Main Research Findings for LGD 3303

1) LGD-3303 is capable of stimulating various aspects of sexual desire in female gonadectomized rats.

2) LGD-3303 enhances BMD, BMC and muscles mass without eliciting negative side effects in male and female gonadectomized rats.

Selected Data

1) 4 different experiments were conducted by researchers Kudwa et. Al, utilizing 2-3 month old female Sprague-Dawley rats purchases from Charles River Laboratories. The subjects were kept on a 12 hour dark, 12 hour light schedule and provided with a free range of food and water. Sexually active female subjects were separated into groups of 3 while sexually active male subjects were housed individually. The experimental animals were housed in same treatment pairs. All female test subjects underwent a bilateral ovariectomy; the rats were given a week to recover from surgery and assigned to treatment groups for experimentation [1].

In experiments 1 and 2, researchers attempted to determine the difference in sexual preference in rats with and without sexual experience, after treatment with LGD 3303 versus DHT. DHT was administered to the subjects through a 2-cm SILASTIC brand capsule packed with crystalline DHT placed under the skin. LGD-3303 was administered to the rats through an oral savage in a suspension of Tween 80, polyethylene glycol-400, and 0.1% carboxyl-methyl cellulose. In experiment 3, 100 mg/kg of the LGD-3303 antagonist, flutamide was administered via subcutaneous injection in order to compare male sexual preference in LGD-3303-treated animals before and after administration of the antagonist.
Additionally, a single 2 mg/kg dose of DHT was subcutaneously injected into the test subjects. After the experimentation period the animals were euthanized so blood and brains could be collected for plasma DHT analysis [1].

Furthermore, sexual preference tests were performed in experiments 1-3 under red-light conditions during the 12-hour dark period. The apparatus developed to test sexual preference consisted of a center open-field arena with stimulus areas positioned opposite each and separated from the open-field by wire mesh. This structure is important to note as it allows for chemosensory and visual/auditory interaction between the subjects without direct physical interaction. In all experiments the testing began 3 hours after the beginning of the 12-hour dark period. The animals were set into the center of the open-field arena while their behavior was observed by an overhead camera. The tests took place over the course of 10 minutes while the subject was exposed to a sexually active male, and an OVX female, primed to facilitate optimal displays of sexual behavior [1].

In experiment 4, sexual behavior tests were conducted under red-light conditions during the 12-hour dark period. However, the peripheral injections and oral gavage were administered during the 12-hour light portion. The testing occurred in a plexiglass, circular area; stud males were habituated to the area an hour before testing while low levels of sexual behavior were induced in female subjects by administration of estradiol benzoate and progesterone. Researchers chose to induce low levels of sexual function in order to see how sexual behavior is affected by administration of LGD-3303 by itself. Hops, darts, and ear wiggles were observed and marked as proceptive behavior. Number of mount attempts by male subjects and number of lordosis displays by the female subjects were recorded as well [1].

2) Researchers Vadja et. Al conducted two separate studies in order to examine the anabolic effects on muscle and bone elicited by treatment with LGD-3303. The first portion of this study included female Sprague-Dawley rats that were approximately 3 months old, and weighing 175-200 grams. The rats were housed 2-3 to a cage prior to random surgery assignment. Before treatment began the rats were scheduled to be ovariectomized, or they underwent a sham-operation. In order for sufficient levels of osteopenia to develop the rats were left untreated for seven weeks.

After the 7 week period of no treatment, the female rats were split into 5 treatment groups. The treatment groups were organized by whether they were ovariectomized and what treatment they received; the groups include 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. The treatments were administered to the subjects daily through an oral gavage over an experimental period of 12 weeks [2].

The researchers took a baseline DXA scan before the onset of treatment in the test subjects. The scans were taken of every animal and included the lumbar spine and the right femur. In addition to the DXA scans, Calcein was subcutaneously injected in the animals 3 and 10 days before euthanasia occurred. At week 11 weeks after the onset of treatment, a second DXA scan was taken. A third scan was obtained after the animals were euthanized 12 weeks after the treatment began. Following euthanasia the wet weights of the animals’ gastrocnemius muscles and the inguinal fat pads were recorded. Bones of the animals were harvested in order for further DXA, histomorphometric, and biomechanical analyses to take place [2].

Blood was collected from the subjects at the onset of treatment, after 5 weeks, and after 12 weeks of treatment. Samples were collected by jugular puncture in anesthetized animals in order to measure serum levels of osteocalcin. Osteocalcin levels in the blood were measured via immunoradiometric assay.

DXA scans taken after treatment ended primarily focused on analyzing changes in the mid femur and lumbar region. The scans encompassed specific bone regions: the L3-L5 region of the lumbar spine, and an undetermined rectangular region-of-interest of the mid-femur. When referring to the mid-femur, researchers define the region as 10% of the total femoral length at the center of the femoral shaft.

