Transport of fluorescein in MDCKII-MRP1 transfected cells and mrp1-knockout mice.

The multidrug resistant-associated protein 1 (MRP1) is a membrane-bound transport protein that is involved in the efflux of organic anions and has been implicated in multidrug resistance in cancer. MRP1 has also been reported to be ubiquitously expressed in normal tissues, including the brain. The presence of functional organic anion transporters in the blood-brain and blood-CSF barriers that influence the distribution of various compounds to the brain has long been known. The purpose of this study was to examine the role of MRP1 in the brain distribution of a model organic anion, fluorescein. The substrate specificity of MRP1 for fluorescein was initially determined by examining the accumulation of fluorescein in MDCKII MRP1-transfected cells. The distribution of fluorescein in the brain was then examined in wild-type and mrp1 gene knockout mice. The results show that in MDCKII MRP1-transfected cells, the accumulation of fluorescein was significantly lower (about 40% lower) than that in wild-type MDCKII cells. MRP1 inhibitors such as probenecid, MK-571, and LY402913 enhanced fluorescein accumulation in MDCKII MRP1-transfected cells to a greater extent than in wild-type MDCKII cells. In an in vivo study, after intravenous injection of fluorescein, the fluorescein brain-to-plasma concentration ratio in mrp1 knockout mice was not significantly different than that in wild-type mice. However, when probenecid was co-administered with fluorescein in wild-type mice, the fluorescein brain-to-plasma ratio was significantly increased (1.5-fold). These findings suggest that fluorescein is a substrate for MRP1. Furthermore, the in vivo study also suggests that MRP1 has a limited role in the transport and distribution of fluorescein in the brain. Therefore, other organic anion transport proteins, including the various isoforms of the MRP family, may be responsible for the accumulation and transport of organic anions in the brain.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101M): a novel sulfonylhydrazine prodrug with broad-spectrum antineoplastic activity.

Our laboratory has synthesized and evaluated the anticancer activity of a number of sulfonylhydrazine DNA modifying agents. As a class, these compounds possess broad spectrum antitumor activity, demonstrating significant activity against a variety of experimental murine tumors, including the P388 and L1210 leukemias, B16 melanoma, M109 lung carcinoma, and M5076 reticulum cell sarcoma, as well as against the human LX-1 lung carcinoma xenograft. The current report describes the activity of a more recently synthesized member of this class, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine (101M). 101M was active in mice against the i.p. implanted L1210 leukemia over a wide range of doses and produced long-term survivors when administered as a single i.p. bolus of 10, 20, 40, 60, or 80 mg/kg, demonstrating a wider margin of safety than the nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Curative therapy was achieved with doses of 101M that did not produce depression of the bone marrow. 101M was also highly effective against the L1210 leukemia when administered by the oral route. The ability of 101M to penetrate the blood-brain barrier and eradicate leukemia cells in the brain was remarkable (>6 log kill). This agent was also curative against L1210 variants resistant to cyclophosphamide, BCNU, or melphalan. Mice implanted with the murine C26 colon carcinoma were also cured by two injections of 10 or 20 mg/kg of 101M. Administration of 101M by two different well-tolerated regimens caused complete regression of established human glioblastoma U251 xenografts in 100% of treated mice, and significant responses were also obtained with 101M against advanced murine M109 lung carcinomas in mice. The broad spectrum of anticancer activity of the sulfonylhydrazine prodrug 101M coupled with the wide range of therapeutic safety exhibited by this agent, makes 101M particularly attractive for further development and clinical evaluation.

The pharmacological phenotype of combined multidrug-resistance mdr1a/1b- and mrp1-deficient mice.

