JP2015199762A - Methods, pharmaceutical compositions and kits for use in treatment of adult t-cell leukemia/lymphoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods, pharmaceutical compositions and kits for the use in the treatment of Adult T-cell leukemia/lymphoma, which exhibits a remarkably high response rate with moderate side effects.SOLUTION: The invention provides a pharmaceutical composition in which interferon, such as interferon α, an arsenic compound selected from arsenic, arsenic trioxide (As2O3), melarsoprol, arsenic sulfur derivatives, and the like, and a reverse transcriptase inhibitor, such as zidovudine are combined.

Description

  The present invention relates to methods, pharmaceutical compositions and kits for use in the treatment of adult T-cell leukemia / lymphoma.

  Adult T cell leukemia / lymphoma (ATL) is an aggressive proliferation of mature activated CD4 + T cells combined with human T cell leukocyte virus type I (HTLV-I). After a very long incubation period, leukemia develops and prior to leukemia, HTLV-I infected activated T cells proliferate oligoclonally. These clonal expansions are caused by the expression of the viral transcriptional activation protein Tax, which activates various cellular genes and is involved in the autocrine loop associated with interleukin 2, interleukin 15, and their cognate receptors. ). Due to the clinical characteristics and prognostic diversity of ATL patients, it is subdivided into smoldering, chronic, lymphoma and acute subtypes. In general, patients with aggressive ATL (acute and lymphoma subtypes) have an intrinsic chemoresistance of malignant cells, large tumor burden with multiple organ failure, hypercalcemia, and / or severe T cells. Very poor prognosis due to infectious complications that frequently occur due to immunodeficiency. Patients with painless ATL (ie, chronic or smoldering subtype) have a better prognosis. However, data from Japan indicate that these patients have poor long-term survival if they are managed with a watchful-waiting policy until disease progression or are managed with chemotherapy. It was. In fact, the 4-year survival of chronic ATL is less than 30%.

  A combination of the antiretroviral nucleotide analogues zidovudine (AZT) and interferon alpha (IFN) has been shown to achieve high response rates in ATL patients (Gill PS, Harrington W Jr, Kaplan MH, et al. Treatment). of adult T-cell leukemia-lymphoma with a combination of interferon alfa and zidovudine.N Engl J Med. 1995; 332: 1744-1748 .; Hermine O, Bouscary D, Gessain A, et al. Brief report: treatment of adult T -cell leukemia-lymphoma with zidovudine and interferon alfa.N Engl J Med. 1995; 332: 1749-1751 .; Bazarbachi A, Hermine O. Treatment with a combination of zidovudine and alpha-interferon in naive and pretreated adult T-cell leukemia J Acquir Immune Defic Syndr Hum Retrovirol. 1996; 13 (suppl 1): S186-S190 .; White JD, Wharfe G, Stewart DM, et al. The combination of zidovudine and interferon alpha-2B in the treatment of adult T-cell leukemia / lymphoma. Leuk Lymphoma. 2001; 40: 287-294; He rmine O, Allard I, Levy V, et al. A prospective phase II clinical trial with the use of zidovudine and interferon-alpha in the acute and lymphoma forms of adult T-cell leukemia / lymphoma. Hematol J. 2002; 3: 276 -282.; Matutes E, Taylor GP, Cavenagh J, et al. Interferon alpha and zidovudine therapy in adult T-cell leukaemia lymphoma: response and outcome in 15 patients. Br J Haematol. 2001; 113: 779-784.). However, most patients eventually relapse, which emphasizes the need for new therapeutic approaches.

  Arsenic trioxide (As) is an acute promyelocytic leukemia (APL) (inherent clinical features and specific cytogenetic abnormalities t (15; 17) (this is the PML gene on chromosome 15 and 17 A highly distinct treatment of acute myeloid leukemia (a distinct subtype) characterized by a reciprocal translocation between the retinoic acid receptor α (RAR-α) gene on chromosome . Clinically, As directly targets and degrades the PML / RARA fusion protein and induces clinical remission in APL patients.

  In the ATL cell line, we previously showed that As synergizes with IFN to induce cell cycle arrest and apoptosis (Bazarbachi A, El-Sabban ME, Nasr R, et al. Arsenic). trioxide and interferon-alpha synergize to induce cell cycle arrest and apoptosis in human T-cell lymphotropic virus type I-transformed cells. Blood. 1999; 93: 278-283.). At the molecular level, the As / IFN combination specifically induces proteosomal degradation of HTLV-1 oncoprotein Tax and reversal of NF-κB activation (El-Sabban ME, Nasr R, Dbaibo G, et al. Arsenic-interferon-alpha-triggered apoptosis in HTLV-I transformed cells is associated with tax down-regulation and reversal of NF-kappa B activation.Blood. 2000; 96: 2849-2855 .; Nasr R, Rosenwald A, El-Sabban ME, et al. Arsenic / interferon specifically reverses 2 distinct gene networks critical for the survival of HTLV-1-infected leukemic cells. Blood. 2003; 101: 4576-4582.). Such specific targeting of viral oncoproteins by IFN / As treatment, which recalls As targeting of PML / RAR in APL, provides a strong rationale for IFN / As combination therapy in ATL patients. In that sense, the results of a phase 2 study of As / IFN combination in 7 patients with AZT, IFN and relapsed / refractory aggressive ATL after chemotherapy have been reported previously (Hermine O, Dombret H, Poupon J, et al. Phase II trial of arsenic trioxide and alpha interferon in patients with relapsed / refractory adult T-cell leukemia / lymphoma. Hematol J. 2004; 5: 130-134.). One patient achieved complete remission, 3 achieved partial remission, and 3 progressed. Patients in complete remission (CR) are still alive after 5 years of follow-up. These results indicate that in these selected aggressive ATL patients with poor prognosis, treatment with As and IFN is feasible and show an anti-leukemic effect in vivo. Similarly, transient responses to the As / IFN combination were reported in Japan in two patients with refractory acute ATL (Ishitsuka K, Suzumiya J, Aoki M, Ogata K, Hara S, Tamura K Therapeutic potential of arsenic trioxide with or without interferon-alpha for relapsed / refractory adult T-cell leukemia / lymphoma. Haematologica. 2007; 92: 719-720).

