Phase I trial of SU14813 in patients with advanced solid malignancies.

BACKGROUND: this phase I, open-label, dose-escalation study investigated SU14813, an oral multitargeted tyrosine kinase inhibitor, in adults with solid tumors. PATIENTS AND METHODS: seventy-seven patients received once-daily SU14813, either for 4 weeks followed by 1 week off treatment (schedule 4/1) or continuously [continuous daily dosing (CDD)]. The primary end point was to determine the maximum tolerated dose (MTD). Safety, pharmacokinetics, pharmacodynamics, and efficacy were assessed. RESULTS: MTDs were 200 mg/day on schedule 4/1 and 100 mg/day with CDD. Adverse events included fatigue (64%), diarrhea (61%), nausea (44%), anorexia (43%), and vomiting (42%). SU14813 steady state was attained by day 8. Exposure increased in a generally dose-proportional manner and SU14813 was eliminated with a mean terminal half-life of 9-34 h. Target plasma concentrations (>100 ng/ml SU14813) were achieved and sustained over 12 h at ≥ 100 mg/day. Progression-free survival among the 1 complete responder and 12 partial responders was 1.4-53.2 months. Fifteen patients remained on treatment at 1 year and 3 patients at 2 years. CONCLUSION: SU14813 has manageable safety and tolerability and allows once-daily continuous oral dosing. SU14813 shows dose-proportional pharmacokinetics, with target plasma concentrations achieved at doses ≥ 100 mg/day. Clinically meaningful activity with durable responses was observed, meriting further study.

In vitro modulation of cisplatin accumulation in human ovarian carcinoma cells by pharmacologic alteration of microtubules.

We have previously shown that forskolin and 3-isobutyl-1-methylxanthine (IBMX) increased accumulation of cisplatin (DDP) in DDP-sensitive 2008 human ovarian carcinoma cells in proportion to their ability to increase cAMP. Since the major function of cAMP is to activate protein kinase A, it was conjectured that the stimulation of DDP accumulation was mediated by a protein kinase A substrate. We now show that exposure of 2008 cells to forskolin resulted in phosphorylation of a prominent 52-kD membrane protein. Microsequencing of the band demonstrated it to be human beta-tubulin. Similarly, pretreatment of 2008 cells with the microtubule stabilizing drug taxol increased platinum accumulation in a dose-dependent manner. In 11-fold DDP-resistant 2008/C13*5.25 cells, decreased DDP accumulation was associated with enhanced spontaneous formation of microtubule bundles and decreased expression of beta-tubulin and the tubulin-associated p53 antioncogene relative to 2008 cells. 2008/C13*5.25 cells had altered sensitivity to tubulin-binding drugs, being hypersensitive to taxol and cross-resistant to colchicine. We conclude that pharmacologic alterations of tubulin enhance accumulation of DDP, and that the DDP-resistant phenotype in 2008/C13*5.25 cells is associated with tubulin abnormalities.

Modulation of drug sensitivity by dipyridamole in multidrug resistant tumor cells in vitro.

The concept of overcoming multidrug resistance using modulators is based on the hypothesis that there will be a synergistic interaction between the modulator and the cytotoxic agent. We examined the ability of dipyridamole (DPM) to synergistically enhance drug sensitivity in drug-sensitive KB-3-1 cells and their drug-resistant variants, KB-GRC1 and KBV1 cells, using median effect analysis to produce a quantitative measure of the extent of synergy. The drug-resistant variants were resistant to vinblastine (VBL), colchicine (COL), and etoposide (VP-16) in the order VBL greater than COL greater than VP-16 on the basis of 50% inhibitory concentration values obtained by clonogenic assay with continuous drug exposure. The extent of staining with the monoclonal antibody HYB-241, directed at a Mr 180,000 form of the mdrI gene product, correlated with drug resistance for all three drugs (r greater than or equal to 0.92). DPM and verapamil elevated the steady state content (Css) of VBL, but there was no correlation between elevation of Css and the extent of synergy observed. DPM enhanced the cytotoxicity of VBL and COL in a synergistic manner in KB-GRC1 cells, and in KBV1 cells DPM interacted synergistically with VBL. VPL was synergistic with VBL only in KB-GRC1 cells. No synergy was observed in the parental KB-3-1 line. These data indicate that, although both DPM and verapamil can increase Css in cells not expressing P-glycoprotein, such an increase was not associated with synergy. In cells expressing mdrl, synergy was observed, and it was greatest for the cytotoxic agent for which expression of mdrl produced the greatest fold-resistance and enhancement of Css. However, neither the level of resistance, the level of expression of mdrl, nor the ability of the modulator to alter Css accurately predicted whether the interaction would be truly synergistic. We conclude that additional factors determine the nature of the drug interaction.

