Feasibility of cellular microencapsulation technology for evaluation of anti-human immunodeficiency virus drugs in vivo.

We investigated the feasibility of micro-encapsulation technology for the evaluation of anti-human immunodeficiency virus (HIV) drugs in vivo. The ability to place human cells in microcapsules with semipermeable membranes for implantation into test animals led to the development of this assay. The anti-HIV activity assay involves microencapsulating human T-lymphoblastoid cells sensitive to the cytopathic effects of HIV; the encapsulated cells are then implanted into athymic nude mice and recovered after drug treatment in vivo. A positive antiviral effect of the test substance is indicated by growth or survival of the virus-infected cells in the microcapsules. Several HIV-sensitive cell lines of T-lymphocyte, monocyte, and nonlymphocyte origin were examined for growth in microcapsules in vitro and in vivo. Light and electron microscopic analysis of the capsules and the human cells contained therein revealed the invasion of mouse immune cells and other adverse effects that could not be overcome by any of numerous technical modifications attempted. We conclude that cellular microencapsulation technology is not feasible for in vivo drug-testing protocols because of immunogenic reactions.

Growth support of small B16 melanoma implants with nitrosourea-sterilized fractions of the same tumor.

B16 melanoma cells sterilized in vitro with 1,3-bis(2-chloroethyl)-1-nitrosourea or in vivo with trans-1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea, have been used to enhance the percentage of tumor takes with small s.c. implants of viable cells and to reduce the latent period between tumor implantation and palpability. The admixture of trans-1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea-inactivated cells with viable cell implants reduced the number of cells required to produce tumors in 50% of the animals by approximately 3 log10 units and markedly reduced the time of tumor appearance from implants of up to 10(6) cells. Similar results were obtained with 1,3-bis(2-chloroethyl)-1-nitrosourea-sterilized cells. The growth-supporting effect obtained with the nitrosourea-inactivated cells appeared to be as pronounced as that previously reported, for this tumor system, with radiation-inactivated cells.

Sequence and schedule-dependent synergy of trimetrexate in combination with 5-fluorouracil in vitro and in mice.

The purpose of this study was to determine the conditions for optimum synergistic efficacy of the two-drug combination of trimetrexate and 5-fluorouracil. Synergistic cell killing of Chinese hamster ovary cells in these clonogenic survival assays was observed only when the cells had been exposed to trimetrexate (25 microM) for 2 to 4 h prior to 5-fluorouracil exposure (either 125 or 250 microM). The schedule dependence of the observed synergy in vitro was closely linked to trimetrexate-induced changes in cellular 5-phosphoribosyl 1-pyrophosphate (PRPP) pools. Exposure to 25 microM trimetrexate induced increases in PRPP pools to 398% and 761% of control values at 2 and 4 h, respectively. Methotrexate (20 microM) also increased Chinese hamster ovary cell PRPP content in a time-dependent fashion to values of 280 and 511% of control after 2 and 4 h of drug exposure. Previous in vivo studies demonstrated a modest degree of therapeutic synergy between trimetrexate and 5-fluorouracil against P388 leukemia. Our in vitro results suggested that the degree of synergy seen in vivo could be increased with appropriate schedule changes. Mice were implanted i.p. with 10(6) P388 leukemia cells on Day 0 and were treated with trimetrexate (every 3 h for eight injections; Days 1, 5, and 9) and 5-fluorouracil (Days 1, 5, and 9) as single agents or in combination on one of two schedules; 5-fluorouracil was administered with either the first or the last of the eight trimetrexate doses on Days 1, 5, and 9. Both treatment regimens demonstrated therapeutic synergy but, as predicted from the in vitro data, the "5-fluorouracil last" was superior to the "5-fluorouracil first" sequence. Treatment with the optimal doses on the "5-fluorouracil last" sequence (trimetrexate, 31; 5-fluorouracil, 33 mg/kg/injection) produced an increased life span of 183% and a net reduction in tumor cell burden of 6.7 logs compared with a 111% increased life span (net reduction in tumor burden of 2.6 logs) produced by the most active of the single agents, 5-fluorouracil. Thus the efficacy of the combination of trimetrexate with 5-fluorouracil was sequence and time dependent both in vitro and in vivo. The synergy, observed in vitro and probably in vivo, was linked to a trimetrexate-induced elevation of intracellular PRPP, thus facilitating the production of 5-fluoropyrimidine nucleotides. These data are similar to the sequence and schedule dependency of the methotrexate/5-fluorouracil combination with important differences.(ABSTRACT TRUNCATED AT 400 WORDS)

L-histidinol: preclinical therapeutic studies in combination with antitumor agents and pharmacokinetic studies in mice.

