Initial trial of slow-release testosterone microspheres in hypogonadal men*

FERTILITY AND STERILITY Copyright c 1988 The American Fertility Society Printed in U.S.A. Initial trial of slow-release testosterone microspheres in hypogonadal men* Allen S. Burris, M.D. t Larry L. Ewing, Ph.D.* RichardJ. Sherins, M.D.t National Institute of Child Health and Human Development, Bethesda, and The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland The duration of hormone release from testosterone-loaded poly(dl-lactide-co-glycolide) microspheres was assessed in eight hypogonadal men. Eight weeks before receiving testosterone (T) microspheres, patients were withdrawn from their prior androgen therapy in order to minimize the contribution of that therapy to hormone levels measured following microsphere injection. Blood was obtained for hormone measurements immediately before and weekly for 14 weeks after the administration of the T microspheres. A biphasic release of hormone was observed; a significant increase in total T and free T concentration (compared to preinjection values) was observed during week 1 (P < 0.01) and between weeks 6 and 11 after injection (P < 0.05). A similar pattern was detected in estradiol and dihydrotestosterone concentrations, whereas T -binding globulin levels showed a small, reciprocal fall after the injection of microspheres (P = not significant [NS]). After receiving the T microspheres, seven of eight hypogonadal men reported the return of normal sexual function; the eighth patient withdrew from the study after 5 weeks. It was concluded that T microspheres are a promising new method of androgen replacement therapy for hypogonadal men. Fertil Steril 50-493, 1988 The intramuscular administration of a long-acting testosterone (T) ester such as T enanthate or T cypionate is presently the most effective method for androgen replacement in men with hypogonadism.1 2 However, the pharmacokinetics of the T esters mandate a 1- or 2-week dosing interval in order to sustain the blood concentration of T within a therapeutic range. 3 4 The microencapsulation of a hormone allows for the effective, long-term release of the hormone in vivo. 5 By dispersing the hormon e in a biodegradable polymer matrix, sustained blood levels may be achieved after injection. 6 9 After preliminary studies conducted in primates, 10 we tested T-loaded poly(dl-lactide-co-glycolide) microspheres that were designed to release T over a period of 12 weeks after intramuscular injection. We administered the T microspheres to eight hypogonadal men in order to determine the clinical efficacy of the preparation and duration of T release. Received February 2, 1988; accepted May 10, 1988. *Supported in part by the National Institutes of Health grant HD-07204 and the Population Center grant HP-06268. t Developmental Endocrinology Branch, National Institute of Child Health and Human Development. *Division of Reproductive Biology, The Johns Hopkins School of Hygiene and Public Health. Reprint requests: Richard J. Sherins, M.D., National Institutes of Health, Building 10, Room 10N262, Bethesda, Maryland20892. Subject Selection MATERIALS AND METHODS Eight hypogonadal men between 22 and 57 years of age (41.1 ± 4.2, mean ± standard error of the mean [SEM]) were enrolled in the study (Table 1). Four patients had a diagnosis of isolated hypogonadotropic hypogonadism (IHH), three patients Burris et al. Testosterone microspheres 493

Table 1 Pretreatment Clinical Characteristics of Hypogonadal Men Who Received Testosterone Microspheres Age Patient (yr) Primary diagnosis Other diagnoses Treatment history Serum T" (ng/dl) 1 2 3 4 5 40 IHHb NIDDM Testosterone enanthate 41 22 IHH Bilateral cryptorchidism Testosterone enanthate 13 28 IHH Testosterone enanthate 49 40 IHH Bilateral cryptorchidism Testosterone enanthate 34 57 Pituitary adenoma Hypothyroid hypoadrenal Testosterone enanthate 80 Essential hypertension Hydrocortisone 6 46 Pituitary adenoma Hypothyroid Testosterone enanthate 49 7 54 Pituitary adenoma Hypothyroid hypoadrenal Testosterone enanthate 17 Hydrocortisone 8 42 Testicular failure Essential hypertension Testostero.1e enanthate 29 Hydrochlorothiazide Pindolol 41.1 ±4.2d a Testosterone (T) values reflect concentrations before any androgen replacement. To convert T to nmol/l, multiply by 0.035. b IHH, isolated hypogonadotropic hypogonadism. Noninsulin-dependent diabetes mellitus. d Mean ± standard error of the mean. had hypopituitarism due to a pituitary adenoma, and one patient had post-traumatic testicular failure. Before entering the study, all patients had been receiving T enanthate for more than 2 years, and none reported erectile dysfunction or impaired libido. Their initial, pretreatment mean serum T concentration was 39 ± 8 ng/dl (Table 1), well below the lower limit of normal in our laboratory (250 ng/dl), reflecting low endogenous T secretion. Before entering the study, a medical history was obtained, and physical examination and routine laboratory studies were performed. IHH (patients nos. 1 to 4) and hypogonadism due to a pituitary adenoma (patients nos. 5 to 7) had been previously confirmed by a low serum T level ( <100 ng/dl) and inappropriately low serum gonadotropin levels. Primary gonadal failure, in patient no. 8, had been previously confirmed by a low serum T concentration and elevated serum gonadotropin levels. The study protocol was approved by the National Institutes of Health Institutional Review Board, and informed consent was obtained from each patient before starting the study. Study Design T enanthate was withheld for 8 weeks before receiving the microspheres in order to allow the serum T concentration to fall toward untreated, hypogonadallevels. At the end of the 8-week period of androgen withdrawal, immediately before injecting the T microspheres, blood was obtained for measurement of serum T, free T, dihydrotestosterone (DHT), testosterone-binding globulin (TeBG), and estradiol (E 2) levels. The T microspheres were administered through a 21-gauge needle by deep injection into the gluteal muscles. A total T dose of 630 mg was given to approximate the normal T production rate (5 to 10 mg/day) 1 for 12 weeks. The total injection volume, approximately 6 ml, was given in two separate 3-ml injections in order to minimize discomfort at the injection site. After administration of T microspheres, blood was obtained daily for 7 days and then weekly (on the seventh day of each week) for 14 weeks. At the end of the 14th week a physical examination and routine laboratory tests were repeated, and each patient received an intramuscular injection of T enanthate 200 mg to allow comparison of hormone concentrations achieved by the two different preparations. One week later, a final blood sample was obtained (week 15). Every week throughout the study, patients were asked about any changes in mood or sexual function. Testosterone Microspheres The T microspheres were formulated by Stolle Research and Development Corporation (Cincinnati, OH). The microsphere matrix consisted of poly(dl-lactide-co-glycolide) in an 85:15 molar ratio. The microspheres were loaded with 48% T (by 494 Burris et al. Testosterone microspheres Fertility and Sterility

