Gossypol Acetic Acid Research Articles

Development of Method for Three-Point Data Estimation and SVR-QSAR Model to Screen Anti Cancer Leads

Present work deals with generation of virtual samples as mathematical modeling of empirical data on the basis of empirical data. The generated samples were used for development of QSAR model. The method deals with extrapolation of sample vector in such a manner that there is conservation of the empirical data distribution. The data distribution has been judged with statistical parameters. The method was implemented with anticancer activity of Gossypol acetic acid against BCL2 target for colorectal cancer. Considering the virtual samples only for model development, model training showed a regression coefficient for leave one out cross validation as 0.996 with 66 virtual samples, and a regression coefficient with external test set data (51 samples) as 0.993. External test set data which were never used in the virtual sample generation showed predicted regression coefficient value of >0.61. On the basis of QSAR model, nine compounds were suggested as anti-BCL2 active compounds. The suggested compounds were further validated by docking study with Gossypol acetic acid and 'Tetrahydroisoquinoline amide substituted phenyl pyrazole' cocrystallized with chimeric BCL2-XL (PDBID: 2W3L) protein.

Gossypol acetic acid induces apoptosis in RAW264.7 cellsviaa caspase-dependent mitochondrial signaling pathway

To investigate the effects of gossypol acetic acid (GA) on proliferation and apoptosis of the macrophage cell line RAW264.7 and further understand the possible underlying mechanism responsible for GA-induced cell apoptosis, RAW264.7 cells were treated with GA (25~35 µmol/L) for 24 h and the cytotoxicity was determined by MTT assay, while apoptotic cells were identified by TUNEL assay, acridine orange/ethidium bromide staining and flow cytometry. Moreover, mitochondrial membrane potential (ΔΨm) with Rhodamine 123 and reactive oxygen species (ROS) with DCFH-DA were analyzed by fluorescence spectrofluorometry. In addition, the expression of caspase-3 and caspase-9 was assessed by Western Blot assay. Finally, the GA-induced cell apoptosis was evaluated by flow cytometry in the present of caspase inhibitors Z-VAD-FMK and Ac-LEHD-FMK, respectively. GA significantly inhibited the proliferation of RAW264.7 cells in a dose-dependent manner, and caused obvious cell apoptosis and a loss of ΔΨm in RAW264.7 cells. Moreover, the ROS production in cells was elevated, and the levels of activated caspase-3 and caspase-9 were up-regulated in a dose-dependent manner. Notably, GA-induced cell apoptosis was markedly inhibited by caspase inhibitors. These results suggest that GA-induced RAW264.7 cell apoptosis may be mediated via a caspase-dependent mitochondrial signaling pathway.

Effect of Combination Regimen of Low-dose Gossypol Acetic Acid with Steroid Hormones on Expression of Protein Kinase C alpha (PKC-α) and Cyclin D1 in Rat Testes

Objective To investigate the contraceptive mechanism of combination regimen of low-dose gossypol acetic acid (GA) with steroid hormones [desogestre/ethinylestradiol/testosterone undecanote (DSG/EE/TU)]. Methods Adult male rats were randomly divided into four groups. Group GH: rats were fed orally with gossypol acetic acid (GA, 12.5 mg/kg) and desogestrel (DSG, 0.125 mg/kg)/ethinylestradiol (EE, 0.025 mg/kg)/testosterone undecanoate (TU, 100 mg/kg per day, qd × 4 weeks or 10 weeks); group G: a single dose of GA (12.5 mg/kg per day, qd × 4 weeks or 10 weeks); group H: the same dosage of DSG/EE/TU as in group GH; group C: rats were treated with vehicle (1% methyl cellulose) as the control. Expression of protein kinase C alpha (PKC-α) and cyclin D1 in rat testes were tested mainly by immunohistochemistry (IHC) and Western blotting. Results IHC results showed that protein PKC-α was expressed mainly in interstitial tissue of testis among seminiferous tubule. The expression of PKC-α in groups H and GH at week 10 was decreased greatly compared with that in group C. The protein cyclin D1 was expressed mainly in residual body of seminiferous tubule cavosurface and interstitial tissue among seminiferous tubule of testis. Western blotting results showed that the expression of PKC-α in groups H and GH at week 10 was decreased significantly compared with that in group C (P Conclusion The administration of low-dose gossypol acetic acid with steroid hormones for 10 weeks can decrease the expression of PKC-α greatly.