Therapeutic studies were conducted with L-histidinol, in combination with cyclophosphamide, bischloroethylnitrosourea, 5-fluorouracil, phenylalanine mustard, or cis-platinum(II)diammine dichloride, in several transplantable tumors in mice. These tumor types included murine L1210 P388 leukemias, M5076 sarcoma, mammary 16/C adenocarcinoma, human LOX melanoma, and colon HT-29 adenocarcinoma. Therapeutic benefits of adding L-histidinol to a regimen, compared to the regimen alone, were marginal. Pharmacokinetic studies indicated a rapid clearance of L-histidinol following a bolus dose (250 mg/kg i.p.), peak plasma concentration of 200 micrograms/ml (1.4 mM), and beta phase t1/2 of 12.6 min. Maximum tolerable plasma steady state concentrations with a 24-h infusion (2000 mg/kg/24 h) were no greater than 25 micrograms/ml (0.18 mM).

Preclinical antitumor activity of penclomedine in mice: cross-resistance, schedule dependence, and oral activity against tumor xenografts in brain.

Penclomedine is 3,5-dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine (NSC 338720), an alpha-picoline derivative with p.o. antitumor activity in preclinical leukemia and solid tumor models. Described here are an in vivo cross-resistance profile of penclomedine, treatment schedule dependence studies, and studies exploring the effects of p.o. drug on human tumors xenografted into mouse brain. The latter studies exploited the apparent facile distribution of penclomedine to the central nervous system. Tumor models used included murine leukemia lines selected in vivo for acquired resistance to various antitumor drugs and the human mammary and lung tumor xenografts MX-1 and H82, respectively. The therapeutic effects of p.o. penclomedine against s.c. MX-1 and H82 xenografts were shown to be independent of treatment schedule. Therapeutic activity was comparable when p.o. and parenteral treatments were compared. Lines of P388 leukemia resistant to melphalan, cyclophosphamide, and carmustine were cross-resistant to penclomedine in vivo. Leukemia lines resistant to antimetabolites, DNA binders/intercalators, and vincristine were not cross-resistant to penclomedine. Intracerebrally implanted MX-1 xenografts retained their sensitivity to p.o. penclomedine, and therapeutic activity was at least comparable to that of carmustine, a drug known for its ability to cross the blood-brain barrier. These results demonstrate attributes of penclomedine that are relatively uncommon among currently available antitumor drugs and that are of interest for the anticipated clinical development of this drug.

Efficacy of the quinocarmycins KW2152 and DX-52-1 against human melanoma lines growing in culture kand in mice.

Quinocarmycin monicitrate (KW2152) and its analogue, DX-52-1, demonstrated specificity for melanomas in the National Cancer Institute in vitro human tumor cell line drug screen. In contrast to most cell lines, a 50% reduction in tumor cell burden (as measured protein) at the end of a 48-h drug incubation was produced in five of eight melanoma lines by KW2152 concentrations (LC50s) ranging from 0.49 to 10.93 microM and by DX-52-1 concentrations ranging from 0.71 to 7.33 microM. Using the COMPARE algorithm, the patterns of differential cytotoxicity for both agents at the LC50 level of effect most closely resembled those for actinomycin D, mithramycin, and Adriamycin. In in vivo studies, both KW2152 (40 mg/kg/day) and DX-52-1 (90 mg/kg/day) caused partial and complete regressions of staged s.c.-implanted LOX IMVI melanoma xenografts following i.p. administration on days 5, 9, and 13 and produced tumor growth delays of 231 and 181%, respectively (P < 0.001). Activity was augmented by more prolonged therapy. Statistically significant growth inhibition of SK-MEL-2, UACC-62, UACC-257, and M14, but not SK-MEL-5 and MALME-3M, melanoma xenografts also was observed following every fourth or seventh day i.p. treatments. Based on these findings, DX-52-1 has been selected by the National Cancer Institute for development to clinical trial especially against melanomas. This agent represents one of the first to be selected for preclinical development based on disease-panel specificity discovered in the National Cancer Institute cancer drug screen.

