Effects of orally active taxanes on P-glycoprotein modulation and colon and breast carcinoma drug resistance.

BACKGROUND: The taxane paclitaxel (Taxol) is often of limited efficacy in chemotherapeutic regimens because some cancer cells express high levels of the efflux pump, P-glycoprotein (Pgp), which removes the drug from the cells. The orally active paclitaxel analog IDN-5109 has been reported to overcome Pgp-mediated drug resistance. We tested whether IDN-5109 acts by modulating Pgp activity. METHODS: Human MDA435/LCC6mdr1 and MDA435/LCC6 breast carcinoma cells, which express and do not express Pgp, respectively, were incubated with [3H]IDN-5109 and paclitaxel to determine intracellular drug accumulation. Flow cytometry was used to analyze intracellular retention of two Pgp substrates, rhodamine 123 (Rh-123) and doxorubicin, in both breast carcinoma cell lines and in human colon carcinoma cells (SW-620, DLD1, and HCT-15, whose Pgp levels vary) treated with different taxanes. The effects of IDN-5109 and paclitaxel on tumor growth in vivo were studied with the use of tumors established through xenografts of Pgp-expressing SW-620 and DLD1 cells in severe combined immunodeficiency mice. All statistical tests were two-sided. RESULTS: Pgp-expressing cells treated with IDN-5109 or with the taxane-based drug resistance reversal agent tRA96023, which blocks Pgp activity, retained 8.1- and 9.4-fold more Rh-123 (P =.0001), respectively, and 1.7- and 1.9-fold more doxorubicin (P =.001), respectively, than cells treated with paclitaxel. Non-Pgp-expressing cells treated similarly demonstrated no increased retention of either substrate. MDA435/LCC6mdr1 cells retained 5.3-fold more [3H]IDN-5109 than [3H]paclitaxel after 2 hours (P =.01). IDN-5109 showed statistically significantly higher tumor growth inhibition than paclitaxel against the SW-620 xenograft (P =.003). CONCLUSIONS: IDN-5109 modulates Pgp activity, resulting in superior tumor growth inhibition against Pgp-expressing tumors as compared with paclitaxel. IDN-5109 may broaden the spectrum of taxane use to include colon tumors.

Plasminogen activator content of human tumor and adjacent normal tissue measured with fibrin and non-fibrin assays.

Plasminogen activators are enzymes which convert the zymogen to plasmin, the physiological enzyme for dissolving fibrin. There are two different physiological activator enzymes, urokinase (UK) and tissue plasminogen activator (t-PA, also known as vascular activator). The most striking difference in the behavior of the two activators is the ability of fibrin to augment the activity of t-PA but not of UK. Since tumor and normal tissues have been shown to contain different ratios of UK:t-PA, it can be anticipated that their comparative activator activities measured with fibrinolytic assays would yield different results from those measured with non-fibrin tests. This study was designed to test the validity of earlier conclusions that: (a) the measured activity of t-PA is augmented in fibrinolytic assays when compared with a non-fibrin assay based on azocaseinolysis; and (b) this difference could explain the failure of some laboratories using fibrinolytic assays to detect a difference in activator activity between tumor and normal tissues or to find more activity in the normal tissue. Azocaseinolytic and fibrinolytic (fibrin plate) assays were used to measure activator activity in a series of 14 normal-tumor tissue pairs. Using azocasein tests, cancer tissues were found to contain significantly higher median activities than normal tissues [Wilcoxon test, P less than 0.05; 13.8 versus 3.7 Committee on Thrombolytic Agents (CTA) units/g tissue, respectively], whereas no significant difference was found with fibrin assays (43.5 versus 69.0 CTA units/g tissue, respectively). Of total activator activity, the median percentage of UK was significantly higher in tumor (95%) than in normal tissue (58%). In addition, using azocaseinolysis it was found that the median UK activity was significantly higher in tumor (12.1 CTA units/g) relative to normal (3.51 CTA units/g) tissues, whereas no difference was found for t-PA. To explain these results in tumor and normal tissues, a mathematical model was derived to describe the difference between azocasein and fibrin assays for both purified plasminogen activator enzymes and activator enzymes in tissue extracts. The model fits the data well, confirming in a quantitative manner the hypotheses of the study. In addition, the study revealed that the azocaseinolytic assay was able to measure the full potential activator activity of purified pro-urokinase enzyme. Pro-urokinase activity could not be measured with standard fibrinolytic assays. These results show the importance of selection and interpretation of plasminogen activator assays in studies dealing with malignant transformation.

