Indazole Carboxylic Acid Research Articles

The lonidamine derivative H2-gamendazole reduces cyst formation in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating renal neoplastic disorder with limited treatment options. It is characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. We tested the effectiveness of the indazole carboxylic acid H2-gamendazole (H2-GMZ), a derivative of lonidamine, to inhibit these processes using in vitro and in vivo models of ADPKD. H2-GMZ was effective in rapidly blocking forskolin-induced, Cl--mediated short-circuit currents in human ADPKD cells, and it significantly inhibited both cAMP- and epidermal growth factor-induced proliferation of ADPKD cells. Western blot analysis of H2-GMZ-treated ADPKD cells showed decreased phosphorylated ERK and decreased hyperphosphorylated retinoblastoma levels. H2-GMZ treatment also decreased ErbB2, Akt, and cyclin-dependent kinase 4, consistent with inhibition of heat shock protein 90, and it decreased levels of the cystic fibrosis transmembrane conductance regulator Cl- channel protein. H2-GMZ-treated ADPKD cultures contained a higher proportion of smaller cells with fewer and smaller lamellipodia and decreased cytoplasmic actin staining, and they were unable to accomplish wound closure even at low H2-GMZ concentrations, consistent with an alteration in the actin cytoskeleton and decreased cell motility. Experiments using mouse metanephric organ cultures showed that H2-GMZ inhibited cAMP-stimulated cyst growth and enlargement. In vivo, H2-GMZ was effective in slowing postnatal cyst formation and kidney enlargement in the Pkd1flox/flox: Pkhd1-Cre mouse model. Thus, H2-GMZ treatment decreases Cl- secretion, cell proliferation, cell motility, and cyst growth. These properties, along with its reported low toxicity, suggest that H2-GMZ might be an attractive candidate for treatment of ADPKD.NEW & NOTEWORTHY Autosomal dominant polycystic kidney disease (ADPKD) is a renal neoplastic disorder characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. This study shows that the lonidamine derivative H2-GMZ inhibits Cl- secretion, cell proliferation, and cyst growth, suggesting that it might have therapeutic value for the treatment of ADPKD.

Physiological Characterization of the Transporter-Mediated Uptake of the Reversible Male Contraceptive H2-Gamendazole Across the Blood-Testis Barrier.

The blood-testis barrier (BTB) is formed by a tight network of Sertoli cells (SCs) to limit the movement of reproductive toxicants from the blood into the male genital tract. Transporters expressed at the basal membranes of SCs also influence the disposition of drugs across the BTB. The reversible, nonhormonal contraceptive, H2-gamendazole (H2-GMZ), is an indazole carboxylic acid analog that accumulates over 10 times more in the testes compared with other organs. However, the mechanism(s) by which H2-GMZ circumvents the BTB are unknown. This study describes the physiologic characteristics of the carrier-mediated process(es) that permit H2-GMZ and other analogs to penetrate SCs. Uptake studies were performed using an immortalized human SC line (hT-SerC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Uptake of H2-GMZ and four analogs followed Michaelis-Menten transport kinetics (one analog exhibited poor penetration). H2-GMZ uptake was strongly inhibited by indomethacin, diclofenac, MK-571, and several analogs. Moreover, H2-GMZ uptake was stimulated by an acidic extracellular pH, reduced at basic pHs, and independent of extracellular Na+, K+, or Cl- levels, which are intrinsic characteristics of OATP-mediated transport. Therefore, the characteristics of H2-GMZ transport suggest that one or more OATPs may be involved. However, endogenous transporter expression in wild-type Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and human embryonic kidney-293 (HEK-293) cells limited the utility of heterologous transporter expression to identify a specific OATP transporter. Altogether, characterization of the transporters involved in the flux of H2-GMZ provides insight into the selectivity of drug disposition across the human BTB to understand and overcome the pharmacokinetic and pharmacodynamic difficulties presented by this barrier. SIGNIFICANCE STATEMENT: Despite major advancements in female contraceptives, male alternatives, including vasectomy, condom usage, and physical withdrawal, are antiquated and the widespread availability of nonhormonal, reversible chemical contraceptives is nonexistent. Indazole carboxylic acid analogs such as H2-GMZ are promising new reversible, antispermatogenic drugs that are highly effective in rodents. This study characterizes the carrier-mediated processes that permit H2-GMZ and other drugs to enter Sertoli cells and the observations made here will guide the development of drugs that effectively circumvent the BTB.