Practical spontaneous metastasis model for in vivo therapeutic studies using a human melanoma.

In vivo studies aimed at therapy of spontaneous human tumor metastases have been hampered by the lack of practical experimental models. The LOX amelanotic melanoma model described here represents a transplantation model which rapidly and reproducibly results in spontaneous pulmonary metastasis following s.c. inoculation into athymic mice. Pulmonary lesions can be detected using a simple bioassay procedure which is useful for estimation of metastatic cell killing. Using this model we demonstrate that systemic therapy with cyclophosphamide or dacarbazine can produce metastatic cell killing consistent with complete eradication of established pulmonary metastases. This model may also prove useful for future experimental therapeutic studies aimed at prevention of metastases by manipulating tumor staging interval and treatment schedule.

Antitumor activity of 2-chloroethyl (methylsulfonyl)methanesulfonate (clomesone, NSC 33847) against selected tumor systems in mice.

Clomesone was evaluated for antitumor activity against a spectrum of animal tumor models. Clomesone exhibited significant antitumor activity against the murine L1210 leukemia implanted i.p., s.c., and intracerebrally (i.c.). Activity against s.c.-implanted tumor was largely independent of schedule and route of administration. Therapeutically optimal single-dose treatment (for tumored mice) was less toxic to nontumored mice than therapeutically optimal prolonged treatment. Clomesone also exhibited activity against other murine tumors (P388 leukemia, B16 melanoma, Lewis lung carcinoma, and M5076 sarcoma). It was active against P388 leukemia sublines resistant to cyclophosphamide, L-phenylalanine mustard, and cis-diamminedichloroplatinum(II). No activity was observed against a P388 subline resistant to N,N'-bis(2-chloroethyl)-N-nitrosourea or against Ridgway osteogenic sarcoma, a nitrosourea-resistant murine solid tumor. Clomesone is generally as effective as the chloroethylnitrosoureas against experimental tumor models. Since clomesone does not have the hydroxyethylating and carbamoylating activities of the chloroethylnitrosoureas (which do not appear to contribute to antitumor activity), it would likely be a more toxicologically selective compound. It may prove to be less carcinogenic than the chloroethylnitrosoureas, and it may contribute less target organ toxicity and less interference with the actions of other drugs when used in combinations.

Preclinical antitumor activity of an alpha-picoline derivative, penclomedine (NSC 338720), on human and murine tumors.

Penclomedine, a synthetic alpha-picoline derivative, was identified as a potential antitumor agent in the P388 leukemia prescreen of the National Cancer Institute. Upon further evaluation in the National Cancer Institute in vivo tumor panel, the compound demonstrated good activity against two breast tumors. A single i.p. dose or five daily doses caused partial regressions of advanced-stage s.c. implanted mouse CD8F1 mammary adenocarcinomas. Also, penclomedine administered i.p. on Days 1,5, and 9 caused regression of the human MX-1 mammary carcinoma implanted under the renal capsule of athymic mice. In contrast, penclomedine demonstrated only marginal to moderate activity against the i.p. implanted L1210 leukemia and M5076 sarcoma and was inactive in three additional non-breast tumor models (i.p. B16 melanoma, i.v. Lewis lung carcinoma, and s.c. colon adenocarcinoma 38). Penclomedine administered p.o. and i.p. was equally effective against the subrenal capsule MX-1. Doses given p.o. every fourth day caused complete regression of 39 of 40 advanced-stage s.c. implanted MX-1 tumors but were much less effective against human H82 small cell lung carcinomas (13 of 80 complete regressions). Penclomedine p.o. also inhibited growth of the human MCF-7 and mouse 16/C breast adenocarcinomas. Further studies to support the development of penclomedine to clinical trial are in progress.

2-Chloroethyl-3-sarcosinamide-1-nitrosourea, a novel chloroethylnitrosourea analogue with enhanced antitumor activity against human glioma xenografts.