Application of a new approach for the quantitation of drug synergism to the combination of cis-diamminedichloroplatinum and 1-beta-D-arabinofuranosylcytosine.

This report describes the application of a new approach, the universal response surface approach, to the quantitative assessment of drug interaction, i.e., the determination of synergism, antagonism, additivity, potentiation, inhibition, and coalitive action. The specific drug combination and experimental growth system for this introductory application was that of 1-beta-D-arabinofuranosylcytosine (ara-C) and cisplatin with simultaneous drug exposure (1, 3, 6, 12, or 48 h) against L1210 leukemia in vitro. To quantitate the type and degree of drug interaction, a model was fitted using nonlinear regression to the data from each separate experiment, and parameters were estimated (K. C. Syracuse and W. R. Greco, Proc. Biopharm. Sect. Am. Stat. Assoc., 127-132, 1986). The parameters included the maximum cell density over background in absence of drug, the background cell density in presence of infinite drug, the 50% inhibitory concentrations and concentration-effect slopes for each drug, and a synergism-antagonism parameter, alpha. A positive alpha indicates synergism, a negative alpha, antagonism, and a zero alpha, additivity. Maximal synergy was found with a 3-h exposure of ara-C + cisplatin, with alpha = 3.08 +/- 0.96 (SE) and 2.44 +/- 0.70 in two separate experiments. Four different graphic representations of the raw data and fitted curves provide visual indications of goodness of fit of the estimated dose-response surface to the data and visual indications of the intensity of drug interaction. The universal response surface approach is mathematically consistent with the traditional isobologram approach but is more objective, is more quantitative, and is more easily automated. Although specifically developed for in vitro cancer chemotherapy applications, the universal response surface approach should prove to be useful in the fields of pharmacology, toxicology, epidemiology, and biomedical science in general.

Quantitation of folic acid enhancement of antifolate synergism.

Trimetrexate (TMTX), 5,10-dideazatetrahydrofolate (DDATHF), and 10-propargyl-5,8-dideazafolate (PDDF, CB3717) are antifolates whose primary intracellular targets are dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and thymidylate synthase, respectively. Varying the medium folic acid (PteGlu) concentration over the range of 0.5 to 100 microM increasingly blocks the growth inhibitory effects of the individual antifolates in Manca human lymphoma cells, but increasingly enhances the synergistic interaction of both TMTX + DDATHF and TMTX+ PDDF combinations. Drug interactions were quantitated using the universal response surface approach, which consists of fitting a concentration-effect surface to experimental data with weighted nonlinear regression, enabling the estimation of the synergism parameter, alpha. Estimates for alpha are larger (more intense synergism) for the TMTX + DDATHF combination (alpha = 4.68 +/- 0.66 at 2 microM PteGlu; alpha = 53.6 +/- 3.7 at 40 microM PteGlu) than for the TMTX + PDDF combination (alpha = 0.690 +/- 0.25 at 2 microM PteGlu; alpha = 7.20 +/- 1.8 at 40 microM PteGlu). However, the relative increase brought about by increasing the PteGlu concentration from 2 microM to 40 microM is similar in each instance, 11- and 10-fold, respectively. These experiments suggest that the enhanced cytotoxic interaction brought about by increased PteGlu concentration with the TMTX + DDATHF combination and the TMTX + PDDF combination may share a common mechanism. The dramatic intensity of the synergism between DDATHF and TMTX at 40 microM PteGlu, along with the concept of modulation of antifolate synergism by folates, suggests future in vivo and/or clinical applications of combinations of these (or similar) compounds.

Modeling of the time-dependency of in vitro drug cytotoxicity and resistance.

