(R)-(-)-Gossypol specifically targets the B-cell lymphoma 2 (Bcl-2) family of anti-apoptotic proteins, including Bcl-2, Mcl-1, and Bcl-xL. It binds to the BH3-binding groove of these proteins with high affinity, acting as a BH3 mimetic to neutralize their survival function. The binding affinities (Ki) are 260+/-30 nM for Bcl-2, 170+/-10 nM for Mcl-1, and 480+/-40 nM for Bcl-xL. By inhibiting these pro-survival proteins, it promotes apoptosis in cancer cells. The compound also targets autophagy pathways and has been reported to have activity against additional cellular targets.

The two enantiomers of natural racemic gossypol are (+)-gossypol and (R)-(-)-gossypol (AT-101). The binding affinities of (R)-(-)-gossypol (AT-101) and (+)-gossypol to Bcl-2 or Bcl-xL are similar, although AT-101 is more efficient than (+)-gossypol. The effects of serum in cell culture tests may be the cause of the inhibition of cell growth and activation of apoptosis. In a 6-day MTT experiment, the racemic form of gossypol and each enantiomer were evaluated against UM-SCC-6 and UM-SCC-14A. Between the two cell lines examined, AT-101 showed a higher degree of growth inhibition in comparison to (±)-gossypol as opposed to (+)-gossypol (P<0.001). (±)-gossypol was shown to exhibit moderate growth inhibition; however, this impact was only seen at higher gossypol dosages (10 μM, P<0.0001). (R)-(-)-Gossypol (AT-101) possesses strong anti-head and neck squamous cell carcinoma (HNSCC) cell line activity in vitro and binds to the BH3 binding groove of the Bcl-xL and Bcl-2 proteins with a comparatively high affinity. Furthermore, it has the ability to effectively trigger programmed cell death in HNSCC tumor cells that express functional p53 and eliminate tumor cells that express mutant p53 via distinct processes. When compared to HNSCC cell lines, the amount of AT-101 needed to 50% suppress the development of human fibroblast cell lines was two to ten times more. (R)-(-)-gossypol (AT-101) concentrations were two to three times greater than in HNSCC cell lines in order to 50% decrease human oral keratinocyte development. In a 6-day MTT experiment, 10 UM-SCC cell lines showed a dose-dependent reduction of cell growth in the 0.5 to 10 μM range when treated with (R)-(-)-Gossypol (AT-101). Cell lines exhibit varying degrees of sensitivity; highly sensitive groups have IC50s between 2 and 5 μM, whilst less sensitive groups have IC50s centered around 10 μM [1]. It has been established that (R)-(-)-Gossypol (AT-101) binds to the proteins Bcl-2, Mcl-1, and Bcl-xL, with Ki values of 260±30 nM, 170±10 nM, and 480±40 nM, respectively [2].

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In vitro, (R)-(-)-Gossypol exhibits potent anti-proliferative activity against a range of cancer cell lines. In a 6-day MTT assay, it demonstrated dose-dependent growth inhibition in 10 UM-SCC HNSCC cell lines, with IC50 values ranging from 2 to 10 uM depending on cell line sensitivity. The compound was more effective than the racemic gossypol mixture and the (+)-enantiomer in inhibiting HNSCC cell growth. It effectively triggers programmed cell death in HNSCC tumor cells expressing functional p53 and can also eliminate tumor cells with mutant p53 via distinct mechanisms. The required concentration for 50% inhibition of normal human fibroblast and oral keratinocyte growth was 2-10 times higher than for cancer cell lines, indicating some selectivity.

In vivo, (R)-(-)-Gossypol (AT-101) has demonstrated antitumor efficacy in various preclinical xenograft models. It has shown activity against head and neck squamous cell carcinoma, prostate cancer, and other solid tumors. The compound is orally bioavailable and has been evaluated in clinical trials for multiple cancer indications. As a BH3 mimetic, it induces apoptosis in tumor tissues by neutralizing Bcl-2 family proteins. Its in vivo efficacy is dose-dependent, and it has been studied in combination with other chemotherapeutic agents to enhance antitumor responses.

