ATX-MS-1467 Induces Long-Term Tolerance to Myelin Basic Protein in (DR2 × Ob1)F1 Mice by Induction of IL-10-Secreting iTregs.

INTRODUCTION: Antigen-specific immunotherapy could provide a targeted approach for the treatment of multiple sclerosis that removes the need for broad-acting immunomodulatory drugs. ATX-MS-1467 is a mixture of four peptides identified as the main immune-dominant disease-associated T-cell epitopes in myelin basic protein (MBP), an autoimmune target for activated autoreactive T cells in multiple sclerosis. Previous animal studies have shown that ATX-MS-1467 treatment prevented the worsening of signs of disease in experimental autoimmune encephalitis (EAE) in the humanized (DR2 × Ob1)F1 mouse in a dose-dependent fashion. METHODS AND RESULTS: Our study extends these observations to show that subcutaneous treatment with 100 µg of ATX-MS-1467 after induction of EAE in the same mouse model reversed established clinical disability (p < 0.0001) and histological markers of inflammation and demyelination (p < 0.001) compared with vehicle-treated animals; furthermore, in longitudinal magnetic resonance imaging analyses, disruption of blood-brain barrier integrity was reversed, compared with vehicle-treated animals (p < 0.05). Chronic treatment with ATX-MS-1467 was associated with an enduring shift from a pro-inflammatory to a tolerogenic state in the periphery, as shown by an increase in interleukin 10 secretion, relative to interleukin 2, interleukin 17 and interferon γ, a decrease in splenocyte proliferation and an increase in interleukin 10+ Foxp3- T cells in the spleen. CONCLUSION: Our results suggest that ATX-MS-1467 can induce splenic iTregs and long-term tolerance to MBP with the potential to partially reverse the pathology of multiple sclerosis, particularly during the early stages of the disease. FUNDING: EMD Serono, Inc., a business of Merck KGaA.

The role of human nucleoside transporters in cellular uptake of 4'-thio-beta-D-arabinofuranosylcytosine and beta-D-arabinosylcytosine.

4'-Thio-beta-D-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 microM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-beta-D-arabinofuranosyl cytosine (araC) were low (0.007 +/- 003 and 0.034 +/- 0.003, respectively) compared with that of [3H]uridine (0.317 +/- 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 microM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 +/- 0.003, 0.42 +/- 0.03, and 0.51 +/- 0.11, respectively) and mediated primarily by hENT1 (approximately 74, approximately 65, and approximately 61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 microM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.

Incorporation of OSI-7836 into DNA of Calu-6 and H460 xenograft tumors.

OSI-7836 (4'-thio-beta-D-arabinofuranosylcytosine) is a novel nucleoside analog in phase I clinical development for the treatment of cancer. As with other nucleoside analogs, the proposed mechanism of action involves phosphorylation to the triphosphate form followed by incorporation into cellular DNA, leading to cell death. This hypothesis has been examined by measuring and comparing the incorporation of ara-C, OSI-7836, and gemcitabine (dFdC) into DNA of cultured cells and by investigating the role of deoxycytidine kinase in OSI-7836 toxicity. We report here additional studies in which the role of cell cycling on OSI-7836 toxicity was investigated and incorporation of OSI-7836 into DNA of xenograft tumors measured. The role of the cell cycle was examined by comparing the toxicity of OSI-7836 in A549 NSCLC cells that were either in log phase growth or had reached confluence. A novel validated LC-MS/MS assay was developed to quantify the concentrations of OSI-7836 in DNA from Calu-6 and H460 human tumor xenografts in mice. Results showed that apoptosis induced by OSI-7836 was markedly greater in cycling cells than in confluent non-cycling cells despite only a modest increase in intracellular OSI-7836 triphosphate concentration. The LC-MS/MS assay developed to measure OSI-7836 incorporation into DNA had an on-column detection limit of 0.25 fmol, a quantification limit of 0.5 fmol, and a sensitivity of approximately 0.1 pmol OSI-7836/micromol dThy. Concentrations of OSI-7836 in splenic DNA (0.4 pmol OSI-7836/micromol dThy) averaged fivefold less than the average concentration in Calu-6 and H460 xenograft DNA (3.0 pmol OSI-7836/micromol dThy) following a 400 mg/kg dose of OSI-7836. Concentrations of OSI-7836 in Calu-6 tumor DNA isolated 24 h following a dose of 400, 1000, or 1600 mg OSI-7836/kg were approximately 1.3, 1 and 1.3 pmol OSI-7836/micromol dThy, respectively. Concentrations of OSI-7836 in DNA from H460 and Calu-6 xenografts did not appear to increase during repeated administration of 400 mg OSI-7836/kg on days 1, 4, 7, and 10. The majority of OSI-7836 in DNA from Calu-6 and H460 tumors of mice dosed with 1600 mg/kg was located at internal nucleotide linkages, similar to dFdC and ara-C. In conclusion, cell cycling studies supported the hypothesis that OSI-7836 cytotoxicity is dependent upon DNA synthesis. A validated LC-MS/MS assay was developed that could quantify OSI-7836 in DNA from tissues. The assay was used to show that OSI-7836 was incorporated in internal linkages in tumor DNA in a manner that was dose-independent at the doses tested and did not appear to accumulate during repeated dosing. The results suggest that if DNA incorporation is a toxic event, the relationships between administered dose, DNA incorporation, and toxicity are complex.

