Novel Formulation Strategy to Improve the Feasibility of Amifostine Administration.

PURPOSE: Amifostine (AMF), a radioprotectant, is FDA-approved for intravenous administration in cancer patients receiving radiation therapy (XRT). Unfortunately, it remains clinically underutilized due to adverse side effects. The purpose of this study is to define the pharmacokinetic profile of an oral AMF formulation potentially capable of reducing side effects and increasing clinical feasibility. METHODS: Calvarial osteoblasts were radiated under three conditions: no drug, AMF, and WR-1065 (active metabolite). Osteogenic potential of cells was measured using alkaline phosphatase staining. Next, rats were given AMF intravenously or directly into the jejunum, and pharmacokinetic profiles were evaluated. Finally, rats were given AMF orally or subcutaneously, and blood samples were analyzed for pharmacokinetics. RESULTS: WR-1065 preserved osteogenic potential of calvarial osteoblasts after XRT to a greater degree than AMF. Direct jejunal AMF administration incurred a systemic bioavailability of 61.5%. Subcutaneously administrated AMF yielded higher systemic levels, a more rapid peak exposure (0.438 vs. 0.875 h), and greater total systemic exposure of WR-1065 (116,756 vs. 16,874 ng*hr/ml) compared to orally administered AMF. CONCLUSIONS: Orally administered AMF achieves a similar systemic bioavailability and decreased peak plasma level of WR-1065 compared to intravenously administered AMF, suggesting oral AMF formulations maintain radioprotective efficacy without causing onerous side effects, and are clinically feasible.

Evaluation of a method for measuring the radioprotective metabolite WR-1065 in plasma using chemical derivatization combined with UHPLC-MS/MS.

Hypotension is the dose-limiting side effect of the radio-protective drug Amifostine and results from relaxation of the vascular smooth muscle, which is directly mediated by the active metabolite, WR-1065, of Amifostine. The route of administration (currently FDA-approved only for intravenous administration) and the rapid metabolic conversion of Amifostine combine to yield high systemic levels of WR-1065 and facilitate the onset of hypotension. Research efforts aiming to optimize the delivery of WR-1065 to maintain efficacy while reducing its peak, systemic concentration below levels that induce hypotension are underway. To fully characterize the effect of reduced dose levels and alternative routes of administration of Amifostine on systemic WR-1065 concentrations, improved analytical techniques are needed. We have developed and evaluated a highly sensitive method for measuring WR-1065 in rat plasma that employs chemical derivatization, protein precipitation and UPLC-MS/MS analysis. The method exhibits a limit of quantification (LOQ) of 7.4 nM in plasma, which is a significant improvement over conventional approaches that utilize LC-electrochemical detection (ECD) (LOQ 150 nM or higher). The method was assessed in a pharmacokinetics study in rats administered Amifostine intravenously and via direct jejunal injection (10 mg/kg each route). The bioavailability of WR-1065 was 61.5% after direct jejunal injection indicating rapid conversion and absorption of the metabolite in the intestinal tract. This demonstrates that an oral formulation of Amifostine designed for site-specific release of the drug in the upper GI tract can deliver systemic absorption/conversion to WR-1065, provided that the formulation protects the therapeutic from gastric decomposition in the stomach.

Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells.

Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration.

Ranking the Binding Energies of p53 Mutant Activators and Their ADMET Properties.

The guardian of the genome, p53, is the most mutated protein found in all cancer cells. Restoration of wild-type activity to mutant p53 offers promise to eradicate cancer cells using novel pharmacological agents. Several molecules have already been found to activate mutant p53. While the exact mechanism of action of these compounds has not been fully understood, a transiently open pocket has been identified in some mutants. In our study, we docked twelve known activators to p53 into the open pocket to further understand their mechanism of action and rank the best binders. In addition, we predicted the absorption, distribution, metabolism, excretion and toxicity properties of these compounds to assess their pharmaceutical usefulness. Our studies showed that alkylating ligands do not all bind at the same position, probably due to their varying sizes. In addition, we found that non-alkylating ligands are capable of binding at the same pocket and directly interacting with Cys124. The comparison of the different ligands demonstrates that stictic acid has a great potential as a p53 activator in terms of less adverse effects although it has poorer pharmacokinetic properties.

A phase I clinical and pharmacology study using amifostine as a radioprotector in dose-escalated whole liver radiation therapy.

