RU2815051C2 - Therapeutic uses of arsenic and possible mechanisms of action - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions is based on the fact that the therapeutic effects of arsenic can be enhanced by the introduction of divalent metal ions and in particular Cu²⁺ ions. It is proposed to use a composition containing arsenic trioxide and Cu²⁺ ions to obtain a medicinal product for the treatment of graft-versus-host disease (GVHD), as well as the use of a combination of Cu²⁺ salt and arsenic trioxide to obtain a medicinal product for the treatment of graft-versus-host disease host (GVHD).

EFFECT: use of the above compositions and combinations provides a significant enhancement of the therapeutic effect of arsenic, while the introduction of Cu²⁺ ions alone does not provide any noticeable effect.

14 cl, 28 dwg, 9 tbl, 11 ex

Description

Field of the present invention

The present invention relates to the field of medicine and is based on the unexpected discovery that the therapeutic effects of arsenic can be enhanced by the administration of divalent metal ions and, in particular, Cu ²⁺ ions.

State of the art

Arsenic compounds have been widely used as traditional medicine in many regions of the world for more than two thousand years to treat various diseases such as psoriasis, syphilis or rheumatoid arthritis. Over the long history of these agents, many different arsenic preparations have been developed and used. For example, Fowler's arsenic solution containing 1% potassium arsenite (KAsO ₂ ) was prescribed for many years as a remedy for leukemia and even as a tonic drug.

Some arsenic compounds are particularly toxic and carcinogenic, with side effects such as cirrhosis of the liver, idiopathic portal hypertension, bladder cancer and skin cancer. However, recently, several arsenic compounds have been rediscovered and developed to treat various diseases such as cancer.

In particular, arsenic trioxide (As ₂ O ₃ , also referred to as “ATO” in this text) is one of the most effective new antitumor (“anticancer” or “cytotoxic”) agents. ATO has been approved by the US Food and Drug Administration (FDA) for the treatment of first-line drug-resistant acute promyelocytic leukemia, polytransretinoic acid (ATRA). Arsenic trioxide has been shown to cause apoptosis of tumor cells.

Arsenic trioxide is also known or currently being researched as a treatment for other diseases such as autoimmune diseases.

Bobé et al. (Blood, 108, 13, pp. 3967-3975, 2006) examined the effects of arsenic trioxide in a mouse model of systemic lupus erythematosus. As₂O₃ significantly prolonged the survival of MRL/lpr mice, preventing the development of the syndrome in young mice and almost completely reversed the diagnosed disease in adult animals. The authors suggested that this compound could be successfully used in the treatment of autoimmune diseases such as systemic lupus erythematosus.

In US 8394422, Chelbi-Alix and Bobé proposed the use of arsenic trioxide for the treatment of autoimmune and/or inflammatory diseases.

Kavian et al. (J Immunol. 2012;188(10):5142-9) described the successful use of ATO in a mouse model of GVHD.

More recently, Mayer et al (J Immunol. 2014 Jan 15; 192(2): 763-70) found that arsenic compounds, including the FDA-approved anti-cancer therapeutic arsenic trioxide, are potent inhibitors of caspase-1 and the innate immune response (interleukin 1). and thus can potentially be used to treat the inflammatory components of autoimmune diseases. In addition, Li et al. demonstrated that arsenic trioxide improves Treg and Th17 balance in patients with rheumatoid arthritis, thus being a potentially useful immunomodulator (Int. Immunopharmacology, 2019, Arsenic trioxide improves Treg and Th17 balance by modulating STAT3 in treatment-naïve rheumatoid arthritis patients ).

As previously reported, As ₂ O ₃ -induced cell differentiation occurs as a result of direct binding of As ₂ O ₃ to zinc finger cysteine residues in the RING-finger and B-Box (RBCC) domain of the PML-RARα fusion protein, resulting in enhanced sumoylation /ubiquitination and then degradation of PML-RARα, thereby promoting cellular differentiation leading to clinical remission (Zhang et al., Science. 2010;328(5975):240-3; Chen et al., Blood. 2011 Jun 16; 117(24):6425-37). However, although this explains the beneficial effect of arsenic in this rare form of cancer, it was clearly not a general phenomenon that could explain the effect of As in other cancers for which it was (and still is) tested for effectiveness (more than 100 clinical trials). trials are or have been conducted with arsenic compounds by 2019, as reported in clinicaltrials.gov), in neither autoimmune diseases nor GVHD.

To explain recent observations of the beneficial effect of As on the immune system, a new mechanism of action has been proposed. Indeed, it has become apparent that one of the major consequences of As exposure to a range of cell types in vitro (cell lines or primary cultures, normal or diseased) and in vivo (animal models of autoimmune diseases) is the induction of specific cell death, typically through stimulation of various , although generally hypothetical, pro-apoptotic programs, expectedly leading to changes in cellular subpopulations with often reduced levels of cytokines and modified intercellular communications.

One of the main assumptions was that some oxidative stress pathways were activated due to the production of ROS (ROS) (sometimes interesting to observe that this is not inhibited by conventional antioxidants (such as N-acetyl-L-cysteine, NAC)), which leads to to GSH consumption and other processes that cause cellular stress leading to cell death, especially in pathologically immune activated cells (Bobé et al. 2006, supra and Kavian et al., supra, suggesting plasma dendritic cell death).

To better understand the antitumor mechanism of arsenic, Zhang et al. (PNAS 2015; Vol. 112, No. 49) used a human proteome microarray to identify arsenic-binding proteins. They identified 360 proteins and found that glycolytic proteins are highly enriched. They suggest that glycolysis in general and the rate-limiting enzyme hexokinase-2 of the glycolytic pathway in particular play a key role in mediating the antitumor activity of arsenic.

Moreover, arsenic has been reported to target Pin1, described as a critical “driver” and unique drug target in cancer and other diseases. Kozono et al. suggest that ATO and ATRA, at clinically safe doses, work together to block multiple pathways leading to cancer through direct, reversible inhibition of Pin 1, thus representing a promising approach to combat breast cancer and other diseases (Nat Commun. 2018 Aug 9;9 (1):3069).

However, the detailed mechanism(s) of action of As are far from fully understood.

Common Side Effects of Arsenic

As ₂ O ₃ is a pharmacologically active compound with highly toxic properties at high concentrations or prolonged exposure.

Interestingly, treatment of acute promyelocytic leukemia (with FDA and EMA approval) and SLE (Phase 2a; discontinued) or GVHD (Phase 2, ongoing) results only in reversible side effects with intravenous (IV) ) dose, usually administered at 0.15 mg/kg/day for 1-2 (and sometimes up to 5) months of intravenous treatment.

Side effects are a potentially serious threat to the health of patients, with the most undesirable being transient prolongation of the cardiac QT interval, possibly together with changes in blood electrolyte levels and increased release of liver enzymes. These side effects are carefully monitored and are essentially reversible, but require temporary interruption of treatment for several days and occur in a significant proportion of patients. Typically, even in patients who meet all the strict inclusion/exclusion rules, approximately one third of them will experience side effects.

Thus, there remains a need to reduce the amount of arsenic administered to patients receiving treatment with arsenic trioxide or other arsenic-containing substances to reduce the side effects of the active pharmaceutical ingredient (API; arsenic trioxide or any other arsenic compound) while maintaining the same level of beneficial biological activity .

However, on the one hand, the beneficial effects of arsenic are dose dependent, and the dosages usually taken are already calculated to reduce the associated side effects. On the other hand, as explained above, the mechanism of action of arsenic is only partially determined, and at present only a few observed biochemical and physiological effects are of great significance for assessing the expected effectiveness of treatment. Most important in the field of oncology and immune pathologies is the ability of arsenic to activate cellular stress processes leading to the release of cytokines and specific cell death, as well as positive interference with immune mechanisms related to certain diseases.

Thus, there is currently a need to find drugs that enhance the beneficial effects of arsenic used as a drug without exhibiting the toxic effects of arsenic. This would allow the amount of arsenic salt(s) delivered for the respective treatment to be appropriately reduced or the effectiveness of the treatment to be increased without increasing the amount of said arsenic salt(s) administered.

ROS production and corresponding regulatory antioxidants

In this regard, the inventors have explored the possibility of enhancing the effect of a given dose of API through the concurrent use of drugs or molecules capable of increasing cellular stress.

Cellular stress is mainly achieved by increasing the amount of reactive oxygen species (ROS). The major oxidative pathway of cellular stress is well known with its cascade of steps including:

Preparation of superoxide radical: O ₂ +1 e ^- →O ₂ ^{· -}

Preparation of hydrogen peroxide: O ₂ ^{· -} +O ₂ ^{· -} +2H ⁺ →H ₂ O ₂ +O ₂

Obtaining a hydroxyl radical: H ₂ O ₂ +Fe ²⁺ → ^OH +Fe ³⁺ +OH ^-

The amount of hydrogen peroxide is regulated by heme peroxidases, which accelerate its disproportionation, and glutathione peroxidase (GPx), which catalyzes its reduction by glutathione (GSH).

There are other ROS such as peroxide radicals RO ₂ , hydroperoxides RO ₂ H and alkoxy radicals RO ^· .

The Fenton reaction that destroys H ₂ O ₂ (reaction (iii) above) is usually catalyzed by free ferrous ions and results in the formation of ^· OH. The Fenton reaction is generally believed to be localized to the endoplasmic reticulum or perinuclear sites, but not to mitochondria or other cellular compartments (Liu Q, et al. Proc Natl Acad Sci USA 2004;101:4302-7). Sources of H ₂ O ₂ can be mitochondria (superoxide dismutase reaction), peroxisomes (acyl-CoA oxidation reaction) and beta-amyloid of senile plaques (superoxide dismutase-like reactions). H ₂ O ₂ that eludes the cellular antioxidant machinery such as glutathione peroxidase and catalase can be non-enzymatically converted in the perinuclear localized Fenton reaction and act as an RNA or DNA damaging agent.