  The present invention relates to a combination of an interferon, an arsenic compound and a reverse transcriptase inhibitor for use in the treatment of adult T-cell leukemia / lymphoma.

Detailed description of the invention:
During a prospective phase II study, we investigated the efficacy and safety of treatment with a combination of As, IFN, and AZT in 10 newly diagnosed chronic ATL patients from Mashhad, northeastern Iran. . They show that this combination therapy is feasible and shows a very high response rate with mild side effects.

  In addition, the inventors have demonstrated that arsenic trioxide / interferon alpha association cures Tax-driven mouse ATL in vivo. Unexpectedly, this alliance immediately disables leukemia transplantation to secondary recipients, but the primary tumor continues to grow and only depletes fairly well later. Leukemia-causing cell (LIC) clearance triggered by treatment is abolished by proteasome inhibition. The results are very reminiscent of the eradication of cells causing APL leukemia by treatment-induced PML / RARA degradation. They provide a rational basis for the most recent success of arsenic / interferon treatment in patients, suggesting that only LIC and not the majority of tumors are dependent on continuous oncogene expression.

  Accordingly, the present invention relates to a combination of an interferon, an arsenic compound and a reverse transcriptase inhibitor for use in the treatment of adult T cell leukemia / lymphoma.

  In its broadest sense, the terms “treat” or “treatment” reverse the progression of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition, Means to alleviate, inhibit or prevent.

  “Interferon” or “IFN” as used herein is defined as such in the literature, including for example any type of IFN (type I and type II), and in particular IFNα, IFNβ, IFNω and IFNγ. It is intended to include any molecule. The term interferon as used herein is also intended to encompass salts, functional derivatives, mutants, muteins, fusion proteins, analogs and active fragments thereof. The polypeptide sequence of human interferon alpha is deposited in the database under the accession numbers: AAA52716, AAA52724 and AAA52713. The polypeptide sequence of human interferon β is deposited in the database under accession numbers AAC417702, NP_002167, AAH96152, AAH96153, AAH96150, AAH96151, AAH69314 and AAH36040. The polypeptide sequence of human interferon gamma is deposited in the database under the accession numbers AAB59534, AAM28885, CAA44325, AAK95388, CAA00226, AAP20100, AAP20098, AAK53058 and NP-000610.

  In a preferred embodiment, the interferon is interferon alpha. Interferon α includes recombinant interferon α2a (eg, ROFERON® interferon available from Hoffman-LaRoche, Nutley, NJ) and interferon α2b (eg, intron A available from Schering Corp., Kenilworth, NJ, USA). Interferon), consensus interferon and purified interferon alpha product.

  As used herein, the term “arsenic compound” is intended to include arsenic and any compound having the same biological properties as arsenic. The expression “compound having the same biological properties as arsenic” means any compound that is an inhibitor of a phosphatase such as arsenic and / or capable of creating a covalent adduct by binding to a dithiol group. Then it should be understood.

  In certain embodiments, the arsenic compound is selected from the group consisting of arsenic, arsenic trioxide (As2O3), meralsoprole, and arsenic sulfur derivatives. Since many of the effects of arsenic are mediated by oxidative stress, substances that promote the production of reactive oxygen species such as arsenic are encompassed by the present invention.

  The term “reverse transcriptase inhibitor” as used herein means any compound capable of inhibiting the activity of HTVL-1 reverse transcriptase. The term includes nucleoside analog reverse transcriptase inhibitors, nucleotide analog reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors.

  In certain embodiments, the reverse transcriptase inhibitor is zidovudine (also referred to as AZT, ZDV and azidothymidine, having the trade name Retrovir), didanosine (also referred to as ddI, having the trade names Videx and Videx), sarcitabine (Also referred to as ddC and dideoxycytidine, having the trade name Hivid), stavudine (also known as stavudine, d4T, having the trade names Zerit and Zerit XR), abacavir (also referred to as Abacavir, ABC, having the trade name Ziagen) A nucleoside analog reverse transcriptase inhibitor selected from the group consisting of Emtricitabine (also referred to as FTC and having the trade name Emtriva) and Apricitabine (also referred to as ATC).

  In certain embodiments, the reverse transcriptase inhibitor is tenofovir (also known as tenofovir disoproxil fumarate, having the trade name Viread) and adefovir (also known as bis-POM PMPA, A nucleotide analog reverse transcriptase inhibitor selected from the group consisting of the names Preveon and Hepsera.

  In a particular embodiment, the reverse transcriptase inhibitor comprises the group consisting of efavirenz (having the trade names Sustiva and Stocrin), nevirapine (viramune), delavirdine (having the trade name Rescriptor) and etoravirin (having the trade name Intelence). A more selected non-nucleoside reverse transcriptase inhibitor.

  A further aspect of the present invention relates to an adult T cell leukemia / lymphoma comprising administering to a subject in need of treatment of adult T cell leukemia / lymphoma an amount of IFN, an arsenic compound and a reverse transcriptase inhibitor. It relates to a method for treating.