Modulation of prostaglandin biosynthesis in hypoxic murine mammary adenocarcinoma cells by misonidazole.

Resistance of hypoxic cells to radiation and chemotherapy remains a major limitation to effective therapy of solid tumors. Misonidazole, a 2-nitroimidazole analogue, has been studied extensively as a radiosensitizer of hypoxic cells and has been shown to undergo bioreductive metabolism to exert preferential cytotoxicity against hypoxic cells. We have investigated the effects of misonidazole on the biosynthesis of prostaglandins (PGs) in a murine mammary adenocarcinoma cell line (No. 4526) under aerobic and hypoxic conditions in attempts to exploit modulation of PG levels under hypoxia as a means of improving therapeutic approaches for the treatment of solid tumors. We report a time-dependent inhibition of PG biosynthesis by the suspended cells under hypoxia induced by flushing sealed vials with N2 (1.5 liters/min). After 30 min of hypoxia, PG formation was inhibited by 50%. Indomethacin was able to further inhibit the PG formation in a concentration-dependent manner under hypoxia. Misonidazole, however, selectively increased the PGE2 biosynthesis under hypoxia by 49% at 100 microM. This increase was concentration dependent over the range of 25 to 100 microM and was blocked by indomethacin (0.1 microM). Imidazole, the heterocyclic moiety in misonidazole without the nitro function, had no effect on PG biosynthesis at these concentrations. These data suggest that arachidonic acid metabolism is sensitive to the differential oxygen levels which exist within solid tumors and that PG levels may be modulated by electron-affinic agents in hypoxic tumor cells.

A novel retinoic acid receptor-selective retinoid, ALRT1550, has potent antitumor activity against human oral squamous carcinoma xenografts in nude mice.

We have identified a novel retinoid, ALRT1550, that potently and selectively activates retinoic acid receptors (RARs). ALRT1550 binds RARs with Kd values of approximately equal to 1-4 nM, and retinoid X receptors with low affinities (Kd approximately equal to 270-556 nM). We studied the effects of ALRT1550 on cellular proliferation in squamous carcinoma cells. ALRT1550 inhibited in vitro proliferation of UMSCC-22B cells in a concentration-dependent manner with an IC50 value of 0.22 +/- 0.1 (SE) nM. 9-cis-Retinoic acid (ALRT1057), a pan agonist retinoid that activates RARs and retinoid X receptors, inhibited proliferation with an IC50 value of 81 +/- 29 nM. In vivo, as tumor xenografts in nude mice, UMSCC-22B formed well-differentiated squamous carcinomas, and oral administration (daily, 5 days/week) of ALRT1550, begun 3 days after implanting tumor cells, inhibited tumor growth by up to 89% in a dose-dependent manner over the range of 3-75 micrograms/kg. ALRT1550 (30 micrograms/kg) also inhibited growth of established tumors by 72 +/- 3% when tumors were allowed to grow to approximately equal to 100 mm3 before dosing began. In comparison, 9-cis retinoic acid at 30 mg/kg inhibited growth of established tumors by 73 +/- 5%. Interestingly, retinoids did not appear to alter tumor morphologies in UMSCC-22B tumors. Notably, ALRT1550 produced a therapeutic index of approximately equal to 17 in this model, indicating a separation between doses that inhibited tumor growth and that induced symptoms of hypervitaminosis A. In summary, ALRT1550 potently inhibits cellular proliferation in vitro and in vivo in this squamous cell carcinoma tumor model. These data support additional study of ALRT1550 for its potential for improving anticancer therapy in human clinical trials.

Retinoid-induced suppression of squamous cell differentiation in human oral squamous cell carcinoma xenografts (line 1483) in athymic nude mice.

