Main Research Findings of LGD-4033 SARMs

1) Results of this clinical study reported that treatment with LGD-4033 is considered safe and well-tolerated and has been shown to improve body composition and hormone expression.

2) The research team Hansson et. Al confirmed the presence of 8 different metabolites in the collected plasma and urine samples in the equine animal model.

 

Selected Data From LGD-4033 SARMs Study

How To Measure Varying Doses of LGD-4033 in Healthy Men?

1) Researchers Basaria et. Al conducted a double-blind controlled study to determine the efficacy of varying doses of LGD-4033 in healthy men. All subjects provided written, informed consent and the study was approved by Boston University’s Institutional Review Board. Subjects considered eligible were nonsmoking, generally healthy men, aged 21-50 years old, and with a BMI between 18/32 kg/m^2. Subjects could be disqualified for a number of reasons, such as, previous use of anabolic steroids or presence of prostate-specific antigen in levels greater than 3 ng/mL [1].

The subjects were randomly assigned to the active drug or placebo treatment group in accordance with the previously set randomization protocol. From there, three dose levels of LGD-4033 were administered to the subjects in comparison to a placebo group. The initial dose of LGD-4033 started at 0.1 mg; if the dose level was completed, all safety data was reviewed by a Data and Safety Monitoring Board as well as a Safety Panel to decide if the dose should be elevated to 0.3 mg.

The dose only increased if the compound met the specific safety criteria with no clinically significant occurrences of toxicity. The same procedures were followed for the 0.3 mg dose level, if completed the subjects moved on to the final dose level of 1.0 mg. Each treatment dose of LGD-4033 was administered daily with 8 ounces of water after an overnight fast. Over a 21 day treatment period 20 doses were administered; pharmacokinetic sampling was conducted on day 2, 48 hours after the first dose, so no treatment was administered. After 3 weeks of treatment the subjects were monitored by the research team for 5 additional weeks [1].

In addition to investigating the safety and tolerability of the SARM, the researchers examined how LGD-4033 affected lean body mass (LBM). LBM was measured by DEXA scans, 1 repetition maximum method (1RM), and stair climbing power. The researchers thought it was important to disclaim that 3 weeks may not be a long enough period of time to accurately measure the anabolic effects elicited by the SARM.

1RM was measured through leg press strength. The subjects went through a full body warm up, one set of 5-10 repetitions at 40%-60% of the estimated maximum, followed by proper rest time and progressively heavier sets until the subject reached failure. The last lift completed successfully was recorded as the 1 RM. Stair climbing power was assessed by the 12-step stair-climb test. The subjects would ascend a staircase, with a step rise of 17 cm, as fast as possible. Motion-activated switch-mats were placed on the 8th and 12th steps in order to record time. Power was calculated from the time elapsed during the recorded trials, the subjects body weight, and the vertical distance [1].

The rate of protein synthesis was measured through the collection of blood samples at various points throughout treatment. An 18-gauge catheter was placed into both forearms of each subject. One side was for blood sampling while the other was for tracer infusion. The collection and infusion took place on day 1 and day 20 of the 3 week treatment period and consisted of a constant infusion of phenylalanine maintained over the course of 6 hours. In addition to the venous blood sample, two muscle biopsies from the vastus lateralis were taken 3 hours and 6 hours into the phenylalanine infusion [1].

The muscle samples were weighed out and the proteins were precipitated using 800 uL of 10% sulfosalicylic acid. Additionally, the enrichment of intracellular phenylalanine was dependent on extraction with cation exchange chromatography and gas chromatography. The mixed-muscle proteins were washed, dried, and hydrolyzed over the course of 24 hours; all leftover amino acids were extracted and analyzed. This was followed by the calculation of the mixed-muscle fractional synthesis rate (FSR).

Finally, a hormone assay was completed in order to measure serum levels of testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and sex hormone-binding globulin (SHBG). Serum levels of testosterone were measured using liquid chromatography-tandem mass spectrometry, while serum LH, FSH, and SHBG were measured using two site-directed immunofluorometric assays [1].

2) The research team of Hansson et. Al utilized three female Thoroughbred horses in order to identify how LGD-4033 is metabolized in the body of a horse. The age of all three horses ranged from 3-6 years while the reported weight varied from 447 to 587 kilograms. Before any treatment was administered the horse underwent extensive blood testing and physical examinations in order to confirm the health of the test subject. All tests and analyses performed were done so in accordance with the Clinical Pathology Laboratory of the William R. Pritchard Veterinary Medical Teaching Hospital at the University of California, Davis. No additional supplements or medications were allowed two weeks before treatment began, however, the subjects had ad libitum access to food and water.

In order to administer LGD -4033 and collect the necessary samples, a 14-gauge catheter was placed in the external jugular vein of the subjects, bilaterally. One side was used for drug administration while the other was meant for sample collection. Following catheter placement, the 0.01 mg/kg dose of the SARM was administered to the subjects through intravenous bolus. Following drug administrations, urine and blood samples were collected from the horses in order to identify the presence of LGD-4033 metabolites in the samples.