PURPOSE: Diffuse intrahepatic tumors are difficult to control. Whole-liver radiotherapy has been limited by toxicity, most notably radiation-induced liver disease. Amifostine is a prodrug free-radical scavenger that selectively protects normal tissues and, in a preclinical model of intrahepatic cancer, systemic amifostine reduced normal liver radiation damage without compromising tumor effect. We hypothesized that amifostine would permit escalation of whole-liver radiation dose to potentially control microscopic disease. We also aimed to characterize the pharmacokinetics of amifostine and its active metabolite WR-1065 to optimize timing of radiotherapy. METHODS AND MATERIALS: We conducted a radiation dose-escalation trial for patients with diffuse, intrahepatic cancer treated with whole-liver radiation and intravenous amifostine. Radiation dose was assigned using the time-to-event continual reassessment method. A companion pharmacokinetic study was performed. RESULTS: Twenty-three patients were treated, with a maximum dose of 40 Gy. Using a logistical regression model, compared with our previously treated patients, amifostine increased liver tolerance by 3.3 ± 1.1 Gy (p = 0.007) (approximately 10%) with similar response rates. Peak concentrations of WR-1065 were 25 µM with an elimination half-life of 1.5 h; these levels are consistent with radioprotective effects of amifostine in patients. CONCLUSION: These findings demonstrate for the first time that amifostine is a normal liver radioprotector. They further suggest that it may be useful to combine amifostine with fractionated or stereotactic body radiation therapy for patients with focal intrahepatic cancer.

Clinical pharmacokinetics of amifostine and WR1065 in pediatric patients with medulloblastoma.

PURPOSE: We evaluated the pharmacokinetics of amifostine and WR1065 in pediatric patients with newly diagnosed medulloblastoma to assess the influence of patient covariates, including demographics, clinical characteristics, and genetic polymorphisms, on amifostine and WR1065 pharmacokinetic parameters. EXPERIMENTAL DESIGN: We assessed the pharmacokinetics of amifostine and WR1065 in 33 children who received amifostine (1-minute infusion, 600 mg/m(2)) just before the start of and 3 hours into a 6-hour cisplatin infusion. Serial blood samples were collected after doses 1 (0 hour) and 2 (3 hours) of course 1. Amifostine and WR1065 were quantitated by high performance liquid chromatography with electrochemical detection. A pharmacokinetic model was simultaneously fit to amifostine and WR1065 plasma or whole blood concentration-versus-time data. The influence of demographic, biochemical, and pharmacogenetic covariates on amifostine and WR1065 disposition was evaluated. RESULTS: Body surface area was the primary size-based covariate for amifostine pharmacokinetics explaining 53% and 56% of interindividual variability in plasma and whole-blood amifostine clearance, respectively. The population-predicted values for amifostine clearance, volume, and apparent WR1065 clearance from the plasma data were 107 L/h/m(2), 5.53 L/m(2), and 30.6 L/h/m(2). The population-predicted values for amifostine clearance, volume, and apparent WR1065 clearance from whole blood data were 136 L/h/m(2), 7.23 L/m(2), and 12.5 L/h/m(2). CONCLUSIONS: These results support using body surface area for calculating doses of amifostine in children. Similar to data in adults, amifostine and WR1065 are rapidly cleared from plasma and whole blood in children.

Amifostine (ETHYOL) protects rats from mucositis resulting from fractionated or hyperfractionated radiation exposure.

PURPOSE: The cytoprotective drug amifostine (Ethyol) protects rats from oral mucositis resulting from a single dose of gamma-irradiation. We expanded earlier studies to determine whether multiple doses of amifostine protect against fractionated or hyperfractionated radiation and whether the active metabolite of amifostine (WR-1065) accumulates in tissues upon repeated administration. METHODS AND MATERIALS: Rats received amifostine daily for 5 days in conjunction with a 1-week fractionated radiation schedule and were evaluated for oral mucositis. Rats also received amifostine before the am or pm exposure or b.i.d. in conjunction with hyperfractionated radiation. To determine the pharmacokinetics of WR-1065 after repeated dosing, amifostine was given 5 days a week for 1 or 3 weeks, and rat tissue and plasma were collected at intervals during and after treatment and analyzed for WR-1065. RESULTS: Amifostine protected rats from mucositis resulting from fractionated or hyperfractionated radiation. When the number of days of amifostine administration was reduced, protection was diminished. A dose of 100 mg/kg given in the morning or 2 doses at 50 mg/kg provided the best protection against hyperfractionated radiation. WR-1065 did not accumulate in tissues or tumor upon repeated administration. CONCLUSIONS: Amifostine prevented radiation-induced mucositis in a rat model; protection was dose and schedule dependent.

