Improved Antibacterial Activity of 1,3,4-Oxadiazole-Based Compounds That Restrict Staphylococcus aureus Growth Independent of LtaS Function.

The lipoteichoic acid (LTA) biosynthesis pathway has emerged as a promising antimicrobial therapeutic target. Previous studies identified the 1,3,4 oxadiazole compound 1771 as an LTA inhibitor with activity against Gram-positive pathogens. We have succeeded in making six 1771 derivatives and, through subsequent hit validation, identified the incorporation of a pentafluorosulfanyl substituent as central in enhancing activity. Our newly described derivative, compound 13, showed a 16- to 32-fold increase in activity compared to 1771 when tested against a cohort of multidrug-resistant Staphylococcus aureus strains while simultaneously exhibiting an improved toxicity profile against mammalian cells. Molecular techniques were employed in which the assumed target, lipoteichoic acid synthase (LtaS), was both deleted and overexpressed. Neither deletion nor overexpression of LtaS altered 1771 or compound 13 susceptibility; however, overexpression of LtaS increased the MIC of Congo red, a previously identified LtaS inhibitor. These data were further supported by comparing the docking poses of 1771 and derivatives in the LtaS active site, which indicated the possibility of an additional target(s). Finally, we show that both 1771 and compound 13 have activity that is independent of LtaS, extending to cover Gram-negative species if the outer membrane is first permeabilized, challenging the classification that these compounds are strict LtaS inhibitors.

Pharmacokinetics and tissue distribution of inositol hexaphosphate in C.B17 SCID mice bearing human breast cancer xenografts.

Inositol hexaphosphate (IP(6)) is effective in preclinical cancer prevention and chemotherapy. In addition to cancer, IP(6) has many other beneficial effects for human health, such as reduction in risk of developing cardiovascular disease and diabetes and inhibition of kidney stone formation. Studies presented here describe the pharmacokinetics, tissue distribution, and metabolism of IP(6) following intravenous (IV) or per os (PO) administration to mice. SCID mice bearing MDA-MB-231 xenografts were treated with 20 mg/kg IP(6) (3 µCi per mouse [(14)C]-uniformly ring-labeled IP(6)) and euthanized at various times after IP(6) treatment. Plasma and tissues were analyzed for [(14)C]-IP(6) and metabolites by high-performance liquid chromatography with radioactivity detection. Following IV administration of IP(6), plasma IP(6) concentrations peaked at 5 minutes and were detectable until 45 minutes. Liver IP(6) concentrations were more than 10-fold higher than plasma concentrations, whereas other normal tissue concentrations were similar to plasma. Only inositol was detected in xenografts. After PO administration, IP(6) was detected in liver; but only inositol was detectable in other tissues. After both IV and PO administration, exogenous IP(6) was rapidly dephosphorylated to inositol; however, alterations in endogenous IPs were not examined.

Tumor disposition of pegylated liposomal CKD-602 and the reticuloendothelial system in preclinical tumor models.

Liposomes, such as pegylated-liposomal CKD-602 (S-CKD602), undergo catabolism by macrophages and dendritic cells (DCs) of the reticuloendothelial system (RES). The relationship between plasma and tumor disposition of S-CKD602 and RES was evaluated in mice bearing A375 melanoma or SKOV-3 ovarian xenografts. Area under the concentration-time curves (AUCs) of liposomal encapsulated, released, and sum total (encapsulated + released) CKD-602 in plasma, tumor, and tumor extracellular fluid (ECF) were estimated. A375 and SKOV-3 tumors were stained with cd11b and cd11c antibodies as measures of macrophages and DC. The plasma disposition of S-CKD602 was similar in both xenograft models. The ratio of tumor sum total AUC to plasma sum total AUC was 1.7-fold higher in mice bearing human SKOV-3 xenografts, compared with A375. The ratio of tumor ECF AUC to tumor sum total AUC was 2-fold higher in mice bearing human SKOV-3 xenografts, compared with A375. The staining of cd11c was 4.5-fold higher in SKOV-3, compared with A375 (P < 0.0001). The increased tumor delivery and release of CKD-602 from S-CKD602 in the ovarian xenografts, compared with the melanoma xenografts, was consistent with increased cd11c staining, suggesting that variability in the RES may affect the tumor disposition of liposomal agents.

Efficacy, pharmacokinetics, tisssue distribution, and metabolism of the Myc-Max disruptor, 10058-F4 [Z,E]-5-[4-ethylbenzylidine]-2-thioxothiazolidin-4-one, in mice.