A validated HPLC assay for the determination of R-(−)-gossypol in human plasma and its application in clinical pharmacokinetic studies

R-(-)-gossypol acetic acid (AT-101), a natural BH3 mimetic, is investigated in a Phase I/II clinical trial for the treatment of advanced solid tumor malignancies. Gossypol undergoes rapid degradation in solution phase, which causes major technical difficulty for its quantitation in plasma. We developed and validated a sensitive HPLC assay for pharmacokinetic evaluation of gossypol. Acetonitrile deproteinization method was chosen for sample preparation and Schiff's base derivative, R-(-)-gossypol-diamino-propanol (GDP), was used as internal standard. Chromatographic separation of gossypol in plasma was performed using a Zorbax Eclipse XDB column C(18) at 30 °C. The mobile phase consists of 10 mmol/L KH(2)PO(4) (pH 3.0) and acetonitrile (20:80) at 1.0 mL/min flow rate. Linearity ranged over 56-3585 ng/mL (R(2)=0.9997±0.0003, n=4), and the limit of detection was 28 ng/mL. The intra- and inter-assay precision was less than 13.7% and the bias ranged from -7.4 to 7.0%. The method was successfully applied to characterize the pharmacokinetics of AT-101 in a Phase I clinical trial. The validated assay is accurate, and sensitive with minimum loss and rapid analysis time and suitable for quantification of gossypol for pharmacokinetics evaluation.

Nuclear Area Factor as a Novel Estimate for Apoptosis in Oral Squamous Cell Carcinoma -Treated Cell Line: A Comparative in-vitro Study with DNA Fragmentation Assay

Aims and objectives:The aims of the present study were to introduce a novel modality for estimation of the anti-neoplastic efficacy of chemotherapeutic agents and to compare this technique with one of the most widely used techniques, the DNA fragmentation assay. Methods:Squamous cell carcinoma cell line HEp-2 was utilized in the present study. The cytotoxic drug used in this study was gossypol-acetic acid. Microscopic slides of each drug concentration at definite post-treatment durations were photomicrographed and analyzed using image anazlysis software for the estimation of nuclear area factor. Data were then statistically analyzed. Internucleosomal DNA fragmentation was investigated to assess whether the morphological changes induced by the drug depended on activation of the apoptotic pathway. Results:The data recorded revealed a decrease in the mean values of NAF of HEp-2 cells treated with different concentrations of gossypol over different durations when compared to control cells. DNA fragmentation was not observed in the control groups over different durations of incubation. The study of internucleosomal DNA fragmentation in HEp-2 cells treated with different concentrations of gossypol over different durations revealed absence of DNA laddering that indicated DNA fragmentation with either 50μM or 100μM gossypol concentrations after until 24 hours of incubation with 50μM gossypol concentration. Conclusions:These results suggest that estimation of nuclear area factor (NAF) is a sensitive predictor of the early apoptotic effect of anti-cancer therapy. DNA fragmentation assay is a useful tool for detection of the late apoptotic changes that discriminate apoptotic effects of anti-cancer drugs from necrotic ones.