Retinoids are promising agents for therapy of squamous cancers. In vitro, retinoids decrease expression of differentiation markers in head and neck squamous carcinoma cells. Little information is available on effects of retinoids on head and neck squamous carcinoma cell xenograft growth in vivo. To address this issue, head and neck squamous carcinoma cells (line 1483) were established as xenografts in nude mice. Control tumors grew rapidly with doubling times of 4-6 days to mean volumes of 1696 mm3 after 24 days. Histological analyses indicated the formation of well-differentiated squamous carcinoma cells exhibiting pronounced stratification (basal and suprabasal cells) and keratinization (keratin pearls) with abundant stroma. Cytokeratin 19 expression was restricted to the basal cell layers, and cytokeratin 4 expression was abundant in suprabasal cells. Mice were treated daily with 30 mg/kg 9-cis retinoic acid, 20 mg/kg all-trans-retinoic acid, or 60 mg/kg 13-cis retinoic acid by p.o. gavage on a schedule of 5 days/week over 4 weeks. Low micromolar (1.48-3.67 microM) and nanomolar (200-490 nM) concentrations of 9-cis retinoic acid and all-trans-retinoic acid were measured in plasmas and xenografts, respectively, 30 min after dosing. Retinoid treatment produced a marked suppression of the squamous cell differentiation of tumor cells manifest by decreased keratinization, loss of stratification, and accumulation of basal cells. This was accompanied by large decreases in the number of CK4-positive cells and concomitant increases of CK19-positive cells. REtinoic acid receptor-beta expression was also increased by 2.9-9.7-fold after chronic retinoid treatment. 9-cis retinoic acid and all-trans-retinoic acid decreased tumor volumes by 23 +/- 5 (SE) and 19 +/- 3%, respectively (P < or = 0.05); 13-cis retinoic acid was inactive. These retinoids did not decrease the rate of exponential tumor growth but increased the latent period until exponential growth began. These studies demonstrate that retinoids do not universally decrease tumor growth but profoundly suppress squamous cell differentiation in vivo in this xenograft model.

Phase I/pharmacokinetic study of high-dose progesterone and doxorubicin.

PURPOSE: We developed a new formulation of progesterone that permits administration of up to 10 g of progesterone as a continuous intravenous infusion over 24 hours and conducted a phase I clinical trial to determine whether progesterone could modulate the in vivo cytotoxicity of the P-glycoprotein substrate doxorubicin. PATIENTS AND METHODS: Thirty-four patients with advanced malignancies were treated with increasing doses of progesterone and a fixed dose of 60 mg/m2 of doxorubicin given as an intravenous bolus 2 hours after starting a 24-hour intravenous infusion of progesterone. RESULTS: Progesterone enhanced doxorubicin-induced myelotoxicity in a dose-dependent fashion without altering the pharmacokinetics of doxorubicin. The steady-state plasma concentration of progesterone at a dose level of 4 g was 4.1 +/- 0.9 mumol/L, which was higher than the minimal concentration required to reverse multidrug resistance (MDR) in vitro. CONCLUSION: Progesterone enhanced the hematologic toxicity of doxorubicin without altering its pharmacokinetics, suggesting that progesterone could modulate P-glycoprotein at the level of pluripotent hematopoietic stem cells. Adequate tissue concentrations of progesterone could be achieved in vivo to modulate doxorubicin toxicity in the bone marrow and thus potentially in tumor tissue as well. Selectivity may potentially be gained by using hematopoietic growth factors to offset the enhanced hematologic toxicity of doxorubicin while leaving the enhancement of toxicity to tumor cells unchanged.

Marked antiangiogenic and antitumor efficacy of AG3340 in chemoresistant human non-small cell lung cancer tumors: single agent and combination chemotherapy studies.

Effective therapy is needed to improve the survival of patients with advanced lung cancers. We studied the effects of a selective metalloprotease inhibitor, AG3340, on chemoresistant human non-small cell lung cancer tumors (line MV522) in vivo. Mice bearing s.c. tumors were given twice-daily oral doses of AG3340. As a single agent, AG3340 inhibited angiogenesis (up to 77%) and tumor growth (up to 65%) in a dose-dependent manner at well-tolerated daily doses up to 400 mg/kg/day and induced significant tumor necrosis. In contrast, tumors were relatively insensitive to carboplatin with approximately 25% growth inhibition observed at a maximum tolerated dose of approximately 30 mg/kg/week (given i.p., twice weekly). Carboplatin inhibited tumor growth markedly only at toxic doses, demonstrating a superior therapeutic index of AG3340 to carboplatin in this tumor model. A suboptimal dose of AG3340, when used in combination with an ineffective maximum tolerated dose of carboplatin, resulted in greater tumor growth inhibitions than those produced by either agent alone. Similarly, growth inhibition was enhanced when AG3340 was used in combination with paclitaxel. Cotreatment with carboplatin did not alter AG3340 plasma concentrations achieved acutely after oral dosing. These data demonstrate an antiangiogenic and antitumor effect of AG3340 when used as a single agent and enhanced growth inhibitions when AG3340 is used in combination with cytotoxic agents. These data suggest that treatment with this novel matrix metalloprotease inhibitor may be beneficial in advanced lung cancers and other chemoresistant malignancies.

