Genetic information, genomic technologies, and the future of drug discovery.

The completion of the first draft of the human genome has provided an unprecedented opportunity to understand the genetic and molecular basis of disease. Parallel developments of new biological technologies, such as transcript profiling, allow scientists to examine almost any biological system in high molecular resolution. Contemporary drug discovery research is now focusing on the identification and validation of pharmaceutical targets in the molecular pathways/systems embedded in this information. Novel therapeutic interventions are being developed and evaluated as a result of this research which will be the basis of innovative pharmaceuticals of the future.

The effect of a selective estrogen receptor modulator on the progression of spontaneous autoimmune disease in MRL lpr/lpr mice.

The MRL lpr/lpr mouse strain is an animal model for the autoimmune disorder systemic lupus erythematosus (SLE). Pathologic changes in the mice include a severe proliferative glomerulonephritis, lymph node and spleen enlargement, increase in autoantibody titers, and shortened life spans. In the present investigation, female MRL lpr/lpr mice have been dosed po daily for 7 months with the selective estrogen receptor modulator (SERM) LY139478 (4 mg/kg) or 17alpha-ethinylestradiol (EE2, 1 mg/kg) and compared to vehicle control animals. The LY139478 group had an increase in survival (73% survival at 7 months, P = 0.02) but the EE2-treated animals did not (53% survival at 7 months, P = 0.4) when compared to the control group (32% survival at 7 months). Although there were no reductions in autoantibody levels as determined by anti-DNA antibody ELISA, histological analysis of kidney tissue indicated that both LY139478 and EE2 mitigated the progression of glomerular nephritis which was evident in the controls. In contrast, there were no significant differences in lymph node size although the LY139478 and EE2 groups retained a well-defined sinusoidal region. Finally, flow cytometric analysis documented that thymuses from animals treated for 7 months with LY139478 but not with EE2 contained predominantly CD4+/CD+ T cells consistent with a normal thymic phenotype observed in non-MRL lpr/lpr mouse strains. These studies demonstrate that SERMs may be potentially useful for the treatment of autoimmune disorders.

Reversal of P-glycoprotein-mediated multidrug resistance by a potent cyclopropyldibenzosuberane modulator, LY335979.

Overexpression of P-glycoprotein (Pgp) by tumors results in multidrug resistance (MDR) to structurally unrelated oncolytics. MDR cells may be sensitized to these oncolytics when treated with a Pgp modulator. The present study evaluates LY335979 as a modulator both in vitro and in vivo. LY335979 (0.1 microM) fully restored sensitivity to vinblastine, doxorubicin (Dox), etoposide, and Taxol in CEM/VLB100 cells. LY335979 modulated Dox cytotoxicity even when LY335979 (0.5 microM) was removed 24 h prior to the cytotoxicity assay. LY335979 blocked [3H]azidopine photoaffinity labeling of the M(r) approximately 170,000 Pgp in CEM/VLB100 plasma membranes and competitively inhibited equilibrium binding of [3H]vinblastine to Pgp (Ki of approximately 0.06 microM). Treatment of mice bearing P388/ADR murine leukemia cells with LY335979 in combination with Dox or etoposide gave a significant increase in life span with no apparent alteration of pharmacokinetics. LY335979 also enhanced the antitumor activity of Taxol in a MDR human non-small cell lung carcinoma nude mouse xenograft model. Thus, LY335979 is an extremely potent, efficacious modulator that apparently lacks pharmacokinetic interactions with coadministered anticancer drugs and is, therefore, an exciting new agent for clinical evaluation for reversal of Pgp-associated MDR.

Synthesis and biological evaluation of a new series of sterols as potential hypocholesterolemic agents.

A new series of sterols was synthesized and tested in a CHO cell-based LDL receptor/luciferase (LDLR/Luc) assay to investigate the capability of derepressing the transcription of LDL receptor promoter in the presence of 25-hydroxycholesterol. The effect of various substitutions on antagonizing the repressing effect mediated by 25-hydroxycholesterol was also studied in terms of regio- and stereochemistry, lipophilicity, steric bulk, and pi-electron density. Except 12, compounds active in the primary LDLR/Luc assay were not active in the secondary simian virus 40/luciferase (SV40/Luc) assay, demonstrating the specificity of their in vitro activity. Eight active compounds of various structural types were selected and screened in a [1-14C-acetate]cholesterol biosynthesis inhibition assay; none has shown any interference with the cholesterol biosynthesis in CHO cells. In hypercholesterolemic hamsters, generally, compounds that were active in vitro were active in vivo and vice versa, with the exception of three in vitro inactive compounds: 3 beta-ols 3a' and 3c' as well as 3-ketone 2a. Experimental results from the livers of hamsters revealed that the in vivo conversion of 3a' or 2a to 3a has in part contributed to the observed in vivo activity, and it is also anticipated that 3c' may similarly be converted to 3c in hamsters.