OBJECTIVES: c-Myc is commonly activated in many human tumors and is functionally important in cellular proliferation, differentiation, apoptosis and cell cycle progression. The activity of c-Myc requires noncovalent interaction with its client protein Max. In vitro studies indicate the thioxothiazolidinone, 10058-F4, inhibits c-Myc/Max dimerization. In this study, we report the efficacy, pharmacokinetics and metabolism of this novel protein-protein disruptor in mice. METHODS: SCID mice bearing DU145 or PC-3 human prostate cancer xenografts were treated with either 20 or 30 mg/kg 10058-F4 on a qdx5 schedule for 2 weeks for efficacy studies. For pharmacokinetics and metabolism studies, mice bearing PC-3 or DU145 xenografts were treated with 20 mg/kg of 10058-F4 i.v. Plasma and tissues were collected 5-1440 min after dosing. The concentration of 10058-F4 in plasma and tissues was determined by HPLC, and metabolites were characterized by LC-MS/MS. RESULTS: Following a single iv dose, peak plasma 10058-F4 concentrations of approximately 300 muM were seen at 5 min and declined to below the detection limit at 360 min. Plasma concentration versus time data were best approximated by a two-compartment, open, linear model. The highest tissue concentrations of 10058-F4 were found in fat, lung, liver, and kidney. Peak tumor concentrations of 10058-F4 were at least tenfold lower than peak plasma concentrations. Eight metabolites of 10058-F4 were identified in plasma, liver, and kidney. The terminal half-life of 10058-F4 was approximately 1 h, and the volume of distribution was >200 ml/kg. No significant inhibition of tumor growth was seen after i.v. treatment of mice with either 20 or 30 mg/kg 10058-F4. CONCLUSION: The lack of significant antitumor activity of 10058-F4 in tumor-bearing mice may have resulted from its rapid metabolism and low concentration in tumors.

Modulation of gemcitabine (2',2'-difluoro-2'-deoxycytidine) pharmacokinetics, metabolism, and bioavailability in mice by 3,4,5,6-tetrahydrouridine.

PURPOSE: In vivo, 2',2'-difluoro-2'-deoxycytidine (dFdC) is rapidly inactivated by gut and liver cytidine deaminase (CD) to 2',2'-difluoro-2'-deoxyuridine (dFdU). Consequently, dFdC has poor oral bioavailability and is administered i.v., with associated costs and limitations in administration schedules. 3,4,5,6-Tetrahydrouridine (THU) is a potent CD inhibitor with a 20% oral bioavailability. We investigated the ability of THU to decrease elimination and first-pass effect by CD, thereby enabling oral dosing of dFdC. EXPERIMENTAL DESIGN: A liquid chromatography-tandem mass spectrometry assay was developed for plasma dFdC and dFdU. Mice were dosed with 100 mg/kg dFdC i.v. or orally with or without 100 mg/kg THU i.v. or orally. At specified times between 5 and 1,440 min, mice (n = 3) were euthanized. dFdC, dFdU, and THU concentrations were quantitated in plasma and urine. RESULTS: THU i.v. and orally produced concentrations >4 microg/mL for 3 and 2 h, respectively, whereas concentrations of >1 microg/mL have been associated with near-complete inhibition of CD in vitro. THU i.v. decreased plasma dFdU concentrations but had no effect on dFdC plasma area under the plasma concentration versus time curve after i.v. dFdC dosing. Both THU i.v. and orally substantially increased oral bioavailability of dFdC. Absorption of dFdC orally was 59%, but only 10% passed liver and gut CD and eventually reached the systemic circulation. Coadministration of THU orally increased dFdC oral bioavailability from 10% to 40%. CONCLUSIONS: Coadministration of THU enables oral dosing of dFdC and warrants clinical testing. Oral dFdC treatment would be easier and cheaper, potentially prolong dFdC exposure, and enable exploration of administration schedules considered impractical by the i.v. route.

Plasma and tissue disposition of non-liposomal DB-67 and liposomal DB-67 in C.B-17 SCID mice.

