Main Research Findings

1) SARMs have been shown to promote various forms of functional therapy through tissue-selective activation of androgenic signaling.

2) LGD 2226 exhibited increased anabolic activity on muscle and bone tissue while also improving sexual behavior in male rats.

Selected Data

1) The research team of Shalender Bhasin, MD and Ravi Jasuja, PhD. examined the potential of selective androgen receptor modulators (SARMs) to promote various forms of functional therapy. Previous research has reported that SARMs bind to androgen receptors and display tissue-selective activation of androgenic signaling, leading to anabolism in skeletal muscles and bones. The actions of SARMs are compared to testosterone, the major ligand for androgen receptors. Testosterone is often supplemented to men and women of all ages suffering from androgen deficiency and decreased muscle and bone wasting. However, administration of androgenic compounds such as testosterone is often related to many dose-limiting adverse side effects such as prostate dysfunction, edema, and erythrocytosis. On the other hand, SARM administration has been shown to result in similar anabolic activity without the adverse side effects associated with typical androgen treatment [1].

In order to target functional limitations caused by osteoporosis, aging, and chronic disorders, researchers first attempted to develop a SARM with the desired activity profile and tissue selectivity. The second approach included elucidating the mechanisms of action of androgens on skeletal muscles and the prostate in order to identify signaling molecules downstream of the androgen receptors that are capable of activating hypertrophic pathways in skeletal muscles but not the prostate. When observing the structure of SARMs, the compounds can be categorized into two groups: steroidal and nonsteroidal. Steroidal SARMs are synthesized by modifying the chemical structure of testosterone molecules. For example, substitution of 7-alpha alkyl makes testosterone less susceptible to 5-alpha reduction, thus increasing tissue selectivity with respect to the prostate. This results in the increased anabolic activity in the levator ani muscle and a decreased rate of anabolism in the prostate and seminal vesicles [1].

Researchers at the University of Tennessee and Ligand Pharmaceuticals reported early data regarding the discovery of nonsteroidal SARMs. After publication of the initial findings various other structural categories of SARM pharmacophores were examined. These categories included: aryl-propionamide, bicyclic hydantoin, quinolones, tetrahydroquinoline analogs, benzimidazole, imidazolopyrozole, indole, pyrazoline derivatives, azasteroidal derivatives, and aniline, diaryl aniline, and benzoxazepinones derivatives. The first generation of SARMS was developed by manipulating the structure of aryl propionamide analogs, bicalutamide and hydroxyflutamide. This initial discovery led to copious amounts of research dedicated solely to modifying compound structures in order to promote tissue selectivity and further hone in on the beneficial anabolic activity [1].

2) The research team of Miner et. Al examined the effects of LGD 2226 on the preservation and improvement of bone, muscle, and functioning of sexual behavior in male Sprague Dawley rats. The experiment was split into a two-week study and a six-week study; for the two week study the subjects were orchidectomized and treated with either a vehicle or doses of LGD 2226 ranging from 1, 3, 10, 30, and 100 mg/kg. 24 hours after the last dose the animals were euthanized while the ventral prostate and levator ani muscle were collected, dissected, and weighed. The six week study also included 7-week-old male Sprague Dawley rats, weighing approximately 250 grams. The test subjects were placed in a cage with 2-3 other animals, fed standard rodent chow, and maintained on a strict 12-hours light/12-hours dark schedule. The animals were allowed to acclimate to their surroundings for one week followed by a perivascular injection of the first bone-labeling marking, calcein. The compound was injected at the base of the tail 3 days prior to ORDX [2].

The rats were randomly assigned to one of 7 groups each containing 10 test subjects; the groups were defined as: 1) gonadally intact (baseline control group), 2) sham-operated, 3) ORDX, 4) ORDX treated with 100 mg/kg of fluoxymesterone, 5) ORDX treated with 1 mg/kg of LGD 2226, 6) ORDX treated with 3 mg/kg of LGD 2226, and 7) ORDX treated with 10 mg.kg of LGD 2226. Group 1 was euthanized on day 2 of the experiment and the tibiae and femora were isolated and stored according to proper procedure for later testing. One day 32 and day 40, the remaining rats were administered a dose of tetracycline as a second and third bone-labeling time point. Urine was continuously collected and centrifuged in order to assess the urine supernatants for DPD and creatinine. All rats were euthanized at the end of 6 weeks and weighed both before and after the collection of the femur and tibia of each subjects’ hind legs [2].

Following bone collection histomorphometric analyses of the tibiae were performed while the cortical bone of the tibial diaphysis and the cancellous bone of the proximal tibia metaphysis were measured through the use of a digitizing morphometry system. Transverse section of the cortical bone obtained from the tibio-fibular junction was prepared according to standard procedure in order for the research team to microscopically examine calcein and tetracycline fluorochrome labeling. The recorded measurements included: 1) the rate of periosteal bone formation, 2) the rate of bone mineral apposition, and 3) the rate of periosteal mineralization. The rate of periosteal bone formation was calculated as the area inside of the periosteal surface and outside the calcein label, divided by the labeling period. Bone mineral apposition was defined as the area inside the periosteal surface and outside the calcein label, divided by the product of the labeled surface length. Periosteal mineralization rate was calculated as the area bordered by each tetracycline label, divided by the product of the length of the labeling period and the initial label.