  The term “subject” as used herein refers to mammals such as rodents, felines, canines and primates. Preferably, the subject according to the invention is a human.

  As used herein, the term “active ingredient of the present invention” means an inducer of promyelocytic leukemia protein (PML) expression, an arsenic compound and a reverse transcriptase inhibitor as defined above. Intended.

  The active ingredient of the present invention can be administered in the form of a pharmaceutical composition as defined below.

  Preferably, the active ingredient of the present invention is administered in a therapeutically effective amount.

  “Therapeutically effective amount” means an amount of an active ingredient of the invention sufficient to treat adult T cell leukemia / lymphoma at a reasonable benefit / risk ratio applicable to any medical treatment.

  It will be appreciated that the total daily usage of the active ingredients of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular subject is the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; Age, weight, overall health, sex and diet; administration time, route of administration and route of excretion of the specific active ingredient used; duration of treatment; in combination with or accidentally together with the specific active ingredient used It will depend on various factors such as the drug used; and factors well known in the medical field. For example, it may be sufficient to start dosing the active ingredient at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired therapeutic effect is achieved. Within the skill of the art.

  The envisaged dose may be, for example, preferably 1 to 50 mg per day, preferably 10 to 15 mg per day for arsenic compounds, and 1 to 1500 mg per day and preferably 1 kg body weight per day. 600-1000 mg of reverse transcriptase inhibitor per day, and for IFN at 1-20 million international units (MIU) per day or every other day, preferably 3-9 million international units (MIU) per day possible.

  In a preferred embodiment, the arsenic compounds of the present invention are preferably administered by the intravenous route, the reverse transcriptase inhibitors of the present invention are preferably administered by the oral route, and the IFN is administered by the intramuscular or subcutaneous route.

  According to the present invention, the active ingredients of the present invention can be administered as a combined preparation for simultaneous use, single use or sequential use in the treatment of adult T cell leukemia / lymphoma.

  The active ingredients of the present invention can be combined with a pharmaceutically acceptable excipient and optionally a sustained release matrix such as a biodegradable polymer to form a therapeutic composition.

  The terms “pharmaceutically” or “pharmaceutically acceptable” are molecular entities that do not cause side reactions, allergic reactions, or other adverse reactions when administered to mammals, particularly humans, as appropriate. And composition. Pharmaceutically acceptable carrier or excipient means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any kind of formulation auxiliaries.

  In the pharmaceutical composition of the present invention, the active ingredient of the present invention can be administered to animals and humans in a unit dosage form as a mixture with a conventional pharmaceutical support. Suitable unit dosage forms are tablets, gel capsules, powders, granules and oral route forms such as oral suspensions or solutions, sublingual and buccal dosage forms, sprays, implants, subcutaneous, transdermal, topical Including intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal dosage forms as well as rectal dosage forms.

  Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle for a formulation that can be injected. These are in particular sterile isotonic saline (monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride etc. or a mixture of such salts) or sterile water or saline depending on the situation. It can be a dried product, in particular a lyophilized composition, which allows the composition of an injectable solution by adding water.

  Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be prevented against the contaminating action of microorganisms such as bacteria and fungi.

  Solutions containing the compounds of the present invention as the free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersants can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

  The active ingredients of the present invention can be formulated into compositions as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of the protein) and inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid An acid addition salt formed in Salts formed with free carboxyl groups also include, for example, inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine Can be derived from

  The carrier can also be a solvent or dispersant including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use in the composition of agents which delay absorption, for example, aluminum monostearate and gelatin.

  Sterile injectable solutions are prepared by combining the required amount of the active ingredient of the present invention in a suitable solvent with the various other ingredients listed above and then filter sterilizing as necessary. Generally, dispersants are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is to dry the active ingredient powder from their pre-sterilized filtered solution, plus any additional desired ingredients, vacuum drying and freezing It is a drying technique.

  When formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solution described above, but drug release capsules and the like can also be used.

  For parenteral administration in aqueous solution, for example, the solution should be buffered appropriately as needed, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, those skilled in the art will understand sterile aqueous media that may be used in light of the present disclosure. For example, a dosage can be dissolved in 1 ml of isotonic NaCl solution and added to a 1000 ml subcutaneous infusion solution or injected at the site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Anyone responsible for administration will, in any event, determine the appropriate dose for the individual subject.

  In addition to the active ingredients of the present invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration. Liposome formulations; sustained release capsules; and any other form currently in use.

  The present invention relates to a pharmaceutical composition comprising an IFN, an arsenic compound and a reverse transcriptase inhibitor and optionally a pharmaceutically acceptable excipient.

  The pharmaceutical composition is particularly suitable for the treatment of adult T cell leukemia / lymphoma.

The present invention also provides a) IFN as a combined preparation for simultaneous use, single use or sequential use in the treatment of adult T cell leukemia / lymphoma
b) an arsenic compound and c) a reverse transcriptase inhibitor,
Relates to a kit comprising

  The present invention also relates to a combination of IFN and an arsenic compound to eradicate leukemogenic cells in a subject suffering from adult T-cell leukemia / lymphoma.

  The term “leukemia-causing cell” as used herein is described by Dick JE. (Dick JE. Stem cell concepts renew cancer research. Blood. 2008 Dec 15; 112 (13): 4793-807.) Means cells with specific properties that allow complete leukemia development and self-renewal.

  According to certain embodiments, the combination further comprises a reverse transcriptase inhibitor.

  The invention will be further illustrated by the following figures and examples. However, these examples and drawings should in no way be construed as limiting the scope of the invention.