Co-administration of Mixed Steroid Hormones can Enhance the Recovery of Spermatogenesis Damaged by Gossypol Acetic Acid in Adult Rats

To determine whether co-administration of mixed steriod hormones can enhance the restoration of spermatogenesis damaged by gossypol acetic acid (GA). Adult male Wistar rats were treated daily for 8 weeks with GA at 50mg/kg plus testosterone undercanoate (100 mg/kg)/desogestrel (0.125 mg/kg)/mini-dose ethinylestradiol (0.025 mg/kg) (TU/DSG/EE), followed by a period of 9 weeks for recovery. Control animals were administered the same dose of GA or TU/DSG/EE, and vehicle, respectively. Testis weight, testicular sperm head count and histological analysis were utilized to assess the spermatogenesis. At the end of the 9-week reovery period, in rats given GA alone, spermatogenesis steadily declined. However, when rats received combined hormone administration during GA treatment, this decline was prevented and an complete recovery of spermagenesis occurred. The haploid spermatids and spermatocytes was not to be protected but to be more aggravatedly damaged. The excellent recovery must have resulted from that the hormone treatment could protect the ability of stem spermatogonia to differentialte and evolve progressively into mature spermatozoa. In addition, the concentrations of serum LH, FSH and intratesticular testosteron (ITT) notably decreased after 2 or 8 weeks of treatment, then returned to control levels at the end of 9-week recovery period. Steriod hormone treatment enhaces the recovery of spermatogenesis through preventing seminiferous epithelim from GA-induced destructive damage in rats. The enhanced recovery was closely associated with the marked suppression in intratesticular testosteron (ITT).

Effects of combined administration of low‐dose gossypol with steroid hormones on the mitotic phase of spermatogenesis of rat

Our previous studies suggested that combined low-dose gossypol with steroid hormones possesses a reversible antifertility role on adult male rat. Spermatocyte apoptosis during the meiosis of spermatogenesis is the main reason for the antifertility. This study evaluates the effects of the regimen on the mitosis of spermatogenesis. Thirty-two adult male rats were divided into four groups randomly, Group GH: rats were fed orally with gossypol acetic acid (GA, 12.5 mg/kg d) and desogestrel (DSG, 0.125 mg/kg d)/ethinylestradiol (EE, 0.025 mg/kg d)/testosterone undecanoate (TU, 100 mg/kg d); Group G: a single dose of GA (12.5 mg/kg d); Group H: the same dosage of DSG/EE/TU as in Group GH; Group C: rats were treated with vehicle (1% methyl cellulose) as control. Testes were removed at the 8th week after treatment to assess the weight, volumes, diameter, and degenerated rate of seminiferous tubule, spermatogonia is quantitated by stereological assay. TUNEL assay and proliferating cell nuclear antigen immunohistochemistry were used to determine the germ cell apoptosis and proliferation. The changes of gene expression of glial cell line-derived neurotrophic factor (GDNF) and Ets variant gene 5 (Etv5) were analyzed. The results manifested that the spermatogenesis was suppressed, the type A and intermediate spermatogonia reduced, and spermatocyte and spermatid apoptosis increased both in Group GH and Group H. The expressions of GDNF protein in these two groups were downregulated, but no change was observed in Etv5 expression. However, the numbers of spermatogonia, germ cell apoptosis and proliferation, and the expressions of GDNF and Etv5 were unchanged in Group G compared with Group C. Above all, we concluded that the mitotic phase of spermatogenesis is suppressed by the combination regimen, it might be the steroid hormones in the regimen that caused the reduction of spermatogonia through the downregulation of GDNF.

Spermatotoxicity, biochemical changes and histological alteration induced by gossypol in testicular and hepatic tissues of male rats

