Main Research Findings

1) Ovariectomized, female Sprague-Dawley rats treated with LGD-3303 experienced improved bone mineral content (BMC) and BMD.

2) Orchidectomized, male Sprague-Dawley rats treated with LGD-3303 experienced increased levator ani muscle mass without eliciting negative effects on the prostate.

3) Treatment with LGD-3303 enhances male-directed sexual preference in sexually experienced female rats.

4) Treatment with LGD-3303 increases male-directed preference in hormone-primed, sexually experienced female rats with no prior experience with the testing paradigms.

5) Androgen receptor antagonist, flutamide, blocks the effects elicited by LGD-3303 on male-directed preference in sexually experienced female rats.

6) LGD-3303 treatment results in a dose-dependent increase in proceptive and lordosis behavior exhibited by hormone-primed, sexually experienced female rats.

Selected Data

1) 3 month old female Sprague-Dawley rats were housed 2-3 rats in a cage and then separated into treatment groups to randomly decide which subjects would be undergoing an ovariectomy (OVX) and which would be getting a sham-operation performed. The female rats were left treated for approximately 7 weeks after the surgeries were performed in order for sufficient levels of osteopenia to develop.

Following 7 weeks of no treatment the female rats were split into 5 different treatment groups. The groups included Sham-operation + vehicle, OVX + vehicle, OVX + 3 mg/kg of LGD-3303, OVX + 3 mg/kg of alendronate, and OVX + 3 mg/kg of LGD-33033, + 3 mg/kg of alendronate. All of these treatments were administered to the rats through an oral gavage and were given daily over the course of a 12 week experimental treatment period [1].

The researchers took a baseline DXA scan and administered Calcein to the animals both 3 and 10 days prior to euthanasia. A second DXA scan was taken during treatment week 11 and after week 12 of treatment the animals were euthanized. Wet weights were taken of the gastrocnemius muscle and the inguinal fat pad of the rats while bones were harvested for DXA scans, biomechanical tests, and histomorphometry measurements in order to examine changes between the treatment groups [1] .

2) 7-8 week old male Sprauge-Dawley rats were sorted by body weight and housed 2-3 rats per cage. The males then underwent a sham-operation or an orchidectomy (ODX). In order to ensure sufficient atrophy of the prostate and levator ani muscle due to hypogonadism, the rats were left untreated for 2 weeks. After 2 weeks the rats were given either testosterone propionate or LGD-3303.

The SARM was administered once daily via oral gavage in a suspension of Tween-80, glycol-400, polyethylene, and 0.1% carboxymethyl cellulose in water, or a vehicle. Once daily, testosterone propionate was dissolved in a solution of DMSO, and polyethylene glycol-400 and subcutaneously injected. This was also compared to the test subjects that received a daily subcutaneous injection of a vehicle. After the male rats were treated for 14 days they were euthanized so the researcher could accurately obtain and measure the weight of the prostate and levator ani muscle [1].

3) Test subjects for experiment one were female rats that were ovariectomized prior to treatment. The subjects were given a week to recover and were randomly assigned into four different treatment groups. DHT was administered to the subjects through a SILASTIC brand capsule containing crystalline DHT implanted midscapula. LGD-3303 was administered at doses of 3 and 30 mg/kg via oral gavage. The three experimental treatment groups were compared to a fourth control group. LGD-3303 was administered once daily over the course of 7 days. The dose was given to the subjects approximately 20 hours before the testing took place and 5-6 hours into the 12 hour dark period. In order to familiarize the subjects to the testing apparatus they were administered a pretest after they were administered a single dose of LGD-3303 [2].

Seven days after the pretest the subjects were tested for sexual preference. 4 days after the sexual preference test the animals underwent sexual behavior tests where each animal was injected with a 40 ug/kg dose of estradiol benzoate 48 before the test occurred. On the morning of the test the animals were administered a 1 mg/kg dose of progesterone 3-4 before the testing. After the baseline sexual behavior tests took place the animals were administered LGD-3303 treatments. A second sexual preference took place 7 days after the treatment was initiated in order to examine how LGD-3303 affected sexual preference [2].