Example 1: Phase II study of the efficacy and safety of the combination of arsenic trioxide, interferon alpha and zidovudine in newly diagnosed chronic adult T-cell leukemia / lymphoma (ATL) The results reported below are Scientific papers (Kchour G, Tarhini M, Kooshyar MM, El Hajj H, Wattel E, Mahmoudi M, Hatoum H, Rahimi H, Maleki M, Rafatpanah H, Rezaee SA, Yazdi MT, Shirdel A , de The H, Hermine O, Farid R, Bazarbachi A. Phase 2 study of the efficacy and safety of the combination of arsenic trioxide, interferon alpha, and zidovudine in newly diagnosed chronic adult T-cell leukemia / lymphoma (ATL). 2009 Jun 25; 113 (26): 6528-32. Published in Epub 2009 May 1).

Materials and Methods Patient characteristics: Ten newly diagnosed previously untreated chronic ATL patients participated in this prospective phase II study after submitting informed consent. Patient participation began in 2007. These patients were referred to the blood-oncology department of Ghaem and Imam Reza hospitals, Mashhad University of Medical Sciences. All ATL patients had serological evidence of HTLV-I infection by enzyme-linked immunosorbent assay (ELISA) and HTLV-I positive confirmation by standard polymerase chain reaction (PCR; data not shown) . Flow cytometric analysis of peripheral blood at the time of diagnosis showed that the tumor cells were CD4 +, CD8− and CD25 + ( Table 1 ). Shimoyama classification criteria for ATL (Shimoyama M. Diagnostic criteria and classification of clinical subtypes of adult T-cell leukaemia-lymphoma: a report from the Lymphoma Study Group (1984-87). Br J Haematol. 1991; 79: 428-437) All patients had chronic ATL. Patient characteristics are shown in Table 1 . The study was approved by the Ethics Committee of the Mashhad University of Medical Sciences and patient informed consent was obtained in accordance with the Declaration of Helsinki.

Study design and treatment schedule: Treatment consists of intravenous As (10 mg / day, 5 days / week), subcutaneous IFN (Pooyesh Darou Pharmaceutical, Tehran, Iran; 5 million units / day) and oral AZT (900 mg / day). It was. Initially, arsenic was planned for a period of 60 days. However, after low tolerance in the first patient who received 60 days of As administration with AZT and IFN, the protocol was modified to 30 days of As. In the case of toxicity, AZT and IFN were temporarily interrupted or their doses were reduced to 600 mg / day and 3 million units / day, respectively. The dose of arsenic was not reduced in the case of toxicity, but As treatment was temporarily interrupted. Details regarding treatment dose, treatment duration and treatment interruption are listed in Table 2 . Treatment of non-responders was at the discretion of the investigator.

  Response Criteria: Complete response (CR) is defined as normalization of the complete blood count (CBC), which is associated with the persistence of all measurable tumors for at least 1 month. However, because this situation can be seen in HTLV-I health carriers, patients with less than 5% atypical lymphocyte survival were considered CR. Very good partial response (VGPR) is defined as CBC standardization, which means that all measurable tumor disappearance lasts for at least 1 month, but more than 5% atypical lymphocytes on peripheral blood smears It is related to survival. Partial response (PR) was defined as more than 50% reduction in leukemia cell count and all measurable tumor sizes. No response (NR) was defined as less than 50% reduction in leukemia cell number or any measurable tumor size or as disease progression. Progression free survival (PFS) was defined as the period between the start of treatment and the day of disease progression, death or last follow-up. Overall survival (OS) was defined as the period between the start of treatment and the date of death or last follow-up.

  Provirus amount: HTLV-I virus copy number per microliter of blood was calculated from the number of cells and the average virus copy number per cell assessed by quantitative PCR. For standardization, real-time quantitative PCR was performed on DNA extracted from the peripheral blood mononuclear cells using primers and Taqman probes located on the tax and albumin genes. TaqMan amplification was performed with a reaction volume of 25 μL using qPCR MasterMix (Eurogentec, Leuven, Belgium). Each sample was analyzed in triplicate using 250 ng DNA in each reaction. Start thermal cycle with incubation at 50 ° C for 2 minutes, followed by initial denaturation step at 95 ° C for 10 minutes, then 45 cycles at 95 ° C for 15 seconds and for tax at 58 ° C for 1 minute (for albumin at 60 ° C for 1 minute) .

Results Toxicity and dose adjustment: Toxicity (WHO> 3) occurred in 4 patients ( Table 3 ). Especially at the end of the first month of treatment, most patients experienced hematological toxicity (severity> 1 [6 patients], severity> 3 [3 patients]). Non-hematological toxicity (severity> 1 [7 patients], severity> 2 [1 patient]) included signs of gastrointestinal (nausea and vomiting) and liver (cytolysis and cholestasis). Overall, toxicity caused dose reductions in 7 patients or treatment was temporarily interrupted. In addition to these objective toxicities, we noticed that most patients experienced severe fatigue during the last week of arsenic treatment. This improved rapidly after the arsenic interruption.

As shown in Table 2 , most patients were able to achieve the originally planned period of arsenic treatment (30 days) and were still receiving AZT and IFN maintenance therapy, although the dose was reduced. Overall, AZT was temporarily interrupted in 2 patients or given a reduced dose in 6 patients. Similarly, IFN was temporarily interrupted in 2 patients or given a reduced dose in 5 patients. Finally, As was temporarily interrupted in one patient.

