Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo.

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2's capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD.

Plasma pharmacokinetics and tissue distribution of the breast cancer resistance protein (BCRP/ABCG2) inhibitor fumitremorgin C in SCID mice bearing T8 tumors.

Multidrug resistance (MDR) remains a major obstacle in the treatment of human cancers. The recently discovered breast cancer resistance protein (BCRP/ABCG2) has been found to be an important mediator of chemotherapeutic MDR. Fumitremorgin C (FTC) is a selective and potent inhibitor of BCRP that completely inhibits and reverses BCRP-mediated resistance at micromolar concentrations. We report a study of the pharmacokinetics and tissue distribution of FTC when administered intravenously (IV) at a dose of 25 mg/kg to female SCID mice bearing the BCRP-overexpressing human ovarian xenograft Igrov1/T8 tumors. Plasma pharmacokinetics and tissue distribution of FTC in various organs and tissues were studied. In addition, the effect of FTC administration on the expression of BCRP in T8 tumors was also assessed by RT-PCR. Administration of a single FTC IV dose did not appear to cause any major toxicities. The resulting pharmacokinetic data were fit to a two-compartment model using NONMEM and the FTC clearance was determined to be 0.55 ml/min (25.0 ml/min/kg) with a 56% inter-animal variability. Area under the plasma concentration time curve was determined by Bailer's method and was calculated to be 1128+/-111 microg min/ml. FTC was widely distributed in all tissues assayed with highest concentrations found in lungs, liver and kidney in decreasing order, respectively. FTC did not appear to have any effect on the expression of BCRP in T8 tumors. Less than 2% of the administered dose was recovered in the urine and feces after 24 h, suggesting hepatic metabolism as a primary mechanism of elimination. The current study can be used as a basis for future animal or in vivo studies with FTC designed to further understand the impact of BCRP on drug resistance.

Accounting for quiescent cells in tumour growth and cancer treatment.

A four-state cell-cycle model with explicit G1-phase representation, termed the quiescent-cell model (QCM), has been proposed to represent biologically the G1-phase specific effect of the chemotherapeutic tamoxifen. The QCM was used to model untreated and tamoxifen-treated tumour xenograft data from the literature with equivalent accuracy to previously developed tumour growth models. Open-loop analysis demonstrated that perturbations to the two newly introduced parameters, kG01 and kG10, significantly altered untreated tumour growth predictions. However, the sensitivity did not carry over to closed-loop simulations, where alterations to kD and kGS proved most significant in determining overall controller performance. Additional mismatch studies comparing controllers designed using the QCM to controllers designed with the Gompertz model and saturating-rate, cell-cycle model returned similar performance for a step-wise tumour reduction case study, but the quiescent-cell controller delivered a more aggressive treatment regimen. More importantly, the Gompertz and saturating-rate, cell-cycle controllers were unable to follow a reference trajectory when measurement updates were made biweekly, with both controllers returning tamoxifen dose schedules alternating between the maximum and minimum allowable dose.

Glyoxalase I inhibitors in cancer chemotherapy.

Several recent developments suggest that the GSH-dependent glyoxalase enzyme system deserves renewed interest as a potential target for antitumour drug development. This summary focuses on the design and development of new classes of tumoricidal agents that specifically target this elementary detoxification pathway in order to induce elevated concentrations of cytotoxic methylglyoxal in tumour cells. Special emphasis is placed on structure- and mechanism-based inhibitors of GlxI (glyoxalase I), the first enzyme in the pathway. A new class of bivalent transition-state analogues is described that simultaneously bind the active site on each subunit of the homodimeric human GlxI, resulting in K (i) values as low as 1 nM. Also described is a new family of bromoacyl esters of GSH that function as active-site-directed irreversible inhibitors of GlxI. Newer prodrugs for delivering the GSH-based inhibitors into tumour cells include reactive sulphoxide esters that undergo acyl exchange with endogenous GSH to give the inhibitors, and polymethacrylamide esters of the inhibitors that are potentially tumour-selective on the basis of the "enhanced permeability and retention effect". Finally, a preliminary evaluation of the efficacy of selected GlxI inhibitors in tumour-bearing mice is given.

Plasma pharmacokinetics and tissue distribution of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) in CD2F1 mice1.