PURPOSE: DB-67 is a silatecan, 7-silyl-modified camptothecin, with enhanced lipophilicity and increased blood stability of the active-lactone ring. The generation of a liposomal formulation of DB-67 may be an attractive method of intravenous (IV) administration and may maintain DB-67 in the active-lactone form. We evaluated the tissue and plasma disposition of DB-67 lactone and hydroxy acid after administration of non-liposomal (NL) and liposomal (L) DB-67 in severe combined immunodeficient (SCID) mice. METHODS: NL-DB-67 and L-DB-67 10 mg/kg IV x 1 were administered via a tail vein in SCID mice. After dosing, mice (n = 3 per time point) were euthanized and blood ( approximately 1 ml) and tissue were collected from 5 min to 48 h after administration. DB-67 lactone and hydroxy acid concentrations in plasma and DB-67 total (sum of lactone and hydroxyl acid) concentrations in tissues were determined by high-performance liquid chromatography (HPLC) with fluorescence detection. RESULTS: Clearance of DB-67 lactone after administration of NL-DB-67 and L-DB-67 were 1.6 and 3.5 l/h/m(2), respectively; DB-67 lactone half-lives after administration of NL-DB-67 and L-DB-67 were 1.4 and 0.9 h, respectively. The percentages of DB-67 lactone in plasma after administration of NL-DB-67 and L-DB-67 were 92% and 89%, respectively. Liver, kidney, spleen, and lung tissues had longer exposure times to DB-67 after administration of L-DB-67 compared with NL-DB-67. CONCLUSION: In plasma, the majority of DB-67 remained in the lactone form after administration of NL-DB-67 and L-DB-67. The plasma disposition of DB-67 was similar after administration of NL-DB-67 and L-DB-67, suggesting that most of the DB-67 is immediately released from the L-DB-67 formulation. Following administration of L-DB-67, the higher and longer exposure of DB-67 in the spleen, as compared with NL-DB-67, is consistent with splenic clearance of liposomes by the reticuloendothelial system.

Tumor, tissue, and plasma pharmacokinetic studies and antitumor response studies of docetaxel in combination with 9-nitrocamptothecin in mice bearing SKOV-3 human ovarian xenografts.

PURPOSE: We evaluated the antitumor activity of two different schedules of docetaxel and 9-nitrocamptothecin (9NC) in mice bearing human SKOV-3 ovarian carcinoma xenografts and evaluated the plasma, tissue, and tumor disposition of each agent alone and in combination. EXPERIMENTAL DESIGN: The following treatment groups were evaluated: (1) docetaxel 10 mg/kg IV on days 0 and 7; (2) 9NC 0.67 mg/kg PO qdx5dx2wk; (3) 9NC 0.67 mg/kg PO qdx5dx2wk in combination with docetaxel 10 mg/kg IV on days 0 and 7; and (4) 9NC 0.67 mg/kg PO qdx5dx2wk in combination with docetaxel 10 mg/kg IV on days 4 and 11; (5) vehicle controls for each agent; and (6) no treatment controls. RESULTS: All treatment regimens produced significant antitumor activity as compared with control groups (P < 0.05). Docetaxel administered on days 0 and 7 or on days 4 and 11 in combination with 9NC resulted in similar antitumor activity (P > 0.05). High docetaxel concentrations in tumor were maintained at late time points as compared with plasma and tissues with the retention of docetaxel at 24 h being 132-fold and 15-fold higher in tumor than in plasma and liver, respectively. After administration of 9NC alone, the ratio of the 9-aminocamptothecin (9AC) area under the concentration versus time curve (AUC) to 9NC AUC in plasma and tumor was 0.15 and 1.34, respectively. CONCLUSIONS: The combination of docetaxel and 9NC was effective against SKOV-3 xenografts. The lack of a difference in sequence-dependent antitumor activity may reflect the sensitivity of the SKOV-3 xenograft to 9NC. The factors associated with tumor-specific retention of docetaxel and the ratio of 9NC to 9AC in tumors is unknown.

Plasma pharmacokinetics and oral bioavailability of 3,4,5,6-tetrahydrouridine, a cytidine deaminase inhibitor, in mice.