Biomechanical testing included the removal of the right femur and 5th lumbar vertebrae from the subject. The whole femur was tested to complete fatigue in a three-point-bend test, while the 5th lumbar vertebrae was tested to complete fatigue in compressions. Data was collected at 10 Hz from the cross-head displacement and load cell. The data was analyzed using the software TestWorks 4; MTS. Furthermore, the research team measured maximum load, stiffness, and absorption of energy for each specimen in order to develop a load-deformation curve.

Cortical and cancellous bone samples were taken from the mid-femoral diaphysis and the lumbar spine, respectively, in order to complete histomorphometric testing. Various measurements of bone formation, such as periosteal perimeter and cortical thickness, were recorded in order to determine the anabolic effect LGD-3303 has on bone segments. In the cancellous bone segments obtained from the lumbar spine various measurements of bone formation, such as bone perimeter and tissue area, were recorded as well.

The second experiment conducted throughout the course of this study included 7-8 week old male Sprauge-Dawley rats. The subjects were organized by body weight and housed 2-3 rats per cage prior to the performance of any surgical procedures. The animals were randomly selected to receive either an orchidectomy (ODX) or undergo a sham-operation. Following surgery the rats were left completely untreated, hypogonadic state in order to ensure atrophy of the levator ani and the prostate. After 2 weeks, treatment of either testosterone propionate or LGD-3303 began for the male rats.

Either LGD-3303 or a vehicle treatment was administered to the rats once daily through an oral gavage. The SARM was provided in a suspension of Tween-80, glycol-400, and 0.1% carboxymethyl cellulose in water. For the group of test subjects receiving testosterone propionate, the treatment was administered once daily through subcutaneous injection. Prior to injection, testosterone propionate was dissolved in a solution of polyethylene glycol-400 and DMSO. The effects of testosterone in the experimental rats were compared to a group of rats that received a subcutaneous injection of a vehicle treatment. The male rats were treated with either testosterone propionate or LGD-3303 over the course of 14 days. At the end of the experimental period they were euthanized in order for the researcher to record the weight of the levator ani muscle and the prostate [2].

Discussion

1) Experiment one conducted by researchers Kudwa et. Al examined the ability of LGD 3303 to enhance sexual preference of females with sexual experience for males. ANOVA testing analyzed the time spent by each animal in the male, female, and neutral areas of both experienced and non-experienced animals. Preference tests were performed after 7 days of LGD-3303 dosing; results of the tests reported that there was a significant correlation between sexual experience and experimental treatment. For example, non-experienced females treated with DHT spent more time with males than the other groups. However sexual experience in the same DHT-treated female experience led to a decrease in time spent with the male subjects. The opposite was true in the females treated with LGD-3303; non-experienced female subjects exhibited a decrease in time spent with males while sexual experience actually caused a drastic increase in the time the subjects spent in the male area of the area [1].

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

The results of experiment 1 indicated that LGD-3303 is more effective at improving sexual preference of females for males in sexually experienced female subjects. Experiment 2 was designed to observe changes in male-directed sexual behavior in hormone-primed, sexually experienced female rats that have not been exposed to experimental testing paradigms. After analysis of the data conducted using ANOVA, the results reported that both the 3 and 30 mg/kg doses of LGD-3303 enhanced male preference in females to a significant level. It is important to note that these results were seen in the rats after 1 dose and after 7 doses indicating that the results were elicited in a manner independent of the dosing [1].

Figure 2: Effects of LGD-3303 treatment on sexual preference and time spent on the male side of the arena.

Experiment 3 attempted to determine the effects of LGD-3303 in regards to activation of the androgen receptors. LGD-3303 was administered to sexually experienced female rat subjects; one group received the androgen receptor antagonist, flutamide, while the other did not. LGD-3303 was administered to animals after they were pretreated with flutamide. Experiment 2 revealed that LGD-3303 increased the amount of time females spent in the male areas of the arena, however, this was blocked in the subjects that received a dose of flutamide. LGD-3303 was also found to increase the amount of time hormone-primed female rats spent in the female area. This effect was inhibited in the presence of flutamide. Additionally, the researchers completed the analysis of time spent in the neutral area of the arena and reported that there were no specific effects elicited in either group [1].

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

Finally, experiment 4 examined lordosis behavior in OVX sexually experienced female rats. The rats treated with 0.5 ug of estradiol benzoate did not experience any significant changes in proceptive behavior. However, the rats primed with 2.0 ug of estradiol benzoate exhibited dramatic increases in proceptive behavior.

Figure 4: Effects of the varying doses of LGD-3303 versus DHT, on proceptive behavior in OVX female rats primed with estradiol benzoate.