Response and survival: All patients initially presented with symptomatic chronic ATL. The most common symptoms were cutaneous symptoms with spotted papules, severe itching and skin ulceration. Treatment with As, IFN and AZT resulted in an excellent 100% response rate ( Table 4 ). On day 30, 5 patients achieved PR and 5 patients were VGPR (this was defined as a standardization of CBC and all measurable tumor disappearances last for at least 1 month, but peripheral blood (Related to the survival of more than 5% atypical lymphocytes on the smear). Impressively, skin lesions almost disappeared within 2-4 weeks. Interestingly, in 7 patients where the first and day 30 DNA was available, the HTLV-I proviral load was significantly reduced from an average of 1415 copies / μL blood to 226 copies / μL (P <.05; Table 5 ). All patients continued to improve response ( Table 4 ). In fact, the disease assessment at the last follow-up was that 7 patients had CR and 2 patients had VGPR (6% and 8% atypical lymphocytes were present on the peripheral blood smear, respectively. 1 patient) was PR (after 2 months of follow-up, patient lymphocyte increase decreased from 185000 x 109 / L to 6400 x 109 / L) . After 8 months (range, 2-15 months) of medium-term follow-up, all patients are still alive; no patients relapsed or progressed.

Discussion:
In this prospective phase II study, we show promising clinical results of the As / IFN / AZT combination in 10 newly diagnosed chronic ATL patients from northeastern Iran. 7 patients who achieved CR, 2 patients who achieved VGPR (clinical and biological CR excluding the presence of more than 5% atypical lymphocytes on peripheral blood smears), and PR (2 An excellent 100% response rate was observed, including one who achieved a lymphocyte increase of> 95% after a short period of follow-up for months. This excellent response rate can be explained in part by the presence of AZT and IFN in the As / IFN / AZT triple combination, but published studies (Gill PS, Harrington W Jr, Kaplan MH, et al. Treatment of adult T-cell leukemia-lymphoma with a combination of interferon alfa and zidovudine.N Engl J Med. 1995; 332: 1744-1748 .; Hermine O, Bouscary D, Gessain A, et al. Brief report: treatment of adult T- cell leukemia-lymphoma with zidovudine and interferon alfa.N Engl J Med. 1995; 332: 1749-1751 .; Bazarbachi A, Hermine O. Treatment with a combination of zidovudine and alpha-interferon in naive and pretreated adult T-cell leukemia / Lymphoma patients.J Acquir Immune Defic Syndr Hum Retrovirol. 1996; 13 (suppl 1): S186-S190 .; White JD, Wharfe G, Stewart DM, et al. The combination of zidovudine and interferon alpha-2B in the treatment of adult T-cell leukemia / lymphoma. Leuk Lymphoma. 2001; 40: 287-294 .; Hermine O, Allard I, Levy V , et al. A prospective phase II clinical trial with the use of zidovudine and interferon-alpha in the acute and lymphoma forms of adult T-cell leukemia / lymphoma. Hematol J. 2002; 3: 276-282 .; Matutes E, Taylor GP, Cavenagh J, et al. Interferon alpha and zidovudine therapy in adult T-cell leukaemia lymphoma: response and outcome in 15 patients. Br J Haematol. 2001; 113: 779-784.) Note in particular that the CR rate is less than that observed in this study. Furthermore, the highest response rate was previously reported using high dose AZT and high dose IFN (6 million units / m 2), whereas most of our patients are considerably lower Received a dose of IFN (total dose of 5-3 million units). Finally, the follow-up of our study is relatively short (medium follow-up for 8 months), but no patients relapsed or progressed. In short, these results strongly suggest that As significantly improves the success rate of AZT / IFN.

  The As / IFN / AZT triple combination was feasible due to mild and manageable side effects. Hematological toxicity has forced temporary treatment interruptions or dose reductions in some patients. Non-hematological toxicity was mainly due to gastrointestinal discomfort. However, it should be noted that most patients experienced severe fatigue during the last week of arsenic treatment. This improved rapidly after the arsenic interruption. Therefore, shorter arsenic (3 weeks) may be associated with increased tolerability and should be investigated in future trials. Overall, based on this study, in patients with chronic ATL, the recommended starting dose of 3 substances during the first month of treatment is AZT (900 mg / day), IFN (5 million IU / day) And As (10 mg / day). A dose reduction of AZT (600 mg / day) and IFN (3 million IU / day) should be done in the case of severe toxicity. At the same dose, AZT / IFN should be maintained after this. However, Phase 1 trials are currently recommended to establish maximum tolerated doses for each drug in this highly effective combination regimen.

  Regarding the mechanism of action, we select HTLV-I-infected cells with a combination of As and IFN ex vivo through reversal of constitutive activation of NF-κB and degradation of Tax oncoprotein by the proteasome Has been shown previously to kill. Proteasome-mediated degradation of Tax by As / IFN is reminiscent of proteasome-mediated degradation of PML-RAR by As in APL. After many years of controversy, it has now been established that the viral transcriptional activator Tax plays an important role in initiating the leukemia process as Tax mouse transgenics develop disease with significant ATL characteristics. Yes. Recently, the inventors have demonstrated that As and IFN cooperate to cure mouse ATL derived from these Tax transgenics. Surprisingly, this combination does not trigger immediate growth arrest or apoptosis, but rather selectively eradicates cells that cause leukemia (LIC). This strongly suggests that LIC, rather than the majority of leukemias, is dependent on continuous oncogene expression. Thus, adding As to AZT / IFN may lead to long-term disease eradication and eventual cure through elimination of LIC. The short-term follow-up of our study shows no definitive evidence for overall survival. Long-term follow-up of patients treated with the As / IFN / AZT combination will demonstrate whether this high complete remission rate translates in terms of disease eradication and patient cure.