Inhibition of cholesteryl ester transfer protein in normocholesterolemic and hypercholesterolemic hamsters: effects on HDL subspecies, quantity, and apolipoprotein distribution.

The effects of cholesteryl ester transfer protein (CETP) inhibition on the serum lipoprotein profile in both normocholesterolemic and hypercholesterolemic hamsters has been determined following subcutaneous injection of 12.5 mg/kg of the CETP neutralizing monoclonal antibody, TP2. Inhibition of CETP activity was greater than 60% and resulted in a 30-40% increase in high density lipoprotein (HDL) in both normal and hypercholesterolemic animals. These HDL effects were observed 1 day post-injection, were maximal by 4 days, and returned to control values by 14 days. Inhibition of CETP activity resulted in a decrease in both low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol concomitant with HDL increase, and in hypercholesterolemic animals resulted in increased total serum cholesterol. In addition to the quantitative differences in LDL and HDL, there were significant increases in the size of the HDL, a shift to smaller LDL particles, and changes in apolipoprotein (apo) composition as evaluated by FPLC and Western blot analysis. Large apoA-I-poor and apoE-containing HDL became prevalent in hypercholesterolemic hamsters after CETP inhibition. In addition, the size of the CETP-containing HDL particles increased with inhibition of transfer activity. While these effects were apparent in normocholesterolemic animals, the changes in apolipoprotein distribution and HDL subspecies as detected on native gels were more significant in the hypercholesterolemic animals. The changes in the HDL profile and apolipoprotein distribution after CETP inhibition in hamsters were similar to those reported in CETP-deficient Japanese subjects, suggesting the utility of the hypercholesterolemic hamster as an in vivo model for the understanding of the lipoprotein changes associated with CETP inhibition.

Occlusive arterial thrombosis in cynomolgus monkeys with varying plasma concentrations of lipoprotein(a).

Lipoprotein(a) (Lp[a]) is a newly recognized risk factor for the development of coronary heart disease and stroke in human beings; however, the mechanisms by which Lp(a) increases the risk of coronary heart disease remain unclear. The purpose of this study was to examine the effects of Lp(a) on the occurrence of occlusive arterial thrombosis. Occlusive arterial thrombus formation was examined in 18 cynomolgus monkeys with high plasma Lp(a) concentrations (> 35 mg/dL, n = 6), intermediate Lp(a) concentrations (20-25 mg/dL, n = 6), and low Lp(a) concentrations (< 12 mg/dL, n = 6). A Goldblatt clamp was positioned around the left common carotid artery to produce a stenotic segment, and the artery was pinch-injured with needle holders. A 20-MHz Doppler velocity crystal, placed distal to the stenosis/injury site, was used to detect cyclic flow reductions (indicative of transient thrombosis) or permanent cessation of flow velocity (indicative of more stable occlusive thrombosis). All monkeys with high Lp(a) concentrations developed permanent cessation of flow, whereas only one of six arteries from low-Lp(a) monkeys developed permanent cessation of flow (p < 0.05). Arteries from monkeys with intermediate Lp(a) concentrations developed pronounced cyclic reductions of flow but did not progress to permanent cessation of flow. There were no differences in plasma von Willebrand factor activity among the three groups. Immunohistochemical analysis of the damaged arterial segments indicated incorporation of Lp(a) into the adventitia, media, and intima of arteries from monkeys with low and high plasma Lp(a) concentrations, as well as the presence of an occlusive thrombus in arteries that developed permanent cessation of flow.(ABSTRACT TRUNCATED AT 250 WORDS)

The development and characterization of Vinca alkaloid-resistant Caco-2 human colorectal cell lines expressing mdr-1.