Gossypol acetic acid (GAA) displays anti-fertility and antioxidant behavior. The efficacies of different doses of gossypol acetic acid were investigated in male albino rats. Rats were allocated into four groups: control group and three GAA-treated groups (2–4), that were injected with GAA (5, 10, 20 mg/kg BW, respectively), through inrtaperitonial injection. Treatment of GAA was found to elicit a significant decrease in sperm counting, sperm motility, serum levels of testosterone, luteinizing hormone and follicle-stimulating hormone, whereas, the activities of testicular 17β-hydroxysteroid dehydrogenase and 17-ketosteroid reductase were increased. The activities of serum transaminases and alkaline phosphatase and hepatic glutathione peroxidase; glutathione reductase, superoxide dismutase and glutathione S-transferase and the level of hepatic glutathione were elevated. While, the lipid peroxidation end product; malondialdehyde, nitric oxide, and lipid profile and the activity of hepatic cytochrome P450 were decreased in GAA-treated rats. The histological analysis of liver and testicular tissues showed sever hepatocyte damage in addition to abnormal localization of hepatocytic nuclei. Also, the testicular pathology of GAA-treated rats showed depressed spermatogensis, sertoli cell toxicity and degeneration of seminiferous tubules.

AT‐101 (R‐(−)‐gossypol acetic acid) enhances the effectiveness of androgen deprivation therapy in the VCaP prostate cancer model

Prostate cancer remains a leading cause of cancer death in American men. Androgen deprivation therapy (ADT) is the most common treatment for advanced prostate cancer patients; however, ADT fails in nearly all cases resulting in castration resistant or androgen-insensitive (AI) disease. In many cases, this progression results from dysregulation of the pro-survival Bcl-2 family proteins. Inhibition of pro-survival Bcl-2 family proteins, therefore, may be an effective strategy to delay the onset of AI disease. Gossypol, a small molecule inhibitor of pro-survival Bcl-2 family proteins, has been demonstrated to inhibit AI prostate cancer growth. The apoptotic effect of gossypol, however, has been demonstrated to be attenuated by the presence of androgen in a prostate cancer xenograft mouse model (Vertebral Cancer of Prostate [VCaP]) treated with AT-101 (R-(-)-gossypol acetic acid). This study was undertaken to better understand the in vitro effects of androgen receptor (AR) on AT-101-induced apoptosis. VCaP cells treated with AT-101 demonstrated an increase in apoptosis and downregulation of Bcl-2 pro-survival proteins. Upon AR activation in combination with AT-101 treatment, apoptosis is reduced, cell survival increases, and caspase activation is attenuated. Akt and X inhibitor of apoptosis (XIAP) are downregulated in the presence of AT-101, and AR stimulation rescues protein expression. Combination treatment of bicalutamide and AT-101 increases apoptosis by reducing the expression of these pro-survival proteins. These data suggest that combination therapy of AT-101 and ADT may further delay the onset of AI disease, resulting in prolonged progression-free survival of prostate cancer patients.

Phase II trial of R-(-)-gossypol acetic acid (NSC 726190, AT-101) in patients with recurrent extensive stage small cell lung cancer (ES-SCLC).

e17523 Background: AT-101 is an oral inhibitor of the anti-apoptotic Bcl proteins (Bcl-2, Bcl-XL, Bcl-W, and Mcl-1) and potent inducer of the proapoptotic proteins noxa and puma. This multicenter, single-arm study evaluated the efficacy of AT-101 in patients with recurrent chemosensitive ES-SCLC. Methods: To be eligible, patients must have had chemosensitive ES-SCLC (defined as no progression during and no recurrence < 2 months after completion of first-line platinum-based chemotherapy), ECOG performance status (PS) 0-2, and adequate organ and marrow function. Patients were not eligible if they had symptomatic and/or progressive brain metastases. Treatment consisted of AT-101 20 mg orally daily for 21 out of 28 days each cycle. Tumor evaluations were performed every 8 weeks. AT-101 was continued in the absence of tumor progression or unacceptable toxicity. The primary endpoint was the proportion of confirmed responses (CR, PR) during the first 6 cycles of treatment. Under the two-stage Simon Optimal design, 14 evaluable (for response) patients were enrolled in stage 1, and if ≥ 2 responses were observed, an additional 28 would be enrolled in stage 2. Results: To date, 15 of 42 planned patients were enrolled in 5 sites. Patient characteristics: median age 67.0 years (range 46-76), male 40%, ECOG PS 0 46.7% and 1 53.3%, Caucasian 86.7%, previous radiation 80%. Median number of cycles received was 2 (range 1-6). A planned interim analysis was performed on an intent to treat basis. One patient discontinued treatment after one cycle due to thrombocytopenia and was considered non-evaluable for the primary endpoint. Among the 14 evaluable patients, no objective responses were reported. Three patients (21%) achieved stable disease after two cycles of therapy but have since progressed on treatment. Grade 3 toxicities included anorexia, fatigue, and nausea/vomiting. One patient had a grade 3 leukopenia which was unrelated to treatment. The study met the stopping rule in the first stage of enrollment and was closed to further accrual. Conclusions: This interim analysis indicates that AT-101 is well-tolerated but has little activity in recurrent chemosensitive SCLC. Supported by N01-CM62205. No significant financial relationships to disclose.

