AIDS-related Kaposi's sarcoma: a phase II study of liposomal doxorubicin. The TLC D-99 Study Group.

TLC D-99 is a unique liposomal formulation of doxorubicin that consists of phosphatidyl choline/cholesterol. The objectives of the study were to evaluate safety and efficacy of two doses of TLC D-99 in the treatment of patients with AIDS-related Kaposi's Sarcoma (KS). Forty HIV-infected persons with biopsy-proven KS were randomized to receive TLC D-99 at doses of either 10 (low) or 20 (high) mg/m2 every 2 weeks. Patients assigned to the low-dose arm could be escalated to the high-dose arm if their KS progressed after 3 cycles of therapy. Median age was 35 years (range, 26-47) and median CD4 count was 13 (range, 0-440). Nineteen patients were assigned to receive the low dose, and 21 patients were assigned to the high dose. Partial response occurred in 15% (6 of 40) of the patients or in 5% (1 of 19) and 24% (5 of 21) in the low- and high-dose arms, respectively; stable disease was observed in 65% (26 of 40) or in 68% (13 of 19) and 62% (13 of 21) in the low and high doses, respectively. Neutropenia was the major toxicity and was observed in 68 and 81% of patients with the low- and high-dose arms, respectively; grade 4 neutropenia was observed in 16 and 14%, respectively. Mild alopecia was noted in only 8%. Therefore, TLC D-99 is active against AIDS-related KS, and the response is dose-dependent.

Facilitated transport of methotrexate polyglutamates into lysosomes derived from S180 cells. Further characterization and evidence for a simple mobile carrier system with broad specificity for homo- or heteropeptides bearing a C-terminal glutamyl moiety.

Studies are presented further characterizing a facilitative system transporting methotrexate (MTX) polyglutamates into lysosomes derived from S180 cells. Initial influx of [3H]MTX + G1 (MTX with 1 additional glutamyl residue) exhibited a slightly alkaline pH optimum (pH 7.7) and was moderately temperature-dependent (Q10 27-37 degrees C = 3.1 +/- 0.1). An analysis of the kinetics of intralysosomal accumulation of [3H]MTX + G1 showed saturation kinetics for initial influx, but linear kinetics for the steady-state level of exchangeable [3H]MTX + G1 at different external concentrations of [3H]MTX + G1. In addition, the system exhibited substantial directional asymmetry with respect to the interaction with MTX + G1 during influx and efflux. Accelerated homo- and heteroexchange diffusion was demonstrated for influx of [3H]MTX + G1, while decelerated homoexchange diffusion was demonstrated for efflux of [3H]MTX + G1 following trans-positioning of MTX + G1 or glutamyl-gamma-glutamate in the opposite compartment. These observations were consistent with a single mobile carrier system mediating influx and efflux of this polyglutamate. Based upon an analysis of competitive interactions with [3H] MTX + G1, this system displayed specificity for MTX-gamma-glutamates, folyl-gamma-polyglutamates, alpha- or gamma-glutamyl peptides and heteropeptides bearing a C-terminal gamma-glutamate but not for MTX or glutamate, themselves. Among polyglutamates, gamma-glutamyl chain length was not a significant factor for transport except in the case of MTX polyglutamates. Overall, our results appear to delineate in the lysosomal membrane a simple mobile carrier system with broad specificity for folyl- or non-folyl-bearing peptides responsible for the transport of MTX polyglutamates.

Metabolic turnover of methotrexate polyglutamates in lysosomes derived from S180 cells. Definition of a two-step process limited by mediated lysosomal permeation of polyglutamates and activating reduced sulfhydryl compounds.

