AR/androgen receptor (Ki = 1 nM)
The primary target of LGD-4033 is the androgen receptor (AR), to which it binds with very high affinity (Ki = 1 nM). As a nonsteroidal selective androgen receptor modulator (SARM), LGD-4033 induces conformational changes upon AR binding, promoting coactivator recruitment and activating downstream gene transcription. The tissue selectivity of LGD-4033 arises from differences in coregulator expression profiles across various tissues: it acts as a full agonist in skeletal muscle and bone, promoting muscle protein synthesis and bone formation, while exhibiting partial agonist or antagonist activity in the prostate and seminal vesicles, thereby avoiding prostate hyperplasia and other side effects associated with traditional androgen replacement therapy. This selectivity enables the compound to maintain anabolic efficacy while significantly reducing androgen-related adverse effects.

The use of selective androgen receptor modulators (SARM) in sports is prohibited by the World Anti-Doping Agency (WADA) due to their potential as performance-enhancing drugs, offering an unfair advantage. LGD-4033 is a SARM known for its similarities to anabolic steroids and can be easily purchased online, leading to increased availability and misuse. Adverse analytical findings have revealed the presence of SARMs in dietary supplements. Although LGD-4033 misuse has been reported in human sports over the years, concerns also arise regarding its illicit use in animal sports, including camel racing. Although various studies have investigated the metabolism of LGD-4033 in humans, horse, and other species, there is limited research specifically dedicated to racing camels. The findings indicated the presence of 12 phase I metabolites and 1 phase II metabolite. Hydroxylation was responsible for the formation of the main phase I metabolites that were identified. A glucuronic acid conjugate of the parent drug was observed in this study, but no sulfonic acid conjugate was found. The possible chemical structures of these metabolites, along with their fragmentation patterns, were identified using MS. Conclusions: These findings provide valuable insights into the metabolism of LGD-4033 in camels and aid in the development of effective doping control methods for the detection of SARMs in camel racing[2].

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LGD-4033 exhibits potent and highly selective androgen receptor binding activity in vitro. Radioligand binding assays determined a Ki value of 1 nM for LGD-4033 binding to the human androgen receptor (AR), demonstrating extremely high affinity. In functional transcriptional activation assays, LGD-4033 shows strong agonistic activity in AR-mediated reporter gene assays, with efficacy comparable to the natural androgen dihydrotestosterone (DHT). For other nuclear receptor family members including estrogen receptors (ERα, ERβ), progesterone receptor (PR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR), no significant binding activity was observed at concentrations up to 10 μM, demonstrating excellent receptor selectivity. Metabolic transformation of LGD-4033 has been detected in human seminal vesicle cellular fractions, although metabolic activity is limited, with primary metabolism occurring via CYP450 enzymes in the liver.

In the 3-mg ligandrol group, bone structural properties were improved (trabecular number: 38 ± 8 vs. 35 ± 7 (femur), 26 ± 7 vs. 22 ± 6 (L), 12 ± 5 vs. 6 ± 3 (tibia) and serum phosphorus levels (1.81 ± 0.17 vs.1.41 ± 0.17 mmol/l), uterus (0.43 ± 0.04 vs. 0.11 ± 0.02 g), and heart (1.13 ± 0.11 vs. 1.01 ± 0.08 g) weights were increased compared to the OVX group. Biomechanical parameters were not changed. Low and medium doses did not affect bone tissue and had fewer side effects. Body weight and food intake were not affected by ligandrol; OVX led to an increase in these parameters and worsened all bone parameters. Conclusion: Ligandrol at high dose showed a subtle anabolic effect on structural properties without any improvement in biomechanical properties of osteoporotic bones. Considering side effects of ligandrol at this dose, its further investigation for the therapy of postmenopausal osteoporosis should be reevaluated[1].

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LGD-4033 demonstrates significant anabolic activity in various animal models and clinical trials. In a randomized, double-blind, placebo-controlled clinical trial in healthy male volunteers, 21 days of oral administration of 0.1, 0.3, and 1.0 mg LGD-4033 resulted in dose-dependent increases in lean body mass without significant changes in prostate-specific antigen (PSA), indicating minimal androgenic activity in prostate tissue. In an ovariectomized (OVX) rat model of osteoporosis, 5 weeks of oral administration of 3 mg/kg LGD-4033 improved bone structural parameters, including increased trabecular number and thickness, without improvement in biomechanical properties; serum phosphorus levels, uterine weight, and heart weight were increased in the treatment group, suggesting some side effects at the high dose. The low doses of 0.03 and 0.3 mg/kg were reported to have fewer side effects but no significant effects on bone tissue. In equine studies, in vivo metabolites of LGD-4033 were identified, with eight phase I and phase II metabolites detected, of which a dihydroxylated metabolite represents the major metabolite in human urine.