Nitrosoureas are among the most widely used agents used in the treatment of malignant gliomas. Here, the activity of 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) was compared with that of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), in vivo against s.c. implanted SF-295 and U-251 central nervous system (CNS) tumor xenografts. When given i.v., q4d for 3 doses, to athymic mice bearing s.c. SF-295 tumors, SarCNU, at an optimum of 167 mg/kg/dose, produced 9 tumor-free animals of 10 total animals, 1 regression, and no evidence of overt toxicity (> or =20% body weight loss). With a similar dosing schedule, BCNU produced no tumor-free animals, six regressions, and one drug-related death at its optimum of 30 mg/kg/dose. Furthermore, SarCNU retained high antitumor activity at two lower dose levels, 66 and 45% of the optimal dose, whereas BCNU demonstrated a progressive loss of antitumor activity at lower doses. Following p.o. administration, SarCNU similarly demonstrated antitumor activity that was superior to that of BCNU. In the U-251 CNS tumor model, SarCNU yielded six of six tumor-free animals at 80 mg/kg/dose with i.p. administration q.d. for 5 days, starting on day 14, whereas BCNU, at 9 mg/kg/dose, yielded three of six tumor-free mice and one drug-related death. Again, SarCNU resulted in tumor-free animals at 66 and 45% of its optimal dose and was relatively nontoxic, in contrast to BCNU. Results of testing to date indicate that SarCNU is clearly more effective than BCNU against the human CNS tumors SF-295 and U-251 in vivo. These results encourage the initiation of clinical trials for SarCNU, in an effort to improve therapeutic approaches to glioma, but clinical trials must determine whether superiority of SarCNU in preclinical models can be extrapolated to patients.

Toxicity and anticancer activity of a new triazine antifolate (NSC 127755).

A new triazine folate antagonist, 3-chloro-4-((4-(2-chloro-4-[4,6-diamino-2,2-dimethyl-s-triazin-1(2H)-yl]phenyl)butyl)) benzenesulfonyl fluoride compounded with ethanesulfonic acid (1:1) (NSC 127755), was highly active against four transplantable colon adenocarcinomas (36, 38, 10/A, 12/A) and the Dunning murine ovarian tumor M5076. Treatment schedule studies indicated that a prolonged time of exposure provided optimum antitumor activity for the compound. The combination of NSC 127755 plus 4-amino-1-[5-O-(1-oxohexadecyl)-beta-D-arabinofuranosyl]-2(1H)-pyrimidinone (palmO-ara-C, NSC 135962) was found to have therapeutic synergism against grossly evident colon adenocarcinoma 36.

Antitumor activity of ethyl 5-amino-1,2-dihydro-2-methyl-3-phenyl-pyrido [3,4-b]pyrazin-7-ylcarbamate, 2-hydroxyethanesulfonate, hydrate (NSC 370147) against selected tumor systems in culture and in mice.

Ethyl 5-amino-1,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin- 7-ylcarbamate, 2-hydroxyethanesulfonate, hydrate (NSC 370147) was evaluated for antitumor activity against a spectrum of tumor systems in culture and in mice. NSC 370147 was cytotoxic to a variety of mouse and human cell lines at nanomolar concentrations. The compound exhibited good in vivo antitumor activity against several murine tumors (P388 and L1210 leukemia, colon 11/A and 36, mammary 16/C, and M5076 sarcoma). Activity was largely independent of route of administration but favored a prolonged treatment schedule. NSC 370147 was as active against murine leukemia sublines resistant to Adriamycin, amsacrine, vincristine, melphalan, cisplatin, methotrexate, and CI-920 (a topoisomerase II inhibitor) as against the corresponding parental lines. Only the 1-beta-D-arabinofuranosylcytosine-resistant P388 subline exhibited any cross-resistance to NSC 370147. NSC 370147 has a spectrum of activity similar to that of vincristine and, unlike vincristine, is active against multidrug-resistant cell lines. Therefore, NSC 370147 is a candidate for clinical trial because of its favorable activity compared to vincristine, its effectiveness against multidrug-resistant cells, and its retention of activity for p.o. administration.

Elevated DT-diaphorase activity and messenger RNA content in human non-small cell lung carcinoma: relationship to the response of lung tumor xenografts to mitomycin Cl.