A phase I study of r-(-)-gossypol (AT-101, NSC 726190) in combination with cisplatin (P) and etoposide (E) in patients with advanced solid tumors and extensive-stage small cell lung cancer (ES-SCLC).

e13030 Background: AT-101 [R-(-)-gossypol acetic acid] (AT) is an orally administered BH3 mimetic that lowers the threshold for apoptosis by direct binding to Bcl-2, Bcl-xL, Mcl-1, and Bcl-W. Combinations of AT and cytotoxic drugs demonstrated synergistic antitumor effects in vitro and in vivo SCLC models. We completed a dose-escalation study of AT in combination with cisplatin and etoposide (EP), in which neutropenia was dose- limiting. The objectives of this study were to determine the maximum tolerated dose (MTD)/recommended phase II dose (RP2D) of AT when combined with EP with pegfilgrastim (F) in patients with advanced solid tumors. Methods: Standard phase I eligibility criteria were used. Prior therapy with Bcl2-inhibitors was not allowed. We previously reported the MTD without F established at AT 20 mg BID days (D) 1-3, P 60 mg/m2 D 1, E 100 mg/m2 D 1-3 every 21 days (Leal, JCO 2009; 27(15S), e13502). Patients were enrolled in 3 additional dose levels (DL) with F support (6 mg SQ D 4): DL1a (AT 30 mg BID, P 60 mg/m2, E 100 mg/m2), DL2a (AT 30 mg BID, P 60 mg/m2, E 120 mg/m2), and DL3a (AT 40 mg BID, P 60 mg/m2, E 120 mg/m2). Results: 12 patients were enrolled: 8 men, 4 women. Tumor types: 4 non-small cell lung cancer (NSCLC), 1 ES-SCLC, 2 esophageal, 2 neuroendocrine, 1 prostate, 1 adrenocortical, 1 gallbladder. No dose limiting toxicities occurred. Grade 3/4 treatment-related toxicities included: 2 episodes of diarrhea (1 DL1a, 1 DL3a), and 1, each, of elevated ALT (DL1a), hypokalemia (DL1a), fatigue (DL2a), neutropenia (DL2a), anemia (DL3a), hypophosphatemia (DL3a), and pulmonary embolism (DL3a). Two patients with NSCLC and 1 with neuroendocrine had a PR. Pharmacokinetic data will be available for presentation. Conclusions: The MTD/RP2D with F support was established at AT 40 mg BID on D 1-3, P 60 mg/m2 D 1, and E 120 mg/m2 D 1-3 every 21 days. F support permits full doses of cytotoxics to be administered. Antitumor activity was observed in patients with lung cancer and neuroendocrine tumor. Accrual to an MTD expansion cohort of patients with untreated ES- SCLC is ongoing. This study was supported by NCI UO1 CA062491, SAIC 25XS097, and 1ULRR025011. No significant financial relationships to disclose.