Enhanced antitumor efficacy of cisplatin in combination with ALRT1057 (9-cis retinoic acid) in human oral squamous carcinoma xenografts in nude mice.

Cisplatin (DDP) is commonly used to treat head and neck tumors. Therapy frequently fails due to development of DDP resistance or toxicities associated with DDP therapy. In this study, effects of ALRT1057 [9-cis retinoic acid (9-cis RA)] on DDP cytotoxicity were studied in a human oral squamous carcinoma xenograft model. Mice bearing xenografts were dosed p.o. daily 5 days/week with 30 mg/kg 9-cis RA and/or i.p. twice weekly with 0.3-0.9 mg/kg DDP. Maximum tolerated doses of 9-cis RA and DDP were approximately 60 and >/=2.9 mg/kg, respectively, under their dosing schedules and routes of administration. Control tumors grew rapidly with mean doubling times of 4 +/- 1 days and reached mean volumes of 1982 +/- 199 (SE) mm3 after 24 days. DDP at doses of 0.3, 0.45, and 0.9 mg/kg inhibited tumor growth by 28, 47, and 86%, respectively, 24 days after tumor cell implantation. Thirty mg/kg 9-cis RA inhibited tumor growth by 25%. In combination, 0.3 mg/kg DDP + 30 mg/kg 9-cis RA inhibited tumor growth by 68%; 0.45 mg/kg DDP + 30 mg/kg 9-cis RA inhibited growth by 78%. These decreases were greater than those that would have been produced by either agent summed separately. Of importance, at doses of 9-cis RA that enhanced DDP cytotoxicity, no change in dose tolerance was observed as compared to tolerances observed for either agent alone, indicating that 9-cis RA increased sensitivity to DDP without altering systemic toxicity. In addition, 9-cis RA profoundly altered squamous cell carcinoma phenotypes by suppressing squamous cell differentiation, resulting in tumors with increased numbers of basal cells. In contrast, DDP selectively depleted proliferating basal cells from carcinomas. In combination, morphological changes produced by 9-cis RA alone predominated, suggesting a possible basis for enhanced DDP sensitivity in tumors exposed to both agents. These data demonstrate that 9-cis RA enhances tumor sensitivity to DDP, and suggest that this combination should be tested in Phase I-II clinical trials for its potential for improving anticancer therapy of squamous cell cancers.

Enhancement of the loss of multiple drug resistance by hydroxyurea.

Gene amplification is one mechanism whereby tumor cells can become resistant to antineoplastic agents. Unstably amplified genes occur either on submicroscopic circular pieces of extrachromosomal DNA called episomes or on small acentric chromosomes called double minutes. Double minutes are frequently associated with cells containing amplified drug-resistance genes. Human epidermoid carcinoma KBV1 cells contain unstably amplified mdr1 genes and overexpress P glycoprotein, resulting in decreased intracellular drug accumulation. In this cell line, a nonlethal concentration of hydroxyurea accelerated the rate of loss of vinblastine resistance once vinblastine had been removed from the culture medium. After removal of vinblastine, KBV1 cells exposed to hydroxyurea for the time required to complete 12 cell doublings accumulated more vinblastine than control cells grown in the absence of hydroxyurea. In contrast, hydroxyurea had no effect on vinblastine sensitivity and accumulation in parental drug-sensitive KB-3-1 cells. Hydroxyurea also had no effect on sensitivity to cisplatin in cisplatin-resistant human ovarian carcinoma 2008/C13* cells, indicating that hydroxyurea's effect on drug sensitivity was specific for a drug-resistance phenotype associated with unstably amplified drug-resistance genes. These results indicate that, after removing selective pressure, hydroxyurea accelerates loss of resistance to vinblastine and increases accumulation of vinblastine in KBV1 cells, presumably by accelerating loss of amplified mdr1 genes and thus P glycoprotein.