Two major classes of plasma membrane proteins that actively extrude a wide range of structurally diverse hydrophobic amphipathic antineoplastic agents from cells, with different mechanisms of action, lead to multidrug resistance. To study the importance of these ATP-binding cassette transporters to the toxicity of cancer chemotherapy agents, we have used mice genetically deficient in both the mdr1a and mdr1b genes [mdr1a/1b(-/-) mice], the mrp1 gene [mrp1(-/-) mice], and the combined genes mdr1a/1b and mrp1 [mdr1a/1b(-/-), mrp1(-/-) mice] and embryonic fibroblasts derived from wild-type mice and from the three gene knockout animals. The consequences of export pump deficiencies were evaluated primarily using vincristine and etoposide. Mice deficient in the three genes, mdr1a/1b and mrp1, exhibited a 128-fold increase in toxicity to vincristine and a 3-5-fold increase in toxicity to etoposide; increased toxicity to embryonic fibroblast cells from triple knockout mice also occurred with vincristine and etoposide. Vincristine, which normally does not express toxicity to the bone marrow and to the gastrointestinal mucosa when used at therapeutic doses, caused extensive damage to these tissues in mdr1a/1b(-/-), mrp1(-/-) mice. The findings indicate that the P-glycoprotein and mrpl are compensatory transporters for vincristine and etoposide in the bone marrow and the gastrointestinal mucosa and emphasize the potential for increased toxicities by the combined inhibition of these efflux pumps.

Evaluation of a reproductive toxicity assay using Xenopus laevis: boric acid, cadmium and ethylene glycol monomethyl ether.

Cadmium (Cd), boric acid (BA) and ethylene glycol monomethyl ether (EGME) were evaluated for reproductive and developmental toxicity in Xenopus laevis. Eight reproductively mature adult male and eight superovulated female Xenopus laevis were exposed to at least five separate sublethal concentrations of each material via the culture water for a period of 30 days. Four respective pairs were mated and the offspring evaluated for developmental effects; an evaluation of reproductive status was performed on the remaining four specimens. Ovary pathology, oocyte count, oocyte maturity and maturation capacity (germinal vesicle breakdown, GVBD) and necrosis were evaluated in the female, whereas testis pathology, sperm count, dysmorphology and motility were studied in the male. Based on this assessment, each test material exerted reproductive toxicity in Xenopus laevis, but with varying potencies. Adult female exposure to Cd and EGME particularly, and to a lesser extent to BA, resulted in transgenerational toxicity to the developing progeny. Further, this model appears to be a useful tool in the initial assessment and prioritization of potential reproductive toxicants for further testing.

The leukotriene C(4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC)-dependent mobilization of dendritic cells to lymph nodes.

Adaptive immune responses begin after antigen-bearing dendritic cells (DCs) traffic from peripheral tissues to lymph nodes. Here, we show that DC migration from skin to lymph nodes utilizes the leukotriene C(4) (LTC(4)) transporter multidrug resistance-associated protein 1 (MRP1). DC mobilization from the epidermis and trafficking into lymphatic vessels was greatly reduced in MRP1(-/-) mice, but migration was restored by exogenous cysteinyl leukotrienes LTC(4) or LTD(4). In vitro, these cysteinyl leukotrienes promoted optimal chemotaxis to the chemokine CCL19, but not to other related chemokines. Antagonism of CCL19 in vivo prevented DC migration out of the epidermis. Thus, MRP-1 regulates DC migration to lymph nodes, apparently by transporting LTC(4), which in turn promotes chemotaxis to CCL19 and mobilization of DCs from the epidermis.

Maintenance of retinoic acid receptor alpha pools by granulocyte colony-stimulating factor and lithium chloride in all-trans retinoic acid-treated WEHI-3B leukemia cells: relevance to the synergistic induction of terminal differentiation.