The in vitro binding assay for (R)-(-)-Gossypol typically involves competitive binding studies using fluorescence polarization or radioligand displacement. Purified recombinant Bcl-2, Mcl-1, or Bcl-xL proteins are incubated with a fluorescently labeled BH3 peptide probe and varying concentrations of the test compound. The displacement of the probe is measured, and the Ki values are calculated from the competition curves. For example, the binding affinities are determined to be 260+/-30 nM for Bcl-2, 170+/-10 nM for Mcl-1, and 480+/-40 nM for Bcl-xL. Data are analyzed using nonlinear regression models to determine the inhibition constants.

In vitro cellular assays for (R)-(-)-Gossypol are conducted using cancer cell lines, such as UM-SCC HNSCC cells, plated in 96-well or 6-well plates. Cells are treated with varying concentrations of the compound (typically 0.5-10 uM) for 6 days. Cell viability and proliferation are assessed using standard colorimetric assays such as MTT or MTS. The IC50 values are determined from dose-response curves. Apoptosis is evaluated by measuring caspase activity, Annexin V staining, or DNA fragmentation. The assay is performed in triplicate with appropriate vehicle controls to ensure data reliability.

In vivo animal studies for (R)-(-)-Gossypol are typically performed in mouse xenograft models of human cancer. Immunocompromised mice are implanted subcutaneously with tumor cells (e.g., HNSCC, prostate cancer). When tumors reach a certain size, animals are treated with (R)-(-)-Gossypol via oral gavage at various doses. Tumor growth is monitored by caliper measurements, and tumor tissues are collected at endpoint for histopathological analysis and biomarker assessment (e.g., apoptosis markers, Bcl-2 family protein levels). Standard study designs include vehicle control and positive control groups, with 6-10 animals per group.

Absorption, Distribution and Excretion
Fat-soluble gossypol is readily absorbed from the gastrointestinal tract. It is highly bound to amino acids (especially lysine) and dietary iron. Gossypol's binding, metabolism, and urinary excretion are limited; most is excreted in feces.

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(R)-(-)-Gossypol is a fat-soluble compound that is readily absorbed from the gastrointestinal tract after oral administration. It is highly bound to plasma proteins. The compound has a long elimination half-life due to its high lipophilicity and extensive tissue distribution. It undergoes hepatic metabolism and is excreted primarily via the bile and feces. Pharmacokinetic studies in animals and humans have shown that AT-101 has a large volume of distribution and accumulates in tissues. Its oral bioavailability and slow clearance support once-daily or twice-daily dosing schedules in preclinical and clinical studies.

Toxicity Summary
Gossypol may induce apoptosis by modulating Bax and Bcl-2 proteins. It is also an inhibitor of calcineurin and protein kinase C and has been shown to bind to calmodulin. (L1239) Interactions …This study used roosters (n = 144) from different humoral immune selection strains. Three individuals from each strain were randomly selected and placed in cages and fed a corn-soybean meal (control) diet for 14 days. Then, six cages from each strain were randomly selected and given four different dietary treatments (1000 mg/kg gossypol, 1000 mg/kg silymarin, a mixture of 1000 mg/kg gossypol and silymarin, or a control diet). Body weight and feed intake data were collected for 21 consecutive days, and blood was collected weekly to collect plasma and determine hematocrit. The chickens were then euthanized, and livers were collected for histological and enzyme activity analysis. Weekly endpoints were analyzed using repeated measures and regression analysis. Plasma and liver enzyme activities, as well as histological parameters, were analyzed using analysis of variance (ANOVA). No significant interactions were observed between diets and strains. Chickens fed gossypol and gossypol-silymarin diets ceased gaining weight on day 14 (P < 0.001) and experienced weight loss on day 21 (P < 0.001). These chickens also showed elevated γ-glutamyltransferase levels on day 14; by day 21, their activity further increased (P < 0.001). Histological examination of liver sections revealed significant fatty degeneration (P < 0.001). Furthermore, quinone reductase activities were significantly higher in chickens treated with gossypol and the combined gossypol-silymarin diet compared to the control and silymarin-treated groups (P < 0.001). Silymarin did not alleviate any clinical symptoms of gossypol poisoning.