Decarboxylative Fluorination of Electron-Rich Heteroaromatic Carboxylic Acids with Selectfluor

A transition-metal-free decarboxylative fluorination of electron-rich five-membered heteroaromatics, including furan-, pyrazole-, isoxazole-, thiophene-, indole-, benzofuran- and indazolecarboxylic acids, with Selectfluor is reported. Fluorinated dimer products were observed for nitrogen-containing heteroaromatic carboxylic acids, such as indole and pyrazole. An effective method has been developed to synthesize the monomer of 2- and 3-fluoroindoles with Li2CO3 as base at low temperature.

Lonidamine-ethyl ester-mediated remodelling of the Sertoli cell cytoskeleton induces phosphorylation of plakoglobin and promotes its interaction with α-catenin at the blood-testis barrier.

Several compounds affect male fertility by disrupting the adhesion of germ cells to Sertoli cells, which results in the release of undeveloped germ cells into the seminiferous tubule lumen that are incapable of fertilising the ovum. Indazole carboxylic acids are one class of compounds exhibiting such effects and they have been investigated as non-hormonal contraceptives for potential human use. The aims of this study were to investigate the effects of lonidamine-ethyl ester, an indazole carboxylic acid, on spermatogenesis and cell junctions, in particular, desmosomes. We found two doses of lonidamine-ethyl ester at 50mg kg-1 to disrupt Sertoli-germ cell adhesion. By light and fluorescent microscopy, pronounced changes were observed in the distribution of actin microfilaments and intermediate filaments, as well as in the localisation of plakoglobin, a protein with structural and signalling roles at the desmosome and adherens junction at the blood-testis barrier. Furthermore, immunoblotting and immunoprecipitation experiments using testis lysates revealed a significant upregulation (P<0.01) of plakoglobin and Tyr-phosphorylated plakoglobin. Co-immunoprecipitation experiments showed an increase in the interaction between plakoglobin and fyn proto-oncogene, an Src family non-receptor tyrosine kinase, after treatment, as well as an increase in the interaction between plakoglobin and α-catenin. Taken collectively, these data indicate that a disruption of Sertoli cell and spermatocyte-spermatid adhesion in the seminiferous epithelium by lonidamine-ethyl ester results in the phosphorylation of plakoglobin, thereby promoting its interaction with α-catenin at the blood-testis barrier.

One-Pot Synthesis of Novel 3,5-Disubstituted-1,2,4-oxadiazoles from Indazole Carboxylic Acid Esters and Amidoximes

One-Pot Synthesis of Novel 3,5-Disubstituted-1,2,4-oxadiazoles from Indazole Carboxylic Acid Esters and Amidoximes

Effect of H2-Gamendazole and Other Indazole Carboxylic Acid (ICA) Analogs on Primary Rat Sertoli Cells Cytoskeletal Structure and Elongation Factor 1 Alpha (EEF1A1) Expression and Function.

Effect of H2-Gamendazole and Other Indazole Carboxylic Acid (ICA) Analogs on Primary Rat Sertoli Cells Cytoskeletal Structure and Elongation Factor 1 Alpha (EEF1A1) Expression and Function.

The elongation factor eEF1A is a binding target for a novel, non‐hormonal male contraceptive