Modulation of cisplatin cytotoxicity by permeabilization of the plasma membrane by digitonin in vitro.

Killing of human ovarian carcinoma 2008 cells by cisplatin (DDP) is in direct proportion to the amount of drug entering the cell. DDP and its analogue [3H]dichloro(ethylenediamine)platinum[II] ([3H]-DEP) enter cells relatively slowly. We found that the uptake of [3H]DEP into 2008 cells could be increased by treating the cells briefly with the plasma membrane-selective detergent digitonin. A similar effect was observed in an 11-fold DDP-resistant subline of 2008 cells, designated 2008/C13*5.25. A measurable effect was produced by concentrations as low as 5 microM, and 40 microM digitonin increased [3H]DEP accumulation at 1 hr by 4.4 +/- 0.2- and 6.5 +/- 0.7-fold (means +/- SD) in 2008 and 2008/C13*5.25 cells, respectively. The effect was rapid, occurring within 1 min. Increased [3H]DEP uptake was accompanied by increased platination of DNA (8.5-fold in 2008 cells and 18.5-fold in 2008/C13*5.25 cells), and by enhanced killing of both the DDP-sensitive and -resistant cells that was shown to be synergistic by median effect analysis. The combination index at 50% cell kill was 0.64 +/- 0.14 (values < 1 indicate synergy). We conclude that a brief exposure to digitonin can increase [3H]DEP uptake in vitro, and can overcome the impaired [3H]DEP accumulation associated with acquired DDP resistance. DDP and digitonin interact synergistically to increase tumor cell kill in vitro.

Broad antitumor and antiangiogenic activities of AG3340, a potent and selective MMP inhibitor undergoing advanced oncology clinical trials.

We studied AG3340, a potent metalloproteinase (MMP) inhibitor with pM affinities for inhibiting gelatinases (MMP-2 and -9), MT-MMP-1 (MMP-14), and collagenase-3 (MMP-13) in many tumor models. AG3340 produced dose-dependent pharmacokinetics and was well tolerated after intraperitoneal (i.p.) and oral dosing in mice. Across human tumor models, AG3340 produced profound tumor growth delays when dosing began early or late after tumor implantation, although all established tumor types did not respond to AG3340. A dose-response relationship was explored in three models: COLO-320DM colon, MV522 lung, and MDA-MB-435 breast. Dose-dependent inhibitions of tumor growth (over 12.5-200 mg/kg given twice daily, b.i.d.) were observed in the colon and lung models; and in a third (breast), maximal inhibitions were produced by the lowest dose of AG3340 (50 mg/kg, b.i.d.) that was tested. In another model, AG3340 (100 mg/kg, once daily, i.p.) markedly inhibited U87 glioma growth and increased animal survival. AG3340 also inhibited tumor growth and increased the survival of nude mice bearing androgen-independent PC-3 prostatic tumors. In a sixth model, KKLS gastric, AG3340 did not inhibit tumor growth but potentiated the efficacy of Taxol. Importantly, AG3340 markedly decreased tumor angiogenesis (as assessed by CD-31 staining) and cell proliferation (as assessed by bromodeoxyuridine incorporation), and increased tumor necrosis and apoptosis (as assessed by hematoxylin and eosin and TUNEL staining). These effects were model dependent, but angiogenesis was commonly inhibited. AG3340 had a superior therapeutic index to the cytotoxic agents, carboplatin and Taxol, in the MV522 lung cancer model. In combination, AG3340 enhanced the efficacy of these cytotoxic agents without altering drug tolerance. Additionally, AG3340 decreased the number of murine melanoma (B16-F10) lesions arising in the lung in an intravenous metastasis model when given in combination with carboplatin or Taxol. These studies directly support the use of AG3340 in front-line combination chemotherapy in ongoing clinical trials in patients with advanced malignancies of the lung and prostate.

Regional lymphatic drug exposure following intraperitoneal administration of 5-fluorouracil, carboplatin, and etoposide.

