AT101-Loaded Cubosomes as an Alternative for Improved Glioblastoma Therapy.

INTRODUCTION: AT101, the R-(-)-enantiomer of the cottonseed-derived polyphenol gossypol, is a promising drug in glioblastoma multiforme (GBM) therapy due to its ability to trigger autophagic cell death but also to facilitate apoptosis in tumor cells. It does have some limitations such as poor solubility in water-based media and consequent low bioavailability, which affect its response rate during treatment. To overcome this drawback and to improve the anti-cancer potential of AT101, the use of cubosome-based formulation for AT101 drug delivery has been proposed. This is the first report on the use of cubosomes as AT101 drug carriers in GBM cells. MATERIALS AND METHODS: Cubosomes loaded with AT101 were prepared from glyceryl monooleate (GMO) and the surfactant Pluronic F-127 using the top-down approach. The drug was introduced into the lipid prior to dispersion. Prepared formulations were then subjected to complex physicochemical and biological characterization. RESULTS: Formulations of AT101-loaded cubosomes were highly stable colloids with a high drug entrapment efficiency (97.7%) and a continuous, sustained drug release approaching 35% over 72 h. Using selective and sensitive NMR diffusometry, the drug was shown to be efficiently bound to the lipid-based cubosomes. In vitro imaging studies showed the high efficiency of cubosomal nanoparticles uptake into GBM cells, as well as their marked ability to penetrate into tumor spheroids. Treatment of GBM cells with the AT101-loaded cubosomes, but not with the free drug, induced cytoskeletal rearrangement and shortening of actin fibers. The prepared nanoparticles revealed stronger in vitro cytotoxic effects against GBM cells (A172 and LN229 cell lines), than against normal brain cells (SVGA and HMC3 cell lines). CONCLUSION: The results indicate that GMO-AT101 cubosome formulations are a promising basic tool for alternative approaches to GBM treatment.

Zero-Order Release of Gossypol Improves Its Antifertility Effect and Reduces Its Side Effects Simultaneously.

Gossypol was considered a promising male contraceptive but finally failed due to two side effects: hypokalemia and the irreversibility of its contraceptive effect. Here we demonstrate that sustained zero-order release could be a solution for these problems. The in vitro release of gossypol from gossypol/PEG layer-by-layer films follows a perfect zero-order kinetics. In vivo tests indicate that the films can maintain the plasma drug concentration constant in male SD rats for ∼20 days for a 30-bilayer film. The plasma drug concentration is 2 orders of magnitude lower than the peak plasma drug concentration when administered orally and the daily dose is >50-fold lower than the commonly used contraceptive oral dose. However, significant antifertility effects were still observed. Furthermore, hypokalemia was not observed, and the antifertility effects can be reversed after a recovery period. The results suggest that zero-order release can significantly improve the desired antifertility effect of gossypol and, meanwhile, significantly reduce its side effects. We envision the drug could be developed to be an effective, safe, and reversible male contraceptive by zero-order release.

Valproic Acid Enhances the Anti-tumor Effect of (-)-gossypol to Burkitt Lymphoma Namalwa Cells.

Burkitt lymphoma is a highly aggressive B-cell neoplasm. New therapeutic methods are needed to overcome the adverse effect of intensive chemotherapy regimens. Valproic acid and (-)-gossypol are two kinds of chemical compounds used as new anti-tumor drugs in recent years. To investigate the anti-tumor effect of valproic acid and (-)-gossypol, Burkitt lymphoma Namalwa cells were cultured and treated with valproic acid and (-)-gossypol at different concentrations. The proliferation of Namalwa cells was dramatically suppressed after the combination treatment with 2 mmol/L valproic acid and 5 µmol/L (-)-gossypol. The combined treatment also enhanced intrinsic apoptosis by down-regulating anti-apoptotic protein Mcl-1. Moreover, the autophagy flux significantly increased in Namalwa cells after combined treatment. However, the enhanced autophagy showed little effect on cell survival with present regimen. The results confirmed that combination of valproic acid and (-)-gossypol had synergistic anti-tumor effect to Burkitt lymphoma Namalwa cells. The related mechanisms might include the down-regulation of anti-apoptotic protein Mcl-1 and avianized pro-survival role of autophagy.