PURPOSE: 17-(Allylamino)-17-demethoxygeldanamycin (17AAG) is a benzoquinone ansamycin compound agent that has entered clinical trials. Studies were performed in mice to: (1) define the plasma pharmacokinetics, tissue distribution, and urinary excretion of 17AAG after i.v. delivery; (2) to define the bioavailability of 17AAG after i.p. and oral delivery; and (3) to characterize the concentrations of 17AAG metabolites in plasma and tissue. MATERIALS AND METHODS: All studies were performed in female CD2F1 mice. Preliminary toxicity studies used 17AAG i.v. bolus doses of 20, 40 and 60 mg/kg. Pharmacokinetic studies used i.v. 17AAG doses of 60, 40, and 26.67 mg/kg and i.p. and oral doses of 40 mg/kg. The plasma concentration versus time data were analyzed by compartmental and noncompartmental methods. The concentrations of 17AAG were also determined in brain, heart, lung, liver, kidney, spleen, skeletal muscle, and fat. Urinary drug excretion was calculated until 24 h after treatment. RESULTS: A 60 mg/kg dose of 17AAG, in its initial, microdispersed formulation, caused no changes in appearance, appetite, waste elimination, or survival of treated animals as compared to vehicle-treated controls. Bolus i.v. delivery of 60 mg/kg microdispersed 17AAG produced "peak" plasma 17AAG concentrations between 5.8 and 19.3 micrograms/ml in mice killed 5 min after injection. Sequential reduction of the 17AAG dose to 40 and 26.67 mg/kg resulted in "peak" plasma 17AAG concentrations between 8.9 and 19.0 micrograms/ml, and 4.8 and 6.1 micrograms/ml, respectively. Noncompartmental analysis of the plasma 17AAG concentration versus time data showed an increase in AUC from 402 to 625 and 1738 micrograms/ml.min when the 17AAG dose increased from 26.67 to 40 and 60 mg/kg, respectively. Across the range of doses studied, 17AAG total body clearance varied from 34 to 66 ml/min per kg. Compartmental modeling of the plasma 17AAG concentration versus time data showed that the data were fitted best by a two-compartment, open, linear model. In each study, substantial concentrations of a material, subsequently identified as 17-(amino)-17-demethoxygeldanamycin (17AG), were measured in plasma. A subsequent, lyophilized formulation of 17AAG proved excessively toxic when delivered i.v. at 60 mg/kg. A repeat i.v. study using a 40 mg/kg dose of this new formulation produced peak plasma 17AAG concentrations of 20.2-38.4 micrograms/ml, and a 17AAG AUC of 912 micrograms/ml.min, which was approximately 50% greater than the AUC produced by a 40 mg/kg dose of microdispersed 17AAG. The bioavailabilities of 17AAG after i.p. and oral delivery were 99% and 24%, respectively. Minimal amounts of 17AAG and 17AG were detected in the urine. After i.v. bolus delivery to mice, 17AAG distributed rapidly to all tissues, except the brain. Substantial concentrations of 17AG were measured in each tissue. CONCLUSIONS: 17AAG has excellent bioavailability when given i.p. but only modest bioavailability when given orally and is metabolized to 17AG and other metabolites when given i.v., i.p., or orally. 17AAG is widely distributed to tissues. These pharmacokinetic data generated have proven relevant to the design of recently initiated clinical trials of 17AAG and could be useful in their interpretation.

Pharmacokinetics and tissue distribution of halofuginone (NSC 713205) in CD2F1 mice and Fischer 344 rats.

PURPOSE: Halofuginone (HF) inhibits synthesis of collagen type I and matrix metalloproteinase-2 and is being considered for clinical evaluation as an antineoplastic agent. Pharmacokinetic studies were performed in rodents to define the plasma pharmacokinetics, tissue distribution, and urinary excretion of HF after i.v. delivery and the bioavailability of HF after i.p. and oral delivery. MATERIALS AND METHODS: Studies were performed in CD2F1 mice and Fischer 344 rats. In preliminary toxicity studies in mice single HF i.v. bolus doses between 1.0 and 5.0 mg/kg were used. Pharmacokinetic studies were conducted in mice after administration of 1.5 mg/kg HF. In preliminary toxicity studies in male rats HF i.v. bolus doses between 0.75 and 4.5 mg/kg were used. In pharmacokinetic studies in rats an HF dose of 3.0 mg/kg was used. Compartmental and non-compartmental analyses were applied to the plasma concentration versus time data. Plasma, red blood cells, various organs, and urine were collected for analysis. RESULTS: HF doses > or = 1.5 mg/kg proved excessively toxic to mice. In mice, i.v. bolus delivery of 1.5 mg/kg HF produced "peak" plasma HF concentrations between 313 and 386 ng/ml, and an AUC of 19,874 ng/ml min, which corresponded to a total body clearance (CLtb) of 75 ml/min per kg. Plasma HF concentration versus time data were best fit by a two-compartment open linear model. The bioavailability of HF after i.p. and oral delivery to mice was 100% and 0%, respectively. After i.v. bolus delivery to mice, HF distributed rapidly to all tissues, except brain. HF persisted in lung, liver, kidney, spleen, and skeletal muscle longer than in plasma. In the oral study, HF was undetectable in plasma and red blood cells, but was easily detectable in kidney, liver, and lung, and persisted in those tissues for 48 h. Urinary excretion of HF accounted for 7-11% of the administered dose within the first 72 h after i.v. dosing and 15-16% and 16% of the administered dose within 24 and 48 h, respectively, after oral dosing. There were no observed metabolites of HF in mouse plasma or tissues. In rats, i.v. bolus delivery of 3.0 mg/kg produced a "peak" plasma HF concentration of 348 ng/ml, and an AUC of 43,946 ng/ml min, which corresponded to a CLtb of 68 ml/min per kg. Plasma HF concentration versus time data were best fit by a two-compartment open linear model. After i.v. bolus delivery to rats, HF distributed rapidly to all tissues, with low concentrations detectable in brain and testes. HF was detectable in some tissues for up to 48 h. HF could be detected in rat plasma after a 3 mg/kg oral dose. Peak HF concentration (34 ng/ml) occurred at 90 min, but HF concentrations were less than the lower limit of quantitation (LLQ) by 420 min. Urinary excretion of HF accounted for 8-11% of the administered dose within the first 48 h after i.v. dosing. No HF metabolites were detected in plasma, tissue, or urine. CONCLUSIONS: HF was rapidly and widely distributed to rodent tissues and was not converted to detectable metabolites. In mice, HF was 100% bioavailable when given i.p. but could not be detected in plasma after oral administration, suggesting limited oral bioavailability. However, substantial concentrations were present in liver, kidney, and lungs. HF was present in rat plasma after an oral dose, but the time course and low concentrations achieved precluded reliable estimation of bioavailability. These data may assist in designing and interpreting additional preclinical and clinical studies of HF.