Hormone‐based oral contraceptives were approved for use in women in 1960 and have become a popular form of birth control. But oral contraceptives are not suitable for all women and other methods have a high failure rate when used incorrectly. According to WHO statistics, 42% of pregnancies are unintended, identifying a need for the development of alternative methods of contraception. Gamendazole (GMZ) was identified from a screen of indazole carboxylic acid analogues as a non‐hormonal compound that causes male infertility in rats. H2‐GMZ, a more potent analogue of GMZ, produced 100% infertility after a single 3mg/kg dose, with 100% reversibility within 6 weeks. Eukaryotic elongation factor 1A (eEF1A) was identified as a binding partner of GMZ. To gain insight into the mechanism of GMZ action, we examined the effect of the drug on the canonical and non‐canonical functions of eEF1A. Total translation rates and nucleotide binding were minimally affected, suggesting that GMZ functions via inhibition of eEF1A's actin bundling function. Co‐sedimentation assays using purified yeast actin and yeast or rabbit eEF1A showed inhibition of actin bundling in the presence of 10 uM GMZ. We are currently developing a light scattering assay to determine kinetics of bundling, reversibility of inhibition and effects of adding competing factors. Preliminary data shows 10 uM GMZ inhibits rabbit eEF1A's actin bundling function.

Trifluoro‐3‐hydroxy‐1H‐indazolecarboxylic Acids and Esters from Perfluorinated Benzenedicarboxylic Acids

AbstractTwelve new 3‐hydroxyindazoles, each bearing three fluorine substituents and a CO2R group (R = H, CH3, C2H5) distributed around its 4‐, 5‐, 6‐, and 7‐positions, have been synthesized. They were studied by NMR in solution (1H, 13C, 15N, 19F) and in the solid state (13C, 15N). In solution, all of them are 3‐hydroxy tautomers: the a form. In the solid state, although the 3‐hydroxy tautomers are still the most frequent, there are some cases of indazolin‐3‐ones – the b form – and one example (12ab) of the very rare case in which both tautomers are present.

A Novel Potent Indazole Carboxylic Acid Derivative Blocks Spermatogenesis and Is Contraceptive in Rats after a Single Oral Dose1

Women have historically been the focus for development of new contraceptive methods. The National Institutes of Health, World Health Organization, and Institute of Medicine have stressed the need to develop nonhormonal, nonsteroidal male contraceptive agents. We report results from initial dose-ranging studies of a new indazole carboxylic acid analogue, gamendazole. An infertility rate of 100% was achieved in seven out of seven proven-fertile male rats 3 wk after a single oral dose of 6 mg/kg of gamendazole. Fertility returned by 9 wk in four of seven animals, with typical numbers of normal-appearing conceptuses. A fertility rate of 100% returned in four of six animals that became infertile at a single oral dose of 3 mg/kg of gamendazole. No differences in mating behavior were observed in either of the gamendazole-treated groups versus the control (vehicle-only) group. In the animals that showed reversible infertility, a transient increase in circulating FSH levels coincided with an initial decline in inhibin B levels after administration of gamendazole, but no other significant changes in circulating reproductive hormones were observed. Gamendazole inhibited production of inhibin B by primary Sertoli cells in vitro with a median inhibitory concentration of 6.8 thorn+/- 3.0 (SEM) (3/4)x 10(-10) M, suggesting that Sertoli cells are a primary target. A biotinylated gamendazole analogue revealed cytoplasmic and perinuclear binding of gamendazole in primary Sertoli cells. Gamendazole represents the most potent new oral antispermatogenic indazole carboxylic acid to date. Our results, however, demonstrate that additional dose-finding studies are required to improve reversibility and widen the therapeutic window before more detailed drug development of this potential nonhormonal male contraceptive agent can occur.

Gamendazole, an Orally Active Indazole Carboxylic Acid Male Contraceptive Agent, Targets HSP90AB1 (HSP90BETA) and EEF1A1 (eEF1A), and Stimulates Il1a Transcription in Rat Sertoli Cells1