Targeting anti-apoptotic Bcl-2 by AT-101 to increase radiation efficacy: data from in vitro and clinical pharmacokinetic studies in head and neck cancer.

BACKGROUND: Pro-survival Bcl-2 family members can promote cancer development and contribute to treatment resistance. Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by overexpression of anti-apoptotic Bcl-2 family members. Increased levels of these anti-apoptotic proteins have been associated with radio- and chemoresistance and poor clinical outcome. Inhibition of anti-apoptotic Bcl-2 family members therefore represents an appealing strategy to overcome resistance to anti-cancer therapies. The aim of this study was to evaluate combined effects of radiation and the pan-Bcl-2 inhibitor AT-101 in HNSCC in vitro. In addition, we determined human plasma levels of AT-101 obtained from a phase I/II trial, and compared these with the effective in vitro concentrations to substantiate therapeutic opportunities. METHODS: We examined the effect of AT-101, radiation and the combination on apoptosis induction and clonogenic survival in two HNSCC cell lines that express the target proteins. Apoptosis was assessed by bis-benzimide staining to detect morphological nuclear changes and/or by propidium iodide staining and flow-cytometry analysis to quantify sub-diploid apoptotic nuclei. The type of interaction between AT-101 and radiation was evaluated by calculating the Combination Index (CI) and by performing isobolographic analysis. For the pharmacokinetic analysis, plasma AT-101 levels were measured by HPLC in blood samples collected from patients enrolled in our clinical phase I/II study. These patients with locally advanced HNSCC were treated with standard cisplatin-based chemoradiotherapy and received dose-escalating oral AT-101 in a 2-weeks daily schedule every 3 weeks. RESULTS: In vitro results showed that AT-101 enhances radiation-induced apoptosis with CI's below 1.0, indicating synergy. This effect was sequence-dependent. Clonogenic survival assays demonstrated a radiosensitizing effect with a DEF37 of 1.3 at sub-apoptotic concentrations of AT-101. Pharmacokinetic analysis of patient blood samples taken between 30 min and 24 h after intake of AT-101 showed a dose-dependent increase in plasma concentration with peak levels up to 300-700 ng/ml between 1.5 and 2.5 h after intake. CONCLUSION: AT-101 is a competent enhancer of radiation-induced apoptosis in HNSCC in vitro. In addition, in vitro radiosensitization was observed at clinically attainable plasma levels. These finding support further evaluation of the combination of AT-101 with radiation in Bcl-2-overexpressing tumors.

Identification of glucuronidation and biliary excretion as the main mechanisms for gossypol clearance: in vivo and in vitro evidence.

1. The natural polyphenol gossypol possesses many therapeutic benefits. Here we aim to determine the elimination pathways of gossypol in vivo and in vitro. 2. Metabolite elucidation of gossypol was performed using UPLC-QTOF/MS coupled with Metabolynx analysis. Clearance of gossypol was evaluated in bile duct cannulated rats and in the single-pass perfused rat intestine model. In vitro glucuronidation of gossypol was characterized using liver and intestine microsomes as well as recombinant UDP-glucuronosyltransferase (UGT) enzymes. 3. Analysis of rat plasma, urine, and feces revealed glucuronidation as the only metabolic pathway for gossypol. In bile duct cannulated rats, considerable amounts of glucuronides (G1, G2 and G3; 58.8-83.2% of dose) and parent compound (5.0-20%) were excreted into bile after IV administration. In the perfused rat intestine model, gossypol was well absorbed with a [Formula: see text] (the dimensionless effective permeability) value of 4.4. Significant amounts of glucuronides (G1, G2 and G3) were excreted into the gut lumen (2.5%) and into the bile (4.8%). Biliary excretion of unchanged gossypol (6.0%) was comparable to that of glucuronides. Further, gossypol was subjected to rapid glucuronidation by liver and intestine microsomes. Reaction phenotyping showed that multiple UGT1A enzymes (including UGT1A1, 1A3, 1A7 and 1A8) are mainly responsible for gossypol metabolism. 4. In conclusion, glucuronidation was the only metabolic pathway for gossypol in rats. Excretion of unchanged gossypol into bile was also an important clearance mechanism.

