Main Research Findings

1) The safety assessment and pharmacokinetic profile developed by researchers LoRusso et. Al determined that RAD140 treatment was an efficient treatment that exhibits effective antitumor activity.

2) Researchers Jayaraman et. Al reported that treatment with RAD140 led to enhanced neuroprotection and increased anabolic activity.

3) 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) LoRusso et. Al conducted the first in-human phase 1 study regarding the efficacy of RAD 140’s antitumor activity, specifically in terms of metastatic breast cancer. The primary goal of the study was to determine the safety and tolerability measures of RAD 140 in patients with breast cancer, as well as to find the maximum tolerated dose (MTD) and the recommended dose for expansion (RDE). The second goal of the study was to characterize the pharmacokinetic profile and evaluate the antitumor activity. This study focused on postmenopausal women who had received a diagnosis for inoperable or advanced metastatic ER+/HER2- breast cancer. RAD 140 was administered in a 3 + 3 dose-escalation phase, followed by a pharmacokinetic expansion (PKE) [2].

Approximately 30 patients were involved in the study and sequentially assigned escalating doses of RAD 140 in a 3 + 3 design. The first treatment round of RAD 140 was administered once daily in 50 mg doses. The study proceeded according to the dose-limiting toxicity (DLT) evaluations, in order to determine MTD and RDE the treatment dose of RAD 140 given to the rats incrementally increased in 50 mg doses. Patients who passed through the first DLT evaluation period without experiencing DLT continued on in the drug study with elevated doses. Researchers stopped increasing the doses when 2 DLTs occurred throughout 3-6 patients. The MTD was then determined by the next lowest treatment dose where only 1 DLT occurred in every 6 people [2].

When initially determining the maximum treatment dosage, researchers added an additional patient group in order to obtain safety data and examine the effects of single-dose PK. The subjects were administered an initial dose of the MTD, followed by 7 days with no treatment. Plasma samples were frequently collected during the 7 day waiting period in order for the researchers to form a PK profile. After this sample period occurred the PKE patients began their daily treatment with RAD 140 at the preliminary MTD levels and followed the same study procedures as the initial group.

Safety assessments of RAD 140 included physical examinations, vital signs, blood tests, serum chemistry, and 12-lead electrocardiograms. The researchers were specifically recording any changes in serum levels of cardiac troponin in the patients. The severity of any side effects were observed and graded according to the Common Terminology Criteria for Adverse Events. Tumor assessments were performed at baseline and at every 8 weeks during Cycles 2-12; all subsequent cycles only required a tumor assessment every 3 months.

Additionally, blood samples for the PK characterized were collected both pre-dose administration of RAD 140. Samples were collected Cycle 1 Day 1 at 1, 2, 4, 6, 8, and 24 hours and Cycle 1 Day 15 at 1, 2, 4, 6, and 8 hours. Pre-dose blood samples were collected Cycles 2-6 Day 28. For the additional PKE group, pre-dose plasma samples were gathered at, 1, 2, 4, 6 ,8, 24, 48, 72, 96, and 144 hours after a single dose of Day 1 of PKEs [2].

Researchers obtained baseline levels of serum sex hormone-binding globulin (SHBG), prostate-specific antigen (PSA), testosterone, 11-deoxycorticosterone, follicle-stimulating hormone (FSH), and estradiol. These levels were measured again on Day 28, the end of treatment. Baseline blood samples were also collected Cycle 1 Day 1, and were recorded at the end of the treatment cycle for 6 months. Furthermore, ESR1 mutations in circulating tumor DNA (ctDNA) were examined through the use of Guardant 360 assay. ESR1 fusion gene alterations are also identified by immunohistochemistry reports conducted on 2-year-old tumor samples or fresh tumor biopsies [2].