Pharmacokinetics and antitumor properties in tumor-bearing mice of an enediol analogue inhibitor of glyoxalase I.

PURPOSE: The enediol analogue S-(N-p-chlorophenyl-N-hydroxycarbamoyl)glutathione (CHG) is a powerful, mechanism-based, competitive inhibitor of the methylglyoxal-detoxifying enzyme glyoxalase I. The [glycyl,glutamyl]diethyl ester prodrug form of this compound (CHG(Et)2) inhibits the growth of different tumor cell lines in vitro, apparently by inducing elevated levels of intracellular methylglyoxal. The purpose of this study was to evaluate the pharmacokinetic properties of CHG(Et)2 in plasma esterase-deficient C57BL/6 (Es-1e) mice after intravenous (i.v.) or intraperitoneal (i.p.) administration of bolus doses of CHG(Et)2. In addition, the in vivo antitumor properties of CHG(Et)2 were evaluated against murine B16 melanoma in these mice, and against androgen-independent human prostate PC3 tumor and human colon HT-29 adenocarcinoma in plasma esterase-deficient nude mice. METHODS: Pharmacokinetics were evaluated after either i.v. or i.p. administration of CHG(Et)2 at the maximally tolerated dose of 120 mg/kg to both tumor-free male and female mice and male and female mice bearing subcutaneous B16 tumors. Tissue concentrations of CHG(Et)2, CHG and the [glycyl]monoethyl ester CHG(Et) were measured as a function of time by reverse-phase C18 high-performance liquid chromatography of deproteinized tissue samples. The efficacy of CHG(Et)2 in tumor-bearing mice was evaluated after i.v. bolus administration of CHG(Et)2 at 80 or 120 mg/kg for 5 days each week for 2 weeks, or after 14 days continuous infusion of CHG(Et)2 using Alzet mini-osmotic pumps. Hydroxypropyl-beta-cyclodextrin was used as a vehicle in the efficacy studies. RESULTS: Intravenous administration of CHG(Et)2 resulted in the rapid appearance of CHG(Et)2 in the plasma of tumor-bearing mice with a peak value of 40-60 microM, followed by a first-order decrease with a half-life of about 10 min. There was a corresponding increase in the concentration of inhibitory CHG in the B16 tumors, with a maximum concentration in the range 30-60 microM occurring at 15 min, followed by a decrease to a plateau value of about 6 microM after 120 min. Neither CHG(Et)2 nor its hydrolysis products were detectable in plasma, after i.p. administration of CHG(Et)2 to tumor-free female mice. From the efficacy studies, dosing schedules were identified that resulted in antitumor effects comparable to those observed with the standard antitumor agents Adriamycin (with B16 tumors), cisplatin (with PC3 tumors), and vincristine (with HT-29 tumors). CONCLUSION: This is the first demonstration that a mechanism-based competitive inhibitor of glyoxalase I effectively inhibits the growth of solid tumors in mice when delivered as the diethyl ester prodrug.

Activation of CPT-11 in mice: identification and analysis of a highly effective plasma esterase.

The camptothecin prodrug CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is converted by esterases to yield the potent topoisomerase I poison SN-38 (7-ethyl-10-hydroxycamptothecin). Recently, a mouse strain (Es1(e)) has been identified that demonstrates reduced plasma esterase activity, and we have monitored the ability of plasma from these mice to metabolize CPT-11. Total plasma esterase activity was reduced 3-fold in Esl(e)mice in comparison to control mice, and this resulted in a 200-fold reduction in SN-38 production after incubation with CPT-11 in vitro. In addition, pharmacokinetic studies of CPT-11 and SN-38 in these animals demonstrated approximately 5-fold less conversion to SN-38. However, extracts derived from tissues from Es1(e) animals revealed total esterase activities similar to those of control mice, and these extracts metabolized CPT-11 with equal efficiency. Northern analysis of RNA isolated from organs indicated that the liver was the primary source of Es-1 gene expression and that very low levels of Es-1 RNA were present in Es1(e) mice. These results suggest that the reduced levels of Es-1 esterase present in Es1(e) mice are due to down-regulation of gene transcription, and that this plasma esterase is responsible for the majority of CPT-11 metabolism in mice.

Plasma pharmacokinetics and tissue distribution in CD2F1 mice of Pc4 (NSC 676418), a silicone phthalocyanine photodynamic sensitizing agent.