6,6′-Dimethoxygossypol: molecular structure, crystal polymorphism, and solvate formation

6,6′-Dimethoxygossypol (DMG) is a naturally occurring derivative of gossypol that is found in relatively high concentration in some Gossypium barbadense cotton varieties. Like gossypol, DMG forms an equimolar solvate with acetic acid, but it was not known if, like gossypol, the compound would also form clathrates with other molecules. To test for this, the compound was crystallized from different solvents. Four new structures of DMG were found that include two polymorphic and two solvated forms. The polymorphs include two monoclinic structures with P21/c and C2/c space groups (P1 and P2, respectively). Packing of the DMG molecules P1 is similar to packing of the gossypol molecules in the P1 polymorphic form of gossypol. The DMG molecules in P2 pack in a highly ordered arrangement that has not been previously observed among gossypol structures. DMG forms equimolar solvates with water (S1) and cyclohexanone (S2). Both structures are triclinic with \(P \bar {1}\) space groups. The DMG molecules in S2 assemble in a manner that is similar to the gossypol molecules of gossypol–cyclohexanone (1:1), and the DMG molecules in S1 pack in a manner that is similar to the DMG molecules in DMG–acetic acid (1:1) as well as the gossypol molecules in gossypol–acetic acid (1:1). Although DMG is not as versatile a host compound as gossypol, it still forms solvates under many crystallization conditions. Consequently, some care is needed to be sure that one understands exactly which form is recovered when the compound is isolated.

Mutual anti-oxidative effect of gossypol acetic acid and gossypol–iron complex on hepatic lipid peroxidation in male rats

Gossypol displays anticancer behavior and anti-fertility in males. Male rats were treated with either gossypol acetic acid (GAA) or gossypol–iron complex (GIC). Serum alanine transaminase (ALT) activity elevated of GAA. However, GIC-treated animals showed a decrease in hepatic glutathione (GSH) content with increased malondialdehyde (MDA) content. Whereas, GSH-Px specific activity increased in GAA group. GAA and GIC induce significant increases in the hepatic NEFA with remarkable decrease in the total saturated fatty acids with a significant increase of PUFA. Lipid peroxidation is inhibited by gossypol, which shield lipids against oxidative damage. Phenols are oxidized to phenoxy radicals, which do not permit anti-oxidation due to resonance stabilization. GAA stimulate hydroxyl radicals ( OH) generation and DNA damage. GAA and GIC produce increase in lipid peroxidation as proved by a steep rise in thiobarbituric acid reactive species (TBARS). Controversy of specificity of TBARS towards compounds other than MDA was reported. If TBARS increased, more specific assay to be employed. Assay of lipid classes and fatty acids pattern, reveled the significance of the technique in assessment of lipid peroxidation in tissues. GAA and GIC were powerful inhibitors of lipid peroxidation and exhibit pro- and antioxidant behavior, with less toxicity of GIC.

Histologic changes in channel catfish, Ictalurus punctatus Rafinesque, fed diets containing graded levels of gossypol-acetic acid

A study was performed to evaluate the histologic changes among fingerling channel catfish, Ictalurus punctatus Rafinesque, fed purified diets containing gossypol from gossypol–acetic acid. The catfish were maintained on diets with 0, 300, 600, 900, 1200 or 1500 mg gossypol kg−1 diet for 12 weeks, and histologic samples from the stomach, proximal and distal intestines, pancreas, liver, and spleen were obtained from fish in all groups. Stomach sections exhibited significant gastric gland necrosis in fish fed 600 mg gossypol kg−1 diet or higher. Non-existent-to-mild enterocyte vacuolization loss, inflammatory cell infiltration, and hyperplastic lamina propria were noted in the intestinal sections from gossypol-fed fish, but no significant differences in severity scores were noted. The pancreas from fish fed doses of gossypol above 600 mg gossypol kg−1 diet exhibited significant mild-to-severe necrosis, and livers from fish fed 900 and 1500 mg gossypol kg−1 diet exhibited significantly higher pigment deposition. No other significant histologic differences were observed in the fish fed diets containing gossypol–acetic acid. The data in this study indicates that at least 600–900 mg gossypol kg−1 purified diet can cause statistically significant histologic changes in fingerling channel catfish, suggesting that gossypol should remain at concentrations below 600 mg gossypol kg−1 diet.