Previous studies have demonstrated that combinations of all-trans retinoic acid (ATRA) with either granulocyte-colony stimulating factor (G-CSF) or lithium chloride (LiCl) produced synergistic terminal differentiation of WEHI-3B myelomonocytic leukemia (D(+)) cells. It was found that steady-state retinoic acid receptor alpha (RARalpha) protein levels were markedly reduced in these cells after exposure to ATRA. Because the presence of receptors for a hormone ligand is required for its action, differentiation therapy with ATRA may be self-limiting. The combination of G-CSF with ATRA significantly attenuated the loss of RARalpha protein, and synergistic terminal differentiation occurred. LiCl was more effective than G-CSF in preserving RARalpha pools and synergized with ATRA more strongly than G-CSF. These findings suggested that the prevention of RARalpha protein loss by G-CSF or LiCl in ATRA-treated cells functioned to extend the differentiation response to the retinoid and was responsible, at least in part, for the observed synergism. D(+) cells transfected with an expression plasmid containing RARalpha cDNA had a 6- to 8-fold increase in steady-state RARalpha mRNA compared with vector-transfected cells and showed a 2- to 3-fold increase in RARalpha protein. ATRA caused a reduction, but not a complete loss, of RARalpha protein in these transfectants, which were considerably more responsive than parental D(+) cells to ATRA as a single agent, supporting the concept that the protection of RARalpha pools results in a heightened differentiation response to ATRA.

Triapine (3-aminopyridine-2-carboxaldehyde- thiosemicarbazone): A potent inhibitor of ribonucleotide reductase activity with broad spectrum antitumor activity.

Previous studies from our laboratories have shown that (a) Triapine() is a potent inhibitor of ribonucleotide reductase activity and (b) hydroxyurea-resistant L1210 leukemia cells are fully sensitive to Triapine. In an analogous manner, Triapine was similarly active against the wild-type and a hydroxyurea-resistant subline of the human KB nasopharyngeal carcinoma. Triapine was active in vivo against the L1210 leukemia over a broad range of dosages and was curative for some mice. This agent also caused pronounced inhibition of the growth of the murine M109 lung carcinoma and human A2780 ovarian carcinoma xenografts in mice. Optimum anticancer activity required twice daily dosing due to the duration of inhibition of DNA synthesis which lasted about 10 hr in L1210 cells treated with Triapine in vivo. DNA synthesis in normal mouse tissues (i.e. duodenum and bone marrow) uniformly recovered faster than that in L1210 leukemia cells, demonstrating a pharmacological basis for the therapeutic index of this agent. Triapine was more potent than hydroxyurea in inhibiting DNA synthesis in L1210 cells in vivo, and the effects of Triapine were more pronounced. In addition, the duration of the inhibition of DNA synthesis in leukemia cells from mice treated with Triapine was considerably longer than in those from animals treated with hydroxyurea. Combination of Triapine with various classes of agents that damage DNA (e.g. etoposide, cisplatin, doxorubicin, and 1-acetyl-1,2-bis(methylsulfonyl)-2-(2-chloroethyl)hydrazine) resulted in synergistic inhibition of the L1210 leukemia, producing long-term survivors of tumor-bearing mice treated with several dosage levels of the combinations, whereas no enhancement of survival was found when Triapine was combined with gemcitabine or cytosine arabinoside. The findings demonstrate the superiority of Triapine over hydroxyurea as an anticancer agent and further suggest that prevention by Triapine of repair of DNA lesions created by agents that damage DNA may result in efficacious drug combinations for the treatment of cancer.

Evaluation of the developmental toxicity of thalidomide using frog embryo teratogenesis assay-xenopus (FETAX): biotransformation and detoxification.

The developmental toxicity of thalidomide was evaluated using FETAX (Frog Embryo Teratogenesis Assay - Xenopus). Young X. Laevis embryos were exposed to this compound in each of two concentration-response experiments with and without differently induced exogenous metabolic activation systems (MASs) and/or inhibited MASs. Young male Sprague-Dawley rats were treated with either isoniazid or Aroclor 1254 to induce cytochrome P-450. Several of the rats were subsequently treated with diethyl maleate (DM) to deplete glutathione reserves. Specific aliquots of rat liver microsomes were treated with 3-amino-1,2,4-triazole (ATZ) or alpha-napthoflavone (alpha-N) to selectively inhibit P-450 activity. Bioactivation was indicated by increased developmental toxicity observed in MAS tests. Results obtained indicated that thalidomide was predominantly activated by P-450 isozyne CYP2E1, although weak cross-specificity between CYP1A1/A2 may have existed. Detoxification pathways for thalidomide were investigated by treatment of the MAS with cyclohexene oxide (CHO) and DM to inhibit the epoxide hydrolase and glutathione conjugation pathways, respectively. Results indicated that epoxide hydrolase was primarily responsible for the detoxification of bioactivated thalidomide. Teratogenesis Carcinog. Mutagen. 20:35-47, 2000.

Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; 3-AP): an inhibitor of ribonucleotide reductase with antineoplastic activity.

The enzyme RR catalyzes the conversion of ribonucleoside diphosphates to their deoxyribonucleotide counterparts. RR is critical for the generation of the cytosine, adenine, and guanine deoxyribonucleotide 5'-triphosphate building blocks of DNA, which are present in cells as exceedingly small intracellular pools. Therefore, interference with the function of RR might well result in an agent with significant antineoplastic activity, particularly against rapidly proliferating tumor cells. HUr is the only inhibitor of RR in clinical usage; this agent, however, is a relatively poor inhibitor of the enzyme and has a short serum half-life. Consequently, HUr is a relatively weak anticancer agent. In an effort to develop a more potent inhibitor of RR with utility as an anticancer agent, we have synthesized 3-AP and demonstrated (a) potent inhibition of L1210 leukemia cells in vitro, (b) curative capacity for mice bearing the L1210 leukemia, (c) marked inhibition of RR, and (d) sensitivity of HUr-resistant cells to 3-AP. These findings collectively demonstrate the clinical potential of 3-AP as an antineoplastic agent.

New insights into the biology and pharmacology of the multidrug resistance protein (MRP) from gene knockout models.

Growing interest in the MRP (multidrug resistance protein) gene stems from its importance in multidrug resistance to chemotherapy, its possible use in gene therapy, and its relationship with the glutathione system. The recent generation of mrp gene knockout models in vitro and in vivo is providing information on the mechanism of action and the physiological function(s) of mrp. The importance of mrp in protection of normal tissues from the toxicity of the anticancer agent etoposide has been established. A total block of mrp has been found to be compatible with life, suggesting that MRP inhibitors can be safely used for treating cancer patients. In some sub-classes of leukocytes, mrp contributes to the transport of leukotriene C4, an endogenous glutathione-S-conjugate. However, the baseline expression of mrp does not appear to contribute to the export of glutathione-S-conjugates of alkylating agents, and thus does not exert a protective role against their toxicity. Besides being capable of exporting certain glutathione-S-conjugates, mrp also catalyzes the co-transport of GSH and drug and, presumably, a presently unknown endogenous metabolite(s).

Role of vitamin D3 receptor in the synergistic differentiation of WEHI-3B leukemia cells by vitamin D3 and retinoic acid.

WEHI-3B D- cells differentiate in response to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)2D3 interact to produce synergistic differentiation of WEHI-3B D- cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)2D3 receptor (VDR) and retinoid receptors, RARalpha and RXRalpha, was measured. No VDR was detected in untreated WEHI-3B D- cells; however, RA and 1,25-(OH)2D3 when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARalpha and RXRalpha were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)2D3 produced synergistic differentiation of WEHI-3B D- cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D+ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)2D3 in WEHI-3B D+ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)2D3 and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1, 25-(OH)2D3.

Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood-cerebrospinal-fluid drug-permeability barrier.