For potential clinical extrapolation of in vitro findings, it is of interest to relate the measured effect of an anticancer agent to concentration and exposure time. The Hill model (A. V. Hill, J. Physiol., 40: iv-vii, 1910) is commonly used to describe pharmacodynamic (PD) effects, including drug-induced growth inhibition of cancer cells in vitro. The IC(X)n x T = k relationship, in which IC(X) is the concentration of agent required to reduce cell growth by X%, T is the exposure time, and n and k are estimable parameters, was first applied to bacterial disinfectant action and then was successfully used to model anticancer drug potency as a function of exposure time (D. J. Adams, Cancer Res., 49: 6615-6620, 1989). Our goal was to create a new global PD modeling paradigm to facilitate the quantitative assessment of the growth-inhibitory effect of anticancer agents as a function of concentration and exposure time. Wild-type human ovarian A2780 and ileocecal HCT-8 carcinoma cells and sublines that were resistant to cisplatin (A2780/CP3), doxorubicin (A2780/DX5B), and raltitrexed (RTX) (HCT-8/DW2) were exposed to various anticancer agents, cisplatin, doxorubicin, paclitaxel, trimetrexate, RTX, methotrexate, and AG2034, for periods ranging from 1 to 96 h. Cell growth inhibition was measured with the sulforhodamine B protein dye assay. Patterns of time-dependency of drug potency, slope of the concentration-effect curves, and relative degree of resistance were characterized. Empirical mathematical expressions were built into a global concentration-time-effect model. The global PD model was then fit to the concentration-time-effect data with iteratively reweighted nonlinear regression. Under specific treatment conditions, the examination of the slope and the shape of the concentration-effect curves revealed a large heterogeneity in drug response, e.g., shallow concentration-effect curve or double or triple Hill "roller coaster" concentration-effect curve. These patterns, which were observed at intermediate exposure times in parental and resistant cells for paclitaxel and trimetrexate or only in resistant HCT-8/DW2 cells for RTX, methotrexate, and AG2034, revealed mechanistic insights for the former cases but possible methodological artifacts for the latter cases. The comprehensive PD modeling of the cytotoxic effect of anticancer agents showed that it was possible to modulate drug effect, response heterogeneity, and drug resistance by altering the time of exposure to the agents. This approach will be useful for: (a) describing complex concentration-time-effect surfaces; (b) refining biological interpretations of data; (c) providing insights on mechanisms of drug action and resistance; and (d) generating leads for clinical use of anticancer drugs.

Lack of reciprocity in drug and light dose dependence of photodynamic therapy of pancreatic adenocarcinoma in vitro.

Two human pancreatic cell lines, MIA PaCa 2 and Capan 2, were treated by photodynamic therapy in vitro with Photophrin (0.01-25 micrograms/ml; 24 h) and then light (1-50 J/cm2; lambda = 630 nm). The following model was fit to 6 datasets with weighted nonlinear regression: [sequence: see text] The symbols are: E, cell growth; Econ, control growth in the absence of the combination; B, background signal; m, slope parameter; gamma, interaction parameter; D, concentration of Photofrin; L, light dose; F, fraction of Photofrin not photobleached by the light dose; k, k1, k2, bleaching parameters; A, distribution parameter for biexponential bleaching equation. Simple reciprocity of photosensitizer concentration and light dose was not found; compensation for photobleaching was critical. MIA PaCa2 required the monoexponential bleaching factor, whereas Capan 2 required the biexponential bleaching factor. The greater photosensitivity of MIA PaCa2 over Capan 2 can be best explained not by differences in the interaction parameter but rather by differences in the photobleaching pattern and rate. It may be possible to further enhance the selectivity of photodynamic therapy if differences in photobleaching between different cell types can be exploited by adequate dosimetry.

Toxicity and pharmacokinetics of a new antifolate, 2,4-diamino-5-adamantyl-6-methylpyrimidine, in dogs.

The toxicology of a potentially useful antitumor agent, 2,4-diamino-5-adamantyl-6-methylpyrimidine (DAMP), and its ethanesulfonate salt has been studied in beagle dogs after 1 to 20 doses. Two types of toxicity could be discerned: the acute central nervous system toxicity manifested by vomiting, convulsions, and minor hypothermia; and the antiproliferative toxicity, similar to that of other folate antagonists, manifested by diarrhea, anorexia, loss of body weight, and hematological changes as well as changes in blood chemistry. There is evidence of a cumulative effect of the drug with respect to antiproliferative toxicity. Characteristically, the animals could be protected against the antiproliferative toxicity by simultaneous administration of folinic acid. The pharmacokinetics of the ethanesulfonate salt of DAMP was studied after i.v. administration of sublethal doses (5 mg/kg) of tritium-labeled drug. Sixty-three % of the administered dose was excreted in the urine and 10% was excreted in the feces within 48 hr after drug administration. Thus, about 27% of the drug was not recovered, and it is possible that it persists in the tissues for a period of several days. Analysis of the plasma and urine revealed that DAMP was metabolized rapidly. At least 2 metabolites were found in plasma and urine, one lipophilic and one hydrophilic, the latter being the predominant form. Pharmacokinetic data were successfully fitted to a model consisting of central and peripheral DAMP compartments and a DAMP metabolite compartment. DAMP was very rapidly sequestered in the peripheral compartment with a rapid phase half-life of 23 sec. The slower phase of DAMP plasma disappearance had a half-life of 3 hr. The short plasma half-life and rapid metabolism distinguished this drug from other lipophilic antifolates.