Transport and metabolic turnover of methotrexate (MTX) polyglutamates were examined in lysosomes derived from S180 cells. These studies extend prior work from this laboratory (Barrueco, J. R., and Sirotnak, F. M. (1991) J. Biol. Chem 266, 11732-11737) which described basic properties of a facilitative transport system in lysosomes capable of mediating intralysosomal accumulation of MTX polyglutamates. In the present report, we show that the rate of turnover of MTX polyglutamates in lysosomes, which releases MTX in the extralysosomal space, is limited by the extent of mediated intralysosomal accumulation of the polyglutamate and reduced sulfhydryls that activate the enzyme folylpolyglutamate hydrolase. Evidence is presented that cysteine functions as the naturally occurring reduced sulfhydryl compound in lysosomes being equipotent to 2-mercaptoethanol as an activator of folylpolyglutamate hydrolase. Folylpolyglutamate hydrolase in permeabilized lysosomes from S180 cells exhibited a low pH optimum characteristic of a lysosomal enzyme, was activated at concentrations of reduced sulfhydryl at 0.1 mM and above, and exhibited Km values in the range of 0.2-3 microM that decreased with increase in polyglutamate chain length. Values for Km for MTX polyglutamates of folylpolyglutamate hydrolase activity were 100-200-fold lower than values for Km or Ki for facilitated intralysosomal transport, whereas capacities for both processes were similar. This relationship between the kinetic properties of each process ensures efficient hydrolysis of MTX polyglutamates within the lysosome.

Evidence for the facilitated transport of methotrexate polyglutamates into lysosomes derived from S180 cells. Basic properties and specificity for polyglutamate chain length.

Evidence is presented outlining basic properties of a previously undescribed facilitative transport system mediating transfer of methotrexate (MTX) polyglutamates from the cytoplasmic to the lysosomal compartment of the cell. These experiments were conducted using purified lysosomes prepared from murine S180 cells, and a model substrate ([3H]MTX + G1; methotrexate with 1 additional glutamyl residue) to examine biological aspects as well as pharmacological significance of this process in a tumor cell model. The data, expressed as a function of latent beta-hexosaminidase activity, a measure of lysosomal integrity, show that [3H]MTX + G1 uptake in lysosomes is temperature-dependent, is stimulated specifically by magnesium chloride and potassium chloride with maximal enhancement observed in the presence of both agents together, exhibits Michaelis-Menten saturation kinetics with Km and Vmax values of 346 +/- 39 microM and 2.8 +/- 0.3 pmol/min/unit of beta-hexosaminidase activity, respectively, and is competitively inhibited by longer chain polyglutamates with increasing effectiveness as shown by Ki values of 334 +/- 19, 201 +/- 16, 106 +/- 13, and 42 +/- 8 microM, for MTX + G1, MTX + G2, MTX + G3, and MTX + G4, respectively. In addition, uptake is inversely related to medium osmolarity indicating that the phenomenon we observe represents internalization of the [3H]MTX + G1 and not adsorption to a possible surface binding site. As a whole, the data are consistent with a single mediated transport system shared by all MTX polyglutamates for entry into lysosomes. It is our view that this transport system represents the initial step in the degradation of polyglutamates in the cell. In addition, based on a comparative analysis of the kinetics for hydrolysis and transport, we suggest that it is also the limiting step in this process and, as such, regulates the extent of degradation of the free cellular pools of these compounds.

Differing specificities for 4-aminofolate analogues of folylpolyglutamyl synthetase from tumors and proliferative intestinal epithelium of the mouse with significance for selective antitumor action.

Folylpolyglutamyl synthetase (FPGS), partially purified from murine L1210 leukemia and Sarcoma 180 cells and the proliferative fraction of luminal epithelium from mouse small intestine (the site of limiting toxicity to folate analogues), was examined for its ability to utilize various 4-aminofolates as substrates. For tumor-derived FPGS, aminopterin was the most preferred substrate overall, exhibiting the lowest value for apparent Km and highest Vmax. The other analogues and folic acid exhibited nearly 2-fold lower Vmax. Folic acid exhibited a 3-fold higher Km than aminopterin. Alkylation of aminopterin (methotrexate) or carbon for nitrogen substitution (10-deazaaminopterin) at N-10 increased Km 3- to 6-fold, while alkylation at C10 (10-ethyl-10-deazaaminopterin) restored Km to near equivalency with aminopterin. For FPGS derived from proliferative intestinal epithelium, aminopterin was also the preferred substrate, but the value for Vmax (derived with crude cell-free extract) was 6-fold lower than for tumor cell FPGS. Values for Vmax (derived with partially purified FPGS) for the other 4-aminofolate analogues and folic acid were similar (methotrexate) or 2-fold (10-ethyl-10-deazaaminopterin) and 5-fold (folic acid) lower than for aminopterin. The value for Km derived with aminopterin was similar to that derived for either tumor cell FPGS. The value for folic acid was 2-fold higher, and alkylation of aminopterin (methotrexate) or carbon to nitrogen substitution (10-deazaaminopterin) at N-10 with (10-ethyl-10-deazaaminopterin) or without alkylation markedly increased Km (27-, 90-, and greater than 100-fold, respectively, for methotrexate, 10-ethyl-10-deazaaminopterin, and 10-deazaaminopterin). In other studies, it was found that the diglutamate of aminopterin (aminopterin +G1) was a relatively poor substrate for FPGS derived from all three sources compared with methotrexate diglutamate, both in respect to values for Km and Vmax that were measured in each case. Findings with FPGS derived from L1210 cells were confirmed by high-pressure liquid chromatography analysis of product formation during the reaction with the parent compounds. The significance of the results presented here to the question of relative toxicity and therapeutic activity of these analogues is discussed.