Cytidine analogues such as cytosine arabinoside, gemcitabine, decitabine, 5-azacytidine, 5-fluoro-2'-deoxycytidine and 5-chloro-2'-deoxycytidine undergo rapid catabolism by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU) is a potent CD inhibitor that has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU pharmacokinetics has not been fully characterized, which has impaired the optimal preclinical evaluation and clinical use of THU. Therefore, we characterized the THU pharmacokinetics and bioavailability in mice. Mice were dosed with THU iv (100 mg/kg) or po (30, 100, or 300 mg/kg). Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma pharmacokinetic parameters were calculated compartmentally and non-compartmentally. THU, at 100 mg/kg iv had a 73 min terminal half-life and produced plasma THU concentrations >1 microg/ml, the concentration shown to effectively block deamination, for 4 h. Clearance was 9.1 ml/min/kg, and the distribution volume was 0.95 l/kg. Renal excretion accounted for 36-55% of the THU dose. A three-compartment model fit the iv THU data best. THU, at 100 mg/kg po, produced a concentration versus time profile with a plateau of approximately 10 mug/ml from 0.5-3 h, followed by a decline with an 85 min half-life. The oral bioavailability of THU was approximately 20%. The 20% oral bioavailability of THU is sufficient to produce and sustain, for several hours, plasma concentrations that inhibit CD. This suggests the feasibility of using THU to decrease elimination and first-pass metabolism of cytidine analogues by CD. THU pharmacokinetics are now being evaluated in humans.

Plasma, tumor, and tissue disposition of STEALTH liposomal CKD-602 (S-CKD602) and nonliposomal CKD-602 in mice bearing A375 human melanoma xenografts.

PURPOSE: S-CKD602 is a STEALTH liposomal formulation of CKD-602, a camptothecin analogue. The cytotoxicity of camptothecin analogues is related to the duration of exposure in the tumor. STEALTH liposomal formulations contain lipid conjugated to methoxypolyethylene glycol and have been designed to prolong drug circulation time, increase tumor delivery, and improve the therapeutic index. For STEALTH liposomal formulations of anticancer agents to achieve antitumor effects, the active drug must be released into the tumor extracellular fluid (ECF). EXPERIMENTAL DESIGN: S-CKD602 at 1 mg/kg or nonliposomal CKD-602 at 30 mg/kg was administered once via tail vein to mice bearing A375 human melanoma xenografts. Mice (n = 3 per time point) were euthanized at 0.083 to 24 h, 48 h, and 72 h after S-CKD02 and from 0.083 to 24 h after nonliposomal CKD-602. Plasma samples were processed to measure encapsulated, released, and sum total (encapsulated plus released) CKD-602, and tumor and tissue samples were processed to measure sum total CKD-602. Microdialysis samples of tumor ECF were obtained from 0 to 2 h, 4 to 7 h, and 20 to 24 h after nonliposomal CKD-602 and from 0 to 2 h, 24 to 27 h, 48 to 51 h, and 72 to 75 h after S-CKD602. A liquid chromatography-mass spectrometry assay was used to measure the total (sum of lactone and hydroxyl acid) CKD-602. The area under the concentration-versus-time curves (AUC) from 0 to infinity and time >1 ng/mL in tumor were estimated. RESULTS: For S-CKD602, the CKD-602 sum total AUC in plasma and tumor and the CKD-602 AUC in tumor ECF were 201,929, 13,194, and 187 ng/mL h, respectively. For S-CKD602, 82% of CKD-602 remains encapsulated in plasma. For nonliposomal CKD-602, the CKD-602 AUC in plasma and tumor and the CKD-602 AUC in tumor ECF were 9,117, 11,661, and 639 ng/mL.h, respectively. The duration of time the CKD-602 concentration was >1 ng/mL in tumor ECF after S-CKD602 and nonliposomal CKD-602 was >72 and approximately 20 h, respectively. For S-CKD602, the CKD-602 sum total exposure was 1.3-fold higher in fat as compared with muscle. The ratio of CKD-602 sum total exposure in fat to muscle was 3.8-fold higher after administration of S-CKD602 compared with nonliposomal CKD-602. CONCLUSION: S-CKD602 provides pharmacokinetic advantages in plasma, tumor, and tumor ECF compared with nonliposomal CKD-602 at 1/30th of the dose, which is consistent with the improved antitumor efficacy of S-CKD602 in preclinical studies. The distribution of S-CKD602 is greater in fat compared with muscle whereas the distribution of nonliposomal CKD-602 is greater in muscle compared with fat. These results suggest that the body composition of a patient may affect the disposition of S-CKD602 and released CKD-602.

Pharmacology and antitumor activity of a quinolinedione Cdc25 phosphatase inhibitor DA3003-1 (NSC 663284).