Antitumor activity of Corynebacterium parvum and retinyl palmitate used in combination on the Lewis lung carcinoma.

The effects of Corynebacterium parvum and retinyl palmitate given at various levels, schedules, and routes of administration on primary Lewis lung carcinoma and its metastases have been evaluated in C57BL/6J mice given s.c. inoculations of 5 X 10(5) tumor cells. Single i.v., but not i.p., s.c., or i.m., administration of C. parvum (0.35 mg/mouse given on Days 0, 1, or 3) reduced growth of tumor and prolonged survival time. Retinyl palmitate (3000 IU/mouse/day) given alone i.p. either before, after, or both before and after tumor inoculation showed no effect on tumor growth, survival of mice, or lung metastases. The combination of retinyl palmitate i.p. (6 daily injections of 1500 IU/mouse after tumor implantation) and C. parvum (0.175 mg/mouse) given i.v. resulted in an increase in life span over control of 146% and appeared to be therapeutically synergistic. This combination produced 90-day cures in about 20% of the treated animals, all of which were found to be tumor free. Two nonparametric statistical procedures, the Kruskal-Wallis and the Dunn test, were used to assess the effects of treatments on survival time and tumor growth and may be generally applicable to animal tumor studies. They provide multiple comparisons of different treatments and allow the inclusion of long-term survivors into the analysis.

Relationship between DNA ploidy, glandular differentiation, and tumor spread in human prostate cancer.

DNA ploidy was evaluated by flow cytometry for 45 human prostate carcinomas (34 prostatectomy specimens and 11 biopsies). Twenty tumors (44.4%) contained a distinct aneuploid stem line. All 11 tumors confined to the prostate gland (pathological Stage B) were diploid. The frequency of aneuploidy increased with advancing stage, and most tumors with distant metastases were aneuploid. The degree of glandular differentiation was characterized by the Gleason score. One-third of tumors with a Gleason score of 5 to 6 were aneuploid, whereas over 70% of poorly differentiated tumors with a Gleason score of 9 to 10 were aneuploid. Among diploid tumors, 45.5% were localized carcinomas (Stage B), 36.4% were characterized by invasion outside the prostate (Stage C), and 18.2% formed pelvic nodal or distant metastases (Stages D1 and D2). In nearly two-thirds of patients with aneuploid tumors, pelvic nodal or distant metastases were found. When tumors were classified according to both DNA ploidy and degree of glandular differentiation, then subgroups of tumors with the highest and lowest degree of malignant potential became apparent. Only 7.1% of diploid tumors with a Gleason score of 5 to 6 formed metastases, but 80% of aneuploid tumors with a higher Gleason score (7 to 10) formed metastases. Diploid tumors with higher Gleason scores and aneuploid tumors with lower Gleason scores had intermediate frequencies of metastases. The presence of an aneuploid stem line in prostate carcinomas indicated that the tumor had spread outside the prostate gland or had metastasized. DNA ploidy may be an important prognostic factor for human prostate cancer. DNA ploidy and the degree of glandular differentiation considered together may improve prognostic evaluation of prostate carcinomas.

An in vivo quantitative structure-activity relationship for a congeneric series of pyropheophorbide derivatives as photosensitizers for photodynamic therapy.