Effects of dose and schedule on the efficacy of ethyol: preclinical studies.

The chemo- and radioprotectant drug amifostine (Ethyol; MedImmune, Inc, Gaithersburg, MD) is approved for intravenous (IV) administration; however, the subcutaneous (SC) route is being explored as a practical alternative. We have previously reported equivalence between IV and SC administration using a rat model of radioprotection and active metabolite (WR-1065) tissue pharmacokinetics. To examine the more clinically relevant fractionated and hyperfractionated radiation schedules and the effects of variations in the time of amifostine administration, we expanded these studies to include radioprotection and pharmacokinetic studies of WR-1065 using multiple dosing. To measure radioprotection using a fractionated radioprotection model, rats were given amifostine over a 1-week period at various doses (25 mg/kg, 50 mg/kg, 100 mg/kg; or 162.5 mg/m(2), 325 mg/m(2), 650 mg/m(2), respectively) IV or SC daily 30 minutes before exposure to 7.5 Gy/dose. Rats were fully protected from mucositis at the highest amifostine dose, with protection diminishing as the amifostine was decreased. Equivalent protection was observed whether the drug was given IV or SC. When the number of days of amifostine administration was reduced, protection was diminished. Amifostine also protected against radiation delivered using a 1-week hyperfractionated schedule (4.5 Gy/exposure twice daily), with optimal protection occurring when the drug was administered bid 30 minutes before each exposure (50 mg/kg) or every day before the morning exposure (100 mg/kg). The need for daily dosing to achieve optimal radioprotection was consistent with the tissue pharmacokinetics of the active metabolite. We found that WR-1065 did not accumulate in tissues or in SC-implanted tumors when amifostine was administered daily for 3 weeks. In addition, tissue and tumor levels of WR-1065 declined to baseline 24 hours after each amifostine dose. In a monkey pharmacokinetic model, plasma levels of WR-1065 (characterized by a pronounced spike of WR-1065 immediately after IV administration that was absent when the drug was given SC) were similar to those of humans; however, levels of WR-1065 in the tissues were higher 30 minutes following SC administration and were equivalent 60 minutes following IV or SC administration. These results suggest that maximum tissue levels and protection occur when amifostine is given 30 to 60 minutes before radiation exposure, that treatment breaks reduce the radioprotection by amifostine, and that protection from hyperfractionated radiation is dependent on amifostine dose and schedule.

Phase I trial of a twice-daily regimen of amifostine with ifosfamide, carboplatin, and etoposide chemotherapy in children with refractory carcinoma.

BACKGROUND: Amifostine protects normal tissues against chemotherapy and radiation-induced toxicity without loss of antitumor effects. Evidence suggests that multiple daily doses of amifostine may improve its cytoprotective effects. The purpose of this study was to assess the dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of twice-daily doses of amifostine with ifosfamide, carboplatin, and etoposide (ICE) chemotherapy for children with refractory malignancies and to determine the pharmacokinetic properties of amifostine, WR-1065, and the disulfide metabolites of amifostine. METHODS: Patients with refractory malignancies were treated with amifostine 15 minutes before and 2 hours after chemotherapy with ifosfamide (3 g/m(2) per dose on Days 1 and 2) and carboplatin (635 mg/m(2) on Day 3). Etoposide was administered on Days 1 and 2 (150 mg/m(2)). The starting dose of amifostine was 740 mg/m(2). Pharmacokinetic studies were performed after the first dose of amifostine. RESULTS: Twelve patients received 23 courses of ICE and amifostine. Dose-limiting toxicities for amifostine at 740 mg/m(2) were somnolence and anxiety. The other Grade 3 and 4 toxicities included asymptomatic, reversible hypocalcemia, vomiting, and reversible hypotension. At a dose of 600 mg/m(2), amifostine was well tolerated. Hypocalcemia, due to rapid, transient suppression of parathyroid hormone production, required close monitoring and aggressive intravenous calcium supplementation. Pharmacokinetic studies revealed high interpatient variability with rapid plasma clearance of amifostine and WR-1065. The median elimination half-life of amifostine (9.3 minutes) and WR-1065 (15 minutes) was much shorter than the disulfide metabolites (74.4 minutes). CONCLUSIONS: The recommended pediatric dose of amifostine for a twice-daily regimen is 600 mg/m(2) per dose (1200 mg/m(2)/day) with DLTs of anxiety and somnolence, lower than the previously recommended single dose of 1650 mg/m(2).