Several novel cell lines with variable resistance to Vinca alkaloids have been derived from the Caco-2 human colorectal carcinoma cell line. Parental Caco-2 cells were found by PCR analysis and immunofluorescence studies to produce a low amount of the mdr-1 gene product (P-glycoprotein) that may well be clinically significant. These cells, which were initially highly sensitive to desacetylvinblastine sulfate (DAVLB sulfate) were selected, without mutagenesis, through continuous culture with increasing concentrations of DAVLB sulfate over a 335-day period. This selection resulted in cell lines that displayed an mdr (multiple-drug-resistance) cross-resistance profile that could be reversed with agents such as verapamil and vindoline. During the selection process the amount of mdr-1 mRNA present, the extent of gene amplification and the amount of gp170 expressed all correlated well with the level of drug resistance. However, this correlation does not hold in the absence of selective pressure for the more resistant cell lines where gene amplification and the amount of P-glycoprotein present remained constant while the level of drug resistance and the amount of mdr-1 mRNA present declined. These cell lines are potential models for studying mdr-I gene expression and drug resistance in human epithelial malignancies.

Long-term growth suppression of human glioma xenografts by chemoimmunoconjugates of 4-desacetylvinblastine-3-carboxyhydrazide and monoclonal antibody 9.2.27.

A conjugate of 4-desacetylvinblastine-3-carboxyhydrazide (DAVLBHY) and the glioma-reactive monoclonal antibody (mAb) 9.2.27 induced long-term suppression of tumor growth in athymic nude mice engrafted with U87MG human glioma cells. In vitro, DAVLBHY had the strongest antiproliferative activity (inhibitory concentration at which incorporation of [3H]thymidine is at 50% of untreated control is 2.0 x 10(-9) M) of seven cytotoxic drugs tested and so was chosen for conjugation to mAb 9.2.27, which reacts specifically with the core protein of chondroitin sulfate proteoglycans found in human glioblastomas. After conjugation of DAVLBHY to the carbohydrate residues of mAb 9.2.27 it retained its full binding capacity. For in vivo studies, DAVLBHY and several conjugate derivatives were evaluated by using two dosages of i.v. injections, each starting 2 days after s.c. tumor inoculation. The control tumors reached a volume of nearly 3000 mm3 within 30 days. Tumor growth was delayed by about 20 days with four i.v. injections of 0.5 mg/kg 9.2.27-DAVLBHY, which was slightly superior to the unconjugated drug. Moreover, 9.2.27-DAVLBHY produced a highly significant suppression of growth so that the average tumor volume was only 3% of that observed in untreated controls after 28 days. Four injections of this conjugate at a larger dose, 2.0 mg/kg, prevented recurrence of the tumors for 130 days in all animals tested, thus demonstrating a significant increase in the therapeutic index, since the unconjugated drug provided limited inhibition of tumor growth for only 40 days. The specificity of the antitumor effect was demonstrated in a comparison with the control conjugate, KS1/4-DAVLBHY, which despite partial tumor suppression had only a transient effect. The specific antitumor effect of 9.2.27-DAVLBHY was unexpected, since the target antigen is expressed at a relatively low density (40,000 sites/cell) on U87MG glioma cells.

The human immune response to KS1/4-desacetylvinblastine (LY256787) and KS1/4-desacetylvinblastine hydrazide (LY203728) in single and multiple dose clinical studies.

Monoclonal antibody (MoAb) conjugates have been used to treat a variety of malignancies. The majority of the MoAbs which have been used therapeutically are from murine sources. The infusion of these foreign proteins into humans can be expected to elicit anti-murine antibodies and may be one of the major limitations to the clinical use of murine MoAbs. In these studies, we report on the nature and specificity of the human anti-murine antibody (HAMA) response in patients given single and multiple infusions of the two Vinca alkaloid conjugates of the MoAb KS1/4, which recognizes tumor-associated antigens in a variety of adenocarcinomas. A HAMA response was induced in a majority of the patients receiving infusions of KS1/4 conjugates, regardless of the specific conjugate used or the number of infusions. The magnitude of the response did not appear to be dose related. Antibodies directed to the drug moieties of these conjugates, anti-Vinca alkaloids, were also induced in patients with HAMA responses. The magnitude of the anti-Vinca response paralleled that of the HAMA.

In vivo selection of human tumor cells resistant to monoclonal antibody-Vinca alkaloid immunoconjugates.