Cdc25 protein phosphatases are regulators of cyclin-dependent kinases and are often highly expressed in human malignancies. Few small molecule inhibitors of the Cdc25 phosphatase family have been identified and little is known about their disposition, metabolism or efficacy in xenograft models. In this study, the efficacy, pharmacokinetics, and metabolism of a potent quinolinedione Cdc25 phosphatase inhibitor, DA3003-1, in mice was examined. DA3003-1 inhibited the growth of subcutaneous human colon HT29 xenografts in SCID mice. After a single i.v. dose of 5 mg/kg, DA3003-1 was not detectable in plasma or tissues beyond 5 min. In vitro studies showed that DA3003-1 was rapidly dechlorinated and conjugated to glutathione. Following DA3003-1 treatment of tumor-bearing SCID mice, reduced glutathione concentrations in HT29 tumor were decreased to a greater extent and remained decreased for longer than the reduced glutathione concentrations in liver and kidneys. These studies suggest that the minimal antitumor activity of DA3003-1 in mice may be due to its rapid metabolism.

Noninvasive and nondestructive optical spectroscopic measurement of motexafin gadolinium in mouse tissues: comparison to high-performance liquid chromatography.

Efficient design of anti-cancer treatments involving radiation- and photo-sensitizing therapeutics requires knowledge of tissue-specific drug concentrations. This study investigates the use of the optical pharmacokinetic system (OPS) to measure concentrations of the anti-cancer agent motexafin gadolinium (MGd) in mouse tissues noninvasively and nondestructively using elastic-scattering spectroscopy. The magnitude of MGd absorbance was quantitated by integration of the MGd peak absorbance area, and MGd concentrations were estimated by comparison with standard curves that were validated by high performance liquid chromatography (HPLC). In tissue-simulating phantoms in vitro, MGd peak absorbance area correlated with MGd concentration. Female C.B-17 SCID mice, bearing subcutaneous MDA-MB-231 human breast cancer xenografts, were dosed with 23 mg/kg MGd i.v. At specific times between 5 min and 24h after dosing, noninvasive OPS measurements were made on skin overlaying the subcutaneous tumor and skin on the opposite flank in vivo, and following exsanguination, nondestructive measurements were made on tumor, skin, and internal tissues in situ. OPS measurements on tissues in vivo detected MGd present in both tissue and blood perfusing the tissue. Both the OPS and the HPLC detected selective localization of MGd in malignant tissues compared with surrounding non-malignant tissues, and neither technique detected MGd in brain tissue. Comparison of MGd concentrations measured by HPLC and OPS is complicated by mismatch between measured tissue volumes, heterogeneous spatial distribution of MGd in tissues, and blood-localized MGd at early time points. Tumor-specific MGd concentrations measured by HPLC correlated with those measured by OPS in vivo and in situ. Best fit lines to the concentration estimates (forced through zero) had slopes of 0.900 and 1.185, respectively; however, the variability was significant (r(2)=0.477 and 0.269). The clinical utility of the OPS to quantitate MGd concentrations remains to be validated.

Quantitative determination of the cytidine deaminase inhibitor tetrahydrouridine (THU) in mouse plasma by liquid chromatography/electrospray ionization tandem mass spectrometry.

A number of anticancer drugs are cytidine analogues that undergo metabolic deactivation catalyzed by cytidine deaminase (CD). 3,4,5,6-Tetrahydrouridine (THU) is a potent inhibitor of CD, by acting as a transition-state analogue of its natural substrate cytidine. However, to date its pharmacokinetic properties have not been fully characterized, which has impaired its optimal preclinical evaluation and clinical use. We report a liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for the sensitive, accurate and precise quantitation of THU in mouse plasma. Validation was performed according to FDA guidelines. The assay employed deuterated THU as the internal standard and an acetonitrile protein precipitation step. Separation, based on hydrophilic interaction chromatography, was achieved with an amino column and an isocratic mobile phase of 0.1% formic acid in acetonitrile and water followed by a wash. Chromatographic separation was followed by positive-mode electrospray ionization MS/MS detection in the multiple reaction monitoring (MRM) mode. The assay was accurate (92.5-109.9%) and precise (2.1-9.0%) in the concentration range of 0.2-50 microg/mL. Recovery from plasma was near-complete (92.9-119.3%) and ion suppression was negligible (-17.5 to -0.2%). Plasma freeze/thaw stability (93.1-102.1%), stability for 3 months at -80 degrees C (99.5-110.9%), and stability for 4 h at room temperature (92.1-102.4%) were all in order. This assay is currently being used to quantitate THU in ongoing pharmacokinetic studies. In addition, the assay is expected to be a useful tool in any future studies involving co-administration of THU with cytidine analogues.