SUMMARY OF THE PRESENT INVENTION

The inventors explored the possibility of enhancing the effect of an appropriate dose of an arsenic compound through the parallel use of drugs or molecules capable of increasing cellular stress by activating the Fenton reaction. To this end, they tested several elements that are known to enhance this reaction, starting with the well-known iron salts, as well as others such as Zn, Mn, Mg, Cu, Au, etc.

As described in the experimental section below, they found that, in a completely unexpected and inexplicable way, by only activating the Fenton reaction, Cu ²⁺ ions especially exhibited strong synergistic effects with arsenic, including all the properties sought, i.e. cell production of H ₂ O ₂ , increased induction of apoptosis and physiological effects in a basic and highly significant test of immune system function, the mixed lymphocyte reaction, developed with mouse cells of different genetic origins.

Thus, the present invention relates to a medicinal product containing an arsenic compound and a metal ion selected from the group consisting of Cu ²⁺ , without excluding the more classical Fenton agents such as Au ²⁺ , Fe ²⁺ , Zn ²⁺ , Mn ^{2 +} , Mg ²⁺ and mixtures thereof.

The invention also relates to the use of a combination of a Cu ²⁺ salt and an arsenic compound in the treatment of various diseases, including neoplastic diseases, autoimmune diseases, inflammatory diseases and neurodegenerative diseases, wherein the arsenic compound and the Cu ²⁺ salt are administered to a patient simultaneously or sequentially. With such complex treatment, Cu ²⁺ ions enhance the therapeutic effect of arsenic.

DESCRIPTION OF DRAWINGS

Figure 1: Effect of As ₂ O ₃ at increasing concentrations on H ₂ O ₂ production, GSH production and HL60 cell viability.

Figure 2: Effect of FeSO ₄ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 µM on H ₂ O ₂ production and HL60 cell viability.

Figure 3: Effect of HAuCl ₂ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 μM on H ₂ O ₂ production and viability of HL60 cells.

Figure 4: Effect of ZnSO ₄ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 µM on H ₂ O ₂ production and HL60 cell viability.

Figure 5: Effect of ZnCl ₂ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 µM on H ₂ O ₂ production and HL60 cell viability.

Figure 6: Effect of MnSO ₄ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 µM on H ₂ O ₂ production and HL60 cell viability.

Figure 7: Effect of MnCl ₂ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 μM on H ₂ O ₂ production and viability of HL60 cells.

Figure 8: Effect of CuSO ₄ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 µM on H ₂ O ₂ production and HL60 cell viability.

Figure 9: Effect of CuCl ₂ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 µM on H ₂ O ₂ production and viability of HL60 cells.

Figure 10: Effect of As ₂ O ₃ (1 µM) in combination with CuCl ₂ (1 or 4 µM) on H ₂ O ₂ production and viability of HL60 cells.

Figure 11: Effect of As ₂ O ₃ (1 µM) in combination with increasing concentrations of CuCl ₂ (0.5-4 µM) on GSH production and HL60 cell viability.

Figure 12: Effect of As ₂ O ₃ on the proliferation of spleen cells of C57BL6 mice in the mixed lymphocyte reaction. Y-axis: DO UptiBlue, which measures cellular metabolic activity (and is considered a direct indicator of growth).

Figure 13: Effect of FeSO ₄ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 μM on the proliferation of spleen cells of C57BL6 mice in the mixed lymphocyte reaction.

Figure 14: Effect of HAuCl ₂ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 μM on the proliferation of spleen cells of C57BL6 mice in the mixed lymphocyte reaction.

Figure 15: Effect of ZnSO ₄ and ZnCl ₂ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 μM on the proliferation of spleen cells of C57BL6 mice in the mixed lymphocyte reaction.

Figure 16: Effect of MnSO ₄ and MnCl ₂ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 μM on the proliferation of spleen cells of C57BL6 mice in the mixed lymphocyte reaction.

Figure 17: Effect of CuSO ₄ and CuCl ₂ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 μM on the proliferation of spleen cells of C57BL6 mice in the mixed lymphocyte reaction.

Figure 18: Effect of CuCl ₂ at increasing concentrations, alone or in combination with As ₂ O ₃ at 1 µM on H ₂ O ₂ production and viability of A20 cells.

Figure 19: Effect of As ₂ O ₃ (1 µM) in combination with increasing concentrations of CuCl ₂ (0.5-4 µM) on GSH production and viability of A20 cells.

Figure 20: Effect of AsI ₃ at increasing concentrations on H ₂ O ₂ production, GSH and viability of HL60 cells.

Figure 21: Effect of CuCl ₂ at increasing concentrations, alone or in combination with AsI ₃ at 1 µM on H ₂ O ₂ production and viability of HL60 cells.

Figure 22: Effect of As ₂ O ₃ and AsI ₃ on the proliferation of spleen cells of C57BL6 mice in the mixed lymphocyte reaction.

Figure 23: Effect of CuCl ₂ at increasing concentrations, alone or in combination with AsI ₃ at 1 μM on the proliferation of spleen cells of C57BL6 mice in the mixed lymphocyte reaction.

Figure 24: Effect of treatment on the occurrence of skin abnormalities (alopecia) in chronic GVHD. The figures show the development of alopecia in different experimental groups. A: Syngeneic (8 mice). B: Allogeneic (9 mice - 7 with alopecia). C: Allogeneic + As ₂ O ₃ 2.5 μg/g (9 mice - 3 with alopecia). D: Allogeneic+As ₂ O ₃ 2.5 μg/g+CuCl ₂ 2.5 μg/g (3 mice - none with alopecia). E: Allogeneic + CuCl ₂ in the reaction of mixed lymphocytes μg/g (2 mice - 1 with alopecia). F: Allogeneic+As ₂ O ₃ 2.5 μg/g+CuCl ₂ 10 μg/g (2 mice - none with alopecia). G: Allogeneic+CuCl ₂ 10 µg/g (5 mice - none with alopecia). H: Allogeneic+As ₂ O ₃ 5 µg/g (5 mice - 1 with alopecia).

Figure 25: Effect of treatment on mice weight during GVHD development. A. Syngeneic and allogeneic groups, treated or untreated with As ₂ O ₃ (2.5 µg/g or 5 µg/g, as indicated). B. Syngeneic group and allogeneic groups, treated or not with As ₂ O ₃ (2.5 µg/g and CuCl ₂ 2.5 µg/g). C. Syngeneic group and allogeneic groups, processed or untreated (As ₂ O ₃ 2.5 µg/g and CuCl ₂ 10 µg/g).

Figure 26: Effect of treatment on vasculitis (clinical assessment: ear thickness) during the development of GVHD. A. Ear thickness of mice in syngeneic and allogeneic groups treated or not treated with As ₂ O ₃ at 2.5 μg/g or 5 μg/g. B. Ear thickness of mice in syngeneic and allogeneic groups treated or not with As ₂ O ₃ at 2.5 μg/g and CuCl ₂ at 2.5 μg/g. C. Ear thickness of mice in syngeneic and allogeneic groups treated or not with As ₂ O ₃ at 2.5 μg/g and CuCl ₂ at 10 μg/g.

Figure 27: Effect of treatment on the liver: transaminase concentrations in the blood of each mouse. A. ALAT in the plasma of mice of syngeneic and allogeneic groups depending on treatment. B. ASAT in the plasma of syngeneic and allogeneic mice depending on treatment.

Figure 28: Effect of treatment on mice weight during GVHD development. Mice treated with copper at 0/0.2 μg/g and 0.5 μg/g.

DETAILED DESCRIPTION

As used herein, the terms “treat” and “treatment” mean any reduction in one or more symptoms of a disease, for example, a reduction in the frequency and/or severity of symptoms in an autoimmune disease and/or an increase in survival that results from the administration of a composition in accordance with the invention to patients with cancer. These terms are used, as a rule, to refer to obtaining the desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease and/or may be therapeutic in terms of partial or complete cure of the disease and/or negative consequences associated with the disease. The term “treatment” as used herein applies to any treatment of a disease in a mammal and includes: preventing the occurrence of the disease in a subject who may be predisposed to the disease but does not yet clinically have it; inhibition of the disease, i.e. suspension of its development; or alleviation of the disease, i.e. regression of the disease. The therapeutic agent may be administered before, during, or after the onset of a disease or injury. Of particular interest is the treatment of ongoing disease when treatment stabilizes or reduces the patient's undesirable clinical symptoms.

As used herein, the phrase “reduction in the severity of at least one symptom” refers to a reduction in one or more symptoms of the disease or condition for which the subject is being treated. In some embodiments, the disease or condition being treated is a hematologic malignancy in which one or more symptoms are relieved, including, but not limited to: weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, a swollen or tender abdomen (due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, poor appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).

The term used in this text is “prevent” and similar words such as “prevent”, “prevent”, etc. denotes an approach to preventing, suppressing, or reducing the likelihood of the occurrence or recurrence of a disease or condition. The term also means delaying the onset of a disease or condition or relapse, or delaying the onset or recurrence of symptoms. The term "prevention" and similar words also mean reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to the onset of the disease or condition or relapse.

As used herein, the term “amount” refers to an “effective amount” or “effective amount” of arsenic+metal ion sufficient to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results. In one embodiment, an effective amount refers to an amount of a compound containing arsenic and a metal ion sufficient to prevent, improve one symptom of, or treat a disease, for example, a hematologic malignancy or an autoimmune disease discussed herein.

A “prophylactically effective amount” refers to an amount of an arsenic+metal ion-containing compound effective to achieve the desired prophylactic result. Typically, but not necessarily, since the prophylactic dose is administered to subjects before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

The "therapeutically effective amount" of the arsenic+metal ion compound may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the properties of the agent (arsenic compound) and concomitant agent (metal ion) to cause desired response in an individual. A "therapeutically effective amount" is also an amount in which the therapeutically beneficial effects are greater than any toxic or harmful effects of the agent. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (patient).

