Methylene blue-containing compounds for the treatment of methemoglobinemia - Patent Application 20070233633

Abstract

The present invention provides a novel method for orally treating methemoglobinemia in a subject, comprising administering to a subject a compound of the formula: n A and H n each of B (if present) is a protic acid, which may be the same or different, and where p=1 or 2; q=0 or 1; n=1 or 2; (p+q)×n=2, or a hydrate or solvate thereof, is orally administered to said subject. This administration should provide a total daily oral dose of 4 mg to 60 mg of MT to the subject per day. [Formula 1] TIFF2025509873000030.tif44170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of priority to UK Patent No. 2204185.9, filed on March 24, 2022, and incorporated herein in its entirety.

[0002] Technical Field The present invention relates generally to methods and materials for use in reducing methemoglobinemia or treating methemoglobinemia in a subject. [Background technology]

[0003] Background technology Methemoglobinemia is a rare disorder involving the oxidation of ferrous iron in hemoglobin to ferric iron in methemoglobin (see “Iolascon, Achille, et al. “Recommendations for diagnosis and treatment of methemoglobinemia.” American Journal of Hematology (2021) 96: 1666-1678”).

[0004] Iron(III) [Fe 3+ The presence of iron in the [[state]] state leads to an allosteric change that irreversibly binds oxygen. The corresponding ferrous globin in a tetramer shifts the oxygen dissociation curve of Hb to the left. This shift leads to an increased affinity of ferrous iron for oxygen and thus impaired oxygen release to tissues, resulting in hypoxia with so-called "functional anemia" without a fall in Hb.

[0005] Methemoglobinemia can result from inherited or acquired processes. The acquired form is the most common, primarily due to exposure to substances that directly or indirectly cause the oxidation of Hb. The inherited form is due to autosomal recessive variants in the CYB5R3 gene or autosomal dominant variants in the globin genes, collectively known as hemoglobinopathies.

[0006] Intravenous methylthioninium chloride (MTC, methylene blue) is the standard treatment for methemoglobinemia (see "Skold, A., et al. "Methemoglobinemia." Southern Medical Journal 2011;104:757-761"). The methylthioninium (MT) moiety is converted to oxidized MT + Tau can exist in the reduced or "leuco" form (as in MTC) and in the reduced or "leuco" form as hydromethylthionine (LMT) (see Harrington, CR, et al. "Cellular models of aggregation-dependent template-directed proteolysis to characterize tau aggregation inhibitors for treatment of Alzheimer disease." Journal of Biological Chemistry 2015;290:10862-10875). MT +Thiazine must first be converted to LMT to allow absorption, distribution, and uptake into cells (see Baddeley, TC, et al. “Complex disposition of methylthioninium redox forms determines efficacy in tau aggregation inhibitor therapy for Alzheimer's disease.” Journal of Pharmacology and Experimental Therarapeutics. 2015;352:110-118, and Merker, MP, et al. “Pulmonary endothelial thiazine uptake: separation of cell surface reduction from intracellular reoxidation.” American Journal of Physiology: Lung Cellular and Molecular Physiology. 1997;272:L673-L680). LMT is the active species at the heme site of action, where it is converted from LMT to Fe. 3+ In this process, Fe 3+ Fe 2+ and LMT is reduced to MT +Continual regeneration of LMT via sustained erythrocyte glycolysis allows restoration of normal oxygen-carrying capacity (see Blank, O., et al. “Interactions of the antimalarial drug methylene blue with methemoglobin and heme targets in Plasmodium falciparum : A physico-biochemical study.” Antioxidants and Redox Signaling. 2012;17:544-554,” and Yubisui, T., et al. “Reduction of methemoglobin through flavin at the physiological concentration by NADPH-flavin reductase of human erythrocytes.” Journal of Biochemistry 1980;87:1715-1720).

[0007] However, the role of MTC in the treatment of methemoglobinemia is paradoxical: for example, MTC actually causes acquired methemoglobinemia (Iolascon 2021, see above).

[0008] Methylene blue-vitamin CN-acetyl cysteine ​​(MCN) has been reported to benefit critically ill COVID-19 patients, and one putative mechanism of action of this drug and administration is through the reduction of methemoglobin (metHb) (“Alamdari, Daryoush Hamidi, et al. “Application of methylene blue-vitamin CN-acetyl cysteine ​​for treatment of critically ill COVID-19 patients, report of a phase-I clinical trial.” European Journal of Pharmacology 885 (2020): 173494).