Altered molecular properties of tubulin in a multidrug-resistant variant of Chinese hamster cells selected for resistance to vinca alkaloids.

The basis for the markedly altered intracellular binding of [3H]vincristine in a multidrug-resistant variant (DC-3F/VCRd-5L) of Chinese hamster lung cells (DC-3F) was investigated. Binding of [3H]vincristine by protein in cytosol derived from each cell type exhibited a differing requirement for GTP in MgCl2 containing buffer of low-ionic strength. Binding of [3H]vincristine occurred to cytosolic protein derived from both variant and parental DC-3F cells, but after removal of GTP, binding only occurred to cytosolic protein from parental cells regardless of the presence of added GTP. Although binding by cytosolic protein from parental DC-3F cells did not require GTP, the addition of 0.1 mM GTP increased by two-fold the rate and extent of binding. When cytosol from variant and parental DC-3F cells was incubated with low concentrations of [3H]vincristine in high-ionic strength buffer and analyzed by molecular-sieve HPLC, most of the protein binding [3H]vincristine in parentally derived cytosol eluted as Mr 110,000-115,000 daltons, corresponding to that for dimeric tubulin. The same binding species was not detected in cytosol derived from variant cells. However, these same fractions derived with both parental and variant cytosols contained tubulin as shown by SDS-PAGE and immunoblotting. A smaller peak of [3H]vincristine binding and an amount of tubulin equal to that found in later fractions were found in the void volume during the same HPLC elution runs with cytosol from both variant and parental DC-3F cells. Evidence was also obtained for differences between parental and variant DC-3F cells in beta-tubulin isoforms following isoelectric focusing and immunoblotting. Parental-cell cytosol contains a single isoform of beta-tubulin. However, in variant cell cytosol the same isoform and, in addition, three more basic isoforms were found. These alterations in [3H]vincristine binding and in isoform compositions of beta-tubulin in variant versus parental DC-3F cells may have importance in regard to vincristine resistance in DC-3F cells.

Enhancement of folate analogue transport inward in L1210 cells during methotrexate therapy of leukemic mice: evidence of the nature of the effect, possible host mediation, and pharmacokinetic significance.