Pharmacokinetics, metabolism, and oral bioavailability of the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine in mice.

PURPOSE: In vivo, 5-fluoro-2'-deoxycytidine (FdCyd) is rapidly and sequentially converted to 5-fluoro-2'-deoxyuridine, 5-fluorouracil, and 5-fluorouridine. The i.v. combination of FdCyd and 3,4,5,6-tetrahydrouridine (THU), a cytidine deaminase (CD) inhibitor that blocks the first metabolic step in FdCyd catabolism, is being investigated clinically for its ability to inhibit DNA methyltransferase. However, the full effects of THU on FdCyd metabolism and pharmacokinetics are unknown. We aimed to characterize the pharmacokinetics, metabolism, and bioavailability of FdCyd with and without THU in mice. EXPERIMENTAL DESIGN: We developed a sensitive high-performance liquid chromatography tandem mass spectrometry assay to quantitate FdCyd and metabolites in mouse plasma. Mice were dosed i.v. or p.o. with 25 mg/kg FdCyd with or without coadministration of 100 mg/kg THU p.o. or i.v. RESULTS: The oral bioavailability of FdCyd alone was approximately 4%. Coadministration with THU increased exposure to FdCyd and decreased exposure to its metabolites; i.v. and p.o. coadministration of THU increased exposure to p.o. FdCyd by 87- and 58-fold, respectively. FdCyd exposure after p.o. FdCyd with p.o. THU was as much as 54% that of i.v. FdCyd with i.v. THU. CONCLUSIONS: FdCyd is well absorbed but undergoes substantial first-pass catabolism by CD to potentially toxic metabolites that do not inhibit DNA methyltransferase. THU is sufficiently bioavailable to reduce the first-pass effect of CD on FdCyd. Oral coadministration of THU and FdCyd is a promising approach that warrants clinical testing because it may allow maintaining effective FdCyd concentrations on a chronic basis, which would be an advantage over other DNA methyltransferase inhibitors that are currently approved or in development.

A mass balance and disposition study of the DNA methyltransferase inhibitor zebularine (NSC 309132) and three of its metabolites in mice.

PURPOSE: To elucidate the in vivo metabolic fate of zebularine (NSC 309132), a DNA methyltransferase inhibitor proposed for clinical evaluation in the treatment of cancer. EXPERIMENTAL DESIGN: Male, CD(2)F(1) mice were dosed i.v. with 100 mg/kg 2-[(14)C]zebularine. At specified times between 5 and 1,440 minutes, mice were euthanized. Plasma, organs, carcass, urine, and feces were collected and assayed for total radioactivity. Plasma and urine were also analyzed for zebularine and its metabolites with a previously validated high-pressure liquid chromatography assay. A similar experiment was done with 2-[(14)C]uridine, the proposed primary metabolite of zebularine. RESULTS: Maximum plasma concentrations were 462, 306, 33.6, 21.7, and 11.5 mumol/L for total radioactivity, zebularine, uridine, uracil (each at 5 minutes), and dihydrouracil (at 15 minutes), respectively. Total radioactivity, zebularine, uridine, uracil, and dihydrouracil were rapidly eliminated from plasma, and after 45 minutes, none of the individual compounds could be quantitated by high-pressure liquid chromatography. Plasma data were consistent with sequential conversion of zebularine to uridine, uracil, and dihydrouracil. 2-Pyrimidinone was not observed. Prolonged retention of radioactivity, at concentrations higher than in plasma, was observed in tissues. Recovery of given radioactivity in urine (30.3% of dose), feces (0.4% of dose), cage wash (7.9% of dose), and tissues and carcass (6.1% of dose) after 24 hours implied that up to 55% of radioactivity was expired as (14)CO(2). Comparison of zebularine and uridine pharmacokinetic data indicated that approximately 40% of the zebularine dose was converted to uridine. CONCLUSIONS: Zebularine is extensively and rapidly metabolized into endogenous compounds that are unlikely to have effects at the concentrations observed.

Quantitative determination of zebularine (NSC 309132), a DNA methyltransferase inhibitor, and three metabolites in murine plasma by high-performance liquid chromatography coupled with on-line radioactivity detection.