The blood-brain barrier and a blood-cerebrospinal-fluid (CSF) barrier function together to isolate the brain from circulating drugs, toxins, and xenobiotics. The blood-CSF drug-permeability barrier is localized to the epithelium of the choroid plexus (CP). However, the molecular mechanisms regulating drug permeability across the CP epithelium are defined poorly. Herein, we describe a drug-permeability barrier in human and rodent CP mediated by epithelial-specific expression of the MDR1 (multidrug resistance) P glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP). Noninvasive single-photon-emission computed tomography with 99mTc-sestamibi, a membrane-permeant radiopharmaceutical whose transport is mediated by both Pgp and MRP, shows a large blood-to-CSF concentration gradient across intact CP epithelium in humans in vivo. In rats, pharmacokinetic analysis with 99mTc-sestamibi determined the concentration gradient to be greater than 100-fold. In membrane fractions of isolated native CP from rat, mouse, and human, the 170-kDa Pgp and 190-kDa MRP are identified readily. Furthermore, the murine proteins are absent in CP isolated from their respective mdr1a/1b(-/-) and mrp(-/-) gene knockout littermates. As determined by immunohistochemical and drug-transport analysis of native CP and polarized epithelial cell cultures derived from neonatal rat CP, Pgp localizes subapically, conferring an apical-to-basal transepithelial permeation barrier to radiolabeled drugs. Conversely, MRP localizes basolaterally, conferring an opposing basal-to-apical drug-permeation barrier. Together, these transporters may coordinate secretion and reabsorption of natural product substrates and therapeutic drugs, including chemotherapeutic agents, antipsychotics, and HIV protease inhibitors, into and out of the central nervous system.

Identification of a repressor of the differentiation of WEHI-3B D- leukemia cells.

The WEHI-3B D+ leukemia is a near-diploid differentiation-competent cell line that undergoes myeloid differentiation in response to retinoic acid. WEHI-3B D- cells, derived from WEHI-3B D+ cells, are near tetraploid and not responsive to the differentiation-inducing properties of the retinoid. To gain information on mechanisms that regulate the maturation of these two cell lines, several multiploid cell lines have been established through fusion of WEHI-3B D+ and WEHI-3B D- cells. Studies with the multiploid cell lines have shown that (a) the cellular growth rate decreases with increased DNA ploidy; (b) near-tetraploid D+/+ cells, obtained by fusing WEHI-3B D+ with WEHI-3B D+ cells, remain differentiation-competent, demonstrating that no direct relationship exists between differentiation competency and DNA ploidy; and (c) near-hexaploid D +/- and D -/+ cells, formed by fusion of WEHI-3B D+ with WEHI-3B D- cells, do not respond to differentiation inducers, suggesting the inhibition of the differentiation machinery of WEHI-3B D+ cells by components from maturation-incompetent WEHI-3B D- cells. The scl transcription factor gene is expressed in WEHI-3B D- cells and is absent in WEHI-3B D+ cells. Overexpression of scl by transfection of scl cDNA in WEHI-3B D+ cells markedly decreased the capacity of retinoic acid to induce differentiation, suggesting that scl functions as a repressor of differentiation in WEHI-3B cell lines.

Disruption of the murine MRP (multidrug resistance protein) gene leads to increased sensitivity to etoposide (VP-16) and increased levels of glutathione.

The mrp (multidrug resistance protein) gene has been associated with the multidrug resistance of cancer cells in vitro and in vivo. To gain information on its physiological role, embryonic stem cells were used to generate mice homozygous for a disruption of the mrp gene, resulting in complete abrogation of mrp expression. No physiological abnormalities were observed, at least up to 4 months of age. Viability, fertility, and a range of histological, hematological, and serum-chemical parameters were similar in mrp(+/+) and mrp(-/-) mice. mrp(-/-) mice displayed an increased sensitivity to etoposide phosphate (2-fold) accompanied by greater bone marrow toxicity, whereas the acute toxicity of sodium arsenite was equivalent in mrp(+/+) and mrp(-/-) mice. Tissue levels of glutathione (GSH) were elevated in breast, lung, heart, kidney, muscle, colon, testes, bone marrow cells, blood mononuclear leukocytes, and blood erythrocytes of mrp(-/-) mice and were unchanged in organs known to express little if any mrp, such as the liver and small intestine. The increase in GSH was not due to an increase in the activity of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis. The findings demonstrate that mrp is dispensable for development and growth but exerts a role in drug detoxification and GSH metabolism.