2) Pregnant Sprague-Dawley rats were obtained in order for the research team of Jayaraman et. Al to investigate the neuroprotective effects of RAD140 in response to normal age-related degeneration. The subjects were euthanized and the research team collected the 17-18 day old pups for the preparation of hippocampal neuronal cultures. The hippocampal neuron cultures were plated according to procedure so the research team could accurately complete cell-viability assay. In order to control for sex-related differences, the cultures were gathered from a mix of 12-14 male and female subjects. 1-2 days in vitro the experimentation began; in order to ensure validity, each experiment was conducted with three different cultures [3].

The researchers measured and interpreted data through the use of Western blot analysis and cell-viability assays. In order to perform Western blot analysis, lysates were collected from the hippocampal cultures after exposure to a reducing sample buffer. The Lysates were boiled and centrifuged over the course of 15 minutes, all leftovers products were analyzed through immunoblotting. The researchers also recorded the band densities via Image J software, and graph-plotted relative percent intensity of the phospho-ERK bands [3].

At the end of the treatment period, cell-viability assays were performed on the cultures. In order to accurately count the amount of viable hippocampal neurons, the cultures were stained with vital dye calcein acetoxymethyl ester. A predetermined counting pattern was set by the research team; the samples were separated into 4 separate fields per well and there were three wells per condition in each experiment that was conducted. After counting, the number of viable cells in the experimental cultures were compared to the vehicle-treated control groups.

The second portion of the study conducted by Jayaraman et. Al utilized gonadectomized (GDX), or sham-GDX Sprague-Dawley rats; all of the subjects were 3 months old males that underwent surgery 14 days prior to treatment. The subjects were kept on a 12 hour light, 12 hour dark schedule and were housed by themselves with food and water available ad libitum. A SILASTIC capsule was packed with dry testosterone, capped with silicone glue and implanted into the GDX rats receiving testosterone treatment. For comparison purposes, the same implantation procedures were followed with the vehicle groups, but the capsule was not packed with dry testosterone [3].

Through an oral gavage, 1 mg/kg of RAD140 suspended in 1 mg/mL of 0.5% methyl cellulose was administered to the GDX test subjects. Treatment with RAD140 took place daily over the course of two weeks. 10 mg/kg of kainate was administered to the rats on day 13, this was in comparison to the animals injected with sterile water control. 1 mg/mL of RAD 140 suspended in safflower oil was subcutaneously injected into the rats on the last day of treatment. Subcutaneous injection was used instead of oral gavage considering that kainate injection increases the difficulty of successful administration. After 14 days of treatment the animals were euthanized. The brains of the test subjects were removed and hemisected in order for proper observation. Additionally the researchers removed and dissected the prostate, seminal vesicles, and levator ani in order to examine any changes in the weight of each organ [3].

Immunohistochemistry tests and seizure assessments were used in order to determine the effects elicited by the injection of kainate. The immunohistochemistry test identified the number of immunoreactive cells found in brain samples. Every eight brain sample was stained with a NeuN antibody and mounted on slides in order for further analysis to occur. 2-dimensional cell counts of the ransome samples were used in order to estimate the total number of immunoreactive cells in the CA2/3 hippocam[pal region. The CA2/CA3 region of every eighth brain slice was outlined so an oriented counting frame could be determined. In order to determine a control variable the number of immunoreactive nuclei was divided by the number of section; the resulting number was defined as the percentage of neurons counted in the sham-GDX, non-lesioned group.

The researchers observed the test subjects for 3 hours after the kainate injection in order to observe any changes in exhibited behavior. Additionally, the subjects were examined so the researchers could identify changes in seizure latency or severity. The latency period quickly began after kainate was injected; this was the first appearance of stereotypical seizure-related behavior and is more commonly described as a “wet dog shake”. Severity of the seizure was rated on a scale 0-5: 0=no seizure activity while 5=seizure accompanied by rearing and falling. Additionally, statistical analysis comparing the treatment groups were performed by using ANOVA technology and Fisher’s least significant difference test [3].

3) 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 [4].