Intraperitoneal (i.p.) administration of chemoterapeutic agents results in greater total drug exposures in the peritoneal cavity than in plasma. A study on the drug exposure for i.p. lymphatics of pigs, receiving 5-fluorouracil (5-FU), etoposide (VP-16) and carboplatin (CBDCA) by the i.p. route was conducted. Drug concentrations in peritoneal fluid, plasma, and thoracic duct lymph were monitored over the ensuing 3 h. 5-FU appeared rapidly in thoracic duct, lymph and plasma. The lymph concentration declined after 20 min while the plasma concentration remained stable. CBDCA reached a stable concentration in lymph and plasma after 60 min. VP-16 peaked in the lymph after 20 min, whereas the plasma concentration continued to rise for 150 min; the peritoneal half-life for VP-16 was too long for clearance to be defined. Total drug exposure (AUC) was for 5-FU 5.7-fold greater for lymph than for plasma and for CBDCA equal in both compartments. VP-16 had a 2.1-fold higher AUC for lymph than for plasma. The results indicate that the i.p. route of administration results in a greater exposure of the lower thoracic duct lymph than the plasma to 5-FU, produces only a marginally increased exposure to VP-16, and results in no difference for CBDCA. The efficacy of 5-FU is a function of total drug exposure. The results reported provide a strong rationale for evaluating the adjuvant use of i.p. 5-FU in colorectal and gastric carcinoma.

Inhibition of GSH-dependent PGH2 isomerase in mammary adenocarcinoma cells by allicin.

We have reported that allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI2 synthase. The effect of allicin on glutathione (GSH) dependent PGH2 to PGE2 isomerase is unknown. We therefore studied the effect of allicin on PGE2 biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either 14C-arachidonic acid (AA) or 14C-PGH2, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE2 was predominantly formed with minimal amounts of PGF2 alpha and PGD2. Formation of 6-keto-PGF1 alpha or TXB2 was not detected indicating the absence of TXA2 and PGI2 synthase activity. Indomethacin and ibuprofen inhibited the PGE2 formation (p less than 0.05). The enzymatic PGE2 formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10-1000 microM, inhibited the formation of PGE2 in cells exposed to 2.0 microM 14C-AA for 20 min. and in sonicated cells incubated with 20.0 microM 14C-PGH2 for 2 min (p less than 0.05). Allicin did not alter cyclooxygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH2 to PGE2 isomerase in this adenocarcinoma cell line.

Modulation of vinblastine sensitivity by dipyridamole in multidrug resistant fibrosarcoma cells lacking mdr1 expression.

We examined the ability of dipyridamole (DPM) to act synergistically with vinblastine (VBL) in HT1080 fibrosarcoma cells and a drug-resistant variant, HT1080/DR4, which lacks mdr1 expression, in order to determine whether DPM requires P-glycoprotein to modulate drug sensitivity. Median effect analysis of clonogenic assay was used to produce the combination index (CI50, values less than 1 indicate synergy). DPM was mildly synergistic with VBL producing a CI50 of 0.83 +/- 0.13 for HT1080 cells and 0.80 +/- 0.16 for HT1080/DR4 cells. HT1080 and HT1080/DR4 cells accumulated 6.7 +/- 0.7 and 5.6 +/- 0.9 pmol 3H-VBL mg cells-1 at steady state (Css) and 20 microM DPM elevated the Css by 1.8 and 2.9-fold, respectively. In comparison, the CI50 was 1.1 +/- 0.2 in parental KB-3-1 cells and 0.1 +/- 0.1 in mdr1-expressing KB-GRC1 cells. The KB-3-1 and KB-GRC1 cells had a Css of 3.8 +/- 0.8 and 0.7 +/- 0.2 pmol 3H-VBL mg cells-1, respectively, and DPM elevated the Css by 9.2-fold in KB-GRC1 cells. These studies demonstrate that DPM can produce synergy independently of mdr1 expression but that much greater levels of synergy are achievable in mdr1-expressing tumour cells.

The efficacy of 9-cis retinoic acid in experimental models of cancer.