Gamendazole was recently identified as an orally active antispermatogenic compound with antifertility effects. The cellular mechanism(s) through which these effects occur and the molecular target(s) of gamendazole action are currently unknown. Gamendazole was recently designed as a potent orally active antispermatogenic male contraceptive agent. Here, we report the identification of binding targets and propose a testable mechanism of action for this antispermatogenic agent. Both HSP90AB1 (previously known as HSP90beta [heat shock 90-kDa protein 1, beta]) and EEF1A1 (previously known as eEF1A [eukaryotic translation elongation factor 1 alpha 1]) were identified as binding targets by biotinylated gamendazole (BT-GMZ) affinity purification from testis, Sertoli cells, and ID8 ovarian cancer cells; identification was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Western blot analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not to TEF3 or HBS1, and was competed by unlabeled gamendazole. However, gamendazole did not inhibit nucleotide binding by EEF1A1. Gamendazole binding to purified Saccharomyces cerevisiae HSP82 inhibited luciferase refolding and was not competed by the HSP90 drugs geldanamycin or novobiocin analogue, KU-1. Gamendazole elicited degradation of the HSP90-dependent client proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggest that gamendazole may represent a new class of selective HSP90AB1 and EEF1A1 inhibitors. Testis gene microarray analysis from gamendazole-treated rats showed a marked, rapid increase in three interleukin 1 genes and Nfkbia (NF-kappaB inhibitor alpha) 4 h after oral administration. A spike in II1a transcription was confirmed by RT-PCR in primary Sertoli cells 60 min after exposure to 100 nM gamendazole, demonstrating that Sertoli cells are a target. AKT1, NFKB, and interleukin 1 are known regulators of the Sertoli cell-spermatid junctional complexes. A current model for gamendazole action posits that this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting infertility.

Synthesis and structural characterization of 1- and 2-substituted indazoles: ester and carboxylic acid derivatives.

A series of indazoles substituted at the N-1 and N-2 positions with ester-containing side chains -(CH2)(n)CO2R of different lengths (n = 0-6, 9, 10) are described. Nucleophilic substitution reactions on halo esters (X(CH2)(n)CO2R) by 1H-indazole inalkaline solution lead to mixtures of N-1 and N-2 isomers, in which the N-1 isomer predominates. Basic hydrolysis of the ester derivatives allowed the synthesis of the corresponding indazole carboxylic acids. All compounds were fully characterised by multinuclear NMR and IR spectroscopies, MS spectrometry and elemental analysis; the NMR spectroscopic data were used for structural assignment of the N-1 and N-2 isomers. The molecular structure of indazol-2-yl-acetic acid (5b) was determined by X-ray diffraction, which shows a supramolecular architecture involving O2-H...N1 intermolecular hydrogen bonds.

Potentiation of Antiproliferative Drug Activity by Lonidamine in Hepatocellular Carcinoma Cells

The ability of lonidamine (LND), a derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of anticancer drugs was investigated in two human hepatocarcinoma (HCC) cell lines. The cytotoxicity of drugs used singly, in association or in sequence was evaluated using the Sulforhodamine B (SRB) assay. LND did not appreciably potentiate the effect of antitumor drugs when given before or simultaneously, in either cell line. Conversely, a synergistic interaction was observed in both cell lines when LND was given after conventional drugs. LND produced a moderate decrease in S-phase cell fraction and did not induce apoptosis. Conversely, paclitaxel (TAX) induced an important block in G2 and an increase in apoptosis. Following a 48-h TAX wash out, a progressive passage of cells from G2 to M phase was observed with a corresponding increase in apoptotic cells. Post-treatment with LND increased the cytotoxicity of some antitumor drugs, especially TAX, in hepatocarcinoma cells, possibly by preventing, as an energolytic drug, cell damage repair or by producing an additional effect on microtubule stabilization.

Male fertility: core chemical structure in pharmacological research

Research in male fertility is of interest in connection with both contraception and treatment of sterility. Several compounds have been shown to inhibit fertility by various mechanisms. The most significant of them are reviewed, including some recently described indazole carboxylic acids. Concerning sterility treatment, no effective treatment is so far available, with the exception of hormonal dysfunctions. The hypothesized role of an excess of free radicals in male sterility suggests a potential therapeutic value of free radicals scavengers. In this connection, indazole derivatives appear of special interest, as some of them display an anti-denaturant activity resulting in a peculiar scavenger-like activity. Some synthetic approaches are proposed, leading to new compounds of potential interest either as male contraceptives or in the treatment of male sterility.

Indazole carboxylic acids in male contraception

Two new chemical entities, 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid, were synthesized based on the core structure of lonidamine (1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid). These compounds apparently exert their effects in the testis by perturbing the Sertoli-germ cell adherens junctions causing germ cell loss from the seminiferous epithelium. Recently completed studies in the rat have demonstrated the efficacy, reversibility, and potential use of these two compounds as oral contraceptives for men. Neither compound affected the hypothalamus-pituitary-testicular axis, and both compounds were neither hepatotoxic nor nephrotoxic. These results suggest that these two compounds are safe for further development.