 

Discussions

1) Data collected by researchers BLANK et. Al allowed the team to conclude that treatment with RAD140 is well-tolerated by postmenopausal subjects diagnosed with metastatic breast cancer. 77.3% of the patients involved in this study reported experiencing side effects ranging in severity from Grade 0 to Grade 4. 31.8% of the patients experienced Grade 3 side effects while 0% of the patients experienced Grade 4 side effects. The more frequent side effects exhibited by the patients were increased weight loss, dehydration, and vomiting, as well as elevated levels of aspartate aminotransferase, alanine aminotransferase, and total bilirubin. It is important to note that 41% of the patients reported serious side effects, however, the researchers determined the serious side effects were not related to this study.

In regards to the occurrence of side effects, the researchers noted that 5 out of 22 patients experienced hypophosphatemia. Out of the 5 cases only 2 were measured at Grade 3 severity, while all 5 cases were deemed capable of reversal. All symptoms of hypophosphatemia in the patients were reversed when RAD140 treatment was temporarily stopped. When treatment resumed at a later date there was no identifiable recurrence of the dose-limiting toxicity [2].

Dose-limiting toxicity (DLT) was observed in 6 out of 17 study-eligible patients. The DLT occurrences were all measured as Grade 3, however, they were deemed capable of reversal. The frequency of DLT occurrences were recorded and used to determine the maximum tolerated dose (MTD) and the recommended dose for expansion (RDE). Results of the study reported that both the MTD and RDE values were approximately 100 mg, when administered once daily.

No significant changes were found when the researchers performed the patients’ physical examinations. Serum levels of 11-deoxycorticosterone (11-DOC) were routinely measured in order to report instances of cardiac inflammation or changes in the hormone biosynthetic pathway. None of the treatments administered to the patients resulted in changes in mean and median 11-DOC levels. In addition to 11-DOC the researchers recorded the levels of cardiac troponin and troponin T found in the plasma. The baseline measurements were used as a way to grade severity ranging from baseline to worst post-baseline value. Troponin T levels were recorded at Grade 0 at baseline values, and only increased slightly to Grade 1 following treatment [2].

Any cardiac side effects elicited by RAD140 were only found in 18.2% of test subjects. The primary side effects reported were Grade 1 palpitations, Grade 2 atrial flutter and atrial fibrillation, and Grade 3 sinus tachycardia and ventricular failure. Patients experiencing Grade 3 tachycardia were released from the study for other treatment purposes. However, the researchers were able to conclude that any of the cardiac side effects seen in the patients were not related to treatment with RAD140. Furthermore, the research team observed that there were no adverse androgenic side effects elicited by the SARM; Grade 1 weight gain, acne, and hair growth were the only reported side effects experienced by the patients [2].

Finally, the pharmacokinetic report developed by the research team reported that the initial treatment groups experienced an elevation in plasma concentration for approximately 24 hours after treatment with RAD140. When RAD140 was administered to the additional, single-dose PK cohort group, serum levels of the SARM increased rapidly, followed by a steady decline resulting in secondary and tertiary peaks. The PK evaluation led the researchers to conclude that RAD140 has an acceptable safety profile and is capable of promoting antitumor activity, specifically in pretreated postmenopausal women diagnosed with metastatic breast cancer.

2) Initial results of the study conducted by BLANK et. Al determined that RAD140 elicited neuroprotective effects. Cell cultures exposed to A-beta for 24 hours experienced a 50% decrease in the number of viable neurons found in the cell culture. Pretreating the cultures with 10 nM of testosterone of a DHT an hour before exposure led to a dramatic decrease in cell death. Similar reductions in cell death were seen when the cultures were pretreated with 30 nM of RAD140 an hour before A-beta exposure.

 

Figure 1: Changes in neuron viability in A) testosterone and C) RAD140

Further analysis of the conducted data revealed that the MAPK/ERK is associated with androgen-mediated neuroprotection against cell death. Initially, the neuronal cultures were pretreated with the presence or absence of a MEK inhibitor and exposed to 100 nM of RAD140. It was determined by the research team that RAD140 protects against apoptosis induced by A-beta exposure. That being said, the next step in the study was to observe the inhibitory effects elicited by U0126 MAPK/ERK signaling in RAD140-treated cell cultures. Researchers came to the conclusion that the neuroprotective effects of RAD140 are blocked when cells are pretreated with the inhibitor, U0126 [3].