9-cis retinoic acid (9-cis RA) is a retinoid receptor pan-agonist that binds with high affinity to both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Using a variety of in vivo and in vitro cancer models, we present experimental data that 9-cis RA has activity as a potential chemotherapeutic agent. Treatment of the human promyelocytic leukemia cell line HL-60 with 9-cis RA decreases cell proliferation, increases cell differentiation, and increases apoptosis. Induction of apoptosis correlates with an increase in tissue transglutaminase (type II) activity. In vivo, 9-cis RA induces complete tumor regression of an early passage human lip squamous cell carcinoma xenograft. Finally, 9-cis RA inhibits the anchorage-independent growth of the human breast cancer cell lines MCF-7 and LY2 (an antiestrogen-resistant MCF-7 variant). Transient co-transfection assays indicate that 9-cis RA inhibits estrogen receptor transcription of an ERE-tk-LUC reporter through RAR or RXR receptors. These data suggest that retinoid receptors can antagonize estrogen-dependent transcription and provides one possible mechanism for the inhibition of cell growth by 9-cis RA in breast cancer cell lines. In summary, these findings present evidence that 9-cis RA has a wide range of activities in human cancer models.

Synergistic interaction between cisplatin and taxol in human ovarian carcinoma cells in vitro.

Taxol, a unique tubulin active agent, was found to demonstrate a marked schedule-dependent synergistic interaction with cisplatin (DDP) in the killing of human ovarian carcinoma 2008 cells in vitro as determined by median effect analysis. The interaction was highly synergistic when 19 h taxol exposure was followed by 1 h concurrent exposure to taxol and DDP. The combination indices (CIs) on this schedule were 0.11 +/- 0.1, 0.25 +/- 0.15 and 0.39 +/- 0.14 at 20%, 50% and 80% cell kill respectively. However, the interaction was antagonistic when 1 h exposure to DDP was followed by 20 h exposure to taxol, or when cells were exposed to DDP and taxol for 1 h concurrently. When taxol preceded DDP, synergy was also observed with the 11-fold DDP-resistant 2008/C13*5.25 subline, which yielded CI values of 0.21 +/- 0.02, 0.30 +/- 0.11 and 0.31 +/- 0.17 at 20%, 50% and 80% cell kill respectively. At an IC50 concentration, taxol had no effect on [3H]cis-dichloro(ethylenediamine) platinum uptake, on the permeability of the plasma membrane or on glutathione or metallothionein levels in 2008 or 2008/C13*5.25 cells. Mitotic arrest in these cells was observed only at taxol concentrations well above those required for synergy with DDP, suggesting that the mechanism underlying the synergistic interaction was not a taxol-induced alteration in cell cycle kinetics. Of additional interest was the fact that the 2008/C13*5.25 cells were hypersensitive to taxol, and that this was partially explained by an alteration in the biochemical pharmacology of taxol. Although cellular taxol accumulation reached steady state within 2 h in both cell lines, taxol efflux was slower and the taxol was more extensively bound in 2008/C13*5.25 cells than in 2008 cells. In addition, the 2008/C13*5.25 cells had only 55% of the parental levels of beta-tubulin content. However, in another pair of DDP-sensitive and -resistant ovarian cell lines no taxol hypersensitivity and no change in beta-tubulin content was observed, indicating that the DDP-resistant and taxol-hypersensitive phenotypes do not segregate together. We conclude that taxol interacts synergistically with DDP in a manner that is highly schedule dependent, and that the hypersensitivity of 2008/C13*5.25 cells no taxol is unrelated to the mechanism of synergy. These in vitro observations suggest that drug schedule will be an important determinant of the activity and toxicity of the DDP and taxol drug combination in clinical studies.

Selective modulation of vinblastine sensitivity by 1,9-dideoxyforskolin and related diterpenes in multidrug resistant tumour cells.

The ability of 1,9-dideoxyforskolin (DDF), 1-deoxyforskolin (DF) and forskolin to modulate cellular sensitivity to vinblastine (VBL) was examined in drug-sensitive parental KB-3-1 cells and a multidrug-resistant subline, KB-GRC1, derived by transfection of mdr1. Fifty microM DF and forskolin enhanced the 1 h uptake of VBL by 8.0 +/- 0.7 (s.d.) and 4.7 +/- 2.5-fold, respectively, with 50 microM DDF producing a 13.6 +/- 1.9-fold increase. The greater effect of DDF relative to forskolin indicated that the effect was independent of activation of cAMP, and this was supported by a lack of effect of dibutyryl cAMP on the uptake. The effect of these agents on uptake were < or = 1.4-fold in KB-3-1 cells. DDF selectively inhibited initial efflux in cells expressing a functional P-glycoprotein (PGP), but both forskolin and DDF inhibited the terminal phase of efflux irrespective of PGP expression. Neither agent affected membrane permeability of polarisation and forskolin did not enhance the uptake of VBL in protein-free liposomes. At a non-toxic concentration of 20 microM, DDF and forskolin decreased the IC50 of VBL from 18.9 to 2.7 and 13 nM in KB-GRC1 cells, respectively, and DDF acted synergistically with VBL as shown by median effect analysis [combination index = 0.20 +/- 0.05 (s.d.)]. In contrast, these diterpenes did not affect VBL sensitivity in KB-3-1 cells. These results indicate that the diterpenes modulate VBL sensitivity predominantly by inhibiting PGP-mediated efflux activity.