UCLA-P3 human lung adenocarcinoma cells were grown in nude mice and given repetitive treatments of a monoclonal antibody-Vinca alkaloid immunoconjugate. Although this therapy resulted in a greater than 4-fold reduction in mean tumor mass of the established tumors, some animals experienced a reinitiation of tumor growth after cessation of conjugate treatment. Two such animals were treated again with high doses of monoclonal antibody-Vinca but one of the tumors was no longer regressed by the drug conjugate. The tumor was excised, enzymatically dissociated, and grown in tissue culture. Cultured cells were reimplanted in nude mice and subjected to further therapy with a monoclonal antibody-Vinca conjugate. The resulting tumors were also refractory to the immunoconjugate therapy. This cycle was repeated for a total of three times and resulted in the serial in vivo selection of three conjugate resistant variants. The mechanism responsible for the in vivo resistance of human tumor cells to the monoclonal antibody-Vinca immunoconjugate is unknown but does not appear to involve antigen modulation, altered tumor cell growth rate, or an apparent decrease in tumor targeting in vivo. The resistance was also not accompanied by any detectable elevation in multidrug resistance 1 mRNA or P-glycoprotein expression. Significantly, the resistance pattern was observed only in vivo and was not maintained by cells grown in vitro.

Antitumor activity of the monoclonal antibody-Vinca alkaloid immunoconjugate LY203725 (KS1/4-4-desacetylvinblastine-3-carboxhydrazide) in a nude mouse model of human ovarian cancer.

The activity of the conjugate of monoclonal antibody KS1/4 with 4-desacetylvinblastine-3-carboxhydrazide (KS1/4-DAVLB-HY) was explored in the OVCAR-3 human ovarian xenograft tumor model. Multiple schedules of KS1/4-DAVLB-HY administration were employed, including a comparison of i.p. and i.v. routes of treatment. When inoculates of 6 x 10(7) OVCAR-3 cells were injected i.p. into female athymic nude mice, untreated control animals had a mean survival of 18-34 days, with the development of massive ascites and large intraabdominal tumors. Significant increases in survival were observed in KS1/4-DAVLB-HY conjugate-treated animals with all schedules utilized. Parallel therapy with equivalent doses of free DAVLB-HY or a non-antigen-binding immunoconjugate did not significantly increase the survival of the animals. These data demonstrate that the immunoconjugate KS1/4-DAVLB-HY significantly increases the survival of OVCAR-3 tumor-bearing mice and indicates that this immunoconjugate may be useful in the treatment of human ovarian cancer.

New antitumor monoclonal antibody-vinca conjugates LY203725 and related compounds: design, preparation, and representative in vivo activity.

A method has been developed to allow the direct coupling of the cytotoxic vinca alkaloid 4-desacetylvinblastine-3-carbohydrazide (DAVLB hydrazide) to a variety of murine monoclonal antibodies directed against human solid tumors. Periodate oxidation of carbohydrate residues on the antibodies, followed by reaction with DAVLB hydrazide in aqueous acid affords, in most cases, conjugates with conjugation ratios of 4-6 vincas per antibody in high yield without significantly impairing antigen binding or solubility. The outcome of the conjugation reaction is highly dependent on the concentration of, and time of exposure of the protein to, the oxidant. These conjugates exhibit potent antitumor activity in vivo against a number of human solid tumor-nude mouse xenografts, with efficacy and safety increased over unconjugated DAVLB hydrazide. This antitumor activity is also superior to that of similarly prepared but nontarget tumor binding antibody-DAVLB hydrazide conjugates. MoAb-DAVLB hydrazide conjugates release DAVLB hydrazide in solution in a temperature- and pH-dependent manner. Hydrolytic release of unmodified DAVLB hydrazide from tumor-localized MoAb-DAVLB hydrazide conjugates in vivo may be an important factor in their antitumor activity.

Biosynthesis and secretion of thrombospondin in human melanoma cells.

A polyclonal antisera specific for human platelet thrombospondin (TSP) has been utilized to study the biosynthesis and secretion of TSP in the M21 human melanoma cell line. Pulse-chase indirect immunoprecipitation analysis reveal that human melanoma cells rapidly synthesize and secrete this platelet alpha-granule associated glycoprotein. Topographical analysis of the melanoma cell surface by indirect immunofluorescence indicate that the TSP molecules have no obvious extracellular organization. The implications of thrombospondin synthesis in the metastatic process of melanoma are discussed.