The terms “include” or “comprise” are intended to indicate that the compositions and methods include the listed elements, but do not exclude others. “Consist essentially of” is used to define compositions and methods to the exclusion of other elements having any significant significance to the combination. For example, a composition consisting essentially of the elements defined herein does not exclude other elements that do not significantly affect the basic and novel feature(s) of the claimed invention. “Consists of” means the exclusion of more than trace amounts of other ingredients and essential process steps described. The embodiments defined by each of the transitional terms are within the scope of the present invention.

The term "approximately" used before a numerical value indicates that the value may vary within reasonable limits such as ±10%, ±5% and ±1%. The expression "about x" includes the value "x".

Singular forms include plural forms unless the context indicates otherwise. Thus, "metal ion" would mean "metal ions".

If necessary, other definitions will be provided below.

According to a first aspect, the present invention relates to a medicament that contains an arsenic compound and a metal ion selected from Cu ²⁺ , Au ²⁺ , Fe ²⁺ , Zn ²⁺ , Mn ²⁺ , Mg ²⁺ and mixtures thereof.

Examples of arsenic compounds (usually salts) that can be used as active ingredients in the composition of the invention include As ₂ O ₃ , AsI ₃ , As ₂ O ₅ , As ₄ O ₆ , As ₂ S ₂ , As ₂ S ₃ , As ₂ S ₅ , As ₄ S ₄ and their mixtures.

The drugs of the present invention preferably contain arsenic trioxide or arsenic triiodide together with a metal ion listed above, such as Cu ²⁺ , alone or in combination with any other metal ion selected from Au ²⁺ , Fe ²⁺ , Zn ²⁺ Mn ²⁺ and Mg ²⁺ .

In certain embodiments shown in the examples below, the Cu ²⁺ ions are in the form of a salt, such as copper sulfate (CuSO ₄ ) or copper (II) chloride (CuCl ₂ ).

The actual dosage for ATO in the treatment of hematologic cancers is 0.15 mg/kg/day. The results presented in the experimental section demonstrate that when ATO is used in combination with Cu ²⁺ , its effects are enhanced. Therefore, the same therapeutic efficacy should be obtained with a lower dose of ATO in the presence of Cu ²⁺ as with the currently used ATO drug. Depending on the situation, the physician may also combine with Cu ²⁺ ions the same concentration of ATO that is currently approved in combination to enhance the therapeutic effect of ATO.

In certain embodiments, the composition in accordance with the present invention is formulated such that one daily dose contains from 0.01 to 0.15 mg/kg/day arsenic trioxide (corresponding to 0.1-1.6 μmol/kg arsenic atoms ).

In certain embodiments, the composition in accordance with the present invention is formulated such that one daily dose contains from 0.01 to 0.05 mg/kg/day arsenic trioxide.

In certain embodiments, the composition in accordance with the present invention is formulated such that one daily dose contains from 0.05 to 0.10 mg/kg/day of arsenic trioxide.

In certain embodiments, the composition in accordance with the present invention is formulated such that one daily dose contains from 0.10 to 0.15 mg/kg/day arsenic trioxide.

When an arsenic compound other than arsenic trioxide is used in the present invention, its dosage may be selected such that the number of arsenic atoms administered to the patient is the same as the dosages specified above for arsenic trioxide.

In certain embodiments, the composition in accordance with the present invention is formulated such that one daily dose contains from 0.05 µmol/kg to 10 µmol/kg Cu ²⁺ , preferably from 0.06 µmol/kg to 2 µmol/kg Cu ²⁺ , for example from 0.3 µmol/kg to 1.1 µmol/kg Cu ²⁺ .

In certain embodiments, the composition in accordance with the present invention is formulated such that one daily dose contains about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0 ,12, 0.15, 0.2, 0.25, 0.30, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9 , 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.2, 2.4, 2.6, 2.8, 3 , 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 µmol/kg Cu ²⁺ .

Of course, a skilled practitioner may choose any other copper dosing regimen, such as once every two days, once every three days, or even once a week, to achieve a dose equivalent to the daily dose described above.

Any disease that has already been successfully treated with ATO or any other arsenic compound may benefit from treatment with the above composition because the combination of arsenic and metal ions such as Cu ²⁺ enhances the beneficial effects of arsenic. In addition, diseases that share common pathophysiological mechanisms with diseases and disorders for which arsenic treatment has already been successfully used can be treated using a synergistic combination of arsenic and a metal ion such as Cu ²⁺ , which has not yet been implemented accordingly, because . higher doses of the active substance would be required, at which it is difficult to ensure the necessary safety.

Thus, the present invention relates to the use of the composition described above for the treatment of a disease among:

- tumor diseases,

- autoimmune diseases and

- inflammatory diseases.

The present invention also relates to the use of a combination of a Cu ²⁺ salt (for example, Cu ₂ SO ₄ or CuCl ₂ ) and an arsenic compound (as described above) in the treatment of a disease selected from the group consisting of a tumor disease, an autoimmune disease and an inflammatory disease, in wherein said arsenic compound and said Cu ²⁺ salt are administered to the patient simultaneously or sequentially.

Thus, the present invention also relates to the use of a composition containing an arsenic compound for the treatment of a disease selected from the group consisting of a neoplastic disease, an autoimmune disease and an inflammatory disease, wherein said arsenic compound is administered to a patient in combination with a Cu ²⁺ salt or any other metal ions with similar properties. In certain embodiments, the composition containing the arsenic compound is administered before the composition containing the Cu ²⁺ salt. In certain embodiments, the composition containing the arsenic compound is administered after the composition containing the Cu ²⁺ salt. In both cases, the arsenic compound and the Cu ²⁺ salt are administered at a time interval, preferably not exceeding 12 hours. In certain embodiments, the composition containing the arsenic compound and the composition containing the Cu ²⁺ salt are administered simultaneously. Regardless of the sequence of administration, the arsenic compound and Cu ²⁺ salt are administered either in the same way or in different ways.

In accordance with other aspects, the present invention relates to the use of a metal ion salt, for example a Cu ²⁺ salt, to promote/enhance the therapeutic effects of an arsenic compound as described above. Cu ²⁺ ions or the like can, for example, be used to enhance the therapeutic effects of ATO or any other arsenic compound used in the treatment of a disease such as a tumor disease, an autoimmune disease or, in general, an inflammatory disease. By "stimulating" in the present invention it is meant that, for the same amount of arsenic compound (ATO), the therapeutic effects of arsenic are greatly enhanced when the patient also receives Cu ²⁺ ions, although the administration of Cu ²⁺ ions alone does not produce any noticeable effect. Stimulation or co-action has been demonstrated at least in vitro in Examples 1-9 below (showing a significant increase in cellular H ₂ O ₂ production and apoptosis induction, as well as physiological effects in the mixed lymphocyte response, which is a very important assay for function immune system). The results shown in Example 10 illustrate this synergistic effect in vivo in an animal model.

According to another aspect, the present invention also relates to a method of treating a disease selected from the group consisting of a neoplastic disease, an autoimmune disease, or generally an inflammatory disease in a patient in need thereof, comprising the step of administering to the patient an effective dose of an arsenic compound (as defined above ) and a metal ion selected from Cu ²⁺ , Au ²⁺ , Fe ²⁺ , Zn ²⁺ , Mn ²⁺ , Mg ²⁺ and mixtures thereof. In accordance with a certain embodiment, a Cu ²⁺ salt is administered to the patient.

Depending on the context (the properties of the arsenic compound, the corresponding disease, the stage of the disease and the general parameters of the patient), the doctor selects the dosage and administration regimen of the arsenic compound and Cu ²⁺ salt and the like.

In certain embodiments, the arsenic compound is arsenic trioxide. In this case, the therapeutically effective amount of ATO is not more than 0.30 mg/kg/day, for example, 0.30, 0.25, 0.20 or 0.15 mg/kg/day ATO, and the prophylactically effective amount of ATO is not more than 0.15 mg/kg/day, for example 0.15, 0.10 or 0.05 mg/kg/day, or even less, down to 0.01 mg/kg/day.

In certain embodiments, the patient is administered a daily dose that ranges from 0.01 to 0.15 mg/kg/day of arsenic trioxide for 1 to 80 days.

In certain embodiments, the patient is administered a daily dose that is 0.01 to 0.05 mg/kg/day of arsenic trioxide for 1 to 80 days, with the possibility of retreatment if necessary to achieve a satisfactory level of clinical efficacy .

In certain embodiments, the patient is administered a daily dose that is 0.05 to 0.10 mg/kg/day of arsenic trioxide for 1 to 80 days.

In certain embodiments, the patient is administered a daily dose that is 0.10 to 0.15 mg/kg/day of arsenic trioxide for 1 to 80 days.

In certain embodiments, the patient is administered a daily dose that is from 0.05 µmol/kg to 10 µmol/kg Cu2+, preferably from 0.06 µmol/kg to 2 µmol/kg Cu ²⁺ , for example from 0.3 µmol/ kg to 1.1 µmol/kg Cu ²⁺ as long as he/she receives the arsenic compound. In certain embodiments, the patient receives about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.15, 0.2, 0. 25, 0.30, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1, 3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.2, 2.4, 2.6, 2.8, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 µmol/kg Cu ²⁺ every day while he/she is receiving the arsenic compound.

Cu ²⁺ ions can be, for example, in the form of copper sulfate or copper (II) chloride.

As already mentioned, the arsenic compound and the Cu ²⁺ salt are administered to the patient simultaneously or sequentially, using the same or different routes of administration. Cu ²⁺ salt can be administered to the patient every day, every other day, two or three times a week, or even weekly.

In certain embodiments, the arsenic compound is administered intravenously.

In certain embodiments, the arsenic compound is administered orally.

In certain embodiments, the arsenic compound is administered topically.

In certain embodiments, the arsenic compound is administered in the form of an aerosol.