PURPOSE: Pc4 is a silicone phthalocyanine photosensitizing agent that is entering clinical trials. Studies were undertaken in mice to develop a suitable formulation and analytical methodology for use in pharmacokinetic studies and to define the plasma pharmacokinetics, tissue distribution, and urinary excretion of Pc4 after i.v. delivery. METHODS: An HPLC method suitable for separation and quantification of Pc4 was developed and validated for use in mouse plasma, tissues, and urine. The stability of Pc4 was characterized in a variety of formulations as well as in mouse plasma. Before pursuing pharmacokinetic studies, preliminary toxicity studies were undertaken. These studies utilized Pc4 formulated in diluent 12:0. 154 M NaCl (1:3, v:v). Pharmacokinetic studies involved Pc4 doses of 40 mg/kg, 10 mg/kg and 2 mg/kg administered as i.v. boluses to female, CD2F1 mice. Doses of 40 mg/kg, 10 mg/kg, and 2 mg/kg were studied with drug formulated in diluent 12:0.154 M NaCl (1:3, v:v). Doses of 10 mg/kg and 2 mg/kg were also studied with drug formulated in a vehicle consisting of polyethylene glycol:Tween 80:0. 01 M sodium phosphate buffer, pH 7.0 (40:0.2:59.8, v:v:v). Compartmental and non-compartmental analyses were applied to the plasma concentration-versus-time data. Concentrations of Pc4 were also determined in a variety of tissues, including brain, lung, liver, kidney, skeletal muscle, skin, heart, spleen, and abdominal fat. Urine was collected from animals treated with each of the doses of Pc4 mentioned above, and daily, as well as cumulative drug excretion was calculated until 168 h after treatment. RESULTS: At a dose of 80 mg/kg, two of five male and two of five female mice were dead by 24 h after injection. Pathologic examination revealed gross findings of blue discoloration affecting many tissues, with lungs that were grossly hemorrhagic and very blue-black. Microscopic examination of the lungs revealed mild acute interstitial pneumonia, with perivascular edema and inflammation, and a detectable margination of neutrophils around larger pulmonary blood vessels. Animals sacrificed 14 days after treatment showed mild granulomatous pneumonia, characterized by clusters of multi-nucleated giant cells, with fewer macrophages and neutrophils. The giant cells frequently contained phagocytized particles, which were clear and relatively fusiform. All mice treated with 40 mg/kg or 20 mg/kg survived and returned to pretreatment weight during the 14 days after treatment. Intravenous bolus delivery of Pc4, at a dose of 40 mg/kg, produced "peak" plasma Pc4 concentrations between 7.81 and 8.92 microg/ml in mice killed at 5 min after injection (the earliest time studied after drug delivery). Sequential reduction of the Pc4 dose to 10 mg/kg in diluent 12:0.154 M NaCl (1:3, v:v), 10 mg/kg in polyethylene glycol:Tween 80:sodium phosphate buffer (40:0.2:59.8, v:v:v), 2 mg/kg in diluent 12:0.154 M NaCl (1:3, v:v), and, finally, 2 mg/kg in polyethylene glycol:Tween 80:sodium phosphate buffer (40:0.2:59.8, v:v:v) resulted in "peak" plasma Pc4 concentrations between 2.07 and 3.24, 0.68 and 0.98 microg/ml, and 0.29 and 0.41 microg/ml, respectively. Pc4 persisted in plasma for prolonged periods of time (72-168 h). Non-compartmental analysis of plasma Pc4 concentration-versus-time data showed an increase in area under the plasma Pc4 concentration-versus-time curve (AUC) when the dose of Pc4 increased from 2 mg/kg to 40 mg/kg. Across the 20-fold range of doses studied, total body clearance (CL(tb)) varied from 376 to 1106 ml h(-1) kg(-1). Compartmental modeling of plasma Pc4 concentration versus time data showed the data to be fit best by a two-compartment, open, linear model. Minimal amounts of Pc4 were detected in the urine of mice. After i.v. bolus delivery to mice, Pc4 distributed rapidly to all tissues and persisted in most tissues for the duration of each pharmacokinetic study. Tissue exposure, as measured by AUC, increased in a dose-dependent fash

Plasma pharmacokinetics of butyrate after intravenous administration of sodium butyrate or oral administration of tributyrin or sodium butyrate to mice and rats.