Urine samples were collected right before drug administration, as well as 3, 6, 8, 12, 24, 48, 72, and 96 hours post-administration. After the samples were collected, 100 uL of the urine was mixed with 100 uL of 0.1% formic acid. Through the use of an Oasis HLB 60 mg cartridge, the researchers completed a solid-phase extraction (SPE). 2.0 mL of all urine samples were diluted with 2.0 mL of formic acid; a portion of the samples would then be subjected to hydrolysis, while the remaining samples were diluted further. For hydrolysis to occur, 2 mL of untreated urine was mixed with a phosphate buffer while 100 uL of beta-glucuronidase was applied. Samples were not diluted any further and prepared for SPE. The other group of samples were then conditioned with 2.0 mL MeOH, 2.0 mL of water, and 2.0 mL of formic acid prior to further washing and elution with 5% MeOH. The samples evaporated to dryness and prepared for final analysis [2].

Similar to the procedure followed with urine samples, blood samples were collected immediately before administration of LGD-4033 and at 5, 10, 15, 30, and 45 minutes, as well as 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 18, 24, 48, 72, and 96 hours post-administration. All samples were collected using the jugular vein catheter until 24 hours post-administration; after that point all collections were obtained via direct venipuncture.

For analytical purposes, 1.0 mL of the plasma sample was added to 20 uL of the SARM, S22. The researchers noted that this is a crucial step as it develops an internal standard. The samples were then treated with 4 mL of ice cold acetonitrile, followed by 20 minutes in the refrigerator and 10 minutes of centrifugation. The resulting supernatant was transferred to new testing equipment and reconstituted in 200 uuL of formic acid following evaporation to dryness [2].

 

Discussion

What Effect Did  Varying Doses of LGD-4033 Have in Healthy Men?

1) Overall the study conducted by Bararia et. Al concluded that LGD-4033 was safe and well-tolerated by the test subjects. Any side effects observed were similar between the experimental and placebo groups. Subjects receiving the 1.0 mg treatment of LGD-4033 reported cases of upper respiratory tract infection, however, researchers were able to determine it was not related to the drug study. Researchers reported that when comparing day 1 to day 21, LGD-4033 accumulated in the system in a dose-proportional manner. These findings were supported by serum measurements of LGD-4033 that report a three-fold increase in concentration of the SARM from day 1 to day 21 [1].

The secondary goal of this study was to examine how LGD-4033 affects various aspects of body composition. This study is specifically viewing variations in plasma lipids, lean body mass (LBM), skeletal muscle mass, and fat mass. Results found that in the 0.3 and 1 mg treatment groups, high density lipoprotein (HDL) experienced a significant reduction from baseline levels, while total cholesterol and low density lipoprotein (LDL) cholesterol did not show any significant changes from baseline. HDL levels returned to baseline after treatment had ended. Additionally, triglyceride levels significantly decreased in response to all three of the different treatment doses [1].

There were also significant changes observed in LBM, fat mass, and skeletal muscle mass in response to treatment. LBM increased in response to the 1.0 mg dose of LGD-4033; this treatment group experienced an average increase of 1.21 kg LBM. There were no significant changes observed in fat mass in response to any of varying treatment doses. Researchers noticed slight increases in skeletal muscle mass, however, all experimental values were similar to the placebo group, indicating that variation between treatment groups was insignificant.

Increases in strength in response to LGD-4033 treatment was measured by 1RM test and a stair-climbing test. In the 1.0 mg treatment group average strength increased in the test subjects by approximately 68.3 N. The research team observed a slight improvement in strength, however, the difference from baseline strength levels was deemed not statistically significant. Results of the study reported that the stair-climbing speed and power test exhibited elevations that seemed to be related to dose-related improvements, however, similar to the 1RM max test, changes from baseline were considered insignificant [1].

When observing fractional mixed-muscle protein synthesis rats through phenylalanine infusion, the researchers did not identify any change in plasma phenylalanine concentrations at the 3 hour or 6 hour mark. Baseline levels averaged 6% an hour, when determining FSR, but in terms of the study, there were no significant changes displayed when comparing the 0.3 mg dose of LGD-4033 and the placebo groups. Furthermore, results of the hormonal assay test revealed that total testosterone and SHBG were suppressed in a dose-dependent manner when comparing day 1 to day 21. The researchers also noted that suppression of free testosterone was only seen in the subjects receiving a 1.0 mg dose of LGD-4033. No significant changes were seen in serum LH levels, however, levels of FSH in the 1.0 mg treatment group were found to be suppressed. Hormone levels returned to their baseline values after treatment with LGD-4033 was discontinued [1].

2) The primary focus of this study was to identify any metabolites of LGD-4033 present in the plasma and urine of three Thoroughbred horses, following treatment with the SARM. The ionization and fragmentation of LGD-4033 resulted in formation of the ion [M+HCOO]-. This main ion that was formed is an adduct of the parent compound, LGD-4033, and formic acid; it was detected at an m/z of 383 and could be removed by using higher cone voltage. It is important to note that the use of a higher cone voltage resulted in an in-source fragmentation detected at m/z 267. Retention time of LGD-4033 was measured at 10.77 min. Overall the research team observed that the parent compound of LGD-4033 was only found in the plasma and the hydrolyzed urine samples [2].