Mammalian cell polyamine homeostasis is altered by the radioprotector WR1065.

Mammalian cells become more susceptible to radiation-induced death and mutagenesis when restricted in their production of the natural polyamines putrescine, spermidine and spermine. The effects of polyamine deprivation are reversed by N-(2-mercaptoethyl)-1, 3-diaminopropane (WR1065), a simple aminothiol that has been extensively studied for its radioprotectant properties. Because this compound and its oxidized derivative WR33278 bear some resemblance to the polyamines, it was hypothesized that radioprotection by WR1065 or its metabolites is derived, at least in part, from their ability to supplement the natural polyamines. To evaluate the ability of these aminothiol compounds to emulate polyamine function in intact cells, rat liver hepatoma (HTC) cells were treated with radioprotective doses of WR1065; the ability of this compound to affect various aspects of normal polyamine metabolism was monitored. Although cellular WR1065 was maintained at levels exceeding those of the polyamines, this aminothiol did not have any polyamine-like effect on the initial polyamine biosynthetic enzyme, ornithine decarboxylase, or on polyamine degradative reactions. On the contrary, treatment with relatively low levels of WR1065 resulted in an unexpected increase in putrescine and spermidine synthesis. WR1065 treatment enhanced the stability, and consequently the activity, of ornithine decarboxylase. This stabilization seems to result from a WR1065-induced delay in the synthesis of antizyme, a critical regulatory protein required in the feedback modulation of polyamine synthesis and transport. The increase in cellular spermidine induced by WR1065 might explain its antimutagenic properties, but is probably not a factor in protection against cell killing by radiation. This is the first evidence that compounds can be designed to control polyamine levels by targeting the activity of the regulatory protein antizyme.

The limits to radioprotection of Chinese hamster V79 cells by WR-1065 under aerobic conditions.

Clonogenic survival and drug content for Chinese hamster V79-171 cells incubated in suspension with WR-1065 prior to gamma irradiation have been determined. Factors that might influence the radioprotection by WR-1065 were investigated in control studies. Intracellular drug levels studied ranged between 0-36 nmol per 10(6) cells. In control studies, it was established that extracellular drug toxicity was not significant for cells in suspension at 10(6) per milliliter over short periods but was important when residual drug was present above 2 microM in the final plating of cells. Accumulation of intracellular drug above 30 nmol per 10(6) cells produced significant cytotoxicity in unirradiated cells. Irradiation with doses as high as 150 Gy produced no significant change in the total drug level or the thiol/disulfide ratio, either for the drug in the cells or for the drug in the medium. Preirradiation with 8 Gy did not change the ability of cells to import the drug but did appear to increase the cytotoxicity of the intracellular drug at levels above 25 nmol per 10(6) cells. There was no qualitative difference in the ability of WR-1065 to protect viable cells preirradiated with 8 Gy compared with protection of unirradiated cells. For a given gamma-ray dose from 2 to 40 Gy, there is a limiting value for surviving fraction which cannot be increased by further elevation of the intracellular drug level in V79-171 cells. Such limiting radioprotection was demonstrated for HT-29/SP-ld, HeLa, Me-180-VCII and OV-2008-VI human tumor cells.

Pharmacokinetics of amifostine and its metabolites in patients.