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Non-human toxicity values
Oral LD50 in rats: 2315 mg/kg
Oral LD50 in pigs: 550 mg/kg

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Toxicology data for (R)-(-)-Gossypol indicate that it has a relatively narrow therapeutic window. Common toxicities observed in preclinical studies include gastrointestinal disturbances, fatigue, and hepatotoxicity. In animal studies, high doses have been associated with adverse effects on the reproductive system, particularly affecting sperm production and motility. The compound's mechanism of action, involving the inhibition of anti-apoptotic proteins, can also lead to off-target effects on normal tissues. Comprehensive toxicology studies have been conducted to support its clinical development, but the compound has not been approved for therapeutic use.

[1]. In vitro effects of the BH3 mimetic, (-)-Gossypol, on head and neck squamous cell carcinoma cells. Clin Cancer Res. 2004 Nov 15;10(22):7757-63.

[2]. Apogossypolone, a nonpeptidic small molecule inhibitor targeting Bcl-2 family proteins, effectively inhibits growth of diffuse large cell lymphoma cells in vitro and in vivo. Cancer Biol Ther. 2008 Sep;7(9):1418-26.

Therapeutic Uses
/Experimental Therapy/ Gossypol (C(30)H(30)O(8)) is a polyphenolic compound derived from the cotton plant (Malvaceae family, Gossypium genus). The gossypol molecule contains six phenolic hydroxyl groups and two aldehyde groups, giving it high chemical activity. Gossypol can undergo Schiff base formation, ozone decomposition, oxidation, and methylation reactions to generate various gossypol derivatives. Due to the diverse biological activities of gossypol and its derivatives, including antifertility, antiviral, anticancer, antioxidant, antitrypanosomiasis, antibacterial, and antimalarial activities, it has been a focus of numerous studies. Because the rotation of the naphthalene ring interphase is restricted, gossypol is a chiral compound with two transisomers (i.e., (+)-gossypol and (-)-gossypol), which exhibit different biological activities. Gossypol is a small-molecule Bcl-2 family pro-survival protein inhibitor and has been shown to inhibit the growth of AI prostate cancer. However, in a mouse model of prostate cancer xenograft (vertebral prostate cancer [VCaP]) treated with AT-101 (R-(-)-gossypol acetate), the presence of androgens attenuated the apoptotic effects of gossypol. This study aimed to better understand the in vitro effects of the androgen receptor (AR) on AT-101-induced apoptosis. VCaP cells treated with AT-101 exhibited increased apoptosis and downregulated expression of the pro-survival protein Bcl-2. Combined treatment with AR activation and AT-101 reduced apoptosis, increased cell survival, and attenuated caspase activation. AT-101 downregulated the expression of Akt and the apoptosis inhibitor X (XIAP), while AR stimulation restored the expression of these proteins. Combined treatment with bicalutamide and AT-101 increased apoptosis by reducing the expression of these pro-survival proteins. These data suggest that combined treatment with AT-101 and ADT may further delay the onset of AI disease, thereby prolonging progression-free survival in prostate cancer patients.
/Experimental Treatment/...A series of new and known gossypol bis-Schiff base analogs were synthesized, and their anticancer activity against HeLa, U87, and M85 cells was tested. Results showed that less active (+)-gossypol could be converted into more active derivatives through simple chemical modification. Many more potent compounds were found compared to (-)-gossypol, which may be promising anticancer drugs; some of these compounds showed superior activity against all three cancer cell lines compared to the anticancer drug cisplatin.../Gossypol Analogs/
/Experimental Treatment/ Twenty-seven patients with pathologically confirmed gliomas relapsed after radiotherapy were treated with gossypol 10 mg orally twice daily. Of these, 15 had glioblastoma, 11 had anaplastic astrocytoma, and 1 had recurrent low-grade glioma. Efficacy was assessed every 8 weeks using CT/MRI scans and clinical criteria, including dexamethasone requirements. Treatment continued until disease progression. Two patients achieved partial remission (PR); four patients remained stable for 8 weeks or longer. One patient maintained PR with improved KPS score for 78 weeks. Another patient achieved partial remission for 8 weeks. Toxicity was mild: two patients with prior extensive treatment experienced mild thrombocytopenia, five patients experienced hypokalemia, and three patients experienced grade 2 hepatotoxicity and peripheral edema. In this study, gossypol levels determined by high-performance liquid chromatography (HPLC) were not correlated with efficacy or toxicity. We conclude that gossypol is well tolerated and, although its efficacy is low in a population of patients with recurrent gliomas who have received extensive treatment and have a poor prognosis, it is measurable…
For more complete data on the therapeutic uses of gossypol (7 items in total), please visit the HSDB record page.