An in vivo quantitative structure-activity relationship (QSAR) study was carried out on a congeneric series of pyropheophorbide photosensitizers to identify structural features critical for their antitumor activity in photodynamic therapy (PDT). The structural elements evaluated in this study include the length and shape (alkyl, alkenyl, cyclic, and secondary analogs) of the ether side chain. C3H mice, harboring the radiation-induced fibrosarcoma tumor model, were used to study three biological response endpoints: tumor growth delay, tumor cell lethality, and vascular perfusion. All three endpoints revealed highly similar QSAR patterns that constituted a function of the alkyl ether chain length and drug lipophilicity, which is defined as the log of the octanol:water partition coefficient (log P). When the illumination of tumor, tumor cells, or cutaneous vasculature occurred 24 h after sensitizer administration, activities were minimal with analogs of log P < or = 5, increased dramatically between log P of 5-6, and peaked between log P of 5.6-6.6. Activities declined gradually with higher log P. The lack of activity of the least-lipophilic analogs was explained in large part by their poor biodistribution characteristics, which yielded negligible tumor and plasma drug levels at the time of treatment with light. The progressively lower potencies of the most lipophilic analogs cannot be explained through the overall tumor and plasma pharmacokinetics of photosensitizer because tumor and plasma concentrations progressively increased with lipophilicity. When compensated for differences in tumor photosensitizer concentration, the 1-hexyl derivative (optimal lipophilicity) was 5-fold more potent than the 1-dodecyl derivative (more lipophilic) and 3-fold more potent than the 1-pentyl analog (less lipophilic), indicating that, in addition to the overall tumor pharmacokinetics, pharmacodynamic factors may influence PDT activity. Drug lipophilicity was highly predictive for photodynamic activity. QSAR modeling revealed that direct antitumor effects and vascular PDT effects may be governed by common mechanisms, and that the mere association of high levels of photosensitizer in the tumor tissue is not sufficient for optimal PDT efficiency.

Super in vitro synergy between inhibitors of dihydrofolate reductase and inhibitors of other folate-requiring enzymes: the critical role of polyglutamylation.

The combined action among polyglutamylatable and nonpolyglutamylatable antifolates, directed against dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), and thymidylate synthase (TS), in human ileocecal HCT-8 cells was examined in a 96-well plate growth inhibition assay (96-h continuous drug exposure). An interaction parameter, alpha, was estimated for each of 95 experiments by fitting a seven-parameter model to data with weighted nonlinear regression. In a representative experiment, raising the folic acid concentration in the medium dramatically increased the Loewe synergy for the combination of trimetrexate (TMTX) and the GARFT inhibitor AG2034 (from a mean alpha +/- SE of 1.50 +/- 0.25 at 2.3 microM folic acid to 146 +/- 20 at 78 microM folic acid). Enhancements were also found for combinations of TMTX with the GARFT inhibitors AG2032, Lometrexol, and LY309887, the AICARFT inhibitor AG2009, and the TS inhibitors LY231514 and Tomudex but not with the GARFT inhibitor LL95509 or with the TS inhibitors AG337, ZD9331, and BW1843U89. Replacing TMTX with methotrexate in two-drug mixtures decreased the intensity of Loewe synergy. Examination of isobolograms at different effect levels revealed informative reproducible changes in isobol patterns. No two-drug combinations among inhibitors of GARFT, AICARFT, and TS exhibited Loewe synergy at either 2.3 or 78 microM folic acid. Thus, the ideal requirement for the folic acid-enhanced synergy is that a nonpolyglutamylatable DHFR inhibitor be combined with a polyglutamylatable inhibitor of another folate-requiring enzyme. A hypothesis to explain this general phenomenon involves the critical role of folylpoly-gamma-glutamate synthetase and the effect of the DHFR inhibitor in decreasing the protection by folic acid of cells to the other antifolates.

Initial clinical (phase I) trial of TLC D-99 (doxorubicin encapsulated in liposomes).

A liposome-encapsulated form of doxorubicin (TLC D-99), which was shown in preclinical toxicology to be less toxic to the gastrointestinal tract and myocardium than free doxorubicin, was administered by constant infusion (1.00-1.80 h) to 38 patients in single doses of 20, 30, 45, 60, 75, and 90 mg/m2 every 3 weeks and daily for 3 days at doses of 20, 25, and 30 mg/m2/day. The dose-limiting toxicity was leucopenia: the maximum tolerated doses were one at 90 mg/m2 and three at 25 mg/m2/day. Nausea, vomiting, and stomatitis were minimal or absent at each dose; alopecia was minor. Fever and chills were noted at most of the doses, and malaise was seen in some patients, especially at the higher doses. No hepatic, renal, or other organ toxicities were observed. Clinical cardiac toxicity was not observed in any patient; however, the cumulative doxorubicin dose was greater than 400 mg/m2 in only one patient. There was large variation among patients in estimated pharmacokinetic parameters and profiles. Higher plasma levels and dose intensities were achieved with TLC D-99 than were predicted for free doxorubicin. Liposome-encapsulated doxorubicin was well tolerated and produced less nausea, vomiting, and stomatitis than would be expected with free doxorubicin administered at equally myelosuppressive doses.