The pharmacokinetics of the cytoprotective agent amifostine (EthyolR; WR 2721) and its main metabolites (WR 1065 and the disulphides) were studied in patients participating in two phase I trials concerning carboplatin or cisplatin in combination with amifostine. Patients were treated with a single dose or three doses of amifostine (740 or 910 mg/m2). The single or first dose was given as a 15 min i.v. infusion just before administration of the chemotherapeutic agent. The additional two infusions were administered 2 and 4 h thereafter. Amifostine was rapidly cleared from the plasma, due to, at least in part, the fast conversion into WR 1065. A biphasic decrease with a final half-life of 0.8 h was observed. The active metabolite WR 1065 was cleared from the plasma with a final half-life of 7.3 +/- 3.6 h. The short initial half-life of WR 1065 can be explained by its fast uptake in tissues and the formation of disulphides. The disulphides were cleared with a final half-life of 8.4-13.4 h and were detectable for at least 24 h after treatment. They may serve as an exchangeable pool of WR 1065. The amifostine peak values at the end of each 15 min infusion did not accumulate in the multiple dosing schedule. For WR 1065 a trend towards an increase in the peak levels was observed [C1,max: 47.5 +/- 11.9 microM, C2,max: 79.0 +/- 13.2 microM, C3,max: 84.8 +/- 15.1 microM, (n = 6)], whereas a trend towards a small decrease was observed for the peak levels of the disulphides [C1,max: 184.2 +/- 12.6 microM, C2,max: 175.0 +/- 23.7 microM, C3,max: 166.0 +/- 17.2 microM, (n = 6)]. This latter finding might suggest a saturation of the disulphide formation or a change in the uptake or elimination of WR 1065, which would result in higher WR 1065 levels in plasma and tissues, after multiple doses of amifostine.

Transport of aminothiol radioprotectors into mammalian cells: passive diffusion versus mediated uptake.

Water:n-octanol partition coefficients (KD) were determined for a series of radioprotective thiols to ascertain whether these could be used to estimate reliably their rates of uptake into mammalian cells by passive diffusion. Values of KD determined for thiols in 0.1 M potassium phosphate, pH 7.4, at 22 degrees C were: N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, WRSH), 2.0 x 10(3); dithiothreitol, 1.4; 2-mercaptoethanol, 1.7; cysteamine, 180; 3-mercaptopropanoic acid, 450; mercaptosuccinic acid, 5 x 10(6) (extrapolated value). Predictions of uptake rates by passive diffusion into mammalian cells using these values and values for the membrane diffusion rate derived from empirical evaluation of appropriate values from the literature for erythrocyte permeability paralleled the experimental rates for WR-1065 and dithiothreitol but were about threefold lower. Although the utility of KD values for quantitative prediction of uptake rates is limited, the analysis clearly indicated that uptake of aminothiols having three or more ionized amino groups will not occur at useful rates by passive diffusion. Studies of WR-1065 import by Chinese hamster V79-171 cells at micromolar levels of WR-1065 revealed an uptake that could not be explained by passive diffusion. This uptake was not inhibited by substrates for common amino acid transport systems but was inhibited by polyamines and by 1 mM DTT, which suggested that WR-33278 (WRSSWR) formed by oxidation of WRSH was being transported by a polyamine transport system. This was confirmed by showing that WRSSWR is imported efficiently by V79-171 cells treated with D,L-2-difluoromethylornithine to deplete intracellular polyamines and hence enhance their transport. Spermine inhibited uptake of WRSSWR and WRSSWR inhibited uptake of [14C]spermine, confirming that a common system is involved in the uptake of these similar molecules, both having +4 charge. It was shown that after import WRSSWR is reduced to WRSH and that uptake at low micro-molar concentrations of WRSSWR results in marked cellular concentration of the drug. These results indicate that the spermidine/spermine transport system may also provide a feasible route for import of radioprotective aminothiols bearing net charges of +3 or +4 into mammalian cells.

Bifunctional NHS-BAT ester for antibody conjugation and stable technetium-99m labeling: conjugation chemistry, immunoreactivity and kit formulation.