The enzyme DT-diaphorase (DTD; NAD(P)H:quinone oxidoreductase, EC 1.6.99.2), is an obligate two electron reductase which catalyzes reduction of a broad range of substrates, including quinones. We report here variations in DTD concentrations among different classes of lung tumors known also to vary in their responsiveness to cytotoxic agents. Small cell lung carcinomas (SCLCs) and cell lines derived from them have the low DTD activities and mRNA content characteristic of normal human lung, whereas non-small cell lung carcinomas (NSCLCs) have greatly elevated levels. DTD activity was increased up to 80-fold in NSCLC tumors relative to normal lung and 20-35-fold in NSCLC relative to SCLC cell lines. Increased DTD activity appeared to be a function of the NSCLC phenotype rather than a result of derivation from a cell type rich in DTD, since all histological classes of NSCLC showed this phenotype. In addition, where transfection of SCLC cell lines with the v-Ha-ras protooncogene caused a transition to a NSCLC phenotype, DTD activity was also elevated. Neuroendocrine-positive cells (SCLC, carcinoids, and a few NSCLC lines) typically had far lower DTD activities than did cell lines which lacked neuroendocrine markers (most NSCLC cells and mesotheliomas). High DTD activity may be exploited in the design of drugs which undergo bioreductive activation by this enzyme. Consistent with this, xenografts derived from NSCLC cell lines with high DTD that were grown in athymic nude mice were more susceptible to the antitumor quinone, mitomycin C, than were xenografts derived from SCLC cells containing low DTD. These data provide a mechanistic basis for the rational design of more effective bioreductive antitumor agents for use against NSCLC.

Conformational energy analysis of the substitution of Val for Gly 233 in a functional region of platelet GPIb alpha in platelet-type von Willebrand disease.

Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder in which patient platelets exhibit an abnormally increased binding of circulating von Willebrand factor (vWF). We have recently shown that this abnormality is associated with a point mutation resulting in substitution of Val for Gly 233 in platelet membrane glycoprotein Ib alpha (GPIb alpha), a major component of the platelet GPIb/IX receptor for vWF. To investigate the effect of this substitution on the three-dimensional structure of this region of the protein, we have generated the allowed (low energy) conformations of the region of the GPI alpha protein containing residues 228-238 (with 5 residues on either side of the critical residue 233) with Gly 233 (wild type) and Val 233 (PT-vWD) using the computer program ECEPP (Empirical Conformational Energies of Peptides Program). The wild-type sequence is Tyr-Val-Trp-Lys-Gln-Gly-Val-Asp-Val-Lys-Ala. We find that the Gly 233-containing peptide can exist in two low energy conformers. The lowest energy conformer is a structure containing a beta-turn at Gln 232-Gly 233 while the alternative conformation is an amphipathic helical structure. Only the amphipathic helical structure is allowed for the Val 233-containing peptide which contains a hydrophobic 'face' consisting of Val 229, Val 233 and Val 236 and another hydrophilic surface composed of such residues as Lys 231 and Asp 235. No such surfaces exist for the lowest energy bend conformer for the Gly 233-containing peptide, but do exist in the higher energy helical structure. The amphipathic surfaces in the 228-238 region of the Val 233-containing GPIb alpha protein may associate strongly with complementary surfaces during vWF binding to the GPIb/IX receptor complex and may help explain heightened association of vWF with this receptor in PT-vWD.

Effects of recombinant neutral endopeptidase (EC 3.4.24.11) on the growth of lung cancer cell lines in vitro and in vivo.

Many lung cancers are stimulated by an autocrine/paracrine system of neuroendocrine peptide hormones. Attempts to block this autocrine growth pathway by interactions with specific ligand-receptor binding using monoclonal antibodies and peptide-specific antagonists have been largely unsuccessful because of the heterogeneity of hormone production and receptor expression. In the normal lung, neutral endopeptidase (NEP; CD10, CALLA, enkephalinase, and EC 3.4.24.11) plays a physiological role in degrading biologically active peptides, including all peptides implicated in autocrine growth stimulation of lung cancer. Cigarette smoke decreases the activity of NEP, indicating that the lack of NEP contributes to the dysregulation of the peptide autocrine system. The cloning of the human NEP gene allowed for production of sufficient quantities of recombinant NEP (rNEP) to evaluate its role in inhibiting the growth of lung cancer cells. In this study, we evaluated the ability of rNEP to inactivate the peptides involved in lung cancer signal transduction and to inhibit the growth of lung cancer cells as well as normal lung cells in vitro and in vivo in athymic nude mice. We showed that the growth inhibition of lung cancer cells by rNEP was related to the dose and schedule. Continuous exposure to high doses was required for growth inhibition. These studies confirm the importance of NEP in this autocrine pathway.