4) Experiment 2 examined how acute exposure of LGD-3303 leads to increased mate seeking in sexually experienced female rats. Prior to experimental treatment the animals were ovariectomized and randomly assigned to treatment groups. The subjects were then subject to two sexual behavior tests; a 40 mg/kg dose of estradiol benzoate was administered 48 hours before testing while a 1 mg/kg dose of progesterone was administered the morning of testing, 3-4 hours before the sexual behavior test occurred.

Preference testing followed the sexual behavior testing; the afternoon of the test, the animals were treated with either 1 mg/kg of DHT, 3 or 30 mg/kg of LFD-3303, or a vehicle product. The sexual preference test took place approximately 20 hours after the administration of DHT or LGD-3303 in an attempt to mimic natural changes in androgen levels. The treatment continued for 6 days after the first preference test and the animals were administered a second preference test at the end of the treatment period [2].

5) Experiment 3 examined how administering an androgen receptor antagonist, flutamide, will affect the previously observed improvements of male-directed sexual preference in female rats given LGD-3303. Additionally, the researchers wanted to determine whether or not LGD-3303 works in an androgen receptor-specific manner. All subjects were ovariectomized prior to receiving a daily, 100 mg/kg dose of flutamide or a vehicle. After 3 days the animals were randomly administered either a 2 mg/kg dose of DHT propionate, a 30 mg/kg dose of LGD-3303, or a vehicle agent. DHT propionate and the vehicle groups underwent a control gavage procedure, while the LGD-3303 treatment group was administered a control vehicle injected. Sexual preference was tested approximately 20 hours after treatment administration [2].

6) The final experiment conducted by researchers Kudwa et. Al tested whether LGD-3303 results in increased levels of proceptivity and lordosis behavior in sexually experienced female rats, when in the presence of sexually experienced male rats. The subjects were prepared accordingly, while sexual experience was simulated through the administration of 40 ug/kg doses of estradiol benzoate and progesterone. One week after the induction of sexual experience, the experimental animals were given either 10 or 100 mg/kg doses of LGD-3303, daily, for 9 days [2].

Subjects were then hormone-primed with 0.5 or 2 ug/mg of estradiol benzoate or progesterone and subjected to a sexual behavior test. An initial baseline test took place in order to observe natural occurrences of proceptive and lordosis behavior. 3 days after baseline testing LGD-3303 treatment began; behavior was tested again 1 day after the first dose and 9 days after the first dose. It’s important to note that these doses of estradiol benzoate and progesterone are considered suboptimal and are meant to elicit low levels of sexual behavior so the research team can determine if any observed changes are due to treatment with LGD-3303. The behavior of all subjects was tested 1 day and 9 days after beginning LGD-3303 treatment, as well as 1 week after stopping treatment [2].

Discussion

1) In the female rats that were administered doses of LGD-3303 or a combination of LGD-3303 + alendronate, results reported that there was an increase in body, muscle, and organ weight. The weight of the gastrocnemius muscle was reported to be significantly enhanced in both of the aforementioned groups. Furthermore, while muscle mass increased, weight of the inguinal fat pad decreased in both the LGD-3303 and LGD-3303 + alendronate groups.

Figure 2. Changes in body weight, muscle weight, and inguinal fat pad weight in response to different treatments.

The DXA scans performed by the research team determined that LGD-3303 elicited positive effects on BMC and BMD. The effects of the SARM were identified in both cortical and cancellous bone. At the lumbar spine, a site containing primarily cancellous bone, exhibited increased BMD and BMC as a result of treatment with LGD-3303. It is important to note that only improvements in BMD were considered significant. Similar results were reported in the subjects treated with alendronate, however, the combination treatment of alendronate and LGD-3303 led to an increase in lumbar spine BMD to a level similar to those found in sham-operated, vehicle-treated subjects.