UNLABELLED: Conjugation chemistry and kit formulated binding of the NHS ester of 6-(4'-(4"-carboxyphenoxy)butyl)-2, 10-dimercapto-2,10-dimethyl-4,8-diazaundecane (NHS-BAT ester) to monoclonal antibodies (MAbs) was investigated. The functionalities of the resulting BAT conjugated and 99mTc-labeled MAbs BW 431/26, MAb 425 and bispecific MDX210 (fragment construct) were tested by immunoreactivity and immunoscintigraphy. METHODS: The kinetics and chemistry of the conjugation reaction were monitored by high-performance liquid chromatography, size-exclusion chromatography and positive fast-atom-bombardment mass spectra (FAB-MS). The 99mTc BAT-MAbs were tested with various immunoreactivity assays. The biodistribution of 99mTc-BAT-BW 431/26 in rats was compared with directly labeled BW 431/26. RESULTS: At pH 8.5 and 25 degrees C, the reactivity of the NHS-BAT ester was high with 90% completion after 30 min. The conjugation yield of 19 microM MAb and 228 microM NHS-BAT ester amounted to 30%. Higher NHS-BAT ester concentrations afforded higher BAT-to-MAb ratios. According to FAB-MS, the conjugation competing hydrolysis surprisingly occurred at the NHS ring. Almost quantitative 99mTc labeling was achieved after 5 min at 25 degrees C. Immunoreactivity of the 99mTc-BAT antibodies showed > 90% recovery and proved to be insensitive to BAT-to-MAb ratios of up to 10. The 99mTc-BAT-BW 431/26 showed similar organ distribution but revealed less urinary excretion compared with the directly labeled BW 431/26. Immunoscintigraphy with 99mTc-labeled and BAT-BW 431/26 and BAT-MAb 425 showed the respective biological function in vivo. CONCLUSION: According to straightforward conjugation chemistry, the ease of 99mTc labeling and the application of a simple ultrafiltration technique, the NHS-BAT ester represents a nondestructive, universally applicable biofunctional ligand to introduce stable 99mTc protein binding sites. Kit formulated conjugation/labeling can be performed with little time requirements and laboratory experience.

Pharmacokinetics of amifostine and its metabolites in the plasma and ascites of a cancer patient.

The pharmacokinetics of amifostine, a protector against chemotherapy and radiation-induced toxicities, was investigated in the plasma and ascites of a cancer patient. A high-performance liquid chromatography (HPLC) procedure with electrochemical detection was used to measure amifostine, its active metabolite, WR 1065, and the disulfides (symmetrical plus mixed disulfides). Both amifostine and WR 1065 were rapidly cleared from the plasma (95% and 50% of the peak concentration within 1 h, respectively). The disulfides, which were rapidly formed from WR 1065, were cleared much more slowly (final half-life 13.6 h). Multiple dosing resulted in a tendency toward increasing peak levels of WR 1065 and decreasing peak levels of the disulfides. Only 1% of the delivered dose appeared in the ascites. Therefore, it is not plausible that the presence of ascites or other third spaces would have an impact on the pharmacokinetics of amifostine.

Extracellular: intracellular and subcellular concentration gradients of thiols.

Chinese hamster V79 cells in Eagle's minimum essential medium in vitro at room temperature were incubated with the aminothiol, WR-1065, or glutathione (GSH) at extracellular concentrations of approximately 1 mmol dm-3. Average intracellular concentrations of GSH, cysteine, and WR-1065 were measured by high performance liquid chromatography, and the effective reducing environment near DNA probed by staining the cells with acridine orange (AO) and measuring the delayed fluorescence. Exposure to either thiol resulted in a rapid, 10-fold increase in average intracellular cysteine concentrations (to about 1 mmol dm-3). Adding extracellular GSH after prior depletion of GSH by treatment with L-buthionine sulfoximine (BSO) did not restore intracellular GSH, but intracellular cysteine was elevated 10-fold. These results are ascribed to thiol/disulfide exchange with cystine in the medium. WR-1065 slowly concentrated intracellularly to approximately 160% of the extracellular concentration. Chemical conjugation of GSH in cells decreased the reducing environment near DNA, but BSO treatment altered the uptake of AO. The electrostatic attraction of WR-1065 toward isolated DNA was markedly affected by ionic strength.

Characterization of WR-1065 absorption in the rat small intestine.

A circulating in situ rat small intestine absorption model was used to study the lumenal metabolism and absorption of [14C]WR-1065. WR-1065 was found to be more tissue reactive and toxic than its phosphorylated form, ethiofos, at equimolar perfusate concentrations. The disappearance profiles of the radiolabeled drug and free WR-1065 indicate that WR-1065 is extensively metabolized in the intestinal lumen prior to absorption. Coadministration of disodium ethylenediaminetetraacetic acid enhances the absorption of the free thiol although not to the same extent as seen with ethiofos. Perfusion of WR-1065 in citrate buffer decreased lumenal degradation of the drug but resulted in decreased absorption. The total material converted to WR-1065 portal blood profiles following ethiofos and WR-1065 perfusion were altered possibly due to distribution and metabolism differences. This study coupled with earlier work completed on ethiofos have increased our understanding of the significant barriers to absorption observed following oral administration of these compounds.