This study focuses on the in vitro metabolism of LGD-4033 in homogenized camel liver using liquid chromatography–high-resolution mass spectrometry (LC-HRMS) to identify and characterize the metabolites[2].

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The AR binding affinity of LGD-4033 is determined by radioligand competitive binding assays. The experimental procedure is as follows: Recombinant human androgen receptor (AR) ligand-binding domain (LBD) is incubated with a fixed concentration of radiolabeled ligand (typically ³H-dihydrotestosterone, ³H-DHT, approximately 0.5-1 nM) in a buffer containing 50 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 10% glycerol, and 1 mM DTT. Increasing concentrations of unlabeled LGD-4033 (0.01 nM–10 μM) are added as competitors, and the mixture is incubated overnight (16-18 hours) at 4°C to reach binding equilibrium. After incubation, a charcoal-dextran suspension is added to adsorb free radioligand, followed by centrifugation. The supernatant is collected and bound radioactivity is measured by liquid scintillation counting. Unlabeled DHT is used as a positive control and reference compound (typically set at 100% relative binding affinity). IC₅₀ values are calculated by nonlinear regression analysis, and Ki values are then derived using the Cheng-Prusoff equation. LGD-4033 exhibits a Ki value of 1 nM.

The cellular functional activity of LGD-4033 is assessed using AR-dependent reporter gene assays. Commonly used cell models include human prostate cancer cell lines (such as LNCaP) or African green monkey kidney fibroblast CV-1 cells stably transfected with androgen-responsive reporter constructs. Cells are cultured in DMEM/RPMI-1640 medium containing 10% charcoal-stripped fetal bovine serum (to remove endogenous hormones) in a 5% CO₂ incubator at 37°C. Cells are seeded in 96-well plates (approximately 1 × 10⁴ cells per well), cultured for 24 hours, then switched to serum-free medium and treated with increasing concentrations of LGD-4033 (0.01 nM–10 μM), with DHT as a positive control and vehicle control (DMSO final concentration ≤0.1%). After 24-48 hours of treatment, cells are lysed, luciferase substrate is added, and luciferase activity is measured using a chemiluminescence detector. Cell viability can also be assessed using MTT or CellTiter-Glo assays to exclude cytotoxic interference. Transcriptional activation activity is expressed as a percentage of the maximal effect of DHT, and EC₅₀ values are calculated. Additionally, metabolic stability studies of LGD-4033 in human seminal vesicle S9 fractions (SV-S9) and human liver S9 fractions (HL-S9) can be performed by incubating the compound with S9 fractions and NADPH cofactor, with samples taken at various time points for analysis of remaining parent compound by LC-MS/MS.

Three-month-old Sprague Dawley rats were either ovariectomized (OVX, n = 60) or left intact (NON-OVX, n = 15). After 9 weeks, OVX rats were divided into four groups: untreated OVX (n = 15) group and three OVX groups (each of 15 rats) treated with ligandrol orally at doses of 0.03, 0.3, or 3 mg/kg body weight. After five weeks, lumbar vertebral bodies (L), tibiae, and femora were examined using micro-computed tomographical, biomechanical, ashing, and gene expression analyses[1].

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The in vivo activity of LGD-4033 in animal models is evaluated through various administration routes and experimental paradigms. Using the OVX rat model of osteoporosis as an example: Three-month-old female Sprague Dawley rats undergo bilateral ovariectomy (OVX) or sham surgery. Nine weeks post-surgery (after confirming osteoporosis model establishment), OVX rats are divided into a vehicle control group and LGD-4033 treatment groups (0.03, 0.3, and 3 mg/kg/day), administered daily by oral gavage for 5 consecutive weeks. Body weight and food intake are monitored weekly throughout the experiment. At the end of week 5, animals are euthanized, and lumbar vertebrae (L4-L6), tibiae, and femora are collected for analysis: micro-computed tomography (micro-CT) is used to analyze bone microstructural parameters (trabecular number, trabecular thickness, trabecular separation, etc.); biomechanical testing (three-point bending assay) evaluates bone mechanical properties; ashing determines bone mineral content; and RT-qPCR examines osteogenic gene expression in bone tissue. Serum is collected for calcium, phosphorus, and hormone level measurements, and organs including the uterus, heart, and liver are weighed to calculate organ coefficients. Results showed that the 3 mg/kg group improved bone structure but was accompanied by side effects such as increased uterine weight. In another equine metabolism study, LGD-4033 was administered intravenously or orally, followed by plasma and urine sample collection for metabolite analysis by UHPLC-HRMS, identifying 8 phase I and phase II metabolites.