Characterization of cells obtained by mechanical and enzymatic means from human melanoma, sarcoma, and lung tumors.

Characterization of cells comprising solid tumors will facilitate the rational design of cancer chemotherapy for individual patients. We have prepared cell suspensions from human melanoma, sarcoma, and lung tumors by thinly slicing the tissue with a microtome and scalpels (mechanical release), followed by treatment with a mixture of collagenase II and DNase I (enzymatic release). This method of disaggregation resulted in two cell suspensions for each tumor specimen, and we characterized these suspensions by assessing their dye exclusion capability, ribonucleoside triphosphate pools, cytological profile and clonogenicity in soft agar. The enzymatic method thus yields cells in addition to those obtainable by a mild mechanical procedure, and these cells are similar in cytological profile and clonogenicity in soft agar to those released mechanically. Furthermore, the enzymatically released population is superior to that released mechanically for purposes requiring large numbers of dye-excluding cells having intact ribonucleotide pools.

Photofrin photodynamic therapy can significantly deplete or preserve oxygenation in human basal cell carcinomas during treatment, depending on fluence rate.

At high fluence rates in animal models, photodynamic therapy (PDT) can photochemically deplete ambient tumor oxygen through the generation of singlet oxygen, causing acute hypoxia and limiting treatment effectiveness. We report that standard clinical treatment conditions (1 mg/kg Photofrin, light at 630 nm and 150 mW/cm2), which are highly effective for treating human basal cell carcinomas, significantly diminished tumor oxygen levels during initial light delivery in a majority of carcinomas. Oxygen depletion could be found during at least 40% of the total light dose, but tumors appeared well oxygenated toward the end of treatment. In contrast, initial light delivery at a lower fluence rate of 30 mW/cm2 increased tumor oxygenation in a majority of carcinomas. Laser treatment caused an intensity- and treatment time-dependent increase in tumor temperature. The data suggest that high fluence rate treatment, although effective, may be inefficient.

Breast cancer resistance protein (BCRP/MXR/ABCG2) in acute myeloid leukemia: discordance between expression and function.

Data on breast cancer resistance protein (BCRP, MXR, ABCG2) expression in acute myeloid leukemia (AML) have been inconsistent, possibly due to use of different assays in different studies. BCRP mRNA was studied by the reverse-transcription polymerase chain reaction and BCRP protein expression (BXP-21, BXP-34 or anti-ABCG2 antibody, with anti-CD34 and anti-CD33) and function (fumitremorgin C modulation of mitoxantrone retention) by flow cytometry in eight cell lines and in pretreatment blasts from 31 AML patients. BCRP mRNA levels, antibody staining and function correlated strongly in cell lines (Pearson r values, 0.73-0.97), but not in AML samples. AML sample BCRP mRNA levels were between those in parental 8226 and 35-fold mitoxantrone-resistant 8226/MR20 cells in all but one case, and BCRP mRNA had the wild-type sequence at codon 482 in all. In AML, unlike in cell lines, BCRP protein expression or function, when present, was only detected in small subpopulations. BCRP mRNA and protein expression did not correlate, nor did staining with different BCRP antibodies, and function did not correlate with mRNA nor protein expression. Presence of BCRP only in subpopulations and discordance among BCRP measurements suggest complex biology of BCRP in AML and incomplete modeling by cell lines.

Folic acid-enhanced synergy for the combination of trimetrexate plus the glycinamide ribonucleotide formyltransferase inhibitor 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin -6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034): comparison across sensitive and resistant human tumor cell lines.