Researchers turned their focus to the neuroprotective effects of RAD140 in vivo following the results of previous experimentation. Male rats underwent gonadectomy or a sham gonadectomy (referred to as GDX and sham-GDX, respectively). Androgen treatment took place over the course of two weeks and on day 13 the subjects were administered a single dose of kainate. Initial results reported that the weight of the prostate, seminal vesicles, and levator ani muscles were all significantly diminished following GDX. However, following treatment with testosterone the weight of each three tissues increased to a weight similar to those recorded in the sham-GDX groups.

In comparison to testosterone, treatment with RAD140 increased the weight of the prostate and seminal vesicles to a level considered insignificant. When treated with the SARM, both the weight of the prostate and seminal vesicles measured significantly lower than the weights collected from the sham-GDX and GDX+testosterone groups. Additionally, the levator ani muscle also exhibited a drastic increase in weight after treatment with RAD140. Records of weights collected from the RAD140 treatment groups were similar to the weight of the levator ani in the sham-GDX and GDX + testosterone treatment groups [3].

 

Figure 2: Changes in the weight of A) seminal vesicles, B) prostate, and C) levator ani muscle, in response to the different treatments

When observing kainate-induced neuron death, the neuroprotective effects of RAD140 were thoroughly examined through immunostaining and assessing cell cultures gathered from the CA2/CA3 hippocampal regions. Initial results reported that kainate injected in sham-GDX vehicle-treatment led to a 20% cell loss, however, cell loss was significantly reduced after the androgen treatment was administered. Overall the research team concluded that testosterone and RAD140 elicited potent neuroprotective effects in GDX rats injected with a single-dose of kainate [3].

 

Figure 3: Percentage of cell survival in response to different treatments.

The research team examined how the latency and severity of kainate-induced seizures responded to hormonal manipulations. The initial step was to determine how seizure behavior was measured; the intensity of the seizure behavior was determined by the degree of hippocampal neuron loss. All animals were observed by the research team for 3 hours. The animals treated with kainate had at least one seizure, while the vehicle-treated animals did not exhibit any seizure behavior. The kainate lesioned animals did not result in significant changes or variations in seizure latency or severity between the treatment groups. It is important to note that the GDX + testosterone and GDX + RAD 140 treatment groups experienced a slight reduction in seizure severity, but the change was considered insignificant [3].

 

Figure 4: Changes observed in latency and severity of kainate-induced seizures in response to different treatment groups

3) 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 5: 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 [4].

 

Figure 6: 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] Leung K, Yaramada P, Goyal P, Cai CX, Thung I, Hammami MB. RAD-140 Drug-Induced Liver Injury. Ochsner J. 2022 Winter;22(4):361-365. doi: 10.31486/toj.22.0005. PMID: 36561105; PMCID: PMC9753945.

[2] LoRusso P, Hamilton E, Ma C, Vidula N, Bagley RG, Troy S, Annett M, Yu Z, Conlan MG, Weise A. A First-in-Human Phase 1 Study of a Novel Selective Androgen Receptor Modulator (SARM), RAD140, in ER+/HER2- Metastatic Breast Cancer. Clin Breast Cancer. 2022 Jan;22(1):67-77. doi: 10.1016/j.clbc.2021.08.003. Epub 2021 Aug 20. PMID: 34565686.

[3] Anusha Jayaraman, Amy Christensen, V. Alexandra Moser, Rebekah S. Vest, Chris P. Miller, Gary Hattersley, Christian J. Pike, Selective Androgen Receptor Modulator RAD140 Is Neuroprotective in Cultured Neurons and Kainate-Lesioned Male Rats, Endocrinology, Volume 155, Issue 4, 1 April 2014, Pages 1398–1406, https://doi.org/10.1210/en.2013-1725

[4] 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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