A phase I study of AT-101 with cisplatin and etoposide in patients with advanced solid tumors with an expanded cohort in extensive-stage small cell lung cancer.

BACKGROUND: A phase I, dose-escalation study of AT-101 with cisplatin and etoposide was conducted to determine the maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), safety and pharmacokinetics in patients with advanced solid tumors, with an expanded cohort in patients with extensive-stage small cell lung cancer (ES-SCLC) to assess preliminary activity. METHODS: In the dose escalation portion, increasing doses of AT-101 were administered orally BID on days 1-3 along with cisplatin on day 1 and etoposide on days 1-3 of a 21 day cycle. At the RP2D, an additional 7 patients with untreated ES-SCLC were enrolled. RESULTS: Twenty patients were enrolled in the dose-escalation cohort, and 7 patients with ES-SCLC were enrolled in the expanded cohort. The MTD/RP2D was established at AT-101 40 mg BID days 1-3 with cisplatin 60 mg/m2 and etoposide 120 mg/m2 on day 1 of a 21 day cycle with pegfilgrastim support. Two DLTs of neutropenic fever were seen at dose level 1. After the addition of pegfilgrastim, no additional DLTs were observed. Grade 3/4 treatment-related toxicities included: diarrhea, increased AST, neutropenia, hypophosphatemia, hyponatremia, myocardial infarction and pulmonary embolism. No apparent PK interactions were observed between the agents. Preliminary activity was observed with PRs in patients with ES-SCLC, high-grade neuroendocrine tumor, esophageal cancer and NSCLC. CONCLUSIONS: AT-101 with cisplatin and etoposide is well tolerated with growth factor support. Anti-tumor activity was observed in a variety of cancers including ES-SCLC, supporting further investigation with BH-3 mimetics in combination with standard chemotherapy for ES-SCLC.

Effects of different sources and levels of dietary gossypol on gossypol residues in plasma and milk of lactating cows.

Free gossypol residues in tissues or milk from feeding whole cottonseed and cottonseed meal were measured for their effect on health of dairy cows and humans. Forty lactating cows were randomly assigned to 5 treatments in a 60-d experiment to investigate the effects of sources and dietary level of gossypol on plasma and milk gossypol concentrations in lactating cows. Five experimental diets had identical net energy for lactation and crude protein content on a dry matter (DM) basis. Soybean meal was the main protein ingredient used in the control diet. Cottonseed meal (CSM) or whole cottonseed (WCS) substituted for part of the soybean meal in the other 4 diets. Gossypol levels in the 5 diets were 0 (control), 91.15 mg/kg of DM in CSM1, 117.31mg/kg of DM in CSM2, 385.43 mg/kg of DM in WCS1, and 611.13 mg/kg in WCS2. Yields of 3.5% fat-corrected milk were significantly higher for cows in the WCS2 group; cows in the CSM1 and WCS1 groups showed no differences but both were numerically higher than the control and CSM2 groups. Milk protein concentration was lower for cows consuming WCS1 compared with the control group. Lactose concentration was lower for cows in the CSM2 group compared with the WCS2 group, but no differences were observed among other diets. Aspartate aminotransferase in serum was significantly higher for the WCS2 group compared with the control and WCS1 groups, but no difference was observed with the CSM1 and CSM2 groups. Concentrations of gossypol in plasma and milk of cows in the WCS1 and WCS2 groups were both higher than those of the other groups. No adverse effects were observed on cows fed diets containing 12.0% CSM, and no gossypol was found in plasma and milk. When WCS comprised 15% of the diet DM, yields of 3.5% fat-corrected milk were increased in cows and gossypol was detected in plasma and milk but not at harmful levels.