The tibiae were sectioned transversely at the mid-diaphysis, followed by storage in 5% formic acid and 10% formalin, and further bisection through the midsagittal plane. The prepared products were mounted on slides in order for morphometric analysis to occur. Throughout the examination the research team measured: 1) total bone area, 2) cancellous bone perimeter, 3) single- and double-labeled perimeters, and 4) interlabeled widths. Total bone area was defined as the total area of trabecular bone expressed as a percentage of the total tissue area of the sample site, while the cancellous bone perimeter was defined as the total trabecular surface length found throughout the sample site. These measurements were then used to calculate values for: 1) percent cancellous bone volume and 2) the rate of cancellous bone formation [2].

An additional 16-week bone study took place and followed procedures similar to the 6-week study with small adjustments made to account for the long-term nature of the experiment. 9- month old Sprague Dawley rats weighing approximately 500 grams were housed in individual cages and allowed to acclimate to their new surroundings for 2 weeks. The same number of rats were randomly assigned to the same categories of experimental treatment groups. All injections of the bone-labeling markers were administered in the same manner at the same time, relative to the length of the study. Additionally, vertebrae L1-L5 were excised and prepared for further analysis and determination of bone mineral density and mechanical properties. The mechanical properties were assessed through a compression test that began with the removal of the two epiphyseal ends, posterior pedicle arch, and the spinous process from the whole vertebrae. The average dorsal to ventral diameter, side-to-side diameter, and height was measured while the research team was able to obtain values for maximal load, stiffness, and energy generated by the load and extension curve. The values were used to calculate cross-sectional area, ultimate strength, elastic modulus, and toughness.

In addition to examining changes in bone in response to LGD 2226 or fluoxymesterone, the two compounds were assessed for their ability to improve various parameters of sexual behavior in ORDX hooded Long-Evans rats. Because these animals have been shown to positively respond to androgens, ovariectomized female Sprague Dawley rats were used as stimulus. Baseline behavior was established prior to ORDX; the male rats were tested for consistent sexual behavior with sexually receptive stimulus female rats. Male rats able to copulate were included in this treatment phase of the study. The test subjects were kept sexually active during the period of time between screening and treatment. Following ORDX and implantation of Silastic capsule, the subjects were divided into three treatment groups and orally administered either a vehicle, 100 mg/kg of LGD 2226, or 100 mg/kg fluoxymesterone. Sexual behavior of the male rats in each treatment group was recorded in intervals of 6- or 7-days over the course of the 8-week experimental treatment period [2].

Discussion

1) The research team of Bhasin and Jasuja were able to achieve selectivity of SARMs by elucidating the mechanism of testosterone’s action on the prostate, as well as how molecules farther downstream were associated with activation of AR signaling in skeletal muscle. Analysis of muscle biopsies collected from male test subjects treated with varying doses of testestore revealed that administration of the compound led to hypertrophy in type I and type II muscle fibers. In relation to testosterone dosage, both type I and type II fibers experienced significant changes in cross-sectional areas. It is important to note that there was no change observed in the absolute number or the relative proportion of type I and type II fibers in response to testosterone administration [1].

Hypertrophy of the skeletal muscle was further examined through observation of muscle satellite cells and the myonuclear number. These variables were assessed through the use of electron microscopy, using direct counting and spatial orientation methods at baseline and after 20 weeks of GnRH agonist and testosterone enanthate treatment. Results reported that absolute and percent satellite cell number was significantly greater than baseline after 20 weeks of the test subjects receiving supraphysiologic doses of testosterone. The observed changes in the number of satellite cells correlated with changes in total and free testosterone levels, indicating that muscle fiber hypertrophy induced by testosterone is correlated with an increase in the number of satellite cells and the myonuclear number.

Recent studies have found that both testosterone and DHT are able to promote association between liganded ARs and beta-catenin, its co-activator. Beta-catenin is stabilized by this interaction and enhances translocation into the nucleus and association with TCF-4, as well as the transcriptional activation of Wnt-target genes. Additionally, Testosterone upregulated the expression of follistatin, resulting in increased muscle mass and decreased fat mass. SMAD 7 is also upregulated by testosterone while TGF-beta-mediated SMAD signaling in TGF-beta target genes is downregulated. The connection between testosterone and follistatin expression indicates that the effects of testosterone are cross-communicated from the WNT pathway to the TGF-beta-SMAD pathway. These results further suggest that candidate molecules located downstream of AR and beta-catenin, such as follistatin, have the potential to mediate the effects of testosterone on the muscle and may provide desired selectivity of anabolism. The discovery of these candidate targets allows for further research to be conducted in order to develop selective anabolic drugs [1].