In certain embodiments, the arsenic compound and the Cu ²⁺ salt are in the same pharmaceutical composition. Thus, the present invention relates to a method of administering a pharmaceutical composition containing an arsenic compound and a Cu ²⁺ salt, comprising the steps of providing said pharmaceutical composition and administering said pharmaceutical composition to a subject. This invention also relates to a method of treating an autoimmune disease and, in general, an inflammatory disease or neoplastic disease, such as cancer or a tumor, in a patient in need thereof, comprising the step of administering to the patient a therapeutically effective amount of a pharmaceutical composition containing an arsenic compound and a Cu ²⁺ salt .

Tumor diseases

The term "neoplastic disease" is used to refer to abnormal cellular proliferation in tissues of a subject as compared to normal proliferation in the same tissue type. Neoplasms include benign tumors and malignant tumors (eg, leukemia, colon tumors, or prostate cancer), which are either invasive or noninvasive. Malignant neoplasms differ from benign neoplasms in that the former exhibit a greater degree of anaplasia, or loss of cell differentiation and orientation, and have invasive and metastatic properties.

Cancers that can be treated in accordance with the present invention include solid and non-solid cancers. In certain embodiments, the cancer is a hematologic malignancy. Examples of hematological malignancies that can be treated in accordance with the invention include acute myeloid leukemia; acute non-lymphocytic leukemia; myeloblastic leukemia, promyelocytic leukemia; chronic myelomonocytic leukemia; monocytic leukemia; erythroleukemia; acute neutrophilic leukemia; myelodysplastic syndrome; acute promyelocytic leukemia; chronic lymphocytic leukemia; chronic myeloid leukemia; hairy cell leukemia; myeloproliferative neoplasms; Hodgkin's lymphoma; non-Hodgkin's lymphoma; myeloma; giant cell myeloma; sluggish myeloma; localized myeloma; multiple myeloma; plasma cell myeloma; sclerosing myeloma; single myeloma; smoldering multiple myeloma; nonsecretory myeloma; osteosclerotic myeloma; plasma cell leukemia; solitary plasmacytoma; and extramedullary plasmacytoma.

In certain embodiments, said hematologic malignancy is acute promyelocytic leukemia (APL). In certain embodiments, the APL is newly diagnosed. In certain embodiments, ALI is relapsed or refractory ALI.

Other types of cancer that can be treated in accordance with the present invention include, but are not limited to, carcinomas (eg, squamous cell carcinomas, adenocarcinomas, hepatocellular carcinomas and renal cell carcinomas), particularly bladder, colon, breast, cervical cancers , colon, esophagus, head, kidneys, liver, lungs, neck, ovaries, pancreas, prostate and stomach; benign and malignant melanomas; myeloproliferative diseases; sarcomas, in particular Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcoma, peripheral neuroepithelioma and synovial sarcoma; central nervous system tumors (eg, gliomas, astrocytomas, oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas, pineal tumors, meningiomas, meningeal sarcomas, neurofibromas, and schwannomas); germline tumors (eg, colon cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, cancer liver, colon cancer and melanoma); mixed types of neoplasia, in particular carcinosarcoma and Hodgkin's disease; and tumors of mixed origin, such as Wilms tumor and teratocarcinomas.

Autoimmune and inflammatory diseases

Autoimmune diseases are diseases of the immune system characterized by the production of antibodies (called autoantibodies) that interact with antigens (called autoantigens) originating from the patient's tissues, or diseases that are characterized by the activation of immune cells (eg, cytotoxic cells) to produce antibodies or without her.

Inflammatory diseases include a wide range of disorders and conditions that are characterized by inflammatory processes, either secondary, primary or even unique (predominant over other immune mechanisms).

Autoimmune diseases and inflammatory diseases that can be treated with the combination of an arsenic compound and a metal ion such as Cu ²⁺ according to the invention include, but are not limited to, systemic lupus erythematosus (SLE); acute disseminated lupus erythematosus; Bechet's disease; juvenile arthritis; Fissinger-Leroy-Reiter syndrome; gout; osteoarthritis; polymyositis; myocarditis; autoimmune rheumatoid arthritis; systemic vasculitis; insulin-dependent diabetes mellitus; type I diabetes, inflammatory bowel disease; celiac disease, an autoimmune thyroid disease; Sjogren's syndrome, autoimmune gastritis, ulcerative colitis; Crohn's disease; autoimmune hepatitis, primary biliary cirrhosis; primary sclerosing cholangitis; skin autoimmune diseases; autoimmune dilated cardiomyopathy, multiple sclerosis (and other demyelinating diseases; myasthenia gravis; vasculitis (eg, Takayasu's arteritis and Wegener's granulomatosis); aplastic anemia, any disease associated with non-tumoral lymphoproliferation; B-lymphocytic lymphoma; Simmonds'syndrome; subacute thyroiditis and Hashimoto's disease ; Addison's disease; autoimmune muscle diseases, autoimmune neuromuscular disorders such as ankylosing spondylitis, multiple sclerosis and its various forms, including childhood syndromes and acute disseminated encephalitis; immune-mediated neuropathies; autoimmune testicular diseases, autoimmune ovarian disease, autoimmune uveitis, Graves' disease , psoriasis, ankylosing spondylitis, Addison's disease, Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, autoimmune lung disease such as Wegener's disease and Churg-Strauss syndrome; immunological lung diseases such as asthma, infiltrative pulmonary disease, hypersensitivity lung disease and sarcoidosis; dermatomyositis, including scleroderma and polymyositis; and vitiligo.

The products of the present invention can also be used for the treatment of graft-versus-host disease (GVHD), either as a prophylactic or curative treatment.

Multiple sclerosis

The beneficial effects of arsenic compounds, especially arsenic trioxide, have already been reported in multiple sclerosis (USA 2018/325944). Thus, multiple sclerosis and related syndromes can be treated in accordance with the invention using a combination of arsenic and a metal ion such as Cu ²⁺ .

Specific diseases that can be treated in accordance with the present invention are human diseases or their animal counterparts that have already been successfully treated with ATO, such as acute promyelocytic leukemia, systemic lupus erythematosus (SLE), chronic graft-versus-host disease (GVHD) ), multiple sclerosis (MS), Sjogren's syndrome, rheumatoid arthritis, Crohn's disease, myelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, malignant glioma, myelodysplastic syndrome, multiple myeloma and liver cancer. As explained above, the present invention provides enhanced effects of arsenic in the treatment of these diseases, which allows the use of lower daily doses of ATO or better therapeutic results with the same dosage that is currently used.

Other characteristics of the invention will also become apparent during the following description of the biological assays that have been carried out within the scope of the invention and which provide it with the necessary experimental support without limiting its scope.

EXAMPLES

In the absence of any indication to the contrary, the experimental results shown in Examples 1-9 were obtained using the following materials and methods.

Cells, animals and chemicals

HL60 cell culture : the HL-60 cell line is a European promyelocytic leukemia cell line (ATCC n°CCL-240). This line was maintained throughout the experiment, maintaining 2 × 10⁵ cells per ml in flask (Falcon 250 ml 75 cm², reference: 353135) in RPMI 1640+GlutaMax culture medium Sigma-Aldrich (Saint-Quentin Fallavier, France) containing 10% fetal bovine serum (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), 1% ciprofloxacin and 1% fungizone (Gibco, USA).

A20 cell culture : A20 cell line is a mouse cell line (ATCC n°TIB-208). This line was maintained throughout the experiment, maintaining 2x10⁵ cells per ml in flask (Falcon 250 ml 75 cm², reference: 353135) in RPMI 1640+GlutaMax culture medium Sigma-Aldrich (Saint-Quentin Fallavier, France) containing 10% fetal bovine serum (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), 1% ciprofloxacin, 1% fungizone (Gibco, USA) and 1% 2-mercaptoethanol (Gibco, USA).

Animals : Eight-week-old female BALB/c mice and female C57Bl6 mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France) and provided with food and water ad libitum. Their content met all the necessary requirements. Mouse spleen cells of each type were used in a mixed culture of lymphocytes (KSL model).

Chemical substances:

- The first five divalent cations tested were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France): CuSO ₄ , FeSO ₄ , MnSO ₄ , ZnSO ₄ and HAuCl ₂ (laboratory grade).

- Three other divalent cations tested were purchased from ChemCon (Freiburg, Germany): CuCl ₂ , MnCl ₂ and ZnCl ₂ (GMP grade).

- Arsenic trioxide (Arscimed, quality clinical batch, stock solution 1 mg/ml) is supplied by MEDSENIC (Strasbourg, France).

Statistical analysis

All quantitative data were analyzed using GraphPad Prism 5 using one-way ANOVA and post-hoc (Tukey). P-value <0.05 is considered statistically significant.

Effects As ₂ O ₃ with or without divalent cations for H products ₂ O ₂ and GSH production in cultured HL-60 cells

Cultivation and processing conditions

Cells (5 × 10 ⁵ per well) were seeded in 96-well round bottom plates (Falcon, Corning, Ref: 353077) and incubated for 48 hours under various experimental conditions in a 37°C 5% CO2 incubator. Two plates were prepared in parallel to measure H ₂ 0 ₂ production and viability of HL-60 cells, respectively.

Product measurement H ₂ 0 ₂

After 48 hours, the supernatant was removed, and 50 μl per well of 50 μg/ml monochlorobimane (Sigma-Aldrich Saint-Quentin Fallavier, France) in PBS saline solution was added to the cells under the same treatment conditions (medium, or As ₂ O ₃ with cation or without it).

H ₂ O ₂ production was assessed by spectrofluorimetry using a Packard fusion spectrofluorimeter. Fluorescence intensity was recorded immediately (T0 hours) and after 6 hours of incubation (T6 hours). The fluorescence excitation/emission maxima were 485/530 nm for 2',7'-dichlorodihydrofluorescein diacetate.

GSH Product Measurement

GSH production was assessed by spectrofluorimetry using a Packard fusion spectrofluorimeter. Fluorescence intensity was recorded immediately (T0 hours) and after 6 hours of incubation (T6 hours). The fluorescence excitation/emission maxima were 380/461 nm for monochlorbimane.