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.

Comparison of pharmacokinetic and metabolic profiling among gossypol, apogossypol and apogossypol hexaacetate.

PURPOSE: To characterize the stability, pharmacokinetics and metabolism of analogs of gossypol, apogossypol and apogossypol hexaacetate to provide a basis for comparison. METHODS: Gossypol, apogossypol and apogossypol hexaacetate were incubated in plasma or liver microsomes from various species, or administered to mice, respectively, from which the stability, metabolism and pharmacokinetic profiles of these analogs were quantitatively determined using a liquid chromatography-mass spectrometry (LC/MS/MS) method. RESULTS: In various species of plasma, apogossypol and gossypol exhibited similar stability, while 20-40% of apogossypol hexaacetate was converted into apogossypol with concurrent formation of the corresponding di-, tri-, tetra-, and penta-acetates of apogossypol. (+/-)-Gossypol and (-)-gossypol showed comparable pharmacokinetic profile and oral bioavailability (12.2-17.6%) with some variations of clearance and V (ss) following oral and intravenous administration to mice. At the same molar dose, apogossypol showed delayed T (max)(1 h), a slower clearance rate and less distribution after administration to mice. Mono- and di-glucuronide conjugates of apogossypol were readily observed in mouse plasma following administration. Apogossypol formulated in sesame oil appeared to possess larger AUC and thus higher oral bioavailability than that formulated in cremophor EL:ethanol:saline. In contrast, intravenous apogossypol hexaacetate exhibited highest clearance rate partially due to its conversion into apogossypol. Concomitant with disappearance of apogossypol hexaacetate (iv), apogossypol converted from apogossypol hexaacetate was quantitatively detected, and accounted for approximately 30% of total plasma apogossypol hexaacetate. Oral apogossypol hexaacetate showed no bioavailability with little apogossypol occurring in the plasma. In human and mouse liver microsomes, glucuronide conjugates of apogossypol and its acetates were readily identified with the exception of gossypol glucuronidation. Apogossypol appeared more stable in human and mouse liver microsomal preparations than gossypol and apogossypol hexaacetate. CONCLUSIONS: Apogossypol and gossypol show similar oral and intravenous pharmacokinetic profiles and in vitro stability although apogossypol appears to have a slower clearance rate, larger AUC, and better microsomal stability. Apogossypol hexaacetate converts to apogossypol in both in vitro and in vivo settings and lacks any quantifiable oral bioavailability.

Effects of source of gossypol and supplemental iron on plasma gossypol in Holstein steers.

Four experiments were conducted to evaluate factors influencing concentrations of plasma total gossypol (TG) in 30 Holstein steers fed cottonseed products. At the end of each 28-d experiment, steers were weighed and blood samples were collected and analyzed for plasma TG concentrations. During the entire study, steers did not show any overt signs of gossypol toxicity. In the 28 d before experiment 1, 30 steers with a body weight (BW) of 273 kg were fed a standardization diet with 15.0% Upland whole cottonseed (WCS) that resulted in a mean intake of 9.08 g/d of TG per steer/d and a plasma TG of 1.66 microg/mL. In experiment 1, 30 steers were fed 1 of 5 diets with 15.0% Upland WCS, but different levels of supplemental Fe [0, 150, 300, 450, and 600 mg/kg of diet dry matter (DM)]. Average daily gain was not affected by level of Fe in the diet, but DM intake, plasma TG, and plasma TG response decreased linearly as Fe in diets increased. In experiment 2, steers were fed diets with 15.0% Upland cottonseed as whole, cracked, roasted, cracked-roasted, or extruded. Analysis of the seed revealed that roasting or extrusion markedly reduced free gossypol (FG) content. Minor effects on animal performance were observed, but plasma TG decreased with roasting or extrusion of seeds, with the greatest reduction when the seed was cracked and then roasted. In experiment 3, steers were fed 2 levels of WCS (7.0 or 14.0% of DM) with 3 levels of cottonseed meal (2.8, 5.5, or 8.5% of DM) in the diet. Animal performance was not altered by diet, but plasma gossypol concentrations and responses were greater in steers fed diets with more WCS, because of the greater FG intake. In experiment 4, 24 steers were fed diets with 15.0% cottonseed (Upland or Pima) either as whole or cracked. Pima cottonseed increased TG and FG intakes, which resulted in greater plasma TG concentration and response. Animal response to processing of cottonseed tended to differ according to type of cottonseed. However, feeding Pima and cracking of cottonseed increased gossypol availability and plasma TG concentrations.