[0009] WO 2021 / 224146 relates to the use of hydromethylthionine and related salts (referred to in the document as "LMTX") as a therapeutic agent for reducing hypoxemia in a subject. It is therefore proposed that these salts can be used to reduce hypoxia and treat symptoms or other causes of hypoxia. The effects disclosed in the document are said to be unrelated to any known effects on metHb, and WO 2021 / 224146 describes that high concentrations of LMT (with oral doses in the range of 150-250 mg / day) can result in a measurable increase in metHb. The results described in WO 2021 / 224146 showed that high dosages of the LMTX compound over a period of time systematically increase metHb levels (see Figure 3 in WO 2021 / 224146), yet still reduce hypoxia.

[0010] From the foregoing, it can be seen that providing compounds that can be safely and conveniently used to treat methemoglobinemia (i.e., to reduce the level of metHb in a subject) would provide a useful contribution to the art. Summary of the Invention [Means for solving the problem]

[0011] Disclosure of the Invention The present invention relates to the use of "LMTX" compounds, including LMT, delivered orally in appropriate dosages to treat methemoglobinemia (i.e., to reduce the level of methemoglobin) in a subject. This treatment does not depend on, and is not dependent on, the presence of hypoxia or is absent. This provides a more convenient method for treating methemoglobinemia than the use of intravenous MTC.

[0012] Oral LMTX has not been previously disclosed for the treatment of methemoglobinemia. However,3+ It is the inventors' insight that the nature of the LMT binding interaction with is such that oral treatment at appropriate doses can provide benefit in terms of ferric-globin conversion.

[0013] Oral therapy is applicable to both hereditary and acquired methemoglobinemia. The availability of an oral therapy with a benign safety profile for use in these conditions would represent a valuable addition to currently available treatment options.

[0014] Thus, in one aspect, there is provided a method of treating (or alleviating) methemoglobinemia in a subject, comprising: orally administering to said subject a methylthioninium (MT)-containing compound. A method is disclosed that includes wherein said administration provides a total daily oral dose of 4 mg to 60 mg of MT to the subject per day, optionally divided into two or more doses; As used herein, the MT-containing compound has the formula: [ka] (In the formula, H n A and H n each B, if present, is a protonic acid, which may be the same or different; And p=1 or 2; q=0 or 1; n=1 or 2; (p+q)×n=2. or a hydrate or solvate thereof.

[0015] The total daily dose of MT can be 8 or 10 or 20 or 20.5 or 21 to 50 or 60 mg.

[0016] The total daily dose can be from about 4, 8, 12, 16, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24 mg to approximately 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 mg.

[0017] The total daily dose can be about 8, 12, 16, 20, 20.5, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg.

[0018] An example dosage is 10 to 50 mg.

[0019] Further dosage examples are 20 or 20.5 to 50 mg.

[0020] Further dosage examples are 30-50 mg.

[0021] A further example dosage is 40-50 mg.

[0022] The total daily dose of compound may be administered as divided doses two or three times daily.

[0023] As explained below, when MT is administered multiple times / days, it may be desirable to use a lower total amount within the recited ranges compared to a single daily dose or fewer administrations per day.

[0024] The patient may be an adult human, and the population-based dosages described herein are assumed on that basis (typical weight 50-70 kg). If desired, equivalent dosages can be utilized for subjects outside of this range by using a subject weight factor by dividing the subject's weight by 60 kg to provide a multiplication factor for each subject.

[0025] Subjects for treatment can be characterized or selected by certain criteria.

[0026] Signs, symptoms, and causes of methemoglobinemia used for selection may include any of those in Table 7 (Iolascon 2021, see above).

[0027] In one embodiment, the subject is not hypoxic or suffering from hypoxemia.

[0028] In one embodiment, the subject has a blood oxygen saturation level (SpO2) of greater than 95% on room air.

[0029] For the present invention, the subject must be able to breathe and swallow, as the treatment will be administered orally.

[0030] The methods of the invention may include the step of selecting a subject according to one or more of the criteria described above.

[0031] Thus, in some embodiments, the subject may be a human who has been diagnosed with methemoglobinemia, or where the method comprises making said diagnosis.