Encapsulation of the topoisomerase I inhibitor GL147211C in pegylated (STEALTH) liposomes: pharmacokinetics and antitumor activity in HT29 colon tumor xenografts.

The topoisomerase I inhibitor GL147211C [7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-campto thecin trifluoroacetate], a camptothecin analogue, has significant activity in tumor cell cytotoxicity assays in vitro and antitumor activity in both animal tumor models and human patients. Its toxicity is significant, however, effectively limiting the amount of drug that can be administered and its clinical utility. To determine whether the therapeutic index of GL147211C could be improved, the drug was encapsulated in long-circulating, pegylated (STEALTH) liposomes (SPI-355). The pharmacokinetics and antitumor activity of SPI-355 were compared to those of nonliposomal GL147211C. The plasma pharmacokinetics of SPI-355 in rats were typical of those of other pegylated liposomal formulations, with significantly increased blood circulation time; the dose-corrected area under the curve and Cmax of SPI-355 (10 mg/kg) were 1250- and 35-fold higher, respectively, than those of nonliposomal GL14711C (8.72 mg/kg). The comparative antitumor activity of SPI-355 and nonliposomal GL1472211C was evaluated in nude mice implanted with HT29 colon carcinoma xenografts. SPI-355 was 20-fold more effective than GL147211C in inhibiting tumor growth (1 mg/kg SPI-355 and 20 mg/kg GL147211C) and produced durable complete remissions of tumors at well-tolerated dose levels that were >5-fold lower than the maximally tolerated dose of GL147211C, which induced no durable complete responses. Signs of toxicity were similar between the two drugs, but liposome encapsulation increased the toxicity of drug approximately 4-fold, with increased weight loss and several deaths with SPI-355 (5 mg/kg SPI-355 versus 20 mg/kg GL147211C). Despite the increased toxicity seen with SPI-355, the therapeutic index of the liposomal formulation was increased approximately 5-fold over that of nonliposomal GL147211C, suggesting that such a pegylated liposomal formulation could demonstrate increased therapeutic index in human patients.

Screening for and identification of novel agents directed at renal cell carcinoma.

We were interested in identifying novel agents for renal cell carcinoma (RCC) by screening for activities that model renal tumor biology. Searching for relative renal cell sensitivity and leukemia insensitivity among cytotoxicity profiles in the NCI Drug Screen database, we identified 16 potential agents with renal selectivity. We evaluated the agents in 10 RCC cell lines (of primary and metastatic origin) isolated from 5 patients. The 50% inhibitory concentrations (IC50) in these cell lines ranged from 0.019 +/- 0.013 to 11.4 +/- 0.55 microM and were comparable with values obtained with renal cell lines in the NCI Drug Screen panel. Because RCC are slowly growing tumors, we evaluated the compounds on rapidly (27% S phase) or slowly (6% S phase) growing cells. In contrast to doxorubicin, where cytotoxicity was restricted to rapidly proliferating cells, three compounds (NSC 280074, 281613, and 281817) were more cytotoxic in slowly proliferating cells. NSC 72151 and 268965 were equitoxic for both populations. NSC 94889, 638850, and 630938 were more cytotoxic in rapidly growing cells. In in vitro time exposure studies, four compounds, NSC 268965, 280074, 281613, and 281817, were maximally cytotoxic with as little as 3 h exposure time. From an analysis comparing the p53 genotype of the 60 cell lines of the National Cancer Institute (NCI) Drug Screen with the cytotoxicity profiles for the 16 putative renal compounds, 13 compounds were classified as likely to be indifferent to p53 status. We also developed a panel specificity detection method for the NCI Drug Screen database to evaluate the prevalence of renal sensitive compounds. Of the 16 studied compounds, 14 were among those identified as renal sensitive by the statistical analysis. Lastly, we found reduced tumor growth in mice with established renal human tumor xenografts after treatment with two of the renal active compounds. These studies describe compounds with potential renal activity that are candidates for preclinical development for renal cell carcinoma.

2-Substituted aminopyrido[2,3-d]pyrimidin-7(8H)-ones. structure-activity relationships against selected tyrosine kinases and in vitro and in vivo anticancer activity.