RESULTS 1000 BOO. BOO ) 400 I I 200 0 1 11 13 15 WEEKS Figure 1 Mean (±SEM) serum concentrations of testosterone (T) before (week 0) and after administration oft microspheres. The asterisk denotes values obtained 1 week after the administration oft enanthate. To convert to nmol/l,-multiply by0.035. weight) and were packaged in prefilled syringes, each containing 315 mg oft. The syringes were sterilized with gamma radiation and subsequently stored at room temperature until use. Just prior to injection, the microspheres were suspended in a diluent consisting of 2% carboxymethylcellulose and 1% polysorbate-20 in sterile water. Hormone Measurements Serum was promptly separated from blood samples and stored at -2o c until hormone analysis was performed. All hormone measurements for each patient were performed in a single assay to avoid interassay variation. T and DHT were separated by high-pressure liquid chromatography and subsequently measured by radioimmunoassay (RIA)Y The sensitivity of the assay is 1.0 ng/dl for both T and DHT. Serum E 2 and TeBG were measured by RIA using standard methods. 12 13 The sensitivities of the assays for E 2 and TeBG were 5 to 12 pg/ml and 0.10 JLg/dl, respectively. FreeT was measured by a solid-phase RIA (Diagnostic Products Corporation, Los Angeles, CA); the sensitivity of this assay is 0.06 ng/dl. Every sample was analyzed for both T and DHT, and alternate samples were analyzed for free T, E 2, and TeBG. Statistical Methods Data are shown as mean ± standard error of the mean (SEM). Comparisons between means were evaluated by Student's t-test or by analysis of variance (ANOV A) for multiple means. Statistical significance was defined as a P value less than 0.05. Serum Hormone Concentrations T levels before and after the administration oft microspheres are shown in Figure 1. The mean total T concentration increased from 73 ± 32 to 641 ± 134 ng/dl on the first day after injection (P < 0.01), fell to a nadir of 173 ± 37 ng/dl during the third week, and subsequently rose again between weeks 6 and 11 (P < 0.05 compared with pre-injection concentration). A peak, meant concentration of 799 ± 198 ng/dl was observed at week 8. The mean free T concentrations (Fig. 2) paralleled the mean total T concentrations, with a peak of 2.2 ± 0.3 ng/dl during the first week (P < 0.01) and a prolonged rise between weeks 3 and 11 (P < 0.05); the peak value was 3.3 ± 0.7 at week 9. Mean DHT and E 2 concentrations (Fig. 2) followed a pattern similar to meant, but the postinjection concentrations were not significantly different from the preinjection concentrations. A slight, but not significant, fall in TeBG (Fig. 2) was noted at the time of the peak in T levels. As shown in Figures 1 and 2, the concentrations of total T, free T, and E 2 measured 1 week after T enanthate (week 15) were similar to values observed 6 to 11 weeks after the injection oft microspheres (P = NS). All DHT and TeBG concentrations observed after T microspheres were similar.. } 2 c 16 0 1 3 5 7 9 11 13 15 ;e fnoot! w z if 3!!! "' ::? 2 "'!!! ill1 1!: iii 0.60 WEEKS '_j 0 1 3 5 7 9 11 13 15 Figure 2 Mean (±SEM) serum concentrations of estradiol (E 2), dihydrotestosterone (DHT), free testosterone, and testosterone-binding globulin (TeBG) before (week 0) and after administration oft microspheres. The asterisk denotes values obtained 7 days after the administration oft enanthate (week 15). To convert to SI units, multiply values by the following constants: E 2, 3.671 (pmol/l); free T, 0.035 (nmol/l); DHT, 0.034 (nmol/l). Burris et al. Testosterone microspheres 495