PURPOSE: To define the plasma concentrations of butyrate achieved and the profile of plasma butyrate concentrations versus time in mice and rats treated with tributyrin or sodium butyrate. METHODS: Female CD2F1 mice were treated with tributyrin by oral gavage or with sodium butyrate by i.v. bolus or oral gavage. Oral tributyrin doses delivered to mice were 3.1, 5.2, 7.8, and 10.3 g/kg. Intravenous sodium butyrate doses were 0.31, 0.62, 0.94, and 1.25 g/kg. Oral sodium butyrate was given to mice at 5 g/kg. Subsequently, similar studies were performed in female Sprague-Dawley rats. Rats were given tributyrin by oral gavage at doses of 3.6, 5.2, or 10.3 g/kg or sodium butyrate i.v. at a dose of 500 mg/kg. Plasma butyrate concentrations were determined by gas chromatography. RESULTS: In mice, oral dosing with tributyrin resulted in detectable plasma butyrate concentrations as early as at 5 min after treatment and produced peak plasma butyrate concentrations at between 15 and 60 min after dosing. Peak plasma butyrate concentrations increased proportionally with increasing tributyrin dose, but as the oral tributyrin dose increased there was a greater than proportional increase in the area under the curve of plasma butyrate concentrations versus time (AUC). At a tributyrin dose of 10.3 g/kg, plasma butyrate concentrations peaked at approximately 1.75 mM and remained >1 mM for between 10 and 60 min after dosing. However, approximately 10% of mice treated with this dose died acutely. At a tributyrin dose of 7.8 g/kg, plasma butyrate concentrations reached approximately 1 mM by 15 min after dosing and remained between 0.8 and 1 mM until 60 min after dosing. No mouse treated with this dose died acutely. Mice given tributyrin doses of 5.2 and 3.1 g/kg achieved peak plasma butyrate concentrations of approximately 0.9 and 0.5 mM, respectively, by 45 min after dosing. Plasma butyrate concentrations in these mice remained above 0.1 mM until 120 and 90 min after dosing, respectively. The four i.v. doses of sodium butyrate resulted in plasma concentration-time profiles that also indicated nonlinear pharmacokinetics and were well described by a one-compartment model with saturable elimination. Values recorded for the Michaelis-Menten constant (Km) and the maximal velocity of the process (Vmax) ranged between 1.02 and 5.65 mM and 0.60 and 1.82 mmol/min, respectively. Values noted for the volume of the central compartment (Vc) varied between 0.48 and 0.72 l/kg. At 1.25 g/kg, i.v. sodium butyrate produced peak plasma butyrate concentrations of 10.5-17.7 mM, and plasma butyrate concentrations remained above 1 mM for 20-30 min. Sodium butyrate delivered orally to mice at 5 g/kg produced peak plasma butyrate concentrations of approximately 9 mM at 15 min after dosing and plasma butyrate concentrations exceeding 1 mM for 90 min after dosing. In rats the 10.3-g/kg oral dose of tributyrin produced peak plasma butyrate concentrations of approximately 3 mM by 75 min after dosing and butyrate concentrations exceeding 1 mM from 30 to 90 min after dosing. The plasma butyrate concentrations produced in rats by 5.2- and 3.6-g/kg doses were appropriately lower than those produced by the 10.3-g/kg dose, and there was no evidence of nonlinearity. The 500-mg/kg i.v. dose of sodium butyrate produced peak plasma butyrate concentrations in rats of approximately 11 mM, and the decline in plasma butyrate concentrations with time after dosing was consistent with saturable clearance. CONCLUSION: These studies document the ability to use oral administration of tributyrin to achieve pharmacologically relevant concentrations of butyrate in rodent plasma. They also document the nonlinear nature of butyrate clearance. These data are being used in the design of clinical trials of oral tributyrin in patients with malignancies and hemoglobinopathies.

A new method for rapidly generating inhibitors of glyoxalase I inside tumor cells using S-(N-aryl-N-hydroxycarbamoyl)ethylsulfoxides.

The enediol analogue S-(N-p-chlorophenyl-N-hydroxycarbamoyl)glutathione is a powerful mechanism-based competitive inhibitor of the anticancer target enzyme glyoxalase I. Nevertheless, this compound exhibits limited toxicity toward tumor cells in vitro because it does not readily diffuse across cell membranes. We describe an efficient method for indirectly delivering the enzyme inhibitor into murine leukemia L1210 cells via acyl interchange between intracellular glutathione and the cell-permeable prodrug S-(N-p-chlorophenyl-N-hydroxycarbamoyl)ethylsulfoxide. The second-order rate constant for the acyl-interchange reaction in a cell-free system is 1.84 mM-1 min-1 (100 mM potassium phosphate buffer, 5% ethanol, pH 7.5, 25 degrees C). Incubation of L1210 cells with the sulfoxide in vitro results in a rapid increase in the intracellular concentration of the glyoxalase I inhibitor (kapp = 1. 41 +/- 0.03 min-1 (37 degrees C)) and inhibition of cell growth (GI50 = 0.5 +/- 0.1 microM). This represents an improvement in both efficiency and potency over the dialkyl ester prodrug strategy in which the inhibitor is indirectly delivered into tumor cells as the [glycyl,glutamyl] diethyl or dicyclopentyl esters. The fact that pi-glutathione transferase catalyzes the acyl-interchange reaction between GSH and the sulfoxide suggests that the sulfoxide, or related compounds, might exhibit greater selective toxicity toward tumor cells that overexpress the transferase.

Mechanism-based competitive inhibitors of glyoxalase I: intracellular delivery, in vitro antitumor activities, and stabilities in human serum and mouse serum.

S-(N-Aryl-N-hydroxycarbamoyl)glutathione derivatives (GSC(O)N(OH)C6H4X, where GS = glutathionyl and X = H (1), Cl (2), Br (3)) have been proposed as possible anticancer agents, because of their ability to strongly inhibit the methylglyoxal-detoxifying enzyme glyoxalase I. In order to test this hypothesis, the in vitro antitumor activities of these compounds and their [glycyl,glutamyl] diethyl ester prodrug forms (1(Et)2-3(Et)2) have been examined. All three diethyl esters inhibit the growth of L1210 murine leukemia and B16 melanotic melanoma in culture, with GI50 values in the micromolar concentration range. Cell permeability studies with L1210 cells indicate that growth inhibition is associated with rapid diffusion of the diethyl esters into the cells, followed by enzymatic hydrolysis of the ethyl ester functions to give the inhibitory diacids. In contrast, the corresponding diacids neither readily diffuse into nor significantly inhibit the growth of these cells. Consistent with the hypothesis that cell growth inhibition is due to competitive inhibition of glyoxalase I, preincubation of L1210 cells with 2(Et)2 increases the sensitivity of these cells to the inhibitory effects of exogenous methylglyoxal. Compound 2(Et)2 is much less toxic to nonproliferating murine splenic lymphocytes, possibly reflecting reduced sensitivity to methylglyoxal and/or reduced chemical stability of the diacid inside these cells. Finally, a plasma esterase-deficient murine model has been identified that should allow in vivo testing of the diethyl esters.