Studies are described that sought the basis for a discrepancy in values for a key kinetic parameter of methotrexate transport (influx Vmax) in L1210 cells derived alternately from biochemical or pharmacokinetic measurements. Our results show that, within a short period of time following administration of a therapeutic dose of methotrexate to leukemic mice, influx of this folate analogue measured in L1210 cells removed from these mice was markedly stimulated. Enhancement of [3H]methotrexate influx in these cells was observed within 15 min of drug administration, was maximum (up to 3-fold) within 2 to 3 h, then decreased with time until 24 h when influx was at the control level. Measurements of [3H]methotrexate influx in cells removed from drug-treated mice were made after a period of incubation in drug-free medium to allow for efflux of exchangeable drug. Enhanced influx of [3H]methotrexate was accounted for by an increase in influx Vmax (influx Km was unchanged) and was further enhanced (to a total of 5-fold) by coadministration of leucovorin. Also, enhancement of influx of [3H]methotrexate in L1210 cells did not occur following administration of 1-beta-D-arabinofuranosylcytidine at a therapeutically equivalent dose to leukemic mice or following exposure of these cells to methotrexate or methotrexate with leucovorin during growth in culture. Methotrexate therapy did not affect all transport systems, since the same therapy of leukemic mice had no effect on influx of the purine nucleoside analogue, 9-beta-D-arabinofuranosyl-2-fluoroadenine, in these same L1210 cells. These findings suggest that stimulation of [3H]methotrexate influx in L1210 cells during therapy with this folate analogue was not due to transstimulation during exchange between folate compounds and was not related to the antiproliferative effect of methotrexate on these tumor cells. The coadministration of cycloheximide with methotrexate to leukemic mice at a dose which markedly inhibited 3H-leucine incorporation into L1210 cell protein severely diminished the stimulation of [3H]methotrexate influx. However, in L1210 cells removed from leukemic mice treated with methotrexate, there was no increase compared to control cells in affinity labeling with the N-hydroxysuccinimide ester of [3H]methotrexate. This suggested that the effect of cycloheximide was not on increased synthesis of folate transporter and that increased rate of translocation of folate transporter, rather than increased amount of transporter, accounted for the increase in [3H]methotrexate influx.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis of the 7-hydroxy metabolites of methotrexate and 10-ethyl-10-deazaaminopterin.

The potent folate antagonists methotrexate and 10-ethyl-10-deazaaminopterin are oxidized in vivo to their 7-hydroxy derivatives. A specific cyanation at the C-7 position of the pteridine ring system using diethyl phosphorocyanidate converted the dimethyl esters of methotrexate and 10-ethyl-10-deazaaminopterin to the corresponding 7-cyano dimethyl ester derivatives, which were hydrolyzed to the 7-hydroxy metabolites.

Carrier-mediated transport of folate compounds in L1210 cells. Initial rate kinetics and extent of duality of entry routes for folic acid and diastereomers of 5-methyltetrahydrohomofolate in the presence of physiological anions.

Comparison of the kinetic parameters for influx of highly purified [3H]folic acid versus [3H]methotrexate in L1210 cells under anionic buffer conditions showed a marked discordancy. In addition, the kinetics for influx of [3H]folic acid were unchanged in variant L1210 cells defective in [3H]methotrexate transport. In these variant cells, the Vmax for methotrexate was reduced 17-fold and the Km was increased 3-fold. The results show that [3H]folic acid influx is mediated by a system which has a low affinity, but a 20-fold higher capacity, for folate compounds than the classical high-affinity system mediating [3H]methotrexate influx. Since the latter system also exhibits very low affinity for [3H]folic acid, it would not be expected to contribute significantly to the total influx of [3H]folic acid. The high-capacity system for [3H]folic acid influx is different from that believed to mediate pterin influx in L1210 cells since it was not inhibited by adenine, a potent inhibitor of pterin influx. However, exposure of cells to [3H]folic acid in a nonanionic buffer resulted in marked stimulation of initial influx, and a fraction of influx under these conditions was inhibited by methotrexate. These results suggest that anions modulate the extent of multiplicity of [3H]folic acid influx by their known effects on the high-affinity, reduced folate/methotrexate system. The diastereomers, at carbon 6, of [14C]5-methyltetrahydrohomofolate shared both transport systems. The influx Km for the natural diastereomer was one-half that of the unnatural form for both transport systems. Both diastereomers showed a much greater differential in affinity between the two transport systems than did [3H]folic acid. Our results suggest that an analog which could be effectively transported by the low-affinity/high-capacity route may be useful in the treatment of tumors resistant to methotrexate due to a defective high-affinity/low capacity influx system. We also found that incubation of L1210 cells with [3H]folic acid or the natural diastereomer [14C]5-methyltetrahydrohomofolate for 10 min resulted in the formation of a nonexchangeable fraction of radioactivity amounting to 20-40% of the total accumulation. This non-exchangeable fraction may be explained by the accumulation of metabolites other than polyglutamates. Preloading of cells with methotrexate prior to incubation with [3H]folic acid prevented the accumulation of radioactivity as a nonexchangeable fraction.

Proposed mechanism of therapeutic selectivity for 9-beta-D-arabinofuranosyl-2-fluoroadenine against murine leukemia based upon lower capacities for transport and phosphorylation in proliferative intestinal epithelium compared to tumor cells.