  In aggressive ATL, preliminary results from two acute ATL patients suggest that adding As to AZT / IFN during the induction phase may lead to severe oncolysis syndrome . Therefore, an attractive strategy in that setting is to add arsenic at a low tumor burden after reducing tumor volume to achieve CR in clinical symptoms similar to those with chronic ATL patients, Induction therapy with AZT / IFN.

  In conclusion, treatment of ATL with As, IFN and AZT is feasible and shows excellent response rates with mild toxicity in patients with chronic ATL. Although the follow-up was relatively short (8 months), no patient relapsed and expects that the disappearance of viral replication (AZT) and Tax degradation (As / IFN) may eradicate the disease Increased. These clinical results strengthen the concept of oncogene targeted cancer therapy.

Example 2: Treatment-induced selective disappearance of cells causing leukemia in mouse ATL:
Experimental Procedure Mice: We used the ATL mouse model of Hasegawa (Hasegawa et al., 2006). To test the effects of different target therapies, we have transferred the disease by direct intraperitoneal transfer of 10 ⁶ spleen cells from Tax transgenic mice into SCID mice (Charles River, USA). A quick and reproducible model was established. Tax expression was only detectable at very low levels by Q-PCR. All mouse protocols were approved by the American University of Beirut Institutional Animal Care and Utilization Committee (IACUC). All animals were housed in a special sterile shed. After deep anesthesia with isoflurane, the animals were sacrificed by cervical dislocation.

Treatment: Arsenic trioxide was obtained from Sigma-Aldrich (St Louis, MO, USA) and recombinant human interferon α (IFN) (Roferon®) was obtained from Hoffman-La Roche (Basel, Switzerland). Proteasome inhibitor PS341 or Bortezomib (Velcade®) was purchased from Millenium Pharmaceuticals. For in vivo experiments, mice received intraperitoneal administration of arsenic (5 μg / g / day), subcutaneous administration of IFN (10 ⁶ IU / day), and a mini-osmotic pump of bortezomib (0.5 mg / Kg / day). (Alzet, Charles river). These dosages are comparable to those in other previous mouse models and are expected to result in plasma concentrations similar to those seen in patients (Nasr et al., 2008). None of the different individuals or the combination treatment regimen was toxic in normal SCID mice (100% survival over 3 months; n = 3 for each condition). In ex-vivo experiments, malignant cells derived from Tax transgenic mice were treated with either arsenic (1 μM), IFN (1000 IU) or arsenic / IFN combinations for up to 48 hours in vitro.

  Histopathology and clinical examination: Tissues from both treated and untreated mice were fixed in neutral buffered formalin (Sigma), then embedded in paraffin, sectioned and stained with hematoxylin and eosin (H & E) And examined by an optical microscope. Rat anti-mouse monoclonal antibody Ki-67 (clone TEC-3) was used to assess Ki-67 expression by immunohistochemistry. Peripheral blood smears were prepared using Giemsa staining. Using normal clinical laboratory techniques, white blood cell (WBC) counts, serum calcium and serum lactate dehydrogenase levels were measured.

A terminal deoxynucleotidyl transferase-mediated nick end label (TUNEL) assay was performed on deparaffinized 5 μm sections treated with proteinase K (20 μg mL ⁻¹ ) for 15 minutes at room temperature. Two pathologists TUNEL positive in 3 different microscopic fields for each organ (liver, lung and spleen) tested on an Olympus ProvisAX70 microscope (Olympus Optical) with wide-angle eyepiece number 26.5 Cell number was assessed. At 600 magnification, this wide-angle field-of-view eyepiece provided a field size of 0.244 mm ² . Counting mitosis in the same way, the results were expressed as an average number of apoptotic cells or mitosis per 600 × field of view.

  Cell cycle analysis and TUNEL assay of isolated splenocytes: Splenocytes were collected, filtered, washed twice with cold PBS, fixed in 100% ethanol at -20 ° C and stored at -20 ° C overnight. . Subsequently, the cells were rinsed with PBS, treated with Tris HCl buffer (pH 7.4) containing 1% DNase-free RNase A, and stained with propidium iodide (PI) (60 mg / ml final). A FACScan flow cytometer (Becton Dickinson) was used to determine the cell cycle phase distribution including different DNA volumes. In each sample, 10,000 ungated events were obtained. Analysis of cell cycle distribution (including apoptosis) was performed using CellQuest software (Becton Dickinson).

  A TUNEL assay was also used to monitor splenocyte apoptosis. The assay was performed according to the manufacturer's (Roche) recommendations. Flow cytometry was used to detect and quantify fluorescein-bound dUTP incorporated into the nucleotide polymer. Approximately 10,000 cells were obtained per sample and analyzed using CellQuest software (Becton Dickinson).

  Quantitative PCR: The spleen is collected, real-time PCR or RT-PCR using LightCycler (Roche Diagnostics), and absolute Tax DNA capacity or absolute Tax mRNA using LightCycler FastStart DNA Master kit or Light Cycler RNA Master kit, respectively. Used to quantify expression. We also used the Lightcycler 4.05 software version. Tax primers SK43I (5′-CGGATACCCAGTCTACCGGT-3 ′) and SK44I (5′-GAGCCCGATAACCGCGTCCATCG-3 ′) and probes SK4I-FL (5′-CCCTACTGGCCACCCTCCTAGCGAC-CTGATCGACT -3 ′) was designed in cooperation with Tib-MolBiol. We also used circulating ATL leukemia cells from two patients with acute ATL after informed consent. For electrophoretic mobility shift assay (EMSA), nuclear extracts derived from splenocytes of treated or untreated mice were prepared as described (El-Sabban et al., 2000).