Folic acid (PteGlu)-enhanced intense synergy has been observed between nonpolyglutamylatable dihydrofolate reductase (DHFR) inhibitors and polyglutamylatable inhibitors of other folate-requiring enzymes, such as glycinamide ribonucleotide formyltransferase (GARFT) and thymidylate synthase. Since this phenomenon is potentially therapeutically useful, we explored its universality by examining the combined action of a DHFR inhibitor, trimetrexate (TMQ), with a GARFT inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]++ +thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034), in eight human cultured cell lines. Using a 96-well plate cell growth inhibition assay, four ileocecal adenocarcinoma cell lines [HCT-8, HCT-8/DW2 (Tomudex-resistant), HCT-8/DF2 (Tomudex-/FdUrd-resistant), and HCT-8/50 (adapted to 50 nM PteGlu)], three head and neck carcinoma cell lines [A253, FaDu, and Hep-2/500 (FdUrd-resistant)], and a non-small cell lung carcinoma cell line [H460] were treated for 96 hr with TMQ + AG2034 in the presence of 23 or 40 microM PteGlu. Cell growth was measured with the sulforhodamine B assay at the end of this period. Drug interactions were assessed by fitting a 7-parameter model including a synergism parameter, alpha, to data with weighted nonlinear regression. Isobologram analysis was also applied. At 23 microM PteGlu, cells exhibited similar intensities of Loewe synergy for the combination of TMQ + AG2034. Loewe synergy was abolished in HCT-8/50 cells cultured and studied in 50 nM PteGlu. At 40 microM PteGlu, the intensity of the combined action in all cell lines was increased However, the most intense Loewe synergy was seen with HCT-8, HCT-8/DF2, H460, FaDu, A253, and Hep-2/500 cells, whereas the HCT-8/50 subculture showed less of the phenomenon, and PteGlu enhancement was the least with HCT-8/DW2, a subline deficient in folylpolyglutamate synthetase (FPGS). The universality of the PteGlu-enhanced intense synergy phenomenon is suggested. Impaired FPGS activity and low-folate adaptation prior to treatment significantly lessen the degree of PteGlu enhancement.

In vitro antitumor activity of 9-nitro-camptothecin as a single agent and in combination with other antitumor drugs.

Preclinical studies at Roswell Park Cancer Institute by Minderman, Cao, and Rustum (unpublished results) showed that a combination of SN-38 and 5-FU against HCT-8 human colon carcinoma cells in vitro was synergistic, with the best interaction occurring when the drugs were added sequentially, SN-38 first. Their in vivo studies using HCT-8 tumor xenografts implanted s.c. in nude athymic mice demonstrated superior efficacy for a sequential i.v. administration of CPT-11, 24 hr before 5-FU. On the basis of these studies, our group has begun to evaluate effects of RFS2000 (9-nitro-20(S)-camptothecin) (9-NC) in combination with a series of other antitumor agents. Using a panel of human tumor cell lines including A121 ovarian cancer, HCT-8 colon cancer, H-460 NSCLC, HT-1080 fibrosarcoma, and MCF7 mammary cancer, we found that a 2-hr exposure to 9-NC resulted in ID50 values of < 1.0 microM, whereas continuous exposure to drug resulted in ID50 values of < 1.0 nM. Tumor growth inhibitory activities of 5-FU, gemcitabine, and paclitaxel were determined for comparison. Combinations of these agents were evaluated with 9-NC using the human HCT-8 colon tumor cell line. Concurrent and sequential combinations of 9-NC with 5-FU had some regions of the concentration-effect surface with local synergy and some with local antagonism. However, sequential combination of 9NC or SN-38 followed by 5-FU, 24 hr later appeared to be highly synergistic at high dose-effect levels (i.e., ID90), suggesting that sequential drug administration may be more efficacious at high effect level and that the order of drug addition is very important. Overall, our results were similar to that found earlier by Rustum's group with CPT11 (or SN-38) and 5-FU, suggesting that sequential combination of 9-NC (or other camptothecin analogues) followed by 5-FU has potential for the treatment of cancer in man.

Combined action of paclitaxel and cisplatin against wildtype and resistant human ovarian carcinoma cells.