The DXA scans taken of the mid-femoral diaphysis, a primarily cortical bone site, found that LGD-3303 led to an increase in BMC. Compared to alendronate by itself, effects of LGD-3303 were far more significant. Combination of the two treatments did not lead to a notable difference in BMC compared to LGD-3303 alone. However, the combination treatment was more effective than treating the subjects with only alendronate.

The histomorphoectomy measurements that were recorded revealed that treatment with LGD-3303 increased the rate of periosteal bone formation at the mid-femoral diaphysis. This effect was important to mention considering that the mid-femoral diaphysis is not a typical site of osteoclast remodel. That being said, any growth at this area is due to osteoblast activation and successful anabolic activity. Alendronate treatment did not lead to any significant changes in periosteal bone growth. Overall, the researchers were able to conclude that LGD-3303 leads enhanced BMD and BMC in cortical and cancellous bone sites.

Finally, the biomechanical studies that were conducted reported that LGD-3303, alendronate, and combination treatment, all led to enhanced peak loading at the mid-femur. While the combination LGD-3303 + alendronate treatment exhibited the largest increase in peak loading, differences were not considered significant since all three treatments led to improvements. Furthermore, the biomechanical studies found that all three treatments increased peak compressive load of the lumbar vertebrae. The combination increased the peak compressive load to the greatest extent, however, variations between the results were not considered significant.

Figure 3: Changes in BMC and BMD in response to different treatment variables.

2) The research team reported that administering LGD-3303 or testosterone propionate to ODX male rats resulted in increased anabolic activity in the levator ani muscle in a dose-dependent manner. A 1 mg/kg dose of both testosterone propionate and LGD-3303 were given to the subjects; the levator ani muscle maintained eugonadal levels, indicating that both treatments display similar levels of effectiveness. Research found that the 1 mg/kg dose of testosterone propionate stimulates the ventral prostate up to 50% or eugonadal levels, while a 3 mg/kg dose of testosterone significantly exceeded the eugonadal levels. This indicates that the effects of testosterone propionate are elicited with minimal selective tissue activity.

On the other hand, a 1 mg/kg dose of LGD-3303 was not shown to elicit significant activity levels on the ventral prostate, measured at <5% efficacy. Higher doses of LGD-3303 did not dramatically affect activity levels either. Compared to 50% restoration of the prostate with 1 mg/kg of testosterone, a 100 mg/kg dose of LGD-3303 was required in order to reach 50% restoration of the eugonadal levels. Both LGD-3303 and testosterone propionate promoted muscle growth in the levator ani, however, in the rats administered testosterone propionate the prostate grew at a rate similar to the muscle. Because the prostate grew at a far less significant rate in the rats treated with LGD-3303, the researchers concluded that the SARM acts as a partial agonist on the prostate

Figure 1. Changes in prostate and levator ani weight in response to administration with testosterone propionate or LGD-3303

3) The first experiment conducted by researchers Kudwa et. al observed the effects of LGD-3303 on the sexual preference of female rats with prior sexual experience. The research team recorded the amount of time both sexually experienced and non-experienced test subjects spent in the different areas of the testing arena. The observations were further analyzed through ANOVA testing. Additionally, a sexual preference test was conducted 7 days after the initiation of LGD-3303 treatment begins. Results of these tests reported that there was a notable link between sexual experience and experimental treatment with LGD-3303 and DHT. Female rats with sexual experience treated with DHT experienced a decrease in the amount of time they spent with male test subjects. However, the sexually experienced subjects that were administered LGD-3303 treatment displayed an increase in the amount of time spent in the male areas of the arena. The opposite was true in the non-experienced female rats receiving LGD-3303 treatment; there was a significant decrease in the amount of time spent in the presence of the male test subjects.