Reversible inhibition of spermatogenesis in rats using a new male contraceptive, 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide.

The oral male contraceptive agent 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF2364) is a new analogue of indazole-carboxylic acid. AF2364 was orally administered to rats at 50 mg/kg body weight once weekly for five consecutive weeks. The effects on fertility efficacy, hormonal profile, organ weights, tissue morphology, and serum microchemistry were examined. Complete infertility was noted in rats 29 days after the initial dose of AF2364 and continued until 90 days. Fertility resumed in 25% of the group after 104 days and had resumed in 75% of the rats by the last mating at 197 days. Morphological examination of the testis showed rapid exfoliation of elongated spermatids and the generation of large multinucleated cells 6 days after the first treatment, with depletion of most germ cells after 40 days. Normal spermatogenesis was noted in 95% of the tubules in the animals that were fertile at 210 days. Morphological analysis of the epididymal compartments revealed reduced lumen size, whereas the prostate exhibited an increase in the glandular lumen with a reduction in epithelium height. No morphological changes were detected in the kidney, liver, and cerebrum by light microscopy. Kidney and liver function, as evaluated by serum chemistry, were not affected by the drug treatment. AF2364 did not alter the levels of FSH, and only minimal changes were noted for LH and testosterone, suggesting that the hypothalamic-pituitary-testicular axis was not affected. These results illustrate the potential of AF2364 as a male contraceptive.

Modulation of cytotoxic drug activity by mitotane and lonidamine in human adrenocortical carcinoma cells.

The ability of mitotane, a DDT derivative with adrenotoxic activity, and lonidamine, an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of doxorubicin, epidoxorubicin, cisplatin and VP16 was investigated in a human adrenocortical carcinoma cell line (SW13). A marked variability in cellular response to a 1-h treatment with the individual anticancer agents was observed. The concentrations able to inhibit SW13 cell proliferation by 50% (IC50) were 0.45 microg/ml and 0.4 microg/ml for doxorubicin and epidoxorubicin, respectively, thus indicating a relative sensitivity to anthracyclines. Conversely, the SW13 cell line displayed a marked resistance to cisplatin (IC50, 13.9 microg/ml) and VP16 (IC50, 15 microg/ml). When cells were exposed to anticancer drugs and mitotane simultaneously or in sequence, a positive modulation of anthracycline cytotoxic effects was observed. Although to a lesser extent, mitotane also increased cisplatin activity. Conversely, no potentiation was observed when mitotane was combined with VP16. Lonidamine slightly increased the cytotoxicity of epirubicin and cisplatin as individual agents. Moreover, a supra-additive effect of the three-drug (epidoxorubicin-cisplatin-lonidamine) combination was observed.

Lonidamine as a modulator of taxol activity in human ovarian cancer cells: effects on cell cycle and induction of apoptosis.

The ability of lonidamine (LND), an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxicity of Taxol (TX) was investigated in the A2780 human ovarian cancer cell line. Different cytotoxicity results were obtained as a function of treatment schedule. Specifically, TX followed by LND produced synergistic effects. Conversely, antagonistic effects were recorded when drugs were given simultaneously or according to the opposite sequence. TX induced an oligonucleosomal DNA fragmentation typical of the apoptotic process. The extent and the kinetics of DNA cleavage in samples treated with the taxane alone were similar to those of samples treated with the TX-LND sequence. Activation of Yama protease and degradation of poly (ADP-ribose) polymerase were not observed after individual or combined treatment. LND did not appreciably modify the effect exerted by TX on proteins involved in cell cycle progression (i.e., inhibition of p34cdc2 expression) and apoptosis (i.e., upregulation of wt p53 and transactivation of p21waf1), and only caused a slight induction of the Bax protein. LND alone did not affect tubulin polymerization in A2780 cells and, when administered after a 24 hr TX exposure, did not appreciably alter the extent of tubulin polymerization induced by the taxane. Although additional studies are needed to define the molecular basis of the TX-LND interaction, our results suggest that LND can positively modulate the antitumor activity of TX in ovarian cancer cells and indicate that the energolytic is potentially useful in combination therapy including the taxane in ovarian cancer patients.