Polymerization of the triphosphates of AraC, 2',2'-difluorodeoxycytidine (dFdC) and OSI-7836 (T-araC) by human DNA polymerase alpha and DNA primase.

OSI-7836 (4'-thio-araC, T-araC) is a nucleoside analogue that shows efficacy against solid tumor xenograft models. We examined how the triphosphates of OSI-7836 (T-araCTP), cytarabine (araCTP), and gemcitabine (dFdCTP) affected the initiation of new DNA strands by the pol alpha primase complex. Whereas dFdCTP very weakly inhibited primase, both T-araCTP and araCTP potently inhibited this enzyme. Primase polymerized T-araCTP and araCTP more readily than its natural substrate, CTP, and incorporation resulted in strong chain termination. dFdCTP, araCTP, and T-araCTP inhibited pol alpha competitively with respect to dCTP. When exogenously added primentemplates were used, pol alpha incorporated all three analogues into DNA, and incorporation caused either weak chain termination (dFdCTP), strong termination (araCTP), or extremely strong termination (T-araC). Furthermore, pol alpha polymerized T-araCTP only nine-fold less well than dCTP, whereas it polymerized araCTP and dFdCTP 24- and 83-fold less well, respectively. The presence of these three analogues in the template strand resulted in significant pausing by pol alpha, although the site and severity of pausing varied between the analogues. During the elongation of primase-synthesized primers, a reaction that is thought to mimic the normal sequence of events during the initiation of new DNA strands, pol alpha polymerized all three compounds. However, incorporation of araCTP and dFdCTP resulted in minimal chain termination, while incorporation of T-araCTP still caused extremely strong termination. The implications of these results with respect to how these compounds affect cells are discussed.

OSI-211, a novel liposomal topoisomerase I inhibitor, is active in SCID mouse models of human AML and ALL.

OSI-211 (liposomal lurtotecan), was evaluated using several different dose schedules (1mg/kg, d1-5, 1.75 mg/kg d1, 3, 5 and 6 mg/kg d1, 8) in severe combined immunodeficient (SCID) mouse models of acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) with early treatment (ET, days 6-8) or late treatment (LT, days 15-19), examining early and advanced disease, respectively. Due to the aggressive nature of the Molt-4 model, the ET and LT were accelerated to day 3 or 4 and day 8 post-implant, respectively. For each model, 2 x 10(7) (KBM-3B) or 1 x 10(7) (Molt-4, HL-60 and CEM) leukemia cells were injected intravenously into the tail vein. Each control and test group consisted of eight animals. All three schedules (1mg/kg qd1-5, 1.75 mg/kg d1, 3, 5 and 6 mg/kg d1, 8) increased the life span of OSI-211 treated animals in each model, with a tendency toward improved efficacy with the 6 mg/kg d1, 8 schedule. As a result, the activity of the 6 mg/kg d1, 8 schedule is detailed for each model. ET significantly (P<0.005) increased survival in the KBM-3B model with 86% long-term survivors (LTS). Using PRC analysis, human beta-globin gene sequences in one or several tissues were amplified in all but 3 LTS, suggesting minimal residual disease in 26 of the 29 LTS. LT also significantly (P<0.005) improved average life span in the KBM-3B model, with an average ILS=196+/-11% and one LTS. Treatment of HL-60 leukemia animals significantly (P<0.005) increased life span, with an ILS=213+/-9% and two LTS for ET, and with an ILS=219+/-4% and no LTS for LT. Treatment of Molt-4 animals, the most aggressive leukemia model tested, significantly (P<0.005) increased life span, with an average ILS=181+/-3% and no LTS for ET and an average ILS=172+/-1% with no LTS for LT. In the CEM model, ET resulted in a significantly (P<0.005) improved ILS=244+/-24% with one LTS. In comparison to OSI-211, treatment with DaunoXome, the liposomal formulation of daunorubicin, a drug with clinical efficacy in AML and ALL, had no effect on survival in the KBM-3B, nor Molt-4 A4 leukemia models when administered at its maximum or near maximum tolerated doses of 3mg/kg d1, 8. These data demonstrate that OSI-211 has potent antileukemia activity in preclinical SCID mouse AML and ALL leukemia models, supporting the clinical investigation of OSI-211 for hematological malignancies.