[0032] The present invention can be applied to treat acquired or hereditary forms of methemoglobinemia. Examples of acquired or hereditary forms of methemoglobinemia are shown in Table 6. Thus, this treatment can be for methemoglobinemia caused by drug exposure (e.g., phenazopyridine (pyridium), sulfamethoxazole, dapsone, aniline, paraquat / monolinuron, nitrates, nitroglycerin, amyl nitrite, isobutyl nitrite, sodium nitrite, benzocaine, lidocaine, prilocaine, or chloramine).

[0033] In some embodiments, the genetic disease is methemoglobinemia type I, II, III, or IV, or HbM disease, or unstable hemoglobin.

[0034] In some embodiments, the subject has a metHb level greater than 20%.

[0035] Symptoms of methemoglobinemia may depend on the rate of metHb formation: for example, patients with lifelong methemoglobinemia may be symptomatic, whereas patients exposed to drugs and toxins that suddenly develop the same levels of methemoglobinemia may be severely ill.

[0036] In some embodiments, the subject has a metHb level as set forth in Table 7. For example, the subject can be symptomatic with a metHb level of greater than 30%, 50%, or 70%.

[0037] In some embodiments, the subject has acute methemoglobinemia.

[0038] In some embodiments, the subject has chronic methemoglobinemia.

[0039] In some embodiments, the genetic disease is autosomal recessive congenital methemoglobinemia.

[0040] WO 2007 / 110627 disclosed certain 3,7-diamino-10H-phenothiazinium salts that are effective as drugs or prodrugs for the treatment of diseases including, generally, Alzheimer's disease and other diseases, such as frontotemporal dementia (FTD), as well as viral diseases. These compounds may also be in the "reduced" or "leuco" form when considering MTC. These leuco methylthioninium compounds were referred to in the document as "LMTX" salts.

[0041] WO 2012 / 107706 describes leuco-methylthioninium bis(hydromethanesulfonate) (LMTM) (WHO INN name: hydromethylthionine): [ka] Other LMTX salts have been described that have superior properties to those listed above, including:

[0042] Preferably, the LMT compound is a "LMTX" compound of the type described in WO 2007 / 110627 or WO 2012 / 107706.

[0043] Thus, the compound has the formula: [ka] or a hydrate or solvate thereof.

[0044] H n A and H n Each B, if present, is a protic acid, which may be the same or different.

[0045] "Protonic acids" are acids that release protons (H + ) donor. Therefore, A in the protonic acid - Or B -is the conjugate base. Thus, protic acids have a pH of less than 7 in water (i.e., a concentration of hydronium ions of 10 per liter). -7 (Molar excess).

[0046] In one embodiment, the salt has the formula: [ka] (where HA and HB are different mono-protic acids) It is a mixed salt having the formula:

[0047] However, preferably the salt is not a mixed salt but has the formula: [ka] (Here, H n Each X is a protic acid, such as a di-protic acid or a mono-protic acid. has.

[0048] In one embodiment, the salt has the formula: [ka] (where H2A is a di-protic acid) has.

[0049] Preferably, the salt is a bismonoprotic acid salt of the formula: [ka] has.

[0050] Examples of protic acids that may be present in the LMTX compounds used herein include the following: Inorganic acids: hydrohalide acids (e.g., HCl, HBr), nitric acid (HNO3), sulfuric acid (H2SO4) Organic acids: Carbonic acid (H2CO3), acetic acid (CH3COOH), methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid.

[0051] Preferred acids are monoprotic acids and the salts are bis(monoprotic acid) salts.

[0052] A preferred MT compound is LMTM: [ka] It is.

[0053] Weight Factor The anhydrous salt has a molecular weight of approximately 477.6. Based on a molecular weight of 285.1 for the LMT core, the weight factor for using this MT compound in the present invention is 1.67. "Weight factor" refers to the relative weight of the pure MT-containing compound to the weight of the MT it contains.

[0054] Other weight coefficients can be calculated for the examples of MT compounds herein, from which corresponding dosage ranges can be calculated.

[0055] Examples of other LMTX compounds are as follows, with their molecular weights (anhydrous) and weight coefficients given: [ka] [ka]

[0056] Thus, the dosages described herein for MT apply mutatis mutandis to these MT-containing compounds, as adjusted for their molecular weight.

[0057] Accumulation factor As will be appreciated by one of skill in the art, for a given daily dosage, more frequent dosing may result in greater accumulation of drug.

[0058] Thus, in certain embodiments of the claimed invention, the total daily dose of the MT compound can be relatively low when administered more frequently (e.g., twice daily [bid] or three times daily [tid]), or high when administered once daily [qd].

