Main Research Findings

1) The process of esterification leads to increased potency and improved efficacy of RAD 150 in comparison to its parent compound, RAD 140.

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

3) RAD140 treatment led to an increased stimulation of the levator ani muscle in a manner similar to testosterone treatment without significant stimulation of the prostate and seminal vesicles.

4) The study conducted by Miller et. Al determined that RAD140 was capable of increasing anabolism in primates without the adverse androgenic side effects associated with testosterone treatment.

 

Selected Data

1) As it was previously mentioned, RAD 150 is the esterified form of RAD 140. RAD 140, like most SARMs, is typically known for its ability to promote anabolic activity in bone and muscle tissue. Current research examines the potential of RAD 140 to enhance neuroprotection and stimulate antitumor activity. As an esterified version of RAD 140, RAD 150 has been shown to elicit similar effects as its parent compound with far more potency. The esterification process proceeds by combining an organic acid (RCOOH) and an alcohol (ROH) to form water and an ester (RCOOR) [1].

Furthermore, esterification reactions occur when a primary alcohol is treated with a carboxylic acid in the presence of sulphuric acid. The resulting compound tends to smell sweet and is generally classified as an ester compound. The chemical reaction typically takes place in 5 steps and follows the format:
CH3COOH + CH3CH2COOH → CH3COOCH2CH3

The esterification process occurs one of three ways: 1) from acid anhydride and alcohol, 2) from acid chloride and alcohol, or 3) from carboxylic acid and alcohol. The reaction between acid anhydride and alcohol is slower than the reaction between acid chloride and alcohol. In order to effectively produce ester compounds the mixture requires the addition of a reagent to warm the fixture. For example 2,6-diiodophenol is commonly used in the acid anhydride reaction to form the ester. The esterification process between acid chloride and alcohol can be performed at room temperature. Finally, the production of esters from carboxylic acid and alcohol requires heating in the presence of an acid catalyst like sulphuric acid [1].

Figure 1: acid anhydride and alcohol

Figure 2: acid chloride and alcohol

Figure 3: carboxylic acid and alcohol

The resulting ester compounds typically have a pleasant smell, leading to widespread usage in the perfume, food flavoring, and cosmetics industries. They are also organic compounds typically found in oils and fats that are able to be used and an organic solvent.

2) 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 [2].

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 [2].

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 [2].

3) Initially, RAD 140 was characterized through the use of in vivo assay in order to determine its efficacy on androgenic activity in both castrated and intact male rats. Young castrated male rats were preferred in this experiment because they still qualified as androgen-naive. Essentially, the rats provided a blank canvas for the researchers to examine the effects elicited by RAD 140 and testosterone on androgenic activity.

Based on the results of the initial experiment the research team of Miller et. Al observed any potential antagonist effects RAD 140 elicits on testosterone propionate (TP). Additionally they examined the changes found in the levator ani muscle when the castrated rats were administered a combination treatment of RAD 140 and TP [3].

In order to gain a full understanding of the androgenic effects of RAD 140 compared to testosterone, the researchers turned their attention to young intact male rats that have only slightly reduced levels of endogenous testosterone. This results in the retention of prostate sensitivity, however, baseline stimulation is similar to levels observed in the castrated rats. The intact rats were given doses of TP and RAD 140 varying from 0.1 mg/kg to 30 mg/kg in order to record the response in muscle growth [3].

4) Researchers Miller et. Al evaluated the effects RAD 140 had in young, male monkeys. The study attempted to identify anabolic changes as well as alterations made to lipid and other chemistry parameters. In order to measure metabolic changes, the weight of the monkeys was recorded since body weight is a strong anabolic androgen marker in nonhuman primates.

The small group sizes allowed the researchers to use the background weight change measured in the weeks leading up to the study to set a baseline body weight. The baseline was then established as the control variable that the experimental groups were being compared to. In addition to body weight, DEXA scans were taken of the monkeys two days before treatment began, and one day after the final dose. For research purposes these days were marked as day 2 and day 29 of the study. These scans were performed in order to show changes in lean tissue and fat as a result of treatment with RAD 140 [3].

Discussion

1) As an esterified version of RAD 140, RAD 150 is considered a superior version of its parents compound due to its increased potency and improved bioavailability. The esterification process also makes RAD 150 far more stable than RAD 140 suggesting that it is more effective and safe. While the benefits of the two compounds are very similar, there is debate regarding how much stronger RAD 150 is compared to RAD 140. Recent evidence suggests that RAD 150 is approximately 10-15% more potent.