Association of WR-1065 with CHO AA8 cells, nuclei, and nucleoids.

The radioprotector WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) has been shown to be the active moiety involved in protecting mammalian cells from the cytotoxic and mutagenic effects of ionizing radiation after administration of WR-1065 or the phosphorylated form, WR-2721. Initial experiments demonstrated that, in our hands, WR-1065 protects Chinese hamster AA8 cells from killing by (a) mechanism(s) other than induction of hypoxia. AA8 cells were then incubated in the presence of [14C]WR-1065 to determine whether association of WR-1065 in vivo was random or targeted to the nucleus or the nuclear matrix. The kinetics of incorporation of labeled material showed rapid incorporation for the first 30 min and little, if any, additional incorporation over the next 2.5 h. Examination of nuclei and nucleoids generated from the AA8 cells indicated that approximately 10% of the drug was localized in the nucleus and the drug that remained was not dislodged with repeated washes of the filters. Association kinetics of the drug with nuclei and nucleoids indicated that there was little increase in drug association with time, suggesting that there may be a limited number of strong association sites in the nucleus, but these sites are either with DNA or with matrix proteins. Exposure of the AA8 cells to 6 Gy of 60Co gamma rays did not significantly alter the association of the drug with AA8 cells. Incubating AA8 cells in [14C]WR-1065 for 30 min and then incubating in drug-free medium indicated that nearly all of the drug was lost from cells within the first 5 min of incubation in drug-free medium. The low level of tightly bound matrix-associated label may be important in generating alterations in matrix organization that have been observed previously in this laboratory.

Pharmacokinetics and disposition of WR-1065 in the rhesus monkey.

The pharmacokinetics of WR-1065 [S-2-(3-aminopropylamino)ethanethiol] were investigated following iv, intraduodenal, and intraportal administrations in the rhesus monkey. Pharmacokinetic parameters were estimated by compartmental modeling of plasma concentration data from 10-min and 120-min iv infusions. Higher apparent volumes of distribution (Vc and Vss) and higher mean residence time (MRT) were observed at the slower infusion rate but a constant total dose. The values reflect a change in the distribution of WR-1065, possibly due to to saturation of binding in plasma and tissue. However, clearance remained unchanged. For a monkey administered approximately twice the 60 mg/kg dose infused over 120 min, data analysis indicates a disproportional increase in AUC and a substantial decrease in clearance. Low and erratic plasma concentrations of free drug (analytically determined without reductive cleavage) were observed following intraduodenal administration of WR-1065, demonstrating the drug's poor oral bioavailability. Results of intraduodenal administrations of radiolabeled drug indicated than an appreciable amount of the radiolabel in the dose reached the systemic circulation. However, after either intraduodenal or iv administration, only 31% of the AUC (radiolabel) could be accounted for as total (free and disulfide-bound) WR-1065 by specific analysis in separate experiments. Low levels of total cysteamine strongly suggest it to be a minor contributor to the disposition of the drug. Free WR-1065 AUC values following intraportal administration were similar to values obtained after iv administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraduodenal administration of ethiofos (WR-2721): dose proportionality study in the rhesus monkey.

A dose progression crossover study of ethiofos (WR-2721) was conducted in three healthy male rhesus monkeys. Each subject was tested with three single intraduodenal doses containing 150, 300, and 600 mg/kg. Blood samples were drawn as a function of time and the concentrations of ethiofos, WR-1065 (free thiol metabolite), and total drug convertible to the free thiol (total WR-1065) were determined by HPLC using electrochemical detection. Ethiofos levels in plasma were usually below quantifiable limits of detection (0.23 mumol/L) at all three dose levels, but free WR-1065 plasma levels increased with increasing dose. Analysis of the free WR-1065 bioavailability values indicated large variability and an unpredictable dose response among subjects. Bound WR-1065 appears to reach saturable levels over the dose range, suggesting a saturable pool of binding sites in plasma. The time-to-peak plasma levels for WR-1065 were variable regardless of the administered dose and ranged from 1.0-2.5 hours. The high variability in the data may be a result of poor permeability or absorption of the parent compound (ethiofos), saturable binding to a variable pool of binding sites in plasma and/or high first-pass metabolism of ethiofos involving the gut lumen, gut wall (epithelium), and liver.