[0059] Treatment and Prevention The term "treatment", as used herein generally in the context of treating a condition, relates to treatments and therapies, whether in humans or animals (e.g., in veterinary applications), that result in some desired therapeutic effect, e.g., inhibition of progression of the condition, including slowing the rate of progression, stopping the rate of progression, reversing the condition, ameliorating the condition, and curing the condition.

[0060] The term "therapeutically effective amount" as used herein relates to an amount of a compound of the present invention, or a material, composition, or dosage form containing said compound, that is effective to produce some desired therapeutic effect commensurate with a reasonable benefit / risk ratio when administered according to a desired treatment regimen. The inventors have demonstrated that the therapeutically effective amount of MT compounds for the diseases of the present invention can be significantly less than previously understood in the art.

[0061] The present invention also encompasses treatment as a prophylactic measure.

[0062] The term "prophylactically effective amount," as used herein, relates to an amount of a compound of the invention, or a material, composition, or dosage form containing said compound, that is effective, when administered in accordance with a desired treatment regimen, to produce some desired prophylactic effect commensurate with a reasonable benefit / risk ratio.

[0063] "Prevention" in the context of this specification should not be understood to draw boundaries around complete success, i.e. complete protection or complete prevention. Rather, prevention in this context refers to treatment administered prior to a condition or prior to the worsening of such a condition, with the goal of maintaining health by helping to delay, alleviate, or avoid that particular condition.

[0064] The term "treatment" includes "combination" treatments and therapies, where two or more treatments and therapies are combined, for example sequentially or simultaneously. These may be symptomatic or disease-modifying treatments.

[0065] The particular combination will be at the discretion of the physician.

[0066] In combination therapy, the agents (i.e., an MT compound as described herein plus one or more other agents) can be administered simultaneously or sequentially, and each can be administered on various dosing schedules and via different routes. For example, when administered sequentially, the agents can be administered closely spaced (e.g., within 5-10 minutes) or more widely spaced (e.g., 1, 2, 3, 4 hours or more, or even longer if needed), with the precise dosing regimen being appropriate to the properties of the therapeutic agents.

[0067] In other embodiments, the treatment is a "monotherapy", ie, the MT-containing compound is not used in combination with another active agent (within the meaning set forth above).

[0068] For the treatment of methemoglobinemia, the MT compound-based treatment regimen described herein will preferably be for a duration appropriate to the disease and symptoms, the particular duration of which will be at the discretion of the physician.

[0069] For example, the duration of treatment 1 to 14, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days 1 to 4, e.g., 1, 2, 3, or 4 weeks It could be.

[0070] In all cases, the duration of treatment will generally be subject to medical advice and review.

[0071] The MT compounds of the present invention, or pharmaceutical compositions comprising same, can be administered orally (or via a nasogastric tube) to the stomach of a subject / patient.

[0072] Typically, in the practice of the invention, the compound will be administered as a composition comprising the compound and a pharma- ceutically acceptable carrier or diluent.

[0073] In some embodiments, the composition is a pharmaceutical composition (e.g., formulation, preparation, pharmaceutical product) comprising a compound as described herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0074] The term "pharmacologically acceptable" as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, and the like, that are suitable for use in contact with the tissues of a subject of interest (e.g., humans), within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0075] In some embodiments, the compositions are pharmaceutical compositions comprising at least one compound as described herein, together with one or more other pharma- ceutically acceptable ingredients known to those of skill in the art, including, but not limited to, pharma- ceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners.

[0076] In some embodiments, the composition further comprises other active agents, for example other therapeutic or prophylactic agents.

[0077] Suitable carriers, diluents, excipients, etc. can be referenced in standard pharmaceutical texts, see, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0078] One aspect of the invention utilizes a dosage unit (e.g., a pharmaceutical tablet or capsule) comprising an MT compound as described herein (e.g., obtained or obtainable by a method as described herein; having a purity as described herein; etc.) and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0079] The "MT compound", although it may be present in relatively small amounts, is intended to be the active agent of the dosage unit, i.e., to have a therapeutic or prophylactic effect with respect to methemoglobinemia. Rather, the other components in the dosage unit will be therapeutically inactive, e.g., carriers, diluents, or excipients.

[0080] Thus, preferably there will be no other active ingredients in the dosage unit other than those associated with the combination therapy described herein, no other drugs intended to have a therapeutic or prophylactic effect with respect to the disorder for which the dosage unit is intended to be used.