Multiple research-based studies have concluded that SARMs are able to increase muscle mass and reduce fat, however, researchers thought it was important to note that RAD 150 does not reduce fat by targeting fat directly, but rather by raising metabolic rate. RAD 150 increases testosterone leading to improved metabolism and a calorie deficit, ultimately resulting in fat loss. Additionally, evidence suggests that RAD 150 is capable of increasing libido, improving recovery from exercise by increasing protein synthesis, and enhancing physical performance by boosting muscle mass and raising metabolism.

2) 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 [2].

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 [2].

3) In vivo assays found that oral administration of increasing doses of RAD 140 led to significant stimulation of the levator ani muscle. These results are seen with doses as small as 0.03 mg/kg. This is compared to the vehicle-treated control group, and the group receiving TP treatment. While it takes 3 mg/kg of RAD 140 to reach the same level of muscle growth in the levator ani as 1 mg/kg of TP, prostate growth and accompanying side effects were significantly reduced in the rats administered the SARM. The researcher thought it was important to note that RAD 140 did not produce a high level of stimulation in the prostate or seminal vesicles, regardless of how high the dose was [3].

Figure 4: Prostate weight versus weight of the levator ani muscle in response to different treatments.

When examining the antagonist effects of RAD 140 against testosterone propionate in the the researchers found that doses greater than 10 mg/kg of RAD 140 antagonizes the effects elicited by 1 mg/kg of TP. However, it is important to note that the antagonistic effects of RAD 140 were seen only in the seminal vesicles; co-administering RAD 140 with TP actually led to an increase in the effects of TP, specifically in the levator ani muscle. The research team of Miller et. Al were able to conclude that in doses of 0.3-1 mg/kg, RAD 140 elicits antagonistic effects for 1 mg/kg of testosterone. A slight reduction in stimulation due to TP was seen in the prostate when co-administered with RAD 140, however, the changes were deemed not of statistical significance. Overall, RAD 140 acts as an agonist to the levator ani and a partial antagonist to the seminal vesicles, and to a lesser extent, the prostate [3].

Figure 5: Visual representation of the tissue-selective activity of RAD 140

Finally, results of RAD 140 treatment in intact male rats found that the SARM increased the weight of the levator ani with no stimulation of the prostate. Weight of the levator ani muscle was shown to increase with the smallest dose of RAD 140 administered, 0.1 mg/kg. The lack of prostate stimulation was observed for every dose of RAD 140 ranging from 0.1 mg/kg to 30 mg/kg. A notable detail of the study explains that a 30 mg/kg dose of RAD 140 was needed in order to reach the same levels of prostate growth elicited with a 0.5 mg/kg dose of TP. When the subjects were given doses 0.3 mg/kg, the researchers reported that RAD 140 resulted in muscle efficacy at levels similar to that of TP administered in doses of 0.5 mg/kg [3].

FIgure 6: Weight of the levator ani in response to varying doses of TP and RAD 140

4) Anabolic activity was assessed through measuring the body weight of the monkeys. After the monkeys were administered with a 0.01, 0.1, or 1 mg/kg doses of RAD 140, body weight was found to increase. The more significant increases in body weight were elicit by the 0.1 and 1 mg/kg dose of RAD 140 while the 0.01 mg/kg dose only resulted in a slight elevation in body weight.

Figure 7: Changes in primate body weight in response to the different treatment dosages.

The researchers established a baseline weight to act as the control group, for comparison purposes. The absolute body weight that was found between the treatment treatments groups ranged between 4.26 and 4.29 kg. With doses of 0.1 mg/kg of RAD 140 over the course of 28 days, the primates exhibited a 10% increase in mean weight. Similar effects were seen with the 1 mg/kg treatment group. Furthermore, the DEXA scans established that there was a clear pattern of fat loss changes elicited by RAD 140 treatment. In comparison, muscle was shown to increase in correlation with dose increase. The researchers concluded that the majority of mass increase was due to an increase in lean mass, however, it is important to note that while the trend was identified the differences in tissue weights were not considered statistically significant [3].

Figure 8: Changes in tissue weight elicited by RAD 140 treatment, measured by DEXA scan analysis

 

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] “Esterification (Alcohol & Carboxylic acid) – Reactions Mechanism & Uses with Videos.” Byju’s, https://byjus.com/chemistry/esterification/. Accessed 31 May 2023.

[2] 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.

[3] Miller CP, Shomali M, Lyttle CR, O’Dea LS, Herendeen H, Gallacher K, Paquin D, Compton DR, Sahoo B, Kerrigan SA, Burge MS, Nickels M, Green JL, Katzenellenbogen JA, Tchesnokov A, Hattersley G. Design, Synthesis, and Preclinical Characterization of the Selective Androgen Receptor Modulator (SARM) RAD140. ACS Med Chem Lett. 2010 Dec 2;2(2):124-9. doi: 10.1021/ml1002508. PMID: 24900290; PMCID: PMC4018048.

 

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