Figure 1: Effects of LGD-3303 and DHT treatment on the amount of time spent in male area of the arena by sexually experienced and non-experienced female rats.

4) Due to the promising results of experiment 1, experiment 2 was conducted in order to identify changes in male-directed sexual behavior in female rats. For the purpose of this study the female rats were prepared by inducing sexual experience and hormone priming the animals without exposure to any of the experimental testing paradigms. Following ANOVA analysis of the data collected, the results four that both the 3 and the 30 mg/kg dose of LGD-3303 significantly enhance the preference of males in female test subjects. These results were identified in the female rats after both 1 dose of LGD-3303 was administered and after 7 doses were administered. This is important to note as it indicates that the effects of LGD-3303 were elicited in a manner that was not dependent on the dose administered to the subjects.

Figure 2: Effects of LGD-3303 treatment on male-directed preference and time spent on the male side of the arena by female rats. .

5) The third experiment conducted by the research team examined whether or not the effects of LGD-3303 are dependent on the activation of androgen receptors. Sexually experienced female rats were placed into different treatment groups and were either administered flutamide or a vehicle. Following pretreatment the rats were then given their treatment of DHT, LGD-3303, or a vehicle. The previous experiments concluded that LGD-3303 was able to increase the amount of time the female rats spent in the male areas of the area, however, initial results reported that the dose of flutamide decreased the amount of time the female rats spent in the presence of the males. This same effect was observed in hormone-primed females that were given flutamide. The researchers briefly mentioned that the analysis of the amount of time the subjects spent in the neutral area of the arena reported insignificant changes in either group.

Figure 3: Changes in time spent in male area of the arena by female rats in response to pretreatment with the androgen receptor antagonist, flutamide.

6) The fourth experiment observed changes in proceptive and lordosis behavior exhibited by sexually experienced and ovariectomized female rats. The research team observed a significant increase in instances of proceptive behavior in the female rats that had been hormone primed with 2.0 ug of estradiol benzoate. However, there were no notable effects elicited in the subjects primed with 0.5 ug of estradiol benzoate.

FIgure 4: The effects elicited by doses of DHT versus LGD-3303, on proceptive behavior in female rats primed with estradiol benzoate.

Effects similar to those observed in proceptive behavior were also identified in lordosis behavior exhibited by the female test subjects. When primed with 2 ug of estradiol benzoate there was a dramatic increase in lordosis behavior. It is important to mention that these results were recorded after 9 doses of the treatment were administered to the rats, rather than 1 dose. In the females primed with 0.5 ug of estradiol benzoate there were no discernable differences in lordosis behavior exhibited by the rats. This effect was observed in the test subjects regardless of whether they were administered LGD-3303 or DHT treatment.

Figure 5: Figure 4: The effects elicited by doses of DHT versus LGD-3303, on lordosis behavior in female rats primed with estradiol benzoate.

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] Vadja EG, Hogue A, Griffith KN, Chang WY, Burnett K, Chen Y, Marschke K, Mais DE, Pedram B, Shen Y, Oeveren A, Zhi L, Lopez FJ, Meglasson MD. “Combination Treatment With a Selective Androgen Receptor Modulator (SARM) and a Bisphosphonate Has Additive Effects in Osteopenic Female Rats*.” Journal of Bone and Mineral Research, vol. 4, no. 3, 2009. American Society for Bone and Mineral Research, https://asbmr.onlinelibrary.wiley.com/doi/pdf/10.1359/jbmr.081007.

[2] Kudwa AE, López FJ, McGivern RF, Handa RJ. A selective androgen receptor modulator enhances male-directed sexual preference, proceptive behavior, and lordosis behavior in sexually experienced, but not sexually naive, female rats. Endocrinology. 2010 Jun;151(6):2659-68. doi: 10.1210/en.2009-1289. Epub 2010 Apr 14. PMID: 20392832.

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