The researchers noticed that lordosis also increased significantly in the female rats primed with 2 ug of estradiol benzoate. These results were recorded after the subjects were administered 9 doses of the treatment rather than just 1 dose of treatment. Additionally, there were no significant changes in lordosis behavior that occurred in either the LGD-3303 or the DHT treatment groups, primed with 0.5 ug estradiol benzoate [1].

Figure 5: Effects of the varying doses of LGD-3303 versus DHT, on lordosis behavior in OVX female rats primed with estradiol benzoate.

2) The research team reported an increase of muscle mass measured in the gastrocnemius muscle in the test subjects. The rats also exhibited a noticeable reduction in inguinal fat pad. These results were observed in the rats treated with both LGD-3303, itself or the combination treatment of LGD-3303 + alendronate.

Figure 6: Changes in body weight, muscle weight, and inguinal fat pad weight in response to the different treatments.

In order to determine the effects of LGD-3303 on BMC and BMD, the researchers performed DXA scans of the femur and the lumbar spine. The research mentions that the effects of LGD-3303 were elicited on both cancellous and cortical bone. The lumbar spine is primarily composed of cancellous bone; the scans reported that treatment with LGD-3303 significantly increased BMD in the cancellous bone region. BMC was also elevated, but to a lesser extent. Treatment with LGD-3303 resulted in similar increases in BMD and BMC, however, the combination treatment of LGD-3303 + alendronate raised BMD to a level that was similar to the vehicle-treated, sham-operation test subjects.

In addition to the cancellous bone of the lumbar spine, DXA scans were taken of the mid-femoral diaphysis, a region consisting primarily of cortical bone. The results reported that LGD-3303 led to a significant increase in BMC; this was compared to alendronate treatment alone, and a vehicle treatment. When combining LGD-3303 and alendronate, the recorded effects of the SARM were elicited in a manner similar to that of LGD-3303 by itself. While there wasn’t a large difference between LGD-3303 and the combination treatment, both methods were more effective that treatment with alendronate only [2].

When observing the mid-femoral diaphysis, the histomorphoectomy tests revealed there were significant increases in the rate of periosteal bone formation after treatment with LGD-3303. The researchers concluded that any growth that occurred in this area was due to osteoblast activation and anabolic activity since the mid-femoral diaphysis region is not a site of osteoclast remodel. Results from both cortical and cancellous bone regions reported that treatment with LGD-3303 and LGD-3303 + alendronate led to improvements in BMD and BMC. However, there were no significant changes observed in periosteal bone growth when the subjects were treated with alendronate alone [2].

Biomechanical tests conducted on the femurs of the subjects found that LGD-3303 treatment, alendronate treatment, and combination treatment led to an improvement in peak load mid-femur. All three treatment groups showed enhanced performance; the largest increase in peak loading was seen in the combination treatment, however, differences between the results of the other treatment groups were not great enough to be considered significant. Biomechanical studies conducted on the lumbar spine observed how peak compressive load responded to the different treatments. Similar to the peak loading in the mid-femur, the combination treatment led to the largest increase in peak compressive load in the lumbar spine. Results between the other treatment groups were not great enough to be considered significant [2].

Figure 7: Changes in bone mineral content and bone mineral density in response to different treatments.

The second portion of this study examined how anabolic activity changed in male ODX rats when varying doses of LGD-3303 or testosterone propionate were administered. Subjects received a 1 mg/kg dose of either testosterone propionate of LGD-3303; the researchers conclude that both treatments are equally effective since data shows that the levator ani muscle maintained eugonadal levels. However, the 1 mg/kg dose of testosterone propionate stimulated growth of the ventral prostate up to 50% of the eugonadal levels. The 3 mg/kg dose of testosterone propionate significantly exceeded eugonadal levels, indicating that the effects of testosterone propionate treatment are elicited with minimal selective tissue activity [2].

The results of testosterone propionate treatment are compared to the use of the 1 mg/kg dose of LGD-3303. This dose of the SARM did not lead to increased growth of the ventral prostate, measured at <5% efficacy. There were no drastic changes in ventral prostate growth when LGD-3303 was administered in higher doses. While 1 mg/kg of testosterone propionate resulted in a 50% restoration of the prostate eugonadal levels, a 100 mg/kg dose of LGD-3303 was needed to reach that same level. Overall, the researchers concluded that both testosterone propionate and LGD-3033 are potential of promoting anabolic activity, specifically in the levator ani muscle. However, the data reported that when treated with testosterone propionate the prostate grew at the similar rate as the muscle. When treating the subjects with LGD-3303 the researchers determined that the SARM acts a partial agonist to the prostate resulting in a reduced rate of growth [2].

Figure 8. Changes in prostate and levator ani weight in response to testosterone propionate treatment or LGD-3303 treatment.

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] 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.

[2] 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.

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