A mixture of cottonseed meal, soybean meal and animal byproduct mixture as a fish meal substitute: growth and tissue gossypol enantiomer in juvenile rainbow trout (Oncorhynchus mykiss).

Diets incorporating three different sources of extracted cottonseed meal (CM), soybean meal and an animal protein mixture were evaluated for juvenile rainbow trout. Fish averaging 0.96 g were divided into groups of 30; 3 groups per treatment, and each group was fed one of four diets for a 16-week period. Fish meal (FM) was replaced on a 25% protein basis by each of three different sources of CM from California (CA), Tennessee (TN), and Arkansas (AR), U.S.A. In the three CM-containing diets another 25% soybean meal protein and 50% animal protein mixture were also incorporated to completely replace FM protein. The results of growth rate and feed utilization showed that FM could be entirely replaced by a mixture of plant proteins (CM and soybean meal) and animal by-product proteins. Hematocrit levels were significantly lower in the group fed CM-containing diets than in the control. The findings suggest that CM can be used as a good protein source by the incorporation of at least 15% in diets (25% of fish meal protein replacement), and that the nutritive values of CM in juvenile trout can be different due to their different origin. Significantly higher concentrations of total gossypol were found in faeces of CM-TN (5.8 +/- 0.4 micromol/g) and CM-AR (5.6 +/- 0.6) groups than in that of CM-CA (3.7 +/- 0.4) group. It was documented that gossypol enantiomers, present in an equal proportion in diets, selectively accumulated in liver and bile, whereas equal proportions of (+)- and (-)-enantiomers were found in whole-body and faeces. Depending on CM source, fish can absorb approximately 35-50% of dietary gossypol, and the majority of the absorbed gossypol seemed to be excreted.

Use of expander cottonseed meal in broiler diets formulated on a digestible amino acid basis.

This experiment was designed to evaluate the use of expander cottonseed meal (CSM) in broiler diets formulated on a digestible amino acid basis and to determine the tissue distribution of gossypol enantiomers and their relationship to gossypol consumption. Gossypol is an antinutritional polyphenolic pigment found in cottonseed. A total of 800 1-d-old broilers (Arbor Acres) was randomly divided into 40 groups of 20 birds each and placed in a broiler house with wood shavings litter. The CSM was included in starter and grower diets at 0, 7, 14, 21, and 28% of the diet. Diets were formulated to have similar levels of digestible methionine and lysine as the 0% CSM control diet. At 21 d of age, five birds per pen were randomly selected for the determination of gossypol enantiomers in plasma, liver, heart, and breast muscle. Tissues were again collected at 42 d of age. Results of this experiment indicated that at 21 d of age cumulative body weight and feed-to-BW ratios of birds fed CSM diets were not significantly different from the control. By 35 d of age, feed-to-BW ratios of experimental birds fed the 28% CSM diet were significantly greater than the control birds, and by 42 d, BW were lower than those of the control group. Tissue concentrations of total, (+) and (-) gossypol increased linearly as the level of CSM increased. At 42 d, liver had the most gossypol with a ratio of 87% (+) gossypol to 13% (-) gossypol. Plasma contained 73% (+) gossypol and 27% (-) gossypol. Heart contained 45% (+) gossypol and 55% (-) gossypol. Breast muscle had the least gossypol. Results of this experiment indicated that expander solvent CSM could be fed to broilers at up to 21% of the total diet if amino acid digestibility was considered.