  Statistical analysis: Survival curves were calculated according to Kaplan and Meier's method. Overall survival is defined as the time from ATL cell injection to death from any cause. Mice that were still alive were censored when they were last known to be alive. The analysis was performed using SPSS software version 15.0 (SPSS). The p-value was obtained by log rank statistical analysis.

Results and discussion:
To investigate the in vivo effect of IFNα / As ₂ O ₃ , we established an ATL transplant model in which SCID mice were inoculated with 10 ⁶ ATL spleen cells derived from Tax transgenic mice. As with the original transgenic mice, the recipient developed massive leukocytosis, splenomegaly, hypercalcemia and multi-organ invasion and died within 28 days. Initially, we wondered if these mouse ATL cells responded ex vivo to the IFNα / As ₂ O ₃ combination. IFNα or arsenic triggered apoptosis in less than 20% Tax transgenic cells. These combinations killed more than 80% of cells after overnight ex vivo exposure, but normal mouse lymphocytes were not affected. Just as in the primary human ATL, protein degradation could not be detected by Western blot analysis, so it was not possible to demonstrate Tax degradation by the proteasome in these ATL cells. However, Tax mRNA levels were very similar in primary mouse and human ATL cells, but 1000 times higher in the HuT-102 cell line, where Tax protein was readily detectable.

To investigate any survival benefit, mice were treated with IFNα, As ₂ O ₃ or both on days 6-30, using doses that were effective in other mouse models. While significant survival benefits have been seen in animals receiving IFNα or As ₂ O ₃ alone, animals receiving the IFNα / As ₂ O ₃ combination often often have a second course of treatment. Healed when given. Since the protocol in which mice were treated for 5 days a week never gave any cure, IFNα / As ₂ O ₃ treatment had to be interrupted. Such ex vivo and in vivo superior sensitivity of both human HTLV-I infected ATL cells and mouse Tax-driven ATL to IFNα / As ₂ O ₃ is essential for Tax degradation in response to this combination. It is strongly supported that it is a contributing factor.

In order to analyze the molecular basis for this healing action, we examined cell proliferation and apoptosis in established mouse ATL treated for 3 days in association. A complete reversal of NFκB activation was observed in splenic ATL cells and calcium plasma levels returned to normal. Unexpectedly, this 3 day treatment reduced the number of circulating ATL cells but did not affect spleen weight. With 6 days of treatment, only IFNα resulted in a significant decrease in spleen weight, whereas As ₂ O ₃ or IFNα / As ₂ O ₃ increased it conversely. Thus, in contrast to ex vivo treatment, primary mouse ATL did not cause extensive cell growth arrest or apoptosis in vivo. Interestingly, when mice received a proteasome inhibitor (PS341) in conjunction with IFNα / As ₂ O ₃ , a dramatic increase in the number of WBCs was observed, indicating that proteasome-mediated Tax This is consistent with the notion that degradation contributes to response to treatment.

The inventors then administered the IFNα / As ₂ O ₃ combination for 15, 20, and 24 days starting 6 days after inoculation. At all time points, this treatment moderately affected spleen weight, but resulted in a decrease in circulating ATL cells and reduced micro-metastasis within the liver parenchymal sinusoids. However, pathological examination demonstrated persistent and massive tissue infiltration and high expression of the proliferation marker Ki67. In fact, the frequency of mitosis in leukemia infiltration of the spleen and liver decreased 2-fold, but no significant cell cycle arrest was observed in ATL spleen cells even after 2 weeks of treatment. Since NFκB blockade was expected to make the cells hypersensitive to apoptosis, we analyzed the spleen and liver for TUNEL positive infiltrating tumor cells. A slight increase in in vivo labeling was observed on days 21 and 26, and ex vivo TUNEL labeling of splenic ATL cells increased only on day 26. Analysis of primary recipients sacrificed on day 30 demonstrated significant tumor regression but no clearance. However, on day 180, cured animals were indistinguishable from uninoculated controls. Therefore, IFNα / As ₂ O ₃ induces only late apoptosis, which is likely the cause of eventual ATL eradication.

Such paradoxical uncoupling between ATL cell proliferation under treatment in immunodeficient animals, delayed apoptosis, and eventual complete disappearance of ATL cells clearly demonstrates this combination of conventional Separate from anti-cancer regimens. Therefore, we wondered if the IFNα / As ₂ O ₃ combination could selectively affect ATL LIC and, secondarily, lead to progressive leukemia depletion. Three days after treatment with IFNα / As ₂ O ₃ , similar amounts of splenic leukemia cells were injected into secondary recipients. Inoculated cells did not show any evidence or cell cycle arrest or TUNEL label. However, inoculation from mice treated for 3 days rather than untreated showed a dramatic increase in survival of secondary recipients, indicating that IFNα / As ₂ O ₃ is specific for ATL LIC Suggest targeting. Using the serial dilution method of ATL cells from untreated vs. IFNα / arsenic treated mice, a clear difference was observed in the survival time of the secondary transplant recipients, indicating that IFNα / As ₂ O ₃ Formally demonstrates a dramatic reduction in the amount of LIC in leukemia cell inoculation. Interestingly, late apoptosis was observed in ATL cells taken from untreated secondary transplants inoculated with blasts from IFNα / As ₂ O ₃ treated primary recipients. Furthermore, ATL did not develop in tertiary recipients of ATL cells derived from these mice. Thus, 3 days of IFNα / As ₂ O ₃ exposure in primary mice triggers late ATL cell apoptosis and subsequent disease depletion in otherwise untreated secondary and tertiary transplants. In stark contrast, when proteasome activity was inhibited during the course of IFNα / As ₂ O ₃ treatment in primary recipients, the decrease in LIC capacity was less pronounced in secondary recipients and all tertiary mice died from ATL. .