The metabolism of zebularine (NSC 309132), a novel agent that inhibits DNA methyltransferases, is still uncharacterized. To examine the in vivo metabolism of zebularine, an analytical method was developed and validated (based on FDA guidelines) to quantitate 2-[(14)C]-zebularine and its major metabolites in murine plasma. Zebularine and its metabolites uridine, uracil and dihydrouracil were baseline-separated based on hydrophilic interaction chromatography by using an amino column. The assay was accurate and precise in the concentration ranges of 5.0-100 microg/mL for zebularine, 2.5-50 microg/mL for uridine, 1.0-10 microg/mL for uracil and 0.5-5.0 microg/mL for dihydrouracil. This assay is being used to quantitate zebularine and its metabolites in ongoing pharmacokinetic studies of zebularine.

Selective inhibition of MCF-7(piGST) breast tumors using glutathione transferase-derived 2-methylene-cycloalkenones.

Human glutathione (GSH) transferase (hGSTP1-1) catalyzes the conversion of antitumor 2-crotonyloxymethyl-2-cycloalkenones (COMCs) to highly reactive exocyclic enone alkylating agents. In vitro efficacy studies show that the cytotoxicities of the COMCs directly correlate with the level of expression of GSTP1-1 in MCF-7(piGST) versus MCF-7wt breast tumors, indicating that the exocyclic enones are the actual cytotoxic species. The COMCs are a potentially important new class of prodrugs, which can specifically target multi-drug-resistant tumors overexpressing hGSTP1-1.

Control-relevant modeling of the antitumor effects of 9-nitrocamptothecin in SCID mice bearing HT29 human colon xenografts.

The mathematical model structure selected to describe system behavior is at least partially dependent on the proposed use of the model. In this paper, a pharmacokinetic(PK)/pharmacodynamic (PD) model for use in drug delivery algorithm synthesis is developed. The antitumor agent 9-nitrocamptothecin (9NC) was administered orally to severe combined immunodeficient (SCID) mice bearing subcutaneously implanted HT29 human colon xenografts, and the effect of 9NC on those xenografts was characterized. Different PK model structures were considered in characterizing the dynamics of the drug concentration in the plasma. Akaike's Information Criterion (AIC) was used to select the model structure maximizing fit accuracy while simultaneously minimizing the number of model parameters. The resulting PK model was a set of coupled linear ordinary differential equations able to describe the nonlinear dynamic behavior (e.g. plateauing, etc.) of the drug concentrations observed in the plasma. Pharmacodynamics were modeled by characterizing tumor growth in both the untreated and drug-treated animals. The resulting PK/PD model related drug administration to effect, and this model has a structure that facilitates future control algorithm synthesis. Control algorithms in this context would directly utilize PK/PD model predictions. These predictions would be used to determine the amount and frequency of drug administration in order to reduce the tumor burden without violating clinically relevant constraints. This methodology could then be used to aid the clinician in selecting dose levels and schedules, and extension to patient tailored treatment may eventually be feasible with this approach.

Relationship between plasma exposure of 9-nitrocamptothecin and its 9-aminocamptothecin metabolite and antitumor response in mice bearing human colon carcinoma xenografts.

9-Nitrocamptothecin has completed phase III studies in patients with newly diagnosed and refractory pancreatic cancer; however, the optimal 9-nitrocamptothecin treatment regimen is unclear. We used an intermittent schedule of 9-nitrocamptothecin to evaluate the relationship between plasma exposure of 9-nitrocamptothecin and its 9-aminocamptothecin metabolite and antitumor response in mice bearing human colon carcinoma xenografts. 9-Nitrocamptothecin was given orally at 0.44, 0.67, or 1.0 mg/kg/d qd x 5d x 2 weeks repeated q 4 weeks for two cycles to female C.B-17 SCID mice bearing HT29 or ELC2 human colon xenografts. Pharmacokinetic studies were done after oral administration of 0.67 mg/kg x 1. Serial samples were obtained and 9-nitrocamptothecin and 9-aminocamptothecin lactone concentrations in plasma were determined by high-performance liquid chromatography analysis with fluorescence detection. The areas under plasma concentration versus time curve (AUC) from 0 to infinity for 9-nitrocamptothecin and 9-aminocamptothecin were calculated. The antitumor activity of 9-nitrocamptothecin was dose-dependent in both colon xenografts. At all doses, 9-nitrocamptothecin treatment resulted in significant antitumor activity in both xenografts compared with vehicle-treated and control groups and achieved levels of tumor regression that met criteria (minimum %T/C < or = 40%) for antitumor activity. In mice bearing HT29 xenografts, the 9-nitrocamptothecin and 9-aminocamptothecin lactone AUCs after administration of 9-nitrocamptothecin at 0.67 mg/kg were 41.3 and 5.7 ng/mL h, respectively. The responses seen in these xenograft models occurred at systemic exposures that are tolerable in adult patients. These results suggest that the intermittent schedule of 9-nitrocamptothecin may be an active regimen in patients with colorectal carcinoma.