Antitumor activity, distribution, and metabolism of 13-cis-retinoic acid as a single agent or in combination with tamoxifen in established human MCF-7 xenografts in mice.

PURPOSE: The efficacy of 13-cis-retinoic acid (13-CRA) given as a single agent or in combination with tamoxifen (TAM) was determined in athymic nude mice bearing advanced s.c. MCF-7 human breast cancers. METHODS: 13-CRA alone was given by gavage at doses ranging from 26.4 to 200 mg/kg. TAM alone was given by gavage at doses of 7.5, 15, 30, or 60 mg/kg. For combination studies, each dose of TAM was followed 4 h later by 13-CRA at doses of 25, 50, 100, or 200 mg/kg. All treatments began on day 12 and were continued for 3 weeks. RESULTS: The median time to two doublings recorded for the control and for 13-CRA and TAM given as single agents at the highest dose were 22.2, 29.2, and 54.7 days, respectively. In combination, 100 and 200 mg/kg 13-CRA with 7.5 mg/kg TAM resulted in a delay in tumor growth at least as high as that achieved with highest-dose TAM alone, but the effect was not synergistic. Pharmacokinetic analysis of 13-CRA was performed in plasma, liver, and tumor from mice bearing 0.5- to 2.0 g carcinomas following a single dose of 100 mg/kg 13-CRA. Results showed that 13-CRA was metabolized differently in various tissues, but concentrations of 13-CRA detected in tumor were in the range reported to be active in vitro. all-trans-Retinoic acid (ATRA) concentrations were about 5% of the 13-CRA concentrations detected in plasma, 68% of those found in liver, and 20% of those found in tumor. 4-oxo-CRA represented between 2% and 10% of 13-CRA concentrations detected in plasma and liver but was not detected in tumor. Furthermore there was no difference in peak plasma 13-CRA concentrations found in the same tissues at 30 min after a single dose or after the eighth dose of 100 mg/kg 13-CRA or 13-CRA and TAM. Mean 13-CRA concentrations detected in liver and tumor were 50-90% and 16-30% of plasma peak concentrations, respectively. No difference in 4-oxo-CRA concentration was observed between the treatment groups. CONCLUSIONS: These data suggest that 13-CRA is not effective against established human breast tumor xenografts despite the stability of the pharmacokinetics of 13-CRA and the generation of ATRA as a metabolite. The addition of 13-CRA to TAM did not improve the efficacy of TAM against these estrogen-receptor-positive xenografts.

Tumor-targeted apoptosis by a novel spermine analogue, 1,12-diaziridinyl-4,9-diazadodecane, results in therapeutic efficacy and enhanced radiosensitivity of human prostate cancer.

Interference with polyamine transport and biosynthesis has emerged as an important anticancer strategy involving polyamine analogues and specific inhibitors of key biosynthetic enzymes. Because the prostate gland has a high polyamine content, by using the polyamine transporter for selective uptake into cancer cells, alkylating polyamines are likely to be highly effective against prostatic tumors. We have recently synthesized a novel class of spermine analogues, the lead compound of which has efficacy against human cancer cells (P. S. Callery et al., U. S. patent, 5,612,239, Issued March 17, 1997.). In this study, to investigate the potential therapeutic efficacy of the lead spermine analogue 1,12-diaziridinyl-4, 9-diazadodecane (BIS), against advanced prostate cancer, we examined the in vitro effect and in vivo efficacy of the compound in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. BIS exhibited a dose-dependent cytotoxic effect against prostate cancer cells via induction of apoptosis. Treatment of cells with BIS (1 microM) for 24 h resulted in a significant induction of apoptosis (24%). Exposure of BIS-treated PC-3 prostate cancer cells to gamma-irradiation resulted in a significant increase in the number of cells undergoing apoptosis and a subsequent decrease in the IC50. Furthermore, BIS treatment led to a significant enhancement of loss of clonogenic survival in irradiated prostate cancer cells (both PC-3 and DU-145). In vivo efficacy trials demonstrated a significant antitumor effect of BIS against both PC-3 and DU-145 tumor xenografts in severe combined immunodeficient mice in a dose-dependent pattern at maximally tolerated doses. Terminal transferase end-labeling analysis indicated that BIS-mediated tumor regression in vivo occurs via induction of apoptosis among prostatic tumor cells. These results suggest that the novel spermine analogue BIS: (a) has a potent antitumor effect against prostatic tumors via induction of apoptosis; and (b) increases the radiosensitivity of human prostate cancer cells by decreasing the apoptotic threshold to radiation. This study may have important clinical implications for the manipulation of this antitumor activity of the polyamine analogue for the optimization of the therapeutic efficacy of radiation in patients with advanced prostate cancer.

Metabolism of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) by murine and human hepatic preparations.