Cottonseed with a high (+)- to (-)-gossypol enantiomer ratio favorable to broiler production.

This study was designed to evaluate the relative toxicity of (+)- and (-)-gossypol enantiomers in 0-3-week-old broilers. Treatments consisted of broiler starter diets formulated with either a glandless, which did not contain gossypol, a commercial glanded [62. 2% (+)-gossypol], or a glanded moco [83.2% (+)-gossypol] crushed cottonseed (CCS) (six replicates/treatment) plus a soybean meal negative control. Glandless cottonseed was mixed with the moco cottonseed (2.4% free gossypol) so that both the commercial glanded and moco glanded cottonseeds contained equivalent concentrations of free gossypol (2.0%). The cottonseed treatments were added at 5 and 10% of the diet. Body weights and feed conversions were determined weekly. Body weights and feed-to-gain ratios of broilers fed 5 and 10% glandless CCS and 5% moco CCS were not significantly different. Broilers receiving 10% commercial glanded CCS weighed significantly less than those subjected to all other treatments. Feed-to-gain ratios were significantly higher for broilers receiving 10% commercial glanded and 10% moco CCS as compared to 5% moco and glandless CCS, 10% glandless CCS, and control. Relative liver weights of birds receiving 10% moco CCS were significantly less than those of birds receiving 10% commercial CCS. The data clearly showed that broilers fed moco CCS containing a relatively high (+)- to (-)-gossypol enantiomer ratio performed better than broilers receiving commercial CCS with a lower (+)- to (-)-gossypol enantiomer ratio.

Gossypol treatment of recurrent adult malignant gliomas.

Gossypol, a polyphenolic compound which depletes cellular energy by inhibition of several intracellular dehydrogenases, has been shown to have antiproliferative activity against human glial tumor cell lines in vitro and in nude mouse xenografts. Human trials of gossypol as a male contraceptive have demonstrated safety of long-term administration. We studied the activity of Gossypol 10 mg PO bid in 27 patients with pathologically confirmed glial tumors which had recurred after radiation therapy. Fifteen patients had glioblastoma, 11 patients anaplastic astrocytoma, 1 patient relapsed low grade glioma. Response was assessed every 8 weeks using CT/MRI scan and clinical criteria including decadron requirement. Treatment was continued until disease progression. Two patients had partial response (PR); 4 had stable disease for 8 weeks or more. One patient maintained a PR with improved KPS for 78 weeks. The other had a PR lasting 8 weeks. Toxicity was mild: 2 heavily pretreated patients had mild thrombocytopenia, 5 patients developed hypokalemia, 3 patients developed grade 2 hepatic toxicity and peripheral edema. Gossypol levels measured by HPLC did not correlate with response or toxicity in this study. We conclude that gossypol is well tolerated and has a low, but measurable, response rate in a heavily pretreated, poor-prognosis group of patients with recurrent glioma. The presumed novel mechanism of action, lack of significant myelosuppression, and activity in patients with advance glioma support further study of gossypol as an antineoplastic agent.

Accumulation of gossypol enantiomers in ovine tissues.

Tissue residue levels of gossypol enantiomers in cottonseed-fed and lethally intoxicated lambs were determined by the high-performance liquid chromatography-ultraviolet detection method. Gossypol was derivatized with (+)-2-amino-1-propanol and separated with a reversed-phase C18 column and the elution of analytes was monitored at 254 nm. The highest residue level was found in the liver tissue (318-416 ng total gossypol/mg dry tissue), and the residue of (-)-gossypol was higher than (+)-gossypol in the heart, muscle and spleen. The detection limit was 2 ng, and the detector response of gossypol-amine adducts was linear between 2 and 100 ng enantiomers.