These results strongly suggest that the in vivo effect of the IFNα / As ₂ O ₃ combination in ATL reflects the rapid disappearance of LIC, presumably through Tax proteolysis. To elucidate the mechanism behind the success of As ₂ O ₃ / IFNα / zidovuline treatment in Even when LICs are so abundant that they do not necessarily die, they irreversibly relax self-renewal and inevitably disappear in primary mice or in secondary transplant recipients. Our observations are quite consistent with the fact that one of the first effects of the leukemia oncogene is to proliferate or self-replicate progenitor cells. The molecular pathways involved in Tax degradation have not been elucidated so far, but since Tax is ubiquitinated and SUMOylated, it may share some similarities with those involved in PML degradation. . In addition to Tax loss, PML degradation by As ₂ O ₃ may contribute to LIC cell cycle initiation and depletion, which explains the paradoxical initial increase in mass. However, As ₂ O ₃ decreased the amount of LIC but never removed the disease.

At present, ATL is an incurable disease. Our approach to that treatment relies on the idea that HTLV-I is a direct initiator of ATL. However, the antiviral zidovudine / IFNα combination induces a 35-50% complete response (CR) rate, significantly extending survival compared to drugs that damage DNA, but most patients eventually relapse And die. Furthermore, this regimen does not eradicate LIC, because discontinuation of treatment causes the disease to recur immediately. Previously, the effects of the IFNα / As ₂ O ₃ regimen have been evaluated in patients with multiple relapse IFNα resistance and nevertheless have shown some success. Our latest study in de novo patients with the As ₂ O ₃ / IFNα / zidovudine combination yielded unprecedented results (90% CR, no recurrence ever). How does this mouse data contribute to our understanding of these prospective clinical outcomes? First, they suggest that the IFNα / As ₂ O ₃ association is likely to act via Tax degradation, identifying it as an HTLV-I targeted therapy. It should be noted here that the regimen used in mice did not contain zidovudine because Tax is under the control of the Lck promoter rather than being expressed by a replicable retrovirus. Furthermore, the zidovudine / IFNα combination was not better than IFNα alone. Scheduling issues (7 days a week better than 5 days a week) may also lead to adjustments to the current 5 days per week patient regimen. Second, our study establishes that the selective clearance of LIC is triggered by oncogene degradation, which indicates that these cells are highly susceptible to continuous oncogene expression or function. It suggests that it depends. To the best of our knowledge, these results are the first to elucidate the uncoupling between massive LIC loss and sustained growth of tumor volume. In this regard, the kinetics of disease clearance in As ₂ O ₃ / IFNα / zidovudine treated patients was significantly slower than tumor debulking with cytotoxic drugs. If only a partial response with As ₂ O ₃ / IFNα / zidovudine was observed on day 30, complete remissions developed over the next month, which is in complete agreement with the mouse model. Our mouse results would predict that patients responding to As ₂ O ₃ / IFNα / zidovudine are less likely to relapse and the potential for a promising treatment for ATL is increased. In summary, our findings have a broad impact on the elaborate construction of new cancer treatments based on oncogene degradation and the definition of relevant endpoints in clinical trials using such drugs.

References:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims (14)

  Combination of interferon, arsenic compound and reverse transcriptase inhibitor for use in the treatment of adult T cell leukemia / lymphoma.   The combination according to claim 1, wherein the interferon is interferon α.   The combination according to claim 1 or 2, wherein the arsenic compound is selected from the group consisting of arsenic, arsenic trioxide (As2O3), meralsoprole, and an arsenic sulfur derivative.   The combination according to any one of claims 1 to 3, wherein the reverse transcriptase inhibitor is zidovudine.   A method for treating adult T cell leukemia / lymphoma comprising administering to a subject in need of treatment for adult T cell leukemia / lymphoma an amount of an interferon, an arsenic compound and a reverse transcriptase inhibitor.   6. The method of claim 5, wherein the arsenic compound is administered at a dosage of 1-50 mg per day, preferably 10-15 mg per day.   The method according to claim 5 or 6, wherein the reverse transcriptase inhibitor is administered at a dose of 1 to 1500 mg / kg body weight and 1 to 1500 mg / kg, preferably 600 to 1000 mg / kg body weight.   8. The interferon is administered at a dose of 1-20 million international units (MIU), preferably 3-9 million international units (MIU) per day or every other day. The method described in 1.   9. The method of any of claims 5-8, wherein the arsenic compound is administered by the intravenous route, the reverse transcriptase inhibitor is administered by the oral route, and the interferon is administered by the intramuscular or subcutaneous route. .   10. The method of any of claims 5-9, wherein the interferon, arsenic compound and reverse transcriptase inhibitor are administered as a combined preparation for simultaneous use, single use or sequential use.   A pharmaceutical composition comprising an IFN, an arsenic compound and a reverse transcriptase inhibitor and optionally a pharmaceutically acceptable excipient.   12. A pharmaceutical composition according to claim 11 for use in the treatment of adult T cell leukemia / lymphoma. The present invention also provides a) IFN as a combined preparation for simultaneous use, single use or sequential use in the treatment of adult T cell leukemia / lymphoma
b) an arsenic compound and c) a reverse transcriptase inhibitor,
Relates to a kit comprising   A combination of IFN and an arsenic compound to eradicate leukemogenic cells in a subject suffering from adult T-cell leukemia / lymphoma.