2) Results of the 2-week study conducted in ORDX rats of 8 weeks of age reported that each subject experienced significant anabolic activity in the levator ani muscle after administration of an oral dose of LGD 2226 ranging from 1 mg/kg to 100 mg/kg, or T. While high levels of androgen supplementation typically leads to considerable prostate growth, LGD 2226 was found to significantly reduce prostate growth when compared to T. Only when LGD 2226 was administered in doses of 100 mg/kg did the prostate growth reach a level comparable to the intact animals. However, at 10 mg/kg administration of T had almost double the weight of the prostate. Overall, results of the 2-week study concluded that LGD 222 is able to enhance the development of muscle without increasing growth and stimulation of the prostate and the hypothalamus-pituitary-gonadal axis.

The 6-week study conducted by the research team included administering doses of 1, 3, or 10 mg/kg of LGD 2226 to ORDX rats. The results of the study revealed that there was similar activity recorded for LH expression and the selectivity of muscle versus prostate size in the animals treated with LGD 2226 in comparison to 100 mg/kg of fluoxymesterone. Bone-labeling biomarkers and DPD, a collagen cross-link, were used to measure the structure, strength, and formation of bone and cartilage tissue. Excretion of DPD is considered a valuable marker of bone degradation in cases of androgen deficiency in male rats. The ORDX rats treated with LGD 2226 had decreased levels of DPD detected in their urine, indicating reduced bone resorption. These findings were confirmed by histomorphometric analysis performed on the tibiae of the test subjects. The number of osteoblasts and their total activity was calculated using the gathered histomorphometric data [2].

Bone density and other static and dynamic measurement of cortical bone was not shown to have experienced any significant effects following castration or treatment of LGD 2225. However, cancellous bone experienced a reduction in bone formation and mineral apposition rate in the rats treated with LGD 2226 and fluoxymesterone. The research team was able to conclude that the reduction in bone formation was due to direct coupling between bone resorption and formation in cancellous bone, indicating a reduction in the rate of bone turnover. Additionally, after ORDX the rate of bone turnover was linked to an increase in bone resorption. However, for both of these parameters 3 mg/kg of LGD 2226 was found to effectively normalize the rate of bone resorption without negatively affecting the prostate [2].

A 16-week study was conducted considering that the research team was not able to gather any significant data regarding the effects of LGD 2226 on bone density and formation after only 8 weeks. Initial results found that administration of 100 mg/kg of fluoxymesterone led to an enlarged liver in the test subjects; this side effect was not reported after administration of 10 mg/kg of LGD 2226. 16 weeks after ORDX the research team reported that the substantial loss of bone density seen in the control group was prevented by both fluoxymesterone and LGD 2226. It is important to mention that the 3 and 10 mg/kg doses of the compound increased bone mineral density about levels of sham-treatment animals. The breaking strength and ultimate breaking strength of the tibiae were found to be above sham levels with all doses of LGD 2226; 1 mg/kg of LGD 2226 was equal to 100 mg/kg of fluoxymesterone. Finally, Histomorphometric analysis found that periosteal mineral apposition rate as well as periosteal bone formation were significantly increased after treatment with LGD 2226. Administration of the compound led to stimulation of bone formation and inhibition of bone resorption in order to prevent the loss of cancellous bone in ORDX rats [2].

Male sexual behavior in primates and rats is typically stimulated by the effects of nonaromatizable androgens that act on the brain and periphery. The effects of the androgens are elicited when the animals are concurrently treated with a suboptimal priming dose of estrogen. Male sexual behavior was measured by recording the amount of mount attempts, intromissions, ejaculation, and overall copulatory efficiency. Following implantation of the estrogen-containing silastic capsules and treatment with a vehicle, fluoxymesterone, or LGD 2226, all results were expressed in graphical format. The results found that rats treated with a vehicle compound experienced a decrease in performance and motivational indicators over the course of the experimental period. Both the active compounds of fluoxymesterone and LGD 2226 were shown to increase the number of mounts, intromissions, and ejaculations. LGD 2226 also enhanced copulatory efficiency from week 4 to the end of the study [2].

Figure 1: Changes in male sexual behavior in response to treatment administration.

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Bhasin S, Jasuja R. Selective androgen receptor modulators as function promoting therapies. Curr Opin Clin Nutr Metab Care. 2009 May;12(3):232-40. doi: 10.1097/MCO.0b013e32832a3d79. PMID: 19357508; PMCID: PMC2907129.

[2] Miner JN, Chang W, Chapman MS, Finn PD, Hong MH, López FJ, Marschke KB, Rosen J, Schrader W, Turner R, van Oeveren A, Viveros H, Zhi L, Negro-Vilar A. An orally active selective androgen receptor modulator is efficacious on bone, muscle, and sex function with reduced impact on prostate. Endocrinology. 2007 Jan;148(1):363-73. doi: 10.1210/en.2006-0793. Epub 2006 Oct 5. PMID: 17023534.

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