Transfer of 3H-gossypol to neonatal rats via milk of nursing dams.

Gossypol is a naturally occurring toxin with potent antifertility action in both males and females. Transfer of gossypol via milk from lactating mother to neonates has not been documented. One theory for the lack of such a finding is that gossypol, once tightly bound to milk proteins, is not extractable by conventional extraction methods for HPLC analysis. This study was designed to examine the possibility and dynamics of transmammary transfer of radioactively-labelled gossypol to neonates through nursing. Nursing rats were dosed with fifty microCi of 3H-gossypol in 0.5 ml of dimethyl sulfoxide via oral intubation on Day 10 post-partum. Six hours after treatment, significant amounts of 3H-gossypol were detected in the coagulated milk collected from the stomachs of the neonates as well as in the blood, liver, lung, spleen and a number of other tissues of these pups. While most neonatal tissues showed a steady decline in 3H-gossypol retention over time regardless of neonatal gender, adrenal glands and gonads preferentially accumulated 3H-gossypol in a time- and sex-dependent manner during the 24-hour sampling period. The results demonstrate that 3H-gossypol is transferred via milk from the nursing dams to their neonates. Thus, the gossypol content of milk presents a conceivable threat to neonatal health, especially to the normal function of steroidogenic organs.

Gossypol pharmacokinetics in mid-lactation Brown Swiss dairy cows.

A bolus equivalent to 450 ppm (dosage based on average feed intake for lactating dairy cows of similar mass) of gossypol was administered orally to three Brown Swiss dairy cows in mid lactation daily for a 7-day treatment period. Blood samples were taken during a 2-day pretreatment period, the 7-day treatment period and a 6-day recovery period. The serum recovered from the cows was stored at -20 degrees C until analysis for extractable gossypol content. The highest concentration of gossypol (a mean of 0.53 microgram/ml serum) was attained in all the cows on day 6 of the treatment period, indicating that a steady-state condition had been reached before the end of the treatment period. The gossypol concentrations then gradually declined during the 6-day recovery period but never fell to the zero baseline. The cows exhibited terminal elimination half-lives of 67, 67.5 and 40 h. Gossypol elimination was best described by a bi-exponential decay curve in two cows and a mono-exponential decay curve in the remaining cow.

Stereoselective interaction between gossypol and rat plasma.

Gossypol, a potential male oral contraceptive, is chiral and chemically reactive. The present study was done to learn more about the stereoselective activity of this drug. The isomers were equipotent in hemolyzing erythrocytes in protein-free buffer while (+) gossypol was a more potent hemolysin than (-) in plasma. Both isomers disappeared from buffer at the same rate while (-) disappeared from plasma much faster than (+). Treating plasma with aspirin or DNFB to react with the free amino groups on the protein, slowed the disappearance of (-) gossypol. We conclude that (-) gossypol binds to free amino groups on protein and this stereoselective protein binding may account for some of the pharmacokinetic or pharmacodynamic difference between the two isomers of gossypol.

Distribution of gossypol.

Male rabbits and rats were administered gossypol (20 mg/kg/day) for 12 and 7 weeks respectively. Gossypol was estimated in different organs by the aniline method of Smith. Rat and rabbit spleen accumulated the highest level of gossypol. The lowest amount of gossypol was accumulated in the rabbit brain; the level of gossypol in rat brain was below the detectable limit of our method. Although rabbits were administered gossypol for 12 weeks, the accumulation of gossypol in rabbit testis was much lower than that of the rat testis. Our data suggest that non-sensitivity of rabbit to the antifertility effect of gossypol may be due to poor accumulation of gossypol in the testis. Negligible amount of gossypol in the brain rules out the possibility of involvement of hypothalamus-pituitary axis in the mechanism of action of gossypol on the testis.