Viability of HL-60 cells

The medium was removed and cells were stained with 0.5% crystal violet and 30% ethanol in PBS for 30 minutes at room temperature. After two washes with PBS, the stained cells were suspended in methanol and absorbance was measured at 550 nm using a Packard fusion spectrofluorimeter (as in Ngô et al., Reactive Oxygen Species Controls Endometriosis Progression; The American journal of pathology; 2009, 175(1): 225-34).

H calculation ₂ O ₂ or GSH received within 6 hours:

_H2O2 or _GSH production was calculated in each treatment condition as follows:

Fluorescence intensity [arbitrary units] at T6 hours - fluorescence intensity [arbitrary units] at T0 hours

DO synthesized by viable cells with intact membrane

Processing conditions and related experiments (2 tests)

- Products H ₂ O ₂ or GSH and cell viability:As range₂0₃: 0.5 ; 1 ; 5 and 10 µm

- H ₂ O ₂ production : comparison of 5 divalent cations in 4 concentrations with or without AS ₂ O ₃ (1 µM):

Sigma:

CuSO ₄ : 0.5; 1 ; 2-4 microns

FeSO ₄ : 0.5; 1 ; 2-4 microns

ZnSO ₄ : 6; 12.5; 25-50 microns

_HAuCl2 : 0.125; 0.25; 0.5; 1 µm

MnSO ₄ : 0.125; 0.25; 0.5; 1 µm

Chemcon:

CuCl ₂ : 0.5; 1 ; 2-4 microns

ZnCl ₂ : 6; 12.5; 25-50 microns

MnCl ₂ : 125; 0.25; 0.5; 1 µm

Additional results

- H ₂ O ₂ or GSH production and cell viability: As ₂ 0 ₃ concentration range at 0.5 ; 1 ; 5 and 10 µm with CuCl ₂ at 1 and 4 µm (1 test)

- GSH production and cell viability: As ₂ 0 ₃ (1 µm) with CuCl ₂ at 0.5, 1, 2-4 µm (2 tests)

Effects As ₂ O ₃ with or without divalent cations for H products ₂ O ₂ and GSH production in cultured A20 cells

Conditions for cultivation and treatment

Cells (1 x ¹⁰⁵ per well) were seeded into 96-well round-bottom plates (Falcon, Corning, Ref: 353077) and incubated for 48 hours under various experimental conditions in a 37°C 5% CO2 incubator. Two plates were prepared in parallel to measure H ₂ 0 ₂ production and viability of A20 cells.

Products H ₂ 0 ₂ , GSH and cell viability determined using the same protocols as described above for H60 cells.

Treatment conditions and related experiments : 1 test H ₂ O ₂ -1 GSH test

- H ₂ O ₂ or GSH production and cell viability: As ₂ 0 ₃ (1 µm) with CuCl ₂ at 0.5; 1 ; 2 and 4 µm.

Effect As ₂ O ₃ with or without divalent cations on the proliferation of C57Bl6 CD4 T cells in vitro (cell proliferation in mixed lymphocyte cultures).

The model is based on a culture of mixed lymphocytes obtained from suspensions of spleen cells from a female C57Bl6 mouse and a female BALB/c mouse irradiated to 30 Gy. Spleen cells were mechanically separated, red blood cells were removed by hypotonic lysis (ACK - NH ₄ Cl 0.15M+KHCO ₃ 1mM+Na ₂ EDTA 0.1mM).

UptiBlue dye was used to quantitatively measure cell proliferation in vitro. C57Bl6 cells (responder cells) were selected with irradiated BALB/c cells (stimulator cells) at 6 x 10 ⁵ per well per line in 96-well round bottom plates (Falcon, Corning, Ref: 353077). The mixed cell culture was incubated for 48 hours under various experimental conditions in a 37°C 5% CO ₂ incubator.

After 48 hours, 10 μl of UptiBlue viable cell counting dye (Interchim, ref: UP669413) was added directly to the culture medium, and proliferation of C57 cells was measured using a spectrofluorimeter (fusion microplate fluorometer, Packard) after 24 hours of incubation with UptiBlue dye at 37 °C with 5% CO ₂ .

Processing conditions: presented 2 tests (except ZnCl₂ and MnCl₂)

Sigma:

CuSO ₄ : 0.5; 1 ; 2 and 4 µm

FeSO ₄ : 0.5; 1 ; 2 and 4 µm

ZnSO ₄ : 6; 12.5; 25 and 50 µm

_HAuCl2 : 0.125; 0.25; 0.5; 1 µm

MnSO ₄ : 0.125; 0.25; 0.5; 1 µm

Chemcon:

CuCl ₂ : 0.5; 1 ; 2 and 4 µm µm

ZnCl ₂ : 6; 12.5; 25 and 50 µm

MnCl ₂ :125; 0.25; 0.5; 1 µm

Example 1: Effects As ₂ O ₃ for products H ₂ O ₂ and GSH and HL60 cell viability

The HL60 cell line is a human leukemia cell line. A patient diagnosed with leukemia undergoes chemotherapy to remove leukemia cells and then receives a bone marrow transplant from a healthy patient. In some cases, hematopoietic stem cell transplantation causes chronic graft-versus-host disease (GVHD). First-line therapy for chronic GVHD is based on immunosuppressive drugs (corticosteroids with or without cyclosporine), achieving a satisfactory response in approximately 30% of patients. Medsenic is currently conducting a phase II study to evaluate the effectiveness of adding arsenic trioxide to standard therapy in controlling chronic GVHD and shortening the duration of corticosteroid therapy. In this study, arsenic trioxide was administered to patients at a dose of 0.15 mg/kg/day. One aspect of this study is to determine whether nitrogen trioxide is able to affect possibly surviving leukemia cells that are resistant to chemotherapy, in addition to its effect on the autoimmune features of chronic GVHD, through a process involving the induction of cellular stress on the cells.

Cells are able to respond to cellular stress by inducing the production of reactive oxygen species. In this study, researchers observed the effects of As compounds/As ₂ O ₃ -induced cellular stress in the presence of divalent cations on hydrogen peroxide (H ₂ O ₂ ) production, GSH production and cell viability.

To induce cellular stress, HL60 cells (5 x 10 ⁵ cells/well/strain) were seeded in 96-well plates (Falcon, Corning, Ref: 353077) and incubated for 48 hours in complete medium with or without arsenic trioxide at several concentrations : 0.1; 0.5; 1; 5 and 10 µm (As ₂ O ₃ ) in an incubator at 37°C 5% CO ₂ .

Effects As ₂ O ₃ :

Figure 1 shows the effect of As ₂ O ₃ at increasing concentrations on H ₂ O ₂ production, GSH and cell viability (4 independent tests).

The production of H ₂ O ₂ in the presence of As ₂ O ₃ is not significant, but increases at 5 μm (Figure 1A).

GSH production increases significantly in the presence of As ₂ O ₃ at 0.5, 1 and 5 μM, and disappears at 10 μM (Figure 1C).

HL-60 cell viability was significantly increased in the presence of As ₂ O ₃ at 1, 5, and 10 μM (P<0.05, P<0.001, and P<0.001, respectively; Figures 1B and 1D).

Cell death at 5 and 10 μm explains the decrease in H ₂ O ₂ and GSH production at these As ₂ O ₃ concentrations.

Conclusion

A dose of 1 µM As ₂ O ₃ was chosen to test the effect of the combination of As ₂ O ₃ and metal ions on H ₂ O ₂ production and viability of HL60 cells.

At this concentration, the biological effect of H ₂ O ₂ production is still compensated by GSH production, and cell viability is maintained. Thus, the effect of As ₂ O ₃ can be modulated by the metal ion.

Example 2: Effects of As Combinations ₂ O ₃ and metal ions for H production ₂ O ₂ and viability of HL60 cells

To induce cellular stress, HL60 cells (5 x 10 ⁵ cells/well/strain) were seeded in 96-well plates (Falcon, Corning, Ref: 353077) and incubated for 48 hours in complete medium with or without 1 μM arsenic trioxide ( As ₂ O ₃ ) with or without various cations, or in culture medium alone in a 37°C 5% CO ₂ incubator. The results presented are obtained from 2 independent experiments (except for MnCl ₂ and ZnCl ₂ ), and the cations have different degrees of quality: laboratory purity CuSO ₄ (0.5; 1; 2 and 4 µm), FeSO ₄ (0.5; 1; 2 and 4 µm), ZnSO ₄ (6; 12.5; 25 and 50 µm), HAuCl ₂ (0.125; 0.25; 0.5; 1 µm), MnSO ₄ (0.125; 0.25; 0 .5; 1 µm) and GMP degree of purity (for production) CuCl ₂ (0.5; 1; 2 and 4 µm), ZnCl ₂ (6; 12.5; 25 and 50 µm), MnCl ₂ (125; 0 .25; 0.5; 1 µm).

Effects of As combinations ₂ O ₃ and metal ions :

The results of experiments on HL60 cells for H ₂ 0 ₂ production and cell viability using FeSO ₄ , HAuCl ₂ , ZnSO ₄ , ZnCl ₂ , MnSO ₄ , MnCl ₂ , CuSO ₄ and CuCl ₂ are shown in Figures 2-9, respectively.

According to statistics, significant differences are shown in the figures as follows:

- control cells compared to cells treated with As ₂ O ₃

- control cells compared to cells treated with cations only

- cells treated with As ₂ O ₃ compared to cells treated with As ₂ O ₃₊ cations.

FeSO₄ separately has a significant impact on H production₂O₂ only at 4 µm (test 1) or with As₂O₃(1 µm) at 4 µm (test 1, p<0.001) and at 2 and 4 µm (test 2, p<0.01 and 0.001, respectively (Figure 2, top graphs). FeSO₄ separately and with As₂O₃(1 μm) do not have a significant effect on cell viability (Figure 2, bottom graphs).