17-(Allylamino)-17-demethoxygeldanamycin (17AAG), a compound that is proposed for clinical development, shares the ability of geldanamycin to bind to heat shock protein 90 and GRP94, thereby depleting cells of p185erbB2, mutant p53, and Raf-1. Urine and plasma from mice treated i.v. with 17AAG contained six materials with absorption spectra similar to that of 17AAG. Therefore, in vitro metabolism of 17AAG by mouse and human hepatic preparations was studied to characterize: (a) the enzymes responsible for 17AAG metabolism; and (b) the structures of the metabolites produced. These materials had retention times on high-performance liquid chromatography of approximately 2, 4, 5, 6, 7, and 9 min. When incubated in an aerobic environment with 17AAG, murine hepatic supernatant (9000 x g) produced each of these compounds; the 4-min metabolite was the major product. This metabolism required an electron donor, and NADPH was favored over NADH. Metabolic activity resided predominantly in the microsomal fraction. Metabolism was decreased by approximately 80% in anaerobic conditions and was essentially ablated by CO. Microsomes prepared from human livers produced essentially the same metabolites as produced by murine hepatic microsomes, but the 2-min metabolite was the major product, and the 4-min metabolite was next largest. There was no metabolism of 17AAG by human liver cytosol. Metabolism of 17AAG by human liver microsomes also required an electron donor, with NADPH being preferred over NADH, was inhibited by approximately 80% under anaerobic conditions, and was essentially ablated by CO. Liquid chromatography/mass spectrometry analysis of human and mouse in vitro reaction mixtures indicated the presence of materials with molecular weights of 545, 601, and 619, compatible with 17-(amino)-17-demethoxygeldanamycin (17AG), an epoxide, and a diol, respectively. The metabolite with retention time of 4 min was identified as 17AG by cochromatography and mass spectral concordance with authentic standard. Human microsomal metabolism of 17AAG was inhibited by ketoconazole, implying 3A4 as the responsible cytochrome P450 isoform. Incubation of 17AAG with cloned CYP3A4 produced metabolites 4 and 6. Incubation of 17AAG with cloned CYP3A4 and cloned microsomal epoxide hydrolase produced metabolites 2 and 4, with greatly decreased amounts of metabolite 6. Incubation of 17AAG with human hepatic microsomes and cyclohexene oxide, a known inhibitor of microsomal epoxide hydrolase, did not affect the production of metabolite 4 but decreased the production of metabolite 2 while increasing the production of metabolite 6. These data imply that metabolite 2 is a diol and metabolite 6 is an epoxide. Mass spectral fragmentation patterns and the fact that 17AG is not metabolized argue for the epoxide and diol being formed on the 17-allylamino portion of 17AAG and not on its ansamycin ring. These data have implications with regard to preclinical toxicology and activity testing of 17AAG as well as its proposed clinical development because: (a) production of 17AG requires concomitant production of acrolein from the cleaved allyl moiety; and (b) 17AG, which was not metabolized by microsomes, has been described as being as active as 17AAG in decreasing cellular p185erbB2.

In vitro metabolism by mouse and human liver preparations of halomon, an antitumor halogenated monoterpene.

OBJECTIVES: To characterize the enzymes responsible for and metabolites produced from the metabolism of halomon, a halogenated monoterpene that is isolated from the red algae Portieria hornemanii and has in vitro activity in the NCI screen against brain, renal, and colon cancer cell lines. MATERIALS AND METHODS: Mouse and human liver fractions, prepared by homogenization and differential centrifugation, were incubated with halomon, extracted with toluene, and analyzed by gas chromatography. RESULTS: In the presence of NADPH, mouse-liver 9,000-g supernatant (S9) fractions metabolized halomon, but boiled S9 fractions did not. NADH could not substitute for NADPH. Further separation of murine hepatic S9 fractions produced a microsomal fraction that contained all of the halomon-metabolizing activity; cytosol had none. Carbon monoxide reduced murine hepatic microsomal metabolism of halomon, whereas an anaerobic, N2 environment greatly accelerated the disappearance of halomon. Human hepatic microsomes metabolized halomon and required NADPH to do so. Carbon monoxide completely inhibited human hepatic microsomal metabolism of halomon. Unlike murine hepatic microsomal metabolism of halomon, anaerobic conditions did not enhance the metabolism of halomon by human hepatic microsomes. Neither 100 microM diethyldithiocarbamate, 1 microM quinidine, 100 microM ciprofloxacin, 3 microM ketoconazole, nor 100 microM sulfinpyrazone inhibited the metabolism of halomon by human hepatic microsomes. Both murine and human hepatic microsomes produced a metabolite of halomon. The mass spectrum of this metabolite indicated the loss of one chlorine atom and one bromine atom. CONCLUSIONS: Halomon is metabolized by mouse and human hepatic cytochrome P-450 enzymes, the identities of which remain unknown. Hepatic metabolism of halomon is very consistent with the concentrations of halomon measured in mouse tissues and urine after i.v. administration of the drug.

Plasma pharmacokinetics, bioavailability, and tissue distribution in CD2F1 mice of halomon, an antitumor halogenated monoterpene isolated from the red algae Portieria hornemannii.