While engaged in therapeutic intervention against a number of proliferative diseases, we have discovered the 2-aminopyrido[2, 3-d]pyrimidin-7(8H)-ones as a novel class of potent, broadly active tyrosine kinase (TK) inhibitors. An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. From the lead structure 2, a series of analogues bearing variable substituents at the C-2 position and methyl or ethyl at N-8 was made. Compounds of this series were competitive with ATP and displayed submicromolar to low nanomolar potency against a panel of TKs, including receptor (platelet-derived growth factor, PDGFr; fibroblast growth factor, FGFr; epidermal growth factor, EGFr) and nonreceptor (c-Src) classes. One of the more thoroughly evaluated members was 63 with IC50 values of 0.079 microM (PDGFr), 0.043 microM (bFGFr), 0.044 microM (EGFr), and 0.009 microM (c-Src). In cellular studies, 63 inhibited PDGF-mediated receptor autophosphorylation in a number of cell lines at IC50 values of 0.026-0.002 microM and proliferation of two PDGF-dependent lines at 0.3 microM. It also caused inhibition of soft agar colony formation in three cell lines that overexpress the c-Src TK, with IC50 values of 0.33-1.8 microM. In in vivo studies against a panel of seven xenograft tumor models with known and/or inferred dependence on the EGFr, PDGFr, and c-Src TKs, compound 63 produced a tumor growth delay of 10.6 days against the relatively refractory SK-OV-3 ovarian xenograft and also displayed activity against the HT-29 tumor. In rat oral bioavailability studies, compound 63 plasma concentrations declined in a biexponential manner, and systemic plasma clearance was high relative to liver blood flow. Finally, in rat metabolism studies, HPLC chromatography identified two metabolites of 63, which were proved by mass spectrometry and synthesis to be the primary amine (58) and N-oxide (66). Because of the excellent potency of 63 against selected TKs, in vitro and in vivo studies are underway for this compound in additional tumor models dependent upon PDGFr, FGFr, and c-Src to assess its potential for advancement to clinical trials.

Enhanced melphalan cytotoxicity in human ovarian cancer in vitro and in tumor-bearing nude mice by buthionine sulfoximine depletion of glutathione.

The development of acquired resistance to alkylating agents frequently limits the effectiveness of chemotherapy in the treatment of ovarian cancer. While the resistance to alkylating agents is multifactorial, the association of drug resistance with elevations in glutathione (GSH) is of potential clinical relevance since there exist pharmacologic means to lower intracellular GSH levels. We have used in vitro and in vivo models of human ovarian cancer to demonstrate that selective inhibition of GSH synthesis with L-buthionine-S,R-sulfoximine (L-BSO) leads to a lowering of GSH levels and an increase in cytotoxicity of the alkylating agent melphalan. In the human ovarian cancer cell line NIH:OVCAR-3, derived from a patient clinically refractory to alkylating agents, L-BSO resulted in a 3.6-fold enhancement of melphalan cytotoxicity. This cell line was also adapted for intraperitoneal growth in athymic nude mice. In this in vivo model, in which the mice die of massive ascites and intraabdominal carcinomatosis, L-BSO given orally in drinking water for 5 days decreased GSH levels in the tumor cells by 96% compared to a 79 and 86% reduction in GSH levels in the bone marrow and gastrointestinal mucosa respectively. Lowering of GSH levels with BSO was not accompanied by an increase in lethality for melphalan in non-tumored nude mice. However, in tumor-bearing nude mice, a single melphalan (5 mg/kg) treatment following GSH depletion with L-BSO was markedly superior to treatment with melphalan alone, producing a 72% increase in median survival time. Furthermore, L-BSO treatment of human bone marrow cells prior to melphalan exposure had little effect on melphalan toxicity as assessed in a CFUc-GM assay. These results suggest that treatment with the GSH synthesis inhibitor BSO may preferentially enhance the cytotoxic effects of alkylating agents against human ovarian cancer and overcome acquired resistance.

The use of the serum protein factor XIIIB (F XIIIB) in parentage testing.

Three thousand thirty-eight white and 313 black paternity cases were phenotyped for genetic variants of the coagulation Factor XIIIB (F XIIIB). Gene frequencies obtained by testing with agarose gel isoelectric focusing clearly differentiated the white and black populations. The expected probability of exclusion (P) for white subjects was P = 0.19 and for black subjects P = 0.25. The observed rates of exclusions for F XIIIB in white and black subjects were close to expected values. The results indicate that F XIIIB is a valuable tool in parentage testing, and the procedures are reasonably easy and inexpensive.