Studies have examined transport and phosphorylation of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-Ara-A), a deaminase resistant adenosine analogue, as mechanisms that could mediate the observed therapeutic efficacy of this agent against murine tumor models. Earlier finds by Avramis and Plunkett (Cancer Res., 42: 2587-2591, 1982) showed markedly less accumulation in vivo of administered F-Ara-A as cytotoxic triphosphate in gastrointestinal mucosa and bone marrow compared to P388 cells. We have pursued the basis for this difference in vitro using L1210 ascites and proliferative epithelial cells (85-95% crypt cells) isolated from mouse small intestine as representative sample populations of drug-sensitive tumor and drug-limiting normal regenerative host tissue. Using a rapid sampling technique, linear initial rates of substrate uptake were established at 25 degrees C for radiolabeled F-Ara-A and adenosine at a concentration range of 1-1000 microM. The relationship between velocity of initial transport and substrate concentration is indicative of Michaelis-Menten saturation kinetics for both substrates. Competition studies between F-Ara-A and adenosine suggest a common route of entry for both substrates in crypt epithelial cells. Results from double-reciprocal analysis of the velocity versus concentration data are consistent with a simple carrier-mediated facilitated diffusion process with Km, V25max, and Ki values of 317 +/- 44 (SE) microM, 49 +/- 7 nmol/s/g dry weight, and 301 +/- 34 microM for F-Ara-A, and 264 +/- 14 microM, 44 +/- 5 nmol/s/g dry weight, and 225 +/- 44 microM for adenosine, respectively. The presence of a single low-affinity carrier in the proliferative epithelial cells contrasts sharply with the high affinity (Km, 68 +/- 14 microM; V25max, 48 +/- 4 nmol/s/g dry weight) and low-affinity (Km, 326 +/- 48 microM; V25max, 124 +/- 44 nmol/s/g dry weight) routes of entry documented for L1210 cells. This differential in transport kinetics conveys a 7- to 8-fold greater capacity to L1210 ascites compared with crypt epithelial cells for uptake of the antitumor agent F-Ara-A. At pharmacologically achievable concentrations of F-Ara-A and in view of this differential, influx of F-Ara-A would be more rate limiting to phosphorylation of F-Ara-A in epithelial cells than in L1210 cells. Metabolism studies with L1210 ascites and proliferative intestinal epithelial cells show that intracellular phosphorylation of F-Ara-A is also elevated in L1210 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane transport and the antineoplastic action of nucleoside analogues.

This article summarizes recent studies characterizing nucleoside transport in mammalian cells and discusses evidence for a role of membrane transport in the pharmacologic action of nucleoside analogues. Some of these studies have also addressed the controversy concerning the multiplicity in transport routes. It seems clear that erythrocytes and, perhaps, some other mammalian cells possess a single, broadly specific system for transporting nucleosides. However, substantial evidence from valid studies discriminating between transport and intracellular metabolism suggests that at least some mammalian cells, including some tumor cells, possess more than a single system. Evidence now exists for a determining role of membrane transport of nucleoside analogues in their cytotoxicity and, in the case of one pyrimidine nucleoside (AraC), in therapeutic responsiveness in leukemic patients. There are also numerous examples of transport-related resistance to nucleoside analogues. Included in this article are the results of studies from the authors' laboratory pertaining to the therapeutic activity of the purine nucleoside, FAraA, in murine tumor models. These studies provide evidence for a determining role of both membrane transport and intracellular phosphorylation in the selective antitumor action of this agent against murine leukemia. Substantially increased transport inward of FAraA occurs at pharmacologically achievable concentrations of this agent in tumor cells as compared to drug-limiting, normal proliferative epithelium of the small intestine. The basis for this differential appears to be the kinetic duality of FAraA and adenosine transport inward found in tumor cells, but not in proliferative intestinal epithelial cells. Tumor cells have highly saturable (low influx Km) and poorly saturable (high influx Km) systems for adenosine transport, both of which are shared by FAraA. In contrast, proliferative epithelial cells have only a poorly saturable system for these substrates. If a similar kinetic duality of nucleoside transport is found in other tumor cells certain implications arise concerning the significance of the duality to neoplastic transformation.