Molecular cloning and characterization of a human adenocarcinoma/epithelial cell surface antigen complementary DNA.

A human adenocarcinoma-associated antigen (KSA) defined by the monoclonal antibody KS1/4 has become the focus of several site-directed strategies for tumor therapy. KSA, a 40,000 Da cell surface glycoprotein antigen, is found at a high density in all adenocarcinomas examined to date and in corresponding normal epithelial tissues. Here we describe the cloning and sequencing of overlapping complementary DNA clones which encode the entire KSA as expressed in UCLA-P3, a human lung adenocarcinoma cell line. We have deduced the 314-amino acid sequence and have compared it to the N-terminal amino acid sequence data of the affinity-purified antigen. The KSA is synthesized as a 314-residue-long preproprotein that is then processed to a 232-residue-long antigen. KSA appears to have a single transmembrane domain of 23 residues that separates the highly charged 26-residue cytoplasmic domain from the extracellular domain. The N-terminal region of the propeptide is rich in cysteines and contains three potential N-glycosylation sites. Computer-assisted analyses at both the DNA and protein levels have found no significant similarities of this protein to known sequences, but a GC-rich 5' terminus is evident. Northern blot analysis shows that transcription of KSA can be detected in RNA isolated from normal colon but not in RNA isolated from normal lung, prostate, or liver.

Isolation and characterization of the human adenocarcinoma-associated glycoprotein gp40.

The human adenocarcinoma-associated antigen gp40 is a cell surface glycoprotein recognized by murine monoclonal antibody KS1/4. A KS1/4-Sepharose affinity matrix was utilized to purify gp40 from detergent lysates of either tissue culture cells or nude mouse xenograft tumors of the human lung adenocarcinoma cell line P3-UCLA. This single immunoaffinity chromatography step yielded an antigen preparation of approximately 95% purity which was further characterized by immunochemical and enzymatic techniques. The gp40 molecule was shown to have both complex and high-mannose oligosaccharides comprising some 16% of the apparent molecular weight. The antigen preparation was suitable for gas-phase N-terminal amino acid sequencing and the first 16 residues of the N-terminus were determined. Despite considerable molecular heterogeneity, gp40 shows a single N-terminal sequence.

Characterization of the human tumor and normal tissue reactivity of the KS1/4 monoclonal antibody.

The tissue and tumor distribution of the antigen recognized by monoclonal antibody KS1/4 was determined by a combination of immunoperoxidase techniques, flow cytometric analyses and solid phase enzyme-linked immunoassays. These data document that the KS1/4 antigen is expressed in many epithelial malignancies and normal epithelial surfaces suggesting that this antigen represents an epithelial/epithelial malignancy marker.

Disposition of the monoclonal antibody-vinca alkaloid conjugate KS1/4-DAVLB (LY256787) and free 4-desacetylvinblastine in tumor-bearing nude mice.

The monoclonal antibody-vinca alkaloid conjugate, KS1/4-DAVLB (LY256787), and free 4-desacetylvinblastine (DAVLB) were administered i.v. to male athymic nude mice bearing P3/UCLA human lung adenocarcinoma tumors. Although the plasma pharmacokinetics were similar between LY256787 and DAVLB (terminal plasma half-lives of 62 and 83 hr, respectively), substantial differences in the volumes of distribution and initial redistribution-elimination phases were found. Uptake of LY256787 into tumor was apparent, with maximal radioequivalent concentrations measured 96 hr after dosing; no similar uptake was found after dosing with free DAVLB. The ratios of concentrations of drug radioequivalents in tumor to those in other tissues were generally greater than 1.0 when measured 24 to 48 hr after dosing with LY256787 but were less than 1.0 after free DAVLB. These data support the concept of site-specific delivery to the tumor tissue of the vinca alkaloid by the antibody. Plasma pharmacokinetics and tissue distribution were compared in males and females with a lower dose of LY256787. No sex-related differences in the plasma pharmacokinetics were found (terminal half-lives of 90 and 84 hr in males and females). Some sex-related biodistribution differences occurred. In all studies, the primary route of elimination was fecal. These studies suggest that the KS1/4 monoclonal antibody targets DAVLB to the P3/UCLA human lung adenocarcinoma in vivo in the human xenograft model and that an increased therapeutic index may be achieved with LY256787 over conventional free drug therapy.