Metabolites of LGD-4033 were identified by observing the mass and fragmentation pattern of the parent ion. When analyzing the urine samples collected from the horses, 8 total metabolites were found, ranging from M1a-M5b. Two of the metabolites had only undergone phase I metabolism while the other 6 of them were considered phase II metabolites. M1a-c were denoted by three different peaks with their signals represented at m/z 529.1047. The three metabolites were found to share the same exact mass as well as the same product ions, however, the product ions were detected at different relative intensities. The research team concluded that the metabolites they identified were isomers.

After the urine samples were hydrolyzed through the use of beta-glucuronidase, metabolites M1a-M1c were no longer detected in the samples. This allowed researchers to define M1a-c as glucuronides. However, there were three peaks observed that exhibited masses corresponding to deprotonation of M2 aglycons. The retention times of the three peaks were recorded to be 7.50, 7.94, and 8.75 minutes, respectively. This indicates that the metabolites are represented as diastereomers or positional isomers. The research team noted that the reported results suggested that the metabolites M1a-c underwent a hydroxylation reaction on the pyrrolidine ring. Furthermore, the results of this analysis correspond with the product ions that had previously been reported for LGD-4033 hydroxylated at the pyrrolidine ring [2].

Figure 1: Ion chromatography for the m/z of LGD-4033 and related metabolites found in experimental horse urine samples.

The M2 metabolite identified in the urine sample had a recorded mass of m/z 545.0997. The mass of the metabolite is related to the deprotonation, dihydroxylation, and glucuronidation of LGD-4033. Out of all metabolites observed, M2 exhibited the lowest intensity; the compound was only identifiable after SPE was performed. The detected fragments reported m/z of 185 and m/z of 170, indicating that both of the hydroxyl groups had been positioned on the pyrrolidine ring. Glucuronic acid was successfully removed once the samples had been hydrolyzed by the administration of beta-glucuronidase. Additionally, there was no peak detected for M2 suggesting that the metabolite was not found in the sample after hydrolyzation [2].

Metabolite M3 was detected with an accurate mass of m/z 385.0625. The metabolite is considered trihydroxylated due to the formation of a product ion following the loss of C4H5F3O3, as well as the identification of corresponding product ions for metabolite M1. On the other hand, metabolite M4 was detected with an accurate mass related to the dihydroxylation of LGD-4033. The main product ion was found at m/z 237, however, this was observed after the ion lost water, CO2, and an HCF3 fragment.

The last two metabolites identified, M5a and M5b, were detected with the same masses and fragmentation patterns. The accurate mass of these metabolites was m/z 513.1105; this corresponded to the conjugation of glucuronic acid to LGD-4033, a claim further supported by the presence of a product ion at m/z 337. M5a was no longer detected in the urine after the sample was treated with beta-glucuronidase, however, there was a peak detected for LGD-4033. The intensity of M5b was not reduced at all after treatment with beta-glucuronidase. This suggests that M5b could potentially act as an N-glucuronide, suggesting it is not susceptible to hydrolysis induced by beta-glucuronidase [2].

When identifying metabolites found in the plasma, the research team was able to find the parent compound, LGD-4033, as well as 6 other metabolites, M1a-c, M4, and M5a-b. These metabolites exhibited the same accurate masses and retention times as they did in the urine samples, however, the identifiable peaks displayed higher intensity. This is interesting to note considering that plasma concentrations are typically lower than urine concentrations which often leads to reduced peak intensity. Additionally, the product ions detected matched the observations the researchers made when analyzing the urine samples. In the plasma samples, the highest intensity was reached approximately 15-45 minutes post-administration, suggesting a rapid transformation of LGD-4033 [2].

Figure 2: Peak area ratio for LGD-4033 and metabolites observed in horse 1

 

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] Basaria S, Collins L, Dillon EL, Orwoll K, Storer TW, Miciek R, Ulloor J, Zhang A, Eder R, Zientek H, Gordon G, Kazmi S, Sheffield-Moore M, Bhasin S. The safety, pharmacokinetics, and effects of LGD-4033, a novel nonsteroidal oral, selective androgen receptor modulator, in healthy young men. J Gerontol A Biol Sci Med Sci. 2013 Jan;68(1):87-95. doi: 10.1093/gerona/gls078. Epub 2012 Mar 28. PMID: 22459616; PMCID: PMC4111291.

[2] Hansson A, Knych H, Stanley S, Berndtson E, Jackson L, Bondesson U, Thevis M, Hedeland M. Equine in vivo-derived metabolites of the SARM LGD-4033 and comparison with human and fungal metabolites. J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Feb 1;1074-1075:91-98. doi: 10.1016/j.jchromb.2017.12.010. Epub 2017 Dec 7. PMID: 29334634.

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