Structural and functional relationships of toyocamycin on NPM-translocation.

Toyocamycin is an antitumor antibiotic which has a pyrrolo[2,3-D]pyrimidine aglycone with a -CN substituent on the 5-carbon. Treatment of HeLa cells with toyocamycin induces redistribution of the nuclear phosphoprotein nucleophosmin/B23 (NPM) from nucleoli to nucleoplasm (NPM-translocation) which can be detected by immunofluorescence. NPM-translocation is useful in showing drug effects and in detecting drug-resistant cancer cells. To study which structural features of toyocamycin are important for NPM-translocation, we used toyocamycin analogs in which the 5-position -CN was either deleted (tubercidin) or replaced with a -CONH2 (sangivamycin) or -C(NOH)NH2. HeLa cells were incubated with these analogs for 4 h and assayed for NPM-translocation by immunofluorescence. We found that the analog with the deletion of the -CN group (tubercidin) did not induce translocation while those with replacement of the -CN group with -CONH2 or -C(NOH)NH2 retained the NPM-translocation activity. When these or similar modifications were applied to 7-deazaguanosine, none of the guanosine analogs were effective. These results indicate that modifications at the 5-position of the pyrrolo[2,3-D]pyrimidine ring and a structure similar to adenine rather than guanine are essential for NPM-translocation. Since inhibition of RNA synthesis did not induce NPM-translocation, our results suggest that interference with NPM's binding in nucleoli by these analogs causes NPM-translocation.

ATP depletion affects NPM translocation and exportation of rRNA from nuclei.

Nucleophosmin/B23 (NPM) is a nucleolar phosphoprotein which shifts from nucleoli to the nucleoplasm in cells treated with certain cytotoxic agents (NPM-translocation). NPM requires GTP for localization into nucleoli (J. Biol. Chem. 268, 5823-5827, 1993). To understand more about NPM's dynamic localization, the effects of lowering ATP on NPM-translocation and rRNA synthesis were studied. When the ATP level in HeLa cells was reduced by sodium azide, NPM-translocation was blocked. Similar results were obtained when ATP was depleted by other agents, suggesting that ATP depletion was responsible for the blocking of NPM-translocation. It was found that newly synthesized rRNA accumulated in the nuclei during ATP-depletion. Significantly larger than normal nucleoli were also observed. These results indicate that NPM may be involved in the transportation of newly synthesized ribosomes.

Studies of actinomycin D induced B23-translocation in P388D1 cells implanted in DBA/2 mice.

Nucleophosmin/B23 is a nucleolar phosphoprotein which redistributes from nucleoli to nucleoplasm (B23-translocation) when cells are exposed to certain anticancer drugs, particularly intercalators. The B23-translocation assay has been demonstrated in cell culture to correlate with drug effects and to detect drug-resistant cells. We now report the effect of actinomycin D on B23-translocation in P388D1 cells implanted in DBA/2 mice. B23-translocation was observed in cells after actinomycin D treatment in a dosage- and time-dependent manner. Translocation could be observed within 30 min after drug treatment. Complete B23-translocation with at least 1-day duration was achieved by a single injection of 0.25 mg/kg. Reduced dosages produced partial B23-translocation with shorter durations. These results indicate that B23-translocation may be useful in monitoring drug effects in animals.

Chemotherapeutic characterization in mice of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfinamide (sulfinosine), a novel purine nucleoside with unique antitumor properties.