to those measured 1 week after T enanthate (P = NS). Clinical Course The injection oft microspheres was well-tolerated by the patients. They reported only transient, minimal discomfort at the time of injection, with one exception. Patient no. 8 experienced moderate pain during the injection; the microspheres he received had been suspended in diluent several hours before use, and clumping was noted in the syringe after injection. No other untoward effects were observed during the study. The efficiency of the microspheres injection, determined by measuring residual Tin the syringes after use, was 97.6 ± 0.05%. During the 8-week androgen withdrawal period, before the injection oft microspheres, all patients experienced symptoms of androgen deficiency, particularly describing a reduction in potency and libido. By the third week after the injection oft microspheres, seven of eight patients described the return of normal sexual function. Patient no. 8 did not experience an improvement in libido or potency after receiving the T microspheres and subsequently withdrew from the study after 5 weeks; his serum T concentration was 257 ng/dl at that time. In the seven remaining patients, normal sexual function was sustained during the 13 weeks after the injection oft microspheres (as ascertained by the sexual history). in the syringe; aggregation could account for his suboptimal clinical response. 14 The long-term efficacy and safety of sustained release hormone-loaded microspheres have been demonstrated in trials of progestin-loaded microspheres6'9 and luteinizing hormone-releasing hormone analog-loaded microspheres. 15-17 The biphasic rise in total and free T concentration that followed the injection of T microspheres was similar to the pharmacokinetics of progestin-loaded microspheres in initial trials.6 Subsequent reformulation of the progestin microspheres 8 9 resulted in nearly zero-order hormone release. Accordingly, we postulate that by varying the particle size, hormone content, polymer composition, and dose of sterilizing radiation, the T microspheres can be reformulated to provide near zero-order T release. 14 The results of our initial trial suggest that T microspheres may be a feasible alternative to current methods of long-term androgen replacement in hypogonadal men. By testing different microsphere preparations and by examining the pharmacokinetics of repeated doses of T microspheres, the suitability, for long-term use can be established. Acknowledgments. The authors acknowledge the invaluable nursing support of Ms. Nelly Rivera and Ms. Tannia Cartledge and editorial assistance of Ms. Kathy Shoobridge. They also thank Mrs. Elizabeth Higginbottom and Mr. Tushar Manek for technical assistance and acknowledge PARFR and the Stolle Corporation for supplying the testosterone microspheres. DISCUSSION This study demonstrates that sustained high T concentrations can be attained in hypogonadal men after the administration of T microspheres. Normal total T and free T concentrations were observed between the sixth and eleventh weeks after injection oft microspheres. The subjective, clinical response to the T microspheres paralleled the serum levels of total T and free T. Testosterone, free T, E 2, and DHT levels were comparable to those measured 1 week after a 200-mg dose of T enanthate. 2 4 Excessive hormone release from the T microspheres was not observed, neither was there clinical evidence of androgen- or estrogenmediated side effects such as stimulation of the prostate or breast enlargement. 1 Our experience with patient no. 8 indicates that the microspheres must be mixed in diluent immediately prior to use in order to avoid aggregation of the microspheres REFERENCES 1. Wilson JD, Griffin JE: The use and misuse of androgens. Metabolism 29:1278, 1980 2. Schulte-Beerbiihl M, Nieschlag E: Comparison of testosterone, dihydrotestosterone, luteinizing hormone, and follicle-stimulating hormone in serum after injection of testosterone enanthate or testosterone cypionate. Fertil Steril 33:201, 1980 3. Snyder PJ, Lawrence DA: Treatment of male hypogonadism with testosterone enanthate. J Clin Endocinol Metab 51:1335, 1980 4. Sokol RZ, Palacios A, Campfield LA, Saul C, Swerdloff RS: Comparison of the kinetics of injectable testosterone in eugonadal and hypogonadal men. Fertil Steril 37:425, 1982 5. Beck LR, Pope VZ: Controlled-release delivery systems for hormones: a review of their properties and current therapeutic use. Drugs 27:528, 1984 6. Beck LR, Cowsar DR, Lewis DH, Cosgrove RJ, Riddle CT, Lowry SL, Epperly T: A new long-acting injectable microcapsule system for the administration of progesterone. Fertil Steril 31:545, 1979 7. Beck LR, Cowsar DR, Lewis DH, Gibson JW, Flowers CE: New long-acting injectable microcapsule contraceptive system. Am J Obstet Gynecol135:419, 1979 496 Burris et al. Testosterone microspheres Fertility and Sterility

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