The purpose of the present study was to define the plasma pharmacokinetics, bioavailability, and tissue distribution in mice of halomon, a halogenated monoterpene from Portieria hornemanii that is active in vitro against brain-, renal-, and colon-cancer cell lines. Halomon formulated in cremophor:ethanol:0.154 M NaCl (1:1:6, by vol.) was injected i.v. at 20, 60, 90, or 135 mg/kg into female CD2F1 mice. Doses of 135 mg/kg were also given i.p., s.c., and by enteral gavage to female CD2F1 mice and i.v. to male CD2F1 mice. Plasma halomon concentrations were measured with a gas-chromatography system using electron-capture detection. Halomon concentrations were also determined in the brains, hearts, lungs, livers, kidneys, spleens, skeletal muscles, fat, red blood cells, and, if present, testes of mice given 135 mg/kg i.v. Halomon plasma pharmacokinetics were well fit by a two-compartment, open linear model and were linear between 20 and 135 mg/kg. Population estimates of parameters describing halomon plasma pharmacokinetics in female CD2F1 mice were developed with a standard two-stage technique and also by simultaneous modeling of data from 20-, 60-, 90-, and 135-mg/kg i.v. studies in female mice. Halomon bioavailability was 45%, 47%, and 4% after i.p., s.c., and enteral dosing, respectively. Urinary excretion of the parent compound was minimal. Halomon was distributed widely to all tissues studied but was concentrated and persisted in fat. Halomon concentrations measured in the brain were comparable with concomitant concentrations detected in plasma and most other tissues. These data and models are helpful in the simulation and evaluation of conditions produced by preclinical screening and toxicology studies.

Plasma pharmacokinetics and urinary excretion of the polyamine analogue 1, 19-bis(ethylamino)-5, 10, 15-triazanonadecane in CD2F1 mice.

The pharmacokinetics of 1, 19-bis(ethylamino)-5, 10, 15-triazanonadecane (BE-4-4-4-4) were determined in CD2F1 female mice after administration of i.v. bolus doses of 20 mg/kg (approximately the dose lethal to 10% of the study animals, approximately LD10) as well as 15, 10, and 5 mg/kg and after s.c., i.p., or p.o. doses of 20 mg/kg. BE-4-4-4-4 in plasma and urine was derivatized with dansyl chloride and measured by gradient high-performance liquid chromatography (HPLC) with fluorescence detection. Data were modeled by noncompartmental and compartmental methods. The declines observed in plasma BE-4-4-4-4 concentrations after i.v. delivery of 20, 15, 10, and 5 mg/kg were modeled simultaneously using an interval of 2000 min between doses and were best approximated by a two-compartment, open, linear model. The time courses of plasma BE-4-4-4-4 concentrations after i.p. and s.c. delivery were fit best by a two-compartment, open, linear model with first-order absorption. Peak plasma concentrations of BE-4-4-4-4 measured following an i.v. dose of 20 mg/kg ranged between 30 and 33 microgram/ml, the terminal elimination half-life was 94 min, and the volume of distribution (Vdss) was 850 ml/kg. The plasma pharmacokinetics of BE-4-4-4-4 were linear with dose. BE-4-4-4-4 (0.5 and 2.0 microM) in mouse plasma was approximately 67% protein-bound. Bio-availabilities after i.p., s.c., and p.o. delivery were 40%, 50%, and approximately 3%, respectively. Urinary excretion of parent BE-4-4-4-4 in the first 24 h after dosing accounted for less than 30% of the delivered dose. As BE-4-4-4-4 proceeds toward and undergoes clinical evaluation, the data and analytical method presented herein should prove useful in formulating a dose-escalation strategy and, possibly, evaluating toxicities encountered.

Synthesis and antitumor evaluation of a highly potent cytotoxic DNA cross-linking polyamine analogue, 1,12-diaziridinyl-4,9-diazadodecane.

A diaziridinylspermine analogue, 1,12-diaziridinyl-4,9-diazadodecane (NSC-667005), was synthesized as a bisalkylating agent with a polyamine backbone. DNA cross-linking was detected in the reaction of linearized pBR322 DNA with 1,12-diaziridinyl-4,9-diazadodecane at concentrations comparable with that required for cross-linking by two nitrogen mustard drugs, mechlorethamine and melphalan. A significant increase in life span of female CD2F1 mice bearing L1210 murine leukemia was observed after intravenous administration of 1,12-diaziridinyl-4,9-diazadodecane in doses of less than 2.7 mg/kg, given on days 1, 5, and 9 of treatment.

Retinoic acid nuclear receptor beta inhibits breast carcinoma anchorage independent growth.

Retinoids modulate cellular proliferation and mediate gene function through a series of nuclear receptors. The retinoic acid nuclear receptor beta (RAR beta) plays an important role in the differentiation of a number of cell types. We now demonstrate that RAR beta expression is confined to normal mammary tissue and is not expressed in either immortalized normal or malignant cell lines. Treatment of RAR beta-transfected MDA-MB-231 cells with 1 microM all-trans-retinoic acid (RA) significantly inhibited monolayer growth of the cells which express recombinant RAR beta. RAR beta-expressing MDA-MB-231 cells formed significantly smaller and fewer colonies in soft agar than the mock-transfected cells. Addition of 1 microM RA stimulated colony size and number in the RAR beta-transfected MDA-MB-231 cells. In contrast to the RAR beta-expressing cells, colony formation by the RAR alpha-expressing cells was similar to the mock-transfected controls and the addition of 1 microM RA to the RAR alpha-transfected cells inhibited colony formation. While demonstrating decreased colony formation in agar, RAR beta-expressing MDA-MB-231 cells failed to exhibit decreased growth in SCID mice. Our results show that RAR beta functions as a negative regulator of growth in breast epithelial cells. In addition, the growth of these cells is differentially regulated by RAR alpha and RAR beta which is most likely the result of the modulation of different genes.