HAuCl ₂ alone does not have a significant effect on the production of H ₂ O ₂ ; this effect is limited at 1 μm with As ₂ O ₃ (p < 0.01, test 1) and at 0.5 μm with As ₂ O ₃ (p < 0.01, test 2; Figure 3, top graphs). HAuCl ₂ alone and with As ₂ O ₃ (1 µM) do not have a significant effect on the viability of HL60 cells (Figure 3, bottom graphs).

ZnSO₄And ZnCl₂alone do not have a reproducible effect on H production₂O₂ (Figures 4 and 5, graphs at top). With As₂O₃ (1 µm), ZnSO₄ significantly increases H production₂O₂at 12.5; 25 and 50 µm (p<0.001, only in test 2; Figure 4, top graph on the right). With As₂O₃, ZnSO₄And ZnCl₂do not have a significant and reproducible effect on cell viability (Figures 4 and 5, bottom graphs).

MnSO ₄ has a significant dose-dependent effect on H ₂ O ₂ production at 0.25, 0.5 and 1 µm (tests 1 and 2, figure 6, top graphs) and an additional effect with As ₂ O ₃ at 0.5 and 1 µm (p<0.001; tests 1 and 2; figure 6, top graphs) and even with As ₂ O, at 0.25 (p<0.001; test 2; figure 6, top left graph). MnSO _4, with or without As ₂ O _3, had no real effect on cell viability (Figure 6, bottom graphs).

MnCl₂, with or without As₂O₃, has a dose-dependent effect on H production₂O₂, but the increase is significant only at 1 µm (p<0.001, test 1, figure 7, top graph). MnSO₄, with or without As₂O₃, has no real effect on cell viability (Figure 7, bottom graph).

CuSO ₄ and CuCl ₂ separately do not have a significant effect on the production of H ₂ O ₂ (figures 8 and 9, graphs at the top). The obvious effect of CuSO ₄ on H ₂ O ₂ production in test 2 can be explained by the abnormally low control value. Both CuSO ₄ and CuCl ₂ enhance the effect of As ₂ O ₃ at all tested concentrations (p<0.001, figures 8 and 9, top graphs). CuSO ₄ and CuCl ₂ , with or without As ₂ O ₃ , appear to have no real effect on cell viability (Figures 8 and 9, bottom graphs).

Table 1 summarizes the results obtained for H ₂ O ₂ production in HL60 cells.

Cations Products H ₂ O ₂ :
cations
regarding control Products H ₂ O ₂ :
As ₂ O ₃ +cations
relative to As ₂ O ₃ Cell viability:
cations
regarding control Cell viability:
As ₂ O ₃ +cations
relative to As ₂ O ₃ FeSO ₄
2 tests 4 µm (test 1)
Not significant (test 2) 4 µm (test 1)
2 and 4 µm (test 2) Not significant (tests 1 and 2) Not significant (tests 1 and 2) HAuCl ₂
2 tests Not significant (tests 1 and 2) 1 µm (test 1)
0.5 µm (test 2) Not significant (tests 1 and 2) Not significant (tests 1 and 2) ZnSO ₄
2 tests Not significant (test1)
50 µm (test 2) Not significant (test 1)
12.5, 25 and 50 µm (test 2) Not significant (test1)
25 and 50 µm (test2) Not significant (tests 1 and 2) ZnCl ₂
1 test 12.5 µm Not significantly Not significantly Not significantly MnSO ₄
2 tests 0.25, 0.5, 1 µm (tests 1 and 2) 0.25, 0.5 and 1 µm (test 1)
0.5 and 1 µm (test 2) 0.5 µm (test 1)
Not significant (test 2) Not significant (tests 1 and 2) MnCl ₂
1 test 1 µm 1 µm Not significantly Not significantly CuSO ₄
2 tests Not significant (test 1)
0.5, 1, 2 and 4 µm (test 2) 0.5, 1, 2 and 4 µm
(tests 1 and 2) Not significant (test 1)
4 µm (test 2) Not significant (test 1)
0.5, 1, 2 and 4 µm (test 2) CuCl ₂
2 tests Not significant (tests 1 and 2) 0.5, 1, 2 and 4 µm (test1)
1, 2 and 4 µm (test2) Not significant (tests 1 and 2) Not significant (tests 1 and 2)

Table 1

Note: carried out several tests to verify the effect of sequential As deposition₂O₃, followed by metal ions (or vice versa: metal ions+As₂O₃) or a mixture of two active ingredients. There were no significant differences between test series (data not shown).

Conclusion

Copper ions do not affect the viability of HL60 cells alone or in the presence of As ₂ O _3, but significantly enhance the effect of As ₂ O ₃ on H ₂ 0 ₂ production in a dose-dependent manner. CuCl ₂ was chosen for further analysis because it complies with (and is readily available in industrial quantities) the Good Manufacturing Practice (GMP).

Example 3: Effect of As combination ₂ O ₃ and CuCl ₂ for products H ₂ O ₂ and GSH and HL60 cell viability.

To induce cellular stress, HL60 cells ( ^5x105 cells/well/strain) were seeded in 96-well plates and incubated for 48 hours in complete medium with or without arsenic trioxide at several concentrations (0.1, 0.5, 1; 5 and 10 µm) and with and without CuCl ₂ at 1 and 4 µm in a 37°C 5% CO ₂ incubator.

As combination effect ₂ O ₃ and CuCl ₂ :

CuCl ₂ at 1 and 4 μm significantly increases H ₂ O ₂ production caused by As ₂ O ₃ at all concentrations (Figure 10, top left diagram).

CuCl ₂ at 1 and 4 µM reduces GSH production caused by As ₂ O ₃ mainly at 1 and 5 µM As ₂ O ₃ (Figure 10, bottom diagram on the left).

CuCl ₂ at 1 and 4 μm had no additional or protective effect on HL60 cell viability in the presence of As ₂ O ₃ (Figure 10, right diagram).

Conclusion

CuCl ₂ tested at 1 and 4 μM with a wide range of As ₂ O ₃ concentrations had no additive or protective effect on HL60 cell viability, but did have an additional effect on H ₂ O ₂ production and a negative effect on GSH production.

Example 4: Effect of As combination ₂ O ₃ and CuCl ₂ on GSH production and HL60 cell viability.

To induce cellular stress, HL60 cells ( ^5x105 cells/well/strain) were seeded in 96-well plates and incubated for 48 hours in complete medium with or without arsenic trioxide at 1 μM and with and without _CuCl2 at 0. 5, 1, 2 and 4 µm in an incubator at 37°C 5% 37°C 5% CO ₂ .

As combination effect ₂ O ₃ (1 µm) and CuCl ₂ (0.5, 1, 2 and 4 µm)

As ₂ O ₃ at 1 µM significantly increases GSH production. In test 1, CuCl ₂ reduced the increase in GSH caused by As ₂ O ₃ , but this effect was only observed at higher concentrations (Figure 11, top graphs).

CuCl ₂ alone at any of the 4 concentrations tested did not have a significant effect on GSH production (Figure 11, top graphs).

CuCl ₂ alone at any of the 4 concentrations tested did not have a significant effect on cell viability (Figure 11, bottom graphs).

Table 2 shows the significant results for GSH production.

GSH Products:
cations
regarding control GSH Products:
As ₂ O ₃ +cations
relative to As ₂ O ₃ Cell viability:
cations
regarding control Cell viability:
As ₂ O ₃ +cations
relative to As ₂ O ₃ CuCl2:
(2 tests) Not significant (tests 1 and 2) 4 µm (test 1)
2 and 4 µm (test 2) Not significant (tests 1 and 2) Not significant (tests 1 and 2)

table 2

Conclusion

These results confirm the strong potentiating effect of copper on _{As2O3 -} induced oxidative stress in HL60 cells.

Example 5: Effect of As combination ₂ O ₃ and metal ions on the proliferation of spleen cells of C57BL6 mice in the reaction of mixed lymphocytes

The mixed lymphocyte response is an ex vivo assay of cellular immunity that is performed between two populations of allogeneic lymphocytes (same species but genetically different). The two populations of lymphocytes are incubated together and the resulting response is measured.

In the present invention, the mixed lymphocyte reaction is used as a model of an autoimmune reaction that corresponds to the proliferation of spleen cells in vitro from C57BL6 mice exposed and stimulated to irradiated spleen cells from BalbC mice. This in vitro method mimics the lymphocyte response that occurs in vivo during graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation.

Cell proliferation assayin vitro performed on mouse spleen cells C57BL6 co-cultured with previously irradiatedin vitro (30 Gray) by BALB/c spleen cells.

For the proliferation assay, cells (6 x 10 ⁵ cells/well/strain) were inoculated into 96-well plates (Falcon, Corning, Ref: 353077) and incubated for 48 hours in complete medium with or without arsenic trioxide at 1 μM (As ₂ O ₃ ), with or without various cations, or in culture medium alone in a 37°C 5% CO ₂ incubator. The results presented are from 2 independent experiments and the cations have different quality levels as described in the materials and methods section above.

The results of studies of spleen cells for controls, FeSO ₄ , HAuCl ₂ , Zn ²⁺ salts (ZnSO ₄ and ZnCl ₂ ), Mn ²⁺ salts (MnSO ₄ and MnCl ₂ ) and Cu ²⁺ salts (CuSO ₄ and CuCl ₂ ) are shown in figures 12-17, respectively. According to statistics, significant differences are presented in the figures as follows:

- control cells compared to cells treated with As ₂ O ₃

- control cells compared to cells treated only with cations and

- cells treated with As ₂ O ₃ compared with cells treated with As ₂ O ₃ + cations.

Checking cultivation conditions : Figure 12 shows the proliferation measurement control (4 independent tests): spleen cells from C57BL6 mice only and spleen cells from BALB/c mice only do not proliferate. In contrast, mixed cells exhibit a lymphocyte response and proliferation equal to that obtained in the presence of mitogen (CD3 5µg/ml/CD28 2µg/ml). Arsenic trioxide (1 µM) completely inhibits cell proliferation in the presence of mitogen.