Antitumor efficacy of AG3340 associated with maintenance of minimum effective plasma concentrations and not total daily dose, exposure or peak plasma concentrations.

Oral administration of AG3340, a novel metalloprotease (MMP) inhibitor, suppresses the growth of human colon adenocarcinoma (COLO-320DM) tumors in vivo (Proc Am Assoc Cancer Res 39: 2059, 1998). In this report, we tested the hypothesis that the growth inhibition of these tumors is associated with maintaining minimum effective plasma concentrations of AG3340. Nude mice were given a total oral daily dose of 25 or 200 mg/kg; 6.25 mg/kg was given four times per day (QID) (25 mg/kg/day), and 100 mg/kg was given in two daily doses (BID) (200 mg/kg/day). Peak plasma concentrations (Cmax) of 83 +/- 43 (mean +/- SD) and 1998 +/- 642 ng/ml were detected 30 min after a single dose with 6.25 mg/kg and 100 mg/kg AG3340, respectively. AUC(0-24 h) values estimated from dosing with 25 and 200 mg/kg/day AG3340 were 672 and 10882 ng*h/ml, respectively. Importantly, both regimen inhibited tumor growth equivalently (74 to 82%). Efficacy was also compared at a total daily dose of 25 mg/kg by giving AG3340: QID (6.25 mg/kg per dose), BID (12.5 mg/kg per dose), and once daily (25 mg/kg per dose). The Cmax of these regimens was 83 +/- 43, 287 +/- 175 and 462 +/- 495 ng/ml, respectively. AG3340 did not inhibit tumor growth with the latter two regimens. The efficacy of 6.25 mg/kg QID (25 mg/kg/day) was superior to the efficacy of 25 mg/kg BID (50 mg/kg/day), substantiating the independence of efficacy from the total daily dose and Cmax. Expectedly, peak to trough fluctuations were significantly smaller with the QID regimen than with BID and QD dosing. After 24 h, the trough was greater than 1 ng/ml with QID dosing but was less than 1 ng/ml after QD and BID dosing. These results suggest that the antitumor efficacy of AG3340 was associated with maintaining minimum effective plasma concentrations of AG3340 and demonstrate that the antitumor efficacy of AG3340 was independent of the total daily dose, peak plasma concentration, and drug exposure in this tumor model.

Ventilator-induced lung injury upregulates and activates gelatinases and EMMPRIN: attenuation by the synthetic matrix metalloproteinase inhibitor, Prinomastat (AG3340).

Mechanical ventilation has become an indispensable therapeutic modality for patients with respiratory failure. However, a serious potential complication of MV is the newly recognized ventilator-induced acute lung injury. There is strong evidence suggesting that matrix metalloproteinases (MMPs) play an important role in the development of acute lung injury. Another factor to be considered is extracellular matrix metalloproteinase inducer (EMMPRIN). EMMPRIN is responsible for inducing fibroblasts to produce/secrete MMPs. In this report we sought to determine: (1) the role played by MMPs and EMMPRIN in the development of ventilator-induced lung injury (VILI) in an in vivo rat model of high volume ventilation; and (2) whether the synthetic MMP inhibitor Prinomastat (AG3340) could prevent this type of lung injury. We have demonstrated that high volume ventilation caused acute lung injury. This was accompanied by an upregulation of gelatinase A, gelatinase B, MT1-MMP, and EMMPRIN mRNA demonstrated by in situ hybridization. Pretreatment with the MMP inhibitor Prinomastat attenuated the lung injury caused by high volume ventilation. Our results suggest that MMPs play an important role in the development of VILI in rat lungs and that the MMP-inhibitor Prinomastat is effective in attenuating this type of lung injury.