning and end of the run-in period (Run In 1 and Run In 2), at the end of the first treatment period (Treatment 1), at the end of the first washout period (Wash-Out 1), at the end of the second treatment period (Treatment 2), and at the end of the second washout period (Wash-Out 2). The SalT concentrations are expressed as log10SalT (picomoles per liter) (to convert SalT picomoles per liter to picograms per milliliter, multiply by 0.288). A represents the boys who were Tanner stage G1 at presentation, and B represents the boys who were already in early puberty at presentation.

Pooled median SalT during run-In and washout periods was similar at 9 pg/ml (range, <7 to 122 pg/ml) and 13 pg/ml (range, <7 to 285 pg/ml), respectively. Pooled median SalT during 8- and 12-h treatment was not statistically different at 179 pg/ml (range, 7–3579 pg/ml) and 150 pg/ml (range, <7 to 3473 pg/ml), respectively. SalT concentration was significantly higher during the treatment periods than during the run-in and washouts (P < 0.0001). During the 8-h treatment block, median SalT during the 8-h application and the 16-h application-free periods were 93 pg/ml (range, 40–3579 pg/ml) and 145 pg/ml (range, 7–1518 pg/ml), respectively (NS). During the 12-h treatment block, median SalT during the 12-h application and the 12-h application-free periods were 356 pg/ml (range, 27–3012 pg/ml) and 161 pg/ml (range, 12–3473 pg/ml), respectively. Figure 2 shows the variation in SalT in each individual case.

Short-term growth

The pooled median LLLVs for the different study blocks were as follows: run-in, 0.34 mm/wk (range, -0.06 to 0.92 mm/wk); washout, 0.48 mm/wk (range, 0.05–0.75 mm/wk); 8-h treatment, 0.78 mm/wk (range, 0.42–1.08 mm/wk); and 12-h treatment, 0.6 mm/wk (range, 0.1–0.83 mm/wk). There was no significant difference in LLLV between any of the nontreatment periods. In the treatment blocks, the LLLV (median, 0.64 mm/wk; range, 0.1–1.08 mm/wk) was significantly higher than that during the run-in and washout periods (median, 0.48 mm/wk; range, -0.06 to 0.92 mm/wk) (P < 0.001). During the 8-h treatment blocks, LLLV (0.78 mm/wk) was higher than during the 12-h treatment blocks (0.6 mm/wk), and this achieved statistical significance (P = 0.03). Over 8 wk, the change in LLL as a percentage of the LLL at baseline was 3.4% (range, 2.7–4.4%) and 2.7% (range, 1.4–3.4%) in the 8- and 12-h groups, respectively. LLLV, irrespective of type of therapy, was 0.65 mm/wk (range, 0.38–0.92 mm/wk) in the first 8-wk block (8-h treatment, 4 boys; 12-h treatment, 3 boys) and 0.6 mm/wk (range, 0.1–1.08 mm/wk) in the second 8-wk block (NS) (Fig. 3).

View larger version (25K): [in this window] [in a new window]   FIG. 3. Median LLLV (millimeters per week) throughout the study in boys who were randomized to receive 8-h treatment first, followed by 12-h treatment (n = 4) (A), and in those who received 12-h treatment first, followed by 8-h treatment (n = 3) (B). The error bars denote 25th and 75th centile values.

bALP. Median bALP during the run-in period was 72 U/liter (range, 50–140 U/liter), and the median %bALP during this period was 2.9% (range, -11 to 58%). The main rise in bALP occurred during the first treatment block with a median %bALP of 44.2% (range, -4 to 87%) and a median bALP at the end of the first 8 wk of treatment of 109.9 U/liter (range, 94.1–167 U/liter). There was a smaller %bALP at the end of the second treatment block of 9.8% (range, -4 to 55%) and a median bALP of 126.9 U/liter (range, 91.6–181.5 U/liter). The increases in bALP were not significantly different between the 8- and 12-h treatment periods. Levels of bALP did not decline significantly during the washout periods, with median bALP and %bALP of 96.6 U/liter (range, 88.9–171.5 U/liter) and -0.7% (range, -19 to 5%), and 123.2 U/liter (range, 86.6–159.5 U/liter) and -4.2% (range, -18 to 3%) at the end of the first and second washouts, respectively (Fig. 4).

View larger version (25K): [in this window] [in a new window]   FIG. 4. Median bALP concentrations in boys who were randomized to 8-h treatment first, followed by 12-h treatment (n = 4) (A), and in those who received 12-h treatment first, followed by 8-h treatment (n = 4) (B). The error bars denote 25th and 75th centile values.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Recent advances in testosterone delivery methods have led to the availability of more user-friendly preparations that may be favored by boys who dislike parenteral preparations. In addition, they may also have the advantage of providing a more physiological testosterone profile. There are very scarce reported data on the use of TT in children, although this treatment modality is increasingly being used in clinical practice. The study by De Sanctis et al. (4) in young men with ß-thalassemia included three older teenagers and had an overnight administration protocol similar to ours. They reported physiological levels of testosterone in blood and promotion of growth and virilization.