Modulation by lonidamine on the combined activity of cisplatin and epidoxorubicin in human breast cancer cells

The ability of lonidamine (LND), an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of cisplatin (CDDP) and epidoxorubicin (EPI), singly or in combination, was investigated in two human breast cancer cell lines (MCF7 and T47D). A 72-hr post-incubation with a non-cytotoxic concentration of LND (75 microM) increased the activity of a 1-hr CDDP treatment as well as that of a 1 to 16-hr EPI treatment. A different pattern of interaction among the drugs and modulator was observed as a function of the sequence of drug treatment. Specifically, supra-additive or additive effects of the combination were obtained in the two cell lines according to the different treatment schemes. In particular, the maximum potentiation was observed in MCF7 cells simultaneously exposed to CDDP, EPI and LND for 1 hr and then post-incubated with LND for 72 hr, and in T47 first exposed to EPI and LND, then to CDDP and LND, and finally post-incubated with LND. Flow cytometric analysis of MCF7 cell distribution in the different cycle phases showed that combined treatment with EPI/CDDP/LND was able to stabilize cell cycle perturbations (mainly G2M accumulation) induced by individual agents. The ability of LND to potentiate CDDP and EPI cytotoxicity, and the consideration that LND causes side effects different from those caused by alkylating agents and anthracyclines, make this compound an attractive candidate for multidrug combination therapy in breast cancer.

258 A phase III multi-institutional randomised trial of lonidamine (L) and post-operative radiotherapy (RT) in supratentorial malignant glioma

Lonidamine, an indazole carboxylic acid derivative, was reported in Phase II/III trials to have efficacy as radiopotentiator in malignant glioma. Between October 1990 and August 1994, 191 pts. with supratentorial malignant glioma were randomly allocated to treatment with RT or RT + L, following surgical resection. Prior to randomisation patients were stratified according to age. One patient was ineligible and excluded from the study (not malignant glioma). Patients (pts) in arm A (98 pts) received RT alone (50 Gy whole bram plus 14 Gy coned-down boost to the tumour volume, 2 Gy/day for 5 days a week), those in arm B (92 pts) received RT + L (150 mg 3 times daily for 1 year starting from 3 days before irradiation). The groups were comparable in median age, performance status, TNM classes, sex, residual tumour size after surgery and histologic grade. Median follow up was 49 weeks. Intention to treat analysis failed to demonstrate significance difference in the survival rates and shapes of the survival curves between the two treatment arms. Cumulative survival at 12 and 24 months calculated by the Kaplan Meier method were 50% ± 5% and 13.4% ± 4% for arm A, 49% ± 5% and 13.4% ± 4% for arm B. (P> 0.4). The Cox proportional hazards model confirmed the prognostic variables of age ( P P P

Enhancement of cisplatin activity by lonidamine in human ovarian cancer cells.

The ability of lonidamine, an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxicity of cisplatin was investigated in human ovarian-cancer cell lines sensitive (A2780) or with experimentally induced resistance (A2780/cp8) to the alkylating agent. A 24-hr post-incubation with 300 microM lonidamine significantly potentiated the activity of a 1-hr cisplatin treatment in both cell lines. In particular, the cisplatin IC50 value was reduced 4-fold in the sensitive line and 5-fold in the resistant line. Flow cytometric analysis showed that, in the resistant cell line, lonidamine alone did not affect cell kinetics, but when given after cisplatin it was able to transform the temporary G2 + M cell accumulation induced by the alkylating agent to a persistent block in S/G2 + M. In the A2780/cp8 cell line, lonidamine was also able to significantly enhance the accumulation of cisplatin-induced DNA interstrand cross-links. Our results suggest that lonidamine can positively modulate the anti-tumor activity of cisplatin in ovarian cancer cells and also indicate that the drug is potentially useful in combination therapy including the alkylating agent for ovarian cancer patients.