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Drug Warning
Following clinical trials conducted in China in the 1970s, gossypol was proposed for use as a male contraceptive. This review summarizes numerous formal animal toxicology studies on gossypol and the recovery of fertility in men after discontinuation of gossypol treatment. These studies prompted the World Health Organization (WHO) Special Programme for Research, Development and Research Training in Human Reproduction (HRP) to decide that gossypol is unsuitable as an anti-fertility drug. …Reports indicate that studies conducted in China have confirmed the effectiveness of gossypol as an anti-fertility drug for men. …Research by the International Organization for the Advancement of Chemical Sciences (IOCSD) showed that 40 out of 70 novel high-purity gossypol structural forms had no higher activity than pure gossypol. Experiments in Sprague-Dawley rats and cynomolgus monkeys confirmed that both (-) and (+) gossypol are too toxic for human contraception. Among the side effects associated with gossypol use, the most serious is hypokalemic paralysis, although the reported differences in incidence may be attributed to variations in dietary potassium intake across different regions and genetic susceptibility. On the other hand, two independent studies confirmed the findings regarding the risk of permanent infertility in healthy men of reproductive age, finding an irreversible infertility rate of 25%. Failure to recover after discontinuing gossypol may be related to prolonged treatment duration, high total dose of gossypol, small testicular volume, and elevated follicle-stimulating hormone (FSH) levels...

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(R)-(-)-Gossypol (AT-101) is a natural product-derived BH3 mimetic and a potent inhibitor of Bcl-2, Mcl-1, and Bcl-xL. It has been extensively studied in preclinical models and clinical trials for various cancers, including head and neck squamous cell carcinoma, prostate cancer, and chronic lymphocytic leukemia. As a small molecule, it represents a class of compounds that target the apoptotic pathway. Despite promising preclinical data, clinical development has faced challenges related to toxicity and bioavailability. The compound is used as a research tool to study apoptosis and Bcl-2 family biology.

C₃₀H₃₀O₈

518.5544

518.194

90141-22-3

(R)-(-)-Gossypol acetic acid;866541-93-7;(S)-Gossypol (acetic acid);1189561-66-7;Gossypol (acetic acid);12542-36-8

3503

Light yellow to yellow solid powder

1.4±0.1 g/cm3

707.9±55.0 °C at 760 mmHg

166-167ºC

395.9±28.0 °C

0.0±2.3 mmHg at 25°C

1.742

6.16

6

8

5

38

780

0

QBKSWRVVCFFDOT-UHFFFAOYSA-N

InChI=1S/C30H30O8/c1-11(2)19-15-7-13(5)21(27(35)23(15)17(9-31)25(33)29(19)37)22-14(6)8-16-20(12(3)4)30(38)26(34)18(10-32)24(16)28(22)36/h7-12,33-38H,1-6H3

7-(8-formyl-1,6,7-trihydroxy-3-methyl-5-propan-2-ylnaphthalen-2-yl)-2,3,8-trihydroxy-6-methyl-4-propan-2-ylnaphthalene-1-carbaldehyde

AT101AT 101AT-101R-(-)-gossypol acetic acid

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)