LGD-4033 exhibits favorable pharmacokinetic characteristics in humans. Phase I clinical trials in healthy male volunteers show that LGD-4033 has good oral bioavailability, with rapid absorption and a time to peak concentration (Tmax) of approximately 1-2 hours. The drug has a long elimination half-life (t₁/₂), demonstrating dose-proportional accumulation upon multiple dosing. In clinical studies using once-daily doses of 0.1, 0.3, and 1.0 mg for 21 days, plasma concentrations showed a linear relationship with dose. The metabolism of LGD-4033 primarily relies on hepatic CYP450 enzymes, with major metabolic pathways including hydroxylation and oxidation. The primary metabolite detected in human urine is dihydroxylated LGD-4033. In equine metabolism studies, the parent compound was detectable in plasma for up to 8 hours and in hydrolyzed urine following β-glucuronidase treatment. Dose-dependent suppression of total testosterone, sex hormone-binding globulin (SHBG), high-density lipoprotein (HDL) cholesterol, and triglyceride levels was observed following administration, with all parameters returning to baseline after treatment discontinuation.

LGD-4033 is generally well tolerated in clinical trials, with no drug-related serious adverse events reported. In a 21-day randomized controlled trial involving 76 healthy men, the frequency of adverse events was similar between active treatment and placebo groups. No significant changes in hemoglobin, PSA, aspartate aminotransferase (AST), alanine aminotransferase (ALT), or QT intervals were observed at any dose level (0.1, 0.3, 1.0 mg). However, LGD-4033 administration was associated with dose-dependent suppression of total testosterone, SHBG, HDL cholesterol, and triglyceride levels, with significant suppression of follicle-stimulating hormone (FSH) and free testosterone observed only at the 1.0 mg dose. Hormone levels and lipid profiles returned to baseline within 5 weeks after treatment discontinuation. In a rat model of osteoporosis, increased uterine and heart weights were observed in the 3 mg/kg high-dose group, suggesting potential androgenic side effects. The lower doses of 0.03 and 0.3 mg/kg exhibited fewer side effects. LGD-4033 is not currently approved as a therapeutic agent and is limited to clinical research purposes; it is also listed as a prohibited substance by the World Anti-Doping Agency (WADA).

[1]. Effects of ligandrol as a selective androgen receptor modulator in a rat model for osteoporosis. J Bone Miner Metab. 2023 Nov;41(6):741-751.
[2]. Investigation of in vitro generated metabolites of LGD-4033, a selective androgen receptor modulator, in homogenized camel liver for anti-doping applications. Rapid Commun Mass Spectrom. 2023 Nov 30;37(22):e9633.

Ligandrol is an investigational selective androgen receptor modulator (SARM) for the treatment of conditions such as muscle atrophy and osteoporosis.

C14H12F6N2O

338.25

338.085

C, 49.71; H, 3.58; F, 33.70; N, 8.28; O, 4.73

1165910-22-4

44137686

White to off-white solid powder

1.5±0.1 g/cm3

439.9±45.0 °C at 760 mmHg

219.8±28.7 °C

0.0±1.1 mmHg at 25°C

1.496

4.04

1

9

2

23

468

2

C1C[C@@H](N(C1)C2=CC(=C(C=C2)C#N)C(F)(F)F)[C@H](C(F)(F)F)O

OPSIVAKKLQRWKC-VXGBXAGGSA-N

InChI=1S/C14H12F6N2O/c15-13(16,17)10-6-9(4-3-8(10)7-21)22-5-1-2-11(22)12(23)14(18,19)20/h3-4,6,11-12,23H,1-2,5H2/t11-,12-/m1/s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)