A phase I study of R-(-)-gossypol (AT-101) in combination with cisplatin (P) and etoposide (E) in patients with advanced solid tumors and extensive-stage small cell lung cancer (ES-SCLC)

e13502 Background: AT-101 [R-(-)-gossypol acetic acid] (AT) is an orally-administered BH3 mimetic that lowers the threshold for apoptosis by direct binding to Bcl-2, Bcl-xL, Mc1–1, Bcl-W, and through upregulation of the proapoptotic proteins Noxa and Puma. Bcl- 2 is over-expressed in &gt;80% of SCLC. In vitro study using SCLC cells showed that treatment with EP had synergistic cytotoxic effects in suppression of Bcl-2. This is a phase I dose-escalation study of AT in combination with EP with an expanded cohort of patients with ES-SCLC. This study is being conducted to determine the maximum tolerated dose (MTD), pharmacokinetics and activity of AT with EP ± pegfilgrastim (F) in patients with advanced, refractory solid tumors and/or ES-SCLC. Methods: This study used standard eligibility criteria except patients must not have received prior therapy that inhibits the Bcl-2 family. At dose level 1, patients received P 60 mg/m2 on day 1 and E 100 mg/m2 on days 1, 2, and 3 every 21 days. AT was administered 30 mg orally BID on days 1, 2 and 3 of each cycle. Results: 10 patients have been enrolled; 7 men, 3 women. Tumor types: 6 lung; 2 prostate; 1 head &amp; neck; 1 unknown primary. 2 of 5 patients enrolled at dose level 1 experienced a DLT of neutropenic fever in cycle 1. Three subsequent patients were enrolled to dose level -1 (20 mg BID x 3 days, d1–3) which was well tolerated. Additional patients are being enrolled at dose level 1a (EP+AT with F). Grade 3/4 toxicities related to AT without F at dose level 1 and -1 were as follows: ANC (8), leucopenia (7), febrile neutropenia (2), low hemoglobin (1), thrombocytopenia (1), elevated AST (1), cardiac ischemia/MI (1), diarrhea (1). There were no reported grade 3/4 toxicities in two patients at level 1a. Four patients had stable disease; two progressive disease and four patients were unevaluable. Conclusions: The MTD without F was established at AT 20mg orally BID, P 60 mg/m2 on day 1, and E 100 mg/m2 on days 1, 2, and 3 every 21 days. The MTD with F has not yet been established. Accrual continues at dose level 1a. Subjective and objective evidence of clinical activity has been observed in patients with refractory solid tumors. This study was supported by NCI, UO1 CA062491, SAIC 25XS097 and 1ULRR025011. No significant financial relationships to disclose.

Toxicity of gossypol at antifertility dosages in male rats: statistical analysis of lethal rates and body weight responses.

The lethal rates and body weight responses of male Wistar rats following oral treatment with gossypol acetic acid (GAA) at dosages of 2.5-30 mg/kg were analysed by various statistical procedures. Daily treatment with GAA for 10 weeks was associated with lethal rates significantly different from placebo groups in both the low dose (2.5, 5.0, and 7.5 mg/kg, p less than 0.05) and high dose groups (15, 20 and 30 mg/kg, p less than 0.01). The pronounced mean body weight gain observed in placebo groups, was suppressed in the GAA-treated groups at each dose level after 10 weeks. This indicates decreased appetite and/or lessened feed utilisation caused by GAA activity. GAA administration at a dose of 30 mg/kg every other day exerted identical deleterious effects as daily treatment. As derived from the antifertility responses of the GAA treatment groups it was evident that the toxic activity of GAA was not related to its antifertility efficacy. It is concluded that GAA has remarkable toxicity at antifertility dosages in rats.