Plasma pharmacokinetics, oral bioavailability, and interspecies scaling of the DNA methyltransferase inhibitor, zebularine.

PURPOSE: Zebularine is a DNA methyltransferase inhibitor proposed for clinical evaluation. EXPERIMENTAL DESIGN: We developed a liquid chromatography/mass spectrometry assay and did i.v. and oral studies in mice, rats, and rhesus monkeys. RESULTS: In mice, plasma zebularine concentrations declined with terminal half-lives (t(1/2)) of 40 and 91 minutes after 100 mg/kg i.v. and 1,000 mg/kg given orally, respectively. Zebularine plasma concentration versus time curves (area under the curve) after 100 mg/kg i.v. and 1,000 mg/kg given orally were 7,323 and 4,935 mug/mL min, respectively, corresponding to a total body clearance (CL(tb)) of 13.65 mL/min/kg, apparent total body clearance (CL(app)) of 203 mL/min/kg, and oral bioavailability of 6.7%. In rats, plasma zebularine concentrations declined with t(1/2) of 363, 110, and 126 minutes after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally, respectively. Zebularine areas under the curve after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally were 12,526, 1,969, and 7,612 mug/mL min, respectively, corresponding to a CL(tb) of 3.99 mL/min/kg for 50 mg/kg i.v. and CL(app) of 127 and 66 mL/min/kg for 250 and 500 mg/kg given orally, respectively. Bioavailabilities of 3.1% and 6.1% were calculated for the 250 and 500 mg/kg oral doses, respectively. In monkeys, zebularine t(1/2) was 70 and 150 minutes, CL(tb) was 3.55 and 10.85 mL/min/kg after i.v. administration, and CL(app) was 886 and 39,572 mL/min/kg after oral administration of 500 and 1,000 mg/kg, respectively. Zebularine oral bioavailability was <1% in monkeys. Interspecies scaling produced the following relationship: CL(tb) = 6.46(weight(0.9)). CONCLUSIONS: Zebularine has limited oral bioavailability. Interspecies scaling projects a CL(tb) of 296 mL/min in humans.

N-(2-hydroxypropyl)methacrylamide copolymers of a glutathione (GSH)-activated glyoxalase i inhibitor and DNA alkylating agent: synthesis, reaction kinetics with GSH, and in vitro antitumor activities.

The incorporation of anticancer prodrugs into polyacrylamide conjugates has been shown to improve tumor targeting via the so-called "enhanced permeability and retention" effect. This strategy has now been expanded to include two different classes of glutathione (GSH)-activated antitumor agents prepared by radical polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-methacryloyloxy-methyl-2-cyclohexenone (7) and/or with S-(N-4-chlorophenyl-N-hydroxycarbamoyl-thioethyl)methacrylamide (8), followed by treatment with 3-chloroperoxybenzoic acid, to give the HPMA copolymers of 7 and the 8-sulfoxide, respectively. In aqueous-buffered solution at pH 6.5, GSH reacts rapidly with poly-HPMA-8-sulfoxide (k approximately 2.3 mM(-1) min(-1)) to give S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (1), a tight-binding transition state analogue inhibitor of the antitumor target enzyme glyoxalase I (K(i) = 46 nM), or with poly-HPMA-7 (k approximately 0.02 mM(-1) min(-1)) to give the electrophilic antitumor agent 3-glutathio-2-methylenecyclohexenone (4). Indeed, B16 melanotic melanoma in culture is inhibited by poly-HPMA-8-sulfoxide and by poly-HPMA-7 with IC(50) values of 168 +/- 8 and 284 +/- 5 microM, respectively. These values are significantly greater than those of the unpolymerized prodrugs suggesting that the cytotoxicity of the polymer prodrugs might be limited by slow cellular uptake via pinocytosis. This prodrug strategy should be applicable to a range of different GSH-based antitumor agents.