Note: Several tests were performed to test the effect of sequential precipitation of As ₂ O ₃ followed by metal ions (or vice versa, metal ions + As ₂ O ₃ ) or a solution of the two agents. There were no significant differences between test series (data not shown).

Effect of As combinations ₂ O ₃ and metal ions

FeSO ₄ has no significant effect on cell proliferation, either alone or with As ₂ O ₃ (Figure 13).

HAuCl ₂ reduced cell proliferation alone, but this effect was limited at 1 μM As ₂ O ₃ and only in test 1 (p < 0.001, Figure 14).

ZnSO₄And ZnCl₂reduce cell proliferation alone, but this effect is limited at 50 µM with As₂O₃ and only in test 1 (p<0.01 and p<0.001, respectively, Figure 15).

MnSO₄And MnCl₂significantly reduce cell proliferation alone, but this effect is limited at 1 µM with As₂O₃ and only in test 1 (Figure 16).

CuSO₄And CuCl₂significantly reduce cell proliferation alone, but this effect is limited at 4 µM or 2 and 4 µM with As₂O₃ (p<0.001, Figure 17).

Table 3 shows the results for the reduction in cell proliferation.

Cations Cell proliferation:
Cations relative to control Cell proliferation:
As ₂ O ₃ + cations relative to As ₂ O ₃ FeSO ₄ (2 tests) Not significant (test 1)
0.5 µm (test 2) Not significant (tests 1 and 2) HauCl ₂ (2 tests) 0.125, 0.50 and 1 µm (tests 1 and 2) 1 µm (test 1)
Not significant (test 2) ZnSO ₄ (2 tests) 6, 25 and 50 µm (test 1)
6 & 50 µm (test 2) 50 µm (test 1)
Not significant (test 2) ZnCl ₂ (2 tests) 6 and 50 µm (test 1)
6, 12.5, 25 and 50 µm (test 2) 50 µm (test 1)
Not significant (test 2) MnSO ₄ (2 tests) 0.125, 0.25, 0.50, 1 µm (test 1 and 2) 1 µm (test 1)
Not significant (test 2) MnCl ₂ (2 tests) Not significant (test 1)
0.125, 0.25, 0.50 and 1 µm (test 2) Not significant (tests 1 and 2) CuSO ₄ (2 tests) 0.5, 1, 2 and 4 µm (tests 1 and 2) 2 and 4 µm (test 1)
Not significant (test 2) CuCl ₂ (2 tests) 0.5, 1, 2 and 4 µm (tests 1 and 2) 4 µm (test 1)
2 and 4 µm (test 2)

Table 3

Conclusion

Cu ²⁺ is the most effective metal ion in reducing the mixed lymphocyte response; changing the salt composition (CuSO ₄ or CuCl ₂ ) or quality level does not affect the target effect.

Example 6: Effect of As combination ₂ O ₃ and CuCl ₂ for products H ₂ O _2, GSH and A20 cell viability.

A20 cells are a mouse lymphoma cell line derived from spontaneous neoplasm of Balb/CANN mouse reticulum cells. In this study, arsenic was tested on a mouse cancer cell line to study its effects in the same way as for HL60 cells. Indeed, before future studies in an in vivo mouse model, it is necessary to test the effect of As ₂ O ₃ on these mouse cells in vitro.

To induce cellular stress, A20 cells ( ^1x105 cells/well/strain) were seeded in 96-well plates and incubated for 48 hours in complete medium with or without arsenic trioxide at 1 μM and with and without CuCl2 at 4 concentrations ( 0.5, 1, 2 and 4 µm) in a 37°C 5% CO ₂ incubator.

Statistically significant differences are presented in the figures as follows:

- control cells compared to cells treated with As ₂ O ₃

- control cells compared to cells treated with cations only

- cells treated with As ₂ O ₃ compared to cells treated with As ₂ O ₃₊ cations.

As combination results ₂ O ₃ and CuCl ₂

As ₂ O ₃ at 1 μM significantly increased H ₂ O ₂ production and decreased A20 cell viability (p < 0.001, test 1, Figure 18A), but in test 2, As ₂ O ₃ only significantly decreased A20 cell viability ( p < 0.001, Figure 18B),

CuCl ₂ alone increased H ₂ O ₂ production at any of the 4 concentrations tested 4 (test 1 p<0.001) and at 3 higher concentrations (test 2 p<0.001). CuCl ₂ alone appeared to increase A20 cell viability at 3 or 2 higher concentrations tested (Figure 18).

At any of the 4 concentrations tested, CuCl ₂ enhanced the effect caused by As ₂ O ₃ on H ₂ O ₂ production (Tests 1 and 2, Figure 18, left graphs). In the presence of As ₂ O ₃ , CuCl ₂ slightly but significantly increased the viability of A20 at higher concentrations (Test 1: 2 and 4 and Test 2: 4 µM; Figure 18, right graph).

As ₂ O ₃ at 1 μM significantly increased GSH production and significantly decreased A20 cell viability (p<0.001, test 1, Figure 19).

CuCl ₂ alone significantly reduced GSH production and increased viability of A20 cells at 2 and 4 μM (test 1; Figure 19). In the presence of As ₂ O ₃ , CuCl ₂ significantly reduced arsenic-induced GSH production at higher concentrations and increased A20 cell viability only at 4 μM (test 1; Figure 19).

Tables 4 and 5 display significant results for H ₂ O ₂ production, GSH and viability of A20 cells.

Cations Products H ₂ O ₂ :
cations
regarding control Products H ₂ O ₂ :
As ₂ O ₃ +cations
relative to As ₂ O ₃ Cell viability:
cations
regarding control Cell viability:
As ₂ O ₃ +cations
relative to As ₂ O ₃ CuCl ₂
(2 tests) 0.5, 1, 2 and 4 µm (test1)
1, 2 and 4 µm (test 2) 0.5, 1, 2 and 4 µm (tests 1 and 2) 1, 2 and 4 µm (test 1)
2 and 4 µm (test 2) 2 and 4 µm (test 1)
4 µm (test 2)

Table 4

Cations GSH Products:
cations
regarding control GSH Products:
As ₂ O ₃ +cations
relative to As ₂ O ₃ Cell viability:
cations
regarding control Cell viability:
As ₂ O ₃ +cations
relative to As ₂ O ₃ CuCl ₂
(1 test) 2 and 4 µm 1, 2 and 4 µm 2 and 4 µm 4 µm

Table 5

Conclusion

In the presence of As ₂ O ₃ , CuCl ₂ significantly increased H ₂ O ₂ production and decreased GSH production without any significant effect on A20 cell viability.

Example 7: AsI Effects ₃ for products H ₂ O ₂ , GSH and HL60 cell viability

To induce cellular stress, HL60 cells (5 x 10 ⁵ cells/well/strain) were seeded in 96-well plates (Falcon, Corning, ref: 353077) and incubated for 48 hours in complete medium with AsI ₃ and without AsI ₃ at several concentrations: 0.1; 0.5; 1; 5 and 10 µm in an incubator at 37°C 5% CO ₂ .

AsI effect ₃ for HL60 cells:

Figures 20-23 show the effect of AsI ₃ at increasing concentrations on H ₂ O ₂ and GSH production and cell viability (1 test).

AsI ₃ significantly reduced H ₂ O ₂ production at all concentrations tested (p<0.001, Figure 20A).

AsI ₃ significantly increased GSH production at 0.1, 1 and 5 µM (p<0.01, p<0.001 and p<0.001, respectively, Figure 20B).

AsI ₃ significantly reduced the viability of HL60 cells at 10 μM (p<0.001, Figure 20C).

Conclusion

The dose of AsI ₃ at 1 µM was chosen to test the effect of combinations with CuCl ₂ on H ₂ 0 ₂ production and HL60 cell viability.

Example 8: Effect of AsI combination ₃ and CuCl ₂ for products H ₂ O ₂ and viability of HL60 cells

To induce cellular stress, HL60 cells ( ^5x105 cells/well/strain) were seeded in 96-well plates and incubated for 48 hours in complete medium with or without _AsI3 at 1 μM and with and without _CuCl2 at 0.5. 1, 2 and 4 µm in a 37°C 5% CO ₂ incubator.

Effect of AsI combination ₃ and CuCl ₂ :

AsI ₃ at 1 µM does not affect H ₂ O ₂ production and cell viability (Figure 21).

Regardless of the concentration tested, CuCl ₂ significantly increased the effect of AsI ₃ (at 1 µM) on H ₂ 0 ₂ production (Figure 21, left graph). CuCl ₂ did not affect cell viability in the presence of AsI ₃ (Figure 21, right graph).

Conclusion:

In the presence of AsI ₃ (1 µM), a significant potentiation effect of CuCl ₂ on the production of H ₂ O ₂ is observed, although it is obviously lower than in the presence of As ₂ O _3.

Example 9: AsI Effect ₃ on the proliferative properties of C57Bl6 spleen cells during the mixed lymphocyte reaction (MLR).

Cell proliferation assayin vitro carried out on mouse spleen cells C57BL6 in co-culture with previously irradiated (30 Gray) so-called “stimulating” spleen cells of BALB/c mice.

For proliferation assays, cells (6 x 10 ⁵ cells/well/strain) were seeded in 96-well plates (Falcon, Corning, Ref: 353077) and incubated for 48 hours in complete medium with or without arsenic trioxide (As ₂ O ₃ ) at 1 µM or arsenic triiodide (AsI ₃ ) at 1 µM in the presence of mitogen or copper at increasing concentrations from 0.5 to 4 µM CuCl ₂ .

Cell proliferation was determined spectrofluorimetrically after 24 hours of incubation of cells with 10% UptiBlue (20 μl in 200 μl medium).

The effects of arsenic trioxide (As ₂ O ₃ ) at 1 µM or arsenic triiodide (AsI ₃ ) at 1 µM on cell proliferation in the presence of mitogen are shown in Figure 22. AsI ₃ is significantly active only in test 2 (Figures 22 and 23, graphs on the right ).