PURPOSE: The combination of paclitaxel (PTX) and cisplatin (DDP) shows good clinical efficacy against ovarian cancer. In order to examine the potential cellular basis for this, and provide leads as to how to optimize the combination, we examined the role of sequence of exposure to PTX and DDP on cell growth in vitro. METHODS: Four human ovarian carcinoma cell lines, A121, A2780/WT, A2780/DX5B and A2780/CP3, two human head and neck carcinoma cell lines, A253 and FaDu, and the human ileocecal carcinoma cell line, HCT-8, were treated with PTX + DDP with seven schedules: (A) 96 h exposure to PTX + DDP; (B) 24 h PTX alone, then 72 h PTX + DDP; (C) 4 h DDP alone, then 92 h PTX + DDP; (D) 24 h PTX alone, 4 h DDP alone, then 68 h drug-free; (E) 4 h DDP alone, 24 h PTX alone, then 68 h drug-free; (F) 3 h PTX alone, 1 h DDP alone, then 92 h drug-free; and (G) 1 h DDP alone, 3 h PTX alone, then 92 h drug-free. Each of 66 two-drug experiments included five plates (440 randomly treated wells per experiment). Cell growth was measured by the sulforhodamine B assay. The nature and the intensity of the drug interactions were assessed by fitting a seven-parameter model to data with weighted nonlinear regression, enabling the estimation of an interaction parameter, alpha, with its standard error. RESULTS: Overall there was very little departure from Loewe additivity: 43 experiments showed Loewe additivity, 10 showed Loewe antagonism, and 13 showed slight Loewe synergy. In vitro Loewe synergy was rare, was small when present, and reproducible only for the A121 and HCT-8 cells exposed to schedule D (24 h PTX prior to 4 h DDP). Isobolographic analysis showed complex combined-action surfaces with regions of local Loewe synergy and antagonism. CONCLUSION: It appears unlikely that the good clinical efficacy of the combination is primarily caused by a synergistic interaction at the cellular level.

Phase I clinical trial and human pharmacokinetics of 2,4-diamino-5-adamantyl-6-methyl pyrimidine ethane sulfonate (DAMP-ES): a lipid-soluble antifolate.

A phase I and pharmacokinetic study of a novel lipid-soluble antifolate, 2,4 diamino-5-adamantyl-6-methyl pyrimidine ethane sulfonate (DAMP-ES) has been carried out on two schedules: I--daily x5; II--24-h continuous infusion. In schedule I, doses of 10-90 mg/m2 per day were evaluated. Dose-limiting toxicity was hematologic, but nausea and vomiting, skin rash, diarrhea, anorexia, alopecia, mucositis, and neurotoxicity were also noted. In schedule II, doses of 192 and 240 mg/m2 were evaluated. Dose-limiting toxicity was neurotoxicity, but hematologic toxicity was also marked. Recommended starting doses for phase II studies are 75 mg/m2 per day for 5 days or 192 mg/m2 by continuous infusion for 24 h. Pharmacokinetic studies indicated a beta-phase plasma half-life of 12.4-24 h and a large and variable volume of distribution.

The validation of a new vascular damage assay for photodynamic therapy agents.

The therapeutic effect of photodynamic therapy (PDT: photodynamic sensitizer + light) is partly due to vascular damage. This report describes a new vascular photodamage assay for PDT agents and a validation of the assay. The method described here quantitates changes in tissue blood perfusion based on the relative amount of injected fluorescein dye in treated and untreated tissues. A specially designed fluorometer uses chopped monochromatic light from an argon laser as a source for exciting fluorescein fluorescence. The fluorescent light emitted from the tissue is collected by a six element fiberoptic array, filtered and delivered to a photodiode detector coupled to a phase-locked amplifier for conversion to a voltage signal for recording. This arrangement permits a rather simple, inexpensive construction and allows for the simultaneous use of the argon laser by other investigators. The routine assay for characterizing a specific photosensitizer at a standard dose consists of the sequential allocation of eight mice to a set of different light doses designed to span the dose-response range of fluorescein fluorescence exclusion (measured 8-10 min after fluorescein injection). The assay validation experiment used an anionic photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a at a dose of 0.4 mumol/kg. The parameter estimates (n = 34 mice) from fitting the standard Hill dose-response model to the data were: median fluorescence exclusion light dose FE50 = 275 +/- 8.3 J/cm2 and Hill sigmoidicity parameter m = -3.66 +/- 0.28. Subsets of the full data set randomly selected to simulate a standard eight mice experiment yielded similar parameter estimates. The new assay provides reliable estimates of PDT vascular damage with a frugal sequential experimental design.