The use of saliva for measurement of systemic concentrations of testosterone enables frequent, easy collection of samples by a noninvasive technique. Patients can collect the samples at home, and even very young children can provide an adequate specimen. SalT concentration represents the free plasma fraction that is capable of diffusing across the salivary gland, and therefore, it is the biologically active fraction available to the tissues (11). Other studies have shown that the SalT concentration is independent of saliva flow rate (12), and it correlates with serum free testosterone (13, 14).

The SalT assay used in this study was able to detect circadian variation in our prepubertal and early pubertal boys in the run-in period to the study. TT treatment significantly increased SalT concentration during the application period, and these high concentrations returned to baseline and showed circadian variation after the treatment stopped. SalT levels were higher during the application period than the application-free period during the 24-h cycle, but this difference did not achieve statistical significance. Our studies also show that overnight TT application at the doses used can be transiently associated with very high levels of testosterone. The adult early morning SalT ranges from 179–1,340 pmol/liter for our assay (our unpublished data), whereas the maximum concentration observed in our study was 12,420 pmol/liter. High concentrations of testosterone have also been noted after im depot testosterone that can persist for several days (2, 15). Saliva testosterone studies after administration of oral TU have shown high interindividual variability in the time taken to reach peak levels (16).

Because growth promotion is another objective of testosterone supplementation in boys with delayed growth and puberty, we assessed the effects of TT therapy on short-term growth. Although the majority of growth in boys with delayed puberty occurs in the spine, precise and accurate methods do not exist for assessing short-term growth of the spine. Our measurements show an increase in height in this group of children. Knemometry, the measurement of LLL, is a validated technique that allows reliable assessment of growth patterns in the short-term (17, 18, 19, 20). At the doses used, significant changes in short-term growth of the lower leg were detected. In addition, the changes observed were similar to those observed in previous studies by this group of investigators after oral TU (40 mg daily) or im Sustanon (50 mg, monthly) (6). Although the SalT concentrations did fall, short-term growth was maintained at a higher rate for a longer period, suggesting a sustained effect of testosterone on growth. It is also possible that this maybe due to the onset of endogenous puberty, but the testosterone levels do not support this explanation completely. The increase in short-term growth seemed to be greater during the shorter duration of nightly treatment. Considering that the SalT levels reached during the two periods were similar, this observation requires further study to investigate any relationship between duration of sex steroid exposure, aromatized estradiol concentrations, and longitudinal growth.

The bone remodelling process involves, first, removal of old bone and, then, replacement with new bone (21). TT treatment led to an increase in bone remodelling as reflected in raised bone formation (bALP) and bone resorption (DPD) markers (22, 23). bALP is an established marker of bone formation that has been shown to correlate with osteoblast maturation and bone mineralization. It has a half-life of around 40 h and a day-to-day variability of less than 4%. DPD are products of collagen degradation that are cleared by the kidney and originate mainly from bone (21). An increase in bone turnover markers has also been observed in adult hypogonadal men treated with testosterone (24), and as in our study, changes were more significant at the beginning of treatment and then declined toward baseline. Although we have interpreted the increased bone turnover as a reflection of increased linear growth (25, 26), androgens not only stimulate growth in the pubertal boy but are also responsible for the attainment of peak bone mass. Men with a history of delayed puberty have been shown to have decreased bone mineral density in adulthood (27), and adult studies have also found that there is an improvement in bone mineral density with testosterone replacement therapy, especially when there is a history of hypogonadism (24, 28, 29). Short-term testosterone treatment in boys has previously been shown to stimulate markers of bone formation (30). However, the finding of a sustained rise in bALP after cessation of testosterone therapy and normalization of SalT levels is an important finding and suggests that intermittent testosterone therapy may also have a role in treating osteoporosis secondary to hypogonadism.

As with LLLV, we did not find a difference in markers of bone turnover between the two doses of testosterone used, and there was evidence of a sustained effect on bone turnover after testosterone treatment stopped and testosterone levels were dropping. Similar SalT levels were also achieved with the two different doses of TT. This observation, as well as the observed similarity between the effects of the two doses on short-term growth and markers of bone turnover, may be due to a greater transdermal transfer of testosterone from the adhesive patch over the first few hours of application. This is supported by previous pharmacokinetic studies of other TT preparations in adults that show that peak serum testosterone levels were often achieved by 8 h after application (4, 31, 32). Our data would question the additional clinical value of continuous 24-h application of TT if the majority of testosterone transfer occurs over the first few hours of application. A reduced duration of exposure may be indicated in cases where the skin displays sensitivity to the adhesive.

In summary, overnight TT therapy in boys with delayed puberty can simulate physiological testosterone secretion, increases short-term growth and bone turnover, and is an acceptable method of induction of growth and puberty.

	Acknowledgments

 
We acknowledge the help of Dr. Malcolm Donaldson for referring patients to the study, the Children Living with Inherited Metabolic Diseases and Congenital Adrenal Hyperplasia Support Group for supporting the development of the SalT assay, and Ferring Pharmaceuticals, Limited, for supporting the costs of the bone turnover assays.

	Footnotes

 
Abbreviations: BA, Bone age; bALP, bone alkaline phosphatase; DPD, deoxypyridinoline cross-links; LLL, lower leg length; LLLV, LLL velocity; NS, not significant; SalT, salivary testosterone; TT, transdermal testosterone; TU, testosterone undecanoate.

Received June 24, 2003.

Accepted November 6, 2003.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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