Antifertility Efficacy of Gossypol Acetic Acid in Male Rats

Fertility response and sperm motility of male Wistar rats exposed to various concentrations (2.5 - 30 mg / kg / day) of gossypol acetic acid (GAA) was studied. GAA was given orally. A marked reduction in fertility occurred when rats received more than 15 mg / kg / day of GAA for 5 - 10 weeks (p less than 0.01); dosage below 15 mg had no antifertility effect even after 20 weeks. There is some evidence that GAA administration (30 mg / kg) every other day for 10 weeks had no antifertility effect. A clear relationship between the inhibition of sperm motility and GAA administration was observed (p less than 0.01). The interrelationships between fertility, sperm motility and GAA treatment were analyzed by Yates' chi-square test and Fisher's exact test.

Studies on the male antifertility agent--gossypol acetic acid. V. Effect of gossypol acetic acid on the fertility of male rats.

Gossypol acetic acid was given to male rats at a dose of 7.5 mg/rat/day six days a week for ten weeks. After nine weeks of gossypol treatment no implantation sites were observed in the females mated with gossypol treated males. After ten weeks of gossypol treatment all the spermatozoa in the vas deferens were non-motile. Gossypol treatment did not affect the body weight and the weights of the accessory sex organs. Plasma LH and FSH levels, hCG binding in testis and succinic dehydrogenase (SDH) and adenosine triphosphatase (ATPase) activities in liver, kidney and testis were not affected by gossypol treatment. Histological observations of the testis revealed partial damage to the seminiferous tubules. Single high doses of gossypol did not induce significant changes in the body weight and weights of testis and accessory sex organs. ATPase activity in the testis was reduced significantly after gossypol treatment, the enzyme activity in liver and kidney, was however, affected at high doses only. Gossypol treatment had no effect on the histoarchitecture of the testis. Intratesticular administration of gossypol evoked localized damage in the testis. Gossypol treatment had no effect on I125 FSH binding to the rat testis homogenate in vitro.

Inhibition of sialyl transferase activity by gossypol acetic acid in human seminal plasma

Seventeen aliquots of 15 microliters seminal plasma of human origin were incubated with 25 micrograms, 15 micrograms, 5 micrograms and 2.5 micrograms gossypol acetic acid (GAA), dissolved in BWW containing 0.9% sodium chloride and 0.9% benzyl alcohol, at 37 degrees C for 60 minutes. Following incubation, activity of sialyl transferase (S.T.) was determined by a procedure involving incorporation of radioactive sialic acid into asialofetuin. The activity of S.T., expressed as cpm per hour per 15 microliters seminal plasma was compared to controls consisting in incubation of sperm in BWW containing 0.9% benzyl alcohol and 0.9% sodium chloride. The activity of S.T. in controls represented 61.3 +/- 8.0%-67.7 +/- 8.9% of the activity obtained by incubation with BWW only. GAA was found to exert a dose-dependent inhibition of S.T. activity, ranging from 38.3 +/- 20.6% to 53.4 +/- 19.4% (with 25 micrograms) and from 11.3 +/- 14.8% to 21.9 +/- 14.8% (with 2.5 micrograms).

The endocrine status of gossypol - treated male rats.

Male adult Wistar rats were exposed daily or every other day to oral gossypol acetic acid (GAA) concentrations of 2.5-30 mg/kg for 10-20 weeks. Controls received the GAA-suspension medium or were left completely untreated. The serum concentrations of testosterone, LH and FSH as well as the weight of testis, epididymis, prostate, seminal vesicle, coagulating gland, and pituitary were determined. The accessory sex organs were prepared for light microscopy. Significant antifertility effect in these animals was achieved at GAA-dosage of 15 mg/kg and higher. GAA-administration neither altered the serum hormonal profiles nor the reproductive organ weights in comparison to the controls. Accordingly, light microscopical examination revealed no alterations in the histological picture of prostate, seminal vesicle and coagulating gland when compared with the controls. The results indicate that GAA does not interfere with the hypothalamic-pituitary-gonadal axis in male adult rats.