In preclinical investigations performed in mice, 2-amino-9-beta-D-ribofuranosyl purine-6-sulfinamide (sulfinosine), a novel derivative of 6-thioguanosine (6TGR), was active against six solid tumors and four strains of experimental leukemia. Sulfinosine penetrated the central nervous system more readily than did 6TGR and, when given repeatedly, was much more effective in the treatment of L1210 leukemia, being curative for some mice. Other findings of major interest to us were the different dosing characteristics of sulfinosine and 6TGR, the divergent efficiencies of the two drugs in generating cellular resistance, and the activity of sulfinosine against experimental leukemias refractory to 6TGR and other experimental or clinically used chemotherapeutic agents. The chemotherapeutic characterization of sulfinosine that evolved from these studies suggests that this agent may have unique properties that deserve clinical consideration. Both the dosing characteristics of the drug and its pronounced activity against thiopurine-resistant experimental leukemia favor the possibility that sulfinosine could be used to advantage in the treatment of human leukemia unresponsive to 6-mercaptopurine or 6-thioguanine.

Oxidation of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfenamide to the corresponding 6-sulfonamide facilitates changes in biologic characterization that include activity against thiopurine-refractory experimental leukemia.

Preclinical investigations in vivo revealed unexpected differences in the biological characteristics of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfenamide (sulfenosine, 1) and 2-amino-9-beta-D-ribofuranosylpurine-6-sulfonamide (sulfonosine, 2), two novel but structurally related derivatives of 6-thioguanosine (6TGR). Strikingly, the addition of a fully oxidized sulfur atom at the 6 position of sulfenosine produced a purine derivative (sulfonosine) that was remarkably active against experimental leukemia resistant to treatment with either sulfenosine or 6TGR. This slight structural modification also appeared to influence solubility, scheduling capability, and oral activity as well as penetration of the central nervous system (CNS) and the onset of cellular resistance. These findings underscore the dramatic changes in biologic activity that can be produced by subtle modifications in molecular structure. We trust they may also contribute to the development of improved clinical therapy.

Synthesis and antitumor evaluation in mice of certain 7-deazapurine (pyrrolo[2,3-d]pyrimidine) and 3-deazapurine (imidazo[4,5-c]pyridine) nucleosides structurally related to sulfenosine, sulfinosine, and sulfonosine.

7-Deaza (pyrrolo[2,3-d]pyrimidine) and 3-deaza (imidazo[4,5-c]pyridine) congeners of sulfenosine (5a and 9), sulfinosine (6a and 10), and sulfonosine (7a) have been prepared and evaluated for their antileukemic activity in mice. Amination of 2-amino-7-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidine-4(3H)-th ion e (4a) and its 2'-deoxy analogue (4c) with a chloramine solution gave the corresponding 4-sulfenamides (5a and 5c, respectively), which on selective oxidation with m-chloroperoxybenzoic acid (MCPBA) gave the respective diastereomeric 2-amino-7-beta-D-ribofuranosyl-pyrrolo[2,3-d]pyrimidine-4-sulfinamide (7-deazasulfinosine, 6a) and its 2'-deoxy derivative (6c). A similar amination of 7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine-4(3H)- thione (4b) gave the corresponding 4-sulfenamide derivative (5b). Oxidation of 5b with 1 molar equiv of MCPBA furnished (R,S)-7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine- 4- sulfinamide (6b), whereas use of excess of MCPBA afforded the corresponding sulfonamide derivative (7b). Treatment of 3-deaza-6-thioguanosine (8) with a chloramine solution gave 3-deazasulfenosine (6-amino-1-beta-D- ribofuranosylimidazo[4,5-c]pyridine-4-sulfenamide, 9). Controlled oxidation of 9 with MCPBA afforded 3-deazasulfinosine (10). As gauged by increases in the mean postinoculation life spans of L1210 inoculated mice, none of these nucleosides exhibited biologically significant activity (T/C greater than or equal to 125). Even so, antileukemic activity appeared to be influenced, albeit not uniformly, by structural modifications in the base and carbohydrate moieties of sulfenosine and sulfinosine. Thus, while several of the compounds were lacking in cytotoxic activity, eight others (4c, 5a, 5c, 6a, 6b, 7b, 9, and 10) were estimated to have reduced body burdens of viable L1210 cells by 16-77%.