The results of these experiments on spleen cells for CuCl ₂ with more or less arsenic triiodide (AsI ₃ ) are shown in Figure 23.

In the presence of AsI ₃ , CuCl ₂ significantly reduced the proliferation of C57BL6 spleen cells at 1, 2 and 4 μm (test 1) and at 4 μm (test 2).

Conclusion:

AsI₃ active, but slightly less than As₂O₃, And joint action with CuCl_2,apparently decreases, which probably indicates that the concentration of As ions is important.

Example 10: As Effects ₂ O ₃ and CuCl ₂ in a mouse model of graft-versus-host disease (GVHD)

The association of arsenic trioxide (As ₂ O ₃ ) with copper molecules (CuCl ₂ ) was assessed in a well-established mouse model of chronic sclerodermatous graft-versus-host disease (Arsenic Trioxide Prevents Murine Sclerodermatous Graft-versus-Host Disease, Kavian et al., 2012). In this model, mice (female BALB/c mice and male H-2d mice) previously exposed to sublethal 7.5 Gray irradiation from a Gammacel source and injected with bone marrow (1×10 ⁶ cells) and splenocytes (2×10 ⁶ cells) from mice with low immune compatibility (male B10.D2 H-2b mice).

7 days after transplantation, mice received intraperitoneal injections of iron trioxide, alone or in combination with copper chloride (“copper”), 5 times a week for 5 weeks. An irradiation control group was added to test the effectiveness of irradiation to ensure that transplantation was successful in recipient mice. The syngeneic group was inoculated with splenocytes and bone marrow from mice of the same genetic origin. This control group should not develop chronic GVHD. Once-weekly clinical assessment of signs such as hair loss, weight loss, diarrhea and vasculitis assessed disease progression.

Groups Recipients Donor Number of mice Control irradiation group BALB/c Ø 3 Syngeneni BALB/c BALB/c 9 Allogeneic BALB/c B10.D2 10 Allogeneic+As ₂ O ₃ 2.5 µg/g BALB/c B10.D2 10 Allogeneic + As ₂ O ₃ 2.5 μg/g + CuCl ₂ 10 μg/g BALB/c B10.D2 10 Allogeneic+CuCl ₂ 10 µg/g BALB/c B10.D2 7 Allogeneic + As ₂ O ₃ 2.5 μg/g + CuCl ₂ 2.5 μg/g BALB/c B10.D2 10 Allogeneic+ CuCl ₂ 2.5 µg/g BALB/c B10.D2 7 Allogeneic+As ₂ O ₃ 5 µg/g BALB/c B10.D2 7

Table 6: Experimental groups, with strains of donor and recipient mice and the number of mice in the group

Initial protocol: After 5 weeks, mice were to be euthanized to evaluate the effect of the combination of copper and arsenic trioxide on organs affected by chronic GVHD (skin, lungs, liver...).

Protocol adjustment: After significant loss of mice in the copper groups, injections had to be stopped at the end of the 4th day. Treatment was resumed the following week on day 8 at a different rate until the end of week 5 of treatment. Copper was then administered only 2 times per week instead of 5 times to mice receiving copper, while mice receiving arsenic alone or arsenic plus copper still received 5 ATO injections per week. Under the new treatment conditions, no deaths were observed among copper-treated mice.

Because the number of mice was significantly reduced, we decided to collect only mouse blood to perform transaminase assays, but no organs were collected. These experiments suggest that copper must be reduced to eliminate its toxic effects in mice, at least when it is administered intraperitoneally.

Groups Number of mice at the beginning Number of mice at the end Exposure control 3 0 Syngeneni 9 8 Allogeneic 10 9 Allogeneic+As ₂ O ₃ 2.5 µg/g 10 9 Allogeneic + As ₂ O ₃ 2.5 μg/g + CuCl ₂ 10 μg/g 10 2 Allogeneic+CuCl ₂ 10 µg/g 7 5 Allogeneic + As ₂ O ₃ 2.5 μg/g + CuCl ₂ 2.5 μg/g 10 3 Allogeneic+CuCl ₂ 2.5 µg/g 7 2 Allogeneic+As ₂ O ₃ 5 µg/g 7 5

Table 7: Number of live mice at the end of the experiment compared to the initial number of mice

The results of this experiment are shown in Table 8 below and in Figures 24-27.

Groups Number of mice Number of mice with alopecia Percentage of mice with alopecia Syngeneni 8 0 0% Allogeneic 9 7 77% Allogeneic+As ₂ O ₃ 2.5 µg/g 9 3 33% Allogeneic + As ₂ O ₃ 2.5 μg/g + CuCl ₂ 2.5 μg/g 3 0 0% Allogeneic+CuCl ₂ 2.5 µg/g 2 1 50% Allogeneic + As ₂ O ₃ 2.5 μg/g + CuCl ₂ 10 μg/g 2 0 0% Allogeneic+CuCl ₂ 10 µg/g 5 0 0% Allogeneic+As ₂ O ₃ 5 µg/g 5 1 20%

Table 8: Experimental results (alopecia)

Despite the fact that the number of mice in the group is not enough for reliable statistics, we can still note that none of the mice that received complex treatment with As ₂ O ₃ (2.5 μg/g) and CuCl ₂ (2.5 or 10 µg/g), did not develop alopecia, which is better than in mice receiving only As ₂ O ₃ 2.5 µg/g (33% alopecia), and even better than in mice receiving As ₂ O ₃ 5 µg/g (20% alopecia) (Figure 24). This confirms that the effect of arsenic is enhanced in combination with copper.

Regarding the weight of the mice, there was no obvious difference between the groups at the end of treatment, even though the mice in the syngeneic group appeared to weigh more than the mice in the other groups (Figure 25).

Mice in the allogeneic group show increased ear thickness (vasculitis) compared to mice in the syngeneic group. We also observe that in the allogeneic group + As ₂ O ₃ 2.5 μg/g, vasculitis decreased, and in the allogeneic group + 5 μg/g it decreased even more until it reached the same values as in the syngeneic group (Figure 26).

With regard to the hepatic balance of animals, the results obtained for serum transaminases are not consistent. This may be due to the variable toxicity of repeated intraperitoneal injections of arsenic-copper trioxide, with the liver being exposed to different amounts depending on the location of each injection and therefore having different harmful effects (Figure 27).

Example 11: As Effects ₂ O ₃ and CuCl ₂ in a mouse model of graft-versus-host disease (GVHD) with a lower dosage of CuCl ₂

Based on these results, we decided to evaluate the cytotoxicity of copper. To this end, we administered a lower dose of copper (0.2 μg/g and 0.5 μg/g) 5 times per week to a new series of BALB/c mice for 5 weeks. We monitored the mice every day, monitoring their weight and survival rate to assess copper toxicity.

After 5 weeks, there were no deaths and mean weights were similar between the PBS control group and the 0.2 µg/g and 0.5 µg/g copper groups. Therefore, a dosage of 0.5 μg/g was selected for a new set of experiments using the same protocol as Example 10, but with the conditions described in Table 9 below.

Groups Recipients Donor Number of mice Exposure control BALB/c Ø 3 Syngeneni BALB/c BALB/c 7 Allogeneic BALB/c B10.D2 7 Allogeneic+As ₂ O ₃ 2.5 µg/g BALB/c B10.D2 10 Allogeneic+As ₂ O ₃ 2.5 μg/g+CuCl ₂ 0.5 μg/g BALB/c B10.D2 10 Allogeneic+CuCl ₂ 0.5 µg/g BALB/c B10.D2 10 Allogeneic+As ₂ O ₃ 5 µg/g BALB/c B10.D2 10

Table 9: Experimental groups, with strains of donor and recipient mice and the number of mice in the group

Unfortunately, this series of experiments was interrupted due to COVID-19, due to which the laboratory was forced to suspend work on March 11, 2020. Experiments will continue after the laboratory resumes work.

Claims (14)

1. The use of a composition containing arsenic trioxide and Cu ²⁺ ions to obtain a drug for the treatment of graft-versus-host disease (GVHD). 2. Use according to claim 1, wherein the Cu ²⁺ ions are in the form of a salt selected from the group consisting of copper sulfate and copper(II) chloride. 3. Use according to claim 1, where the specified medicinal product is formulated so that one daily dose contains from 0.01 to 0.10 mg/kg/day of arsenic trioxide. 4. Application according to any one of paragraphs. 1-3, where the specified medicinal product is formulated in such a format that one daily dose contains from 0.05 µmol/kg to 10 µmol/kg Cu ²⁺ . 5. Use according to claim 4, where the specified medicinal product is formulated in such a format that one daily dose contains from 0.06 µmol/kg to 2 µmol/kg Cu ²⁺ . 6. Use according to claim 5, where the specified medicinal product is formulated in such a format that one daily dose contains from 0.3 µmol/kg to 1.1 µmol/kg Cu ²⁺ . 7. Application according to any one of paragraphs. 1-3, wherein said GVHD is chronic graft-versus-host disease (GVHD). 8. Use of a combination of Cu ²⁺ salt and arsenic trioxide to obtain a drug for the treatment of graft-versus-host disease (GVHD). 9. Use according to claim 8, wherein the Cu ²⁺ salt is copper sulfate or copper (II) chloride. 10. Use according to claim 8 or 9, where the specified medicinal product is formulated so that one daily dose contains from 0.01 to 0.10 mg/kg/day of arsenic trioxide. 11. Application according to any one of paragraphs. 8-10, where the specified medicinal product is formulated so that one daily dose contains from 0.05 µmol/kg to 10 µmol/kg Cu ²⁺ . 12. Use according to claim 11, where the specified medicinal product is formulated in such a format that one daily dose contains from 0.06 µmol/kg to 2 µmol/kg Cu ²⁺ . 13. Use according to claim 12, where the specified medicinal product is formulated in such a format that one daily dose contains from 0.3 µmol/kg to 1.1 µmol/kg Cu ²⁺ . 14. Application according to any one of paragraphs. 8-13, wherein said GVHD is chronic graft-versus-host disease (GVHD).

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