[0081] In some embodiments, the dosage unit is a tablet.

[0082] In some embodiments, the dosage unit is a capsule.

[0083] In some embodiments, the capsule is a gelatin capsule.

[0084] In some embodiments, the capsule is an HPMC (hydroxypropyl methylcellulose) capsule.

[0085] The appropriate amount of MT in the composition will depend on how often per day it is taken by the subject or how many units are taken at one time. Thus, dosage units can individually contain less than the total daily dose.

[0086] Exemplary dosage units may contain 10, 20, 30, 40, 50, or 60 mg of MT.

[0087] Using the weight coefficients described or illustrated herein, one of ordinary skill in the art can select the appropriate amount of MT-containing compound for use in an oral formulation.

[0088] As explained above, the MT weight factor for LMTM is 1.67. Because it is convenient to use unitary or simple fractional amounts of active ingredients, a non-limiting example of a LMTM dosage unit can include 17 mg, providing 10 mg of LMT or MT, etc.

[0089] In one embodiment, a unit dosage pharmaceutical composition is provided comprising about 17, 34, 51 mg, etc. of LMTM.

[0090] A unit dosage composition described herein (LMTX compound plus optionally other ingredients) can be provided in a labeled packet accompanied by instructions for its use.

[0091] In one embodiment, the packaging is a bottle, such as those well known in the pharmaceutical arts. A typical bottle can be made from pharmacopoeia grade HDPE (high density polyethylene) with a child-resistant HDPE push-lock closure and can contain a silica gel desiccant, which is present in a sachet or container. The bottle itself can include a label and can be packaged in a cardboard container with instructions for the user and, optionally, an additional copy of the label.

[0092] In one embodiment, the package or packet is a blister pack (preferably one with aluminum cavities and aluminum foil, which is therefore substantially impermeable to moisture), in which case the package may be packaged in a cardboard container with instructions and a label for the user on the container.

[0093] The label or instructions can provide information about treating methemoglobinemia.

[0094] Treatment methods Another aspect of the present invention relates to a method for the treatment of methemoglobinemia, as explained above, comprising administering to a patient in need of treatment a prophylactically or therapeutically effective amount of a compound as described herein, preferably in the form of a pharmaceutical composition.

[0095] Use in methods of treatment Another aspect of the present invention pertains to a compound or composition as described herein for use in a method of treatment of methemoglobinemia in the human or animal body by therapy.

[0096] Use in pharmaceutical manufacturing Another aspect of the present invention pertains to the use of an MT compound or composition as described herein in the manufacture of a medicament for use in the treatment of methemoglobinemia.

[0097] In some embodiments, the medicament is a composition, eg, a dosage composition, as described herein.

[0098] Mixture of oxidized and reduced MT compounds The LMT-containing compounds utilized in the present invention contain oxidized (MT + ) compounds, which may be oxidized (e.g., autoxidized) after synthesis to give the corresponding oxidized form. Thus, if not unavoidable, compositions containing the compounds of the invention will likely contain at least some of the corresponding oxidized compounds as impurities. For example, the "LMT" salts may contain up to 15%, e.g., 10-15%, of MT + It may contain salt.

[0099] When mixed MT compounds are used, the MT dosage can be easily calculated using the molecular weight coefficients of the compounds present.

[0100] Salts and solvates Although the MT-containing compounds described herein are salts per se, they can also be provided in the form of mixed salts (i.e., a compound of the invention combined with another salt). Such mixed salts are intended to be encompassed by the term "and pharma-ceutically acceptable salts thereof." Unless otherwise specified, a reference to a particular compound also includes its salts.

[0101] The compound of the present invention can also be provided in the form of a solvate or hydrate. The term "solvate" is used herein in the usual sense to refer to a complex of a solute (e.g., a compound, a salt of a compound) and a solvent. If the solvent is water, the solvate can be conveniently referred to as a hydrate, for example, a monohydrate, a dihydrate, a trihydrate, a pentahydrate, etc. Unless otherwise specified, any reference to a compound includes its solvate and any hydrate form.

[0102] Of course, solvates or hydrates of the salts of the compounds are also encompassed by the present invention.

[0103] In order to more fully describe and disclose the present invention and the prior art to which the invention pertains, several patents and publications are cited herein. Each of these references is incorporated by reference in its entirety into the present disclosure herein to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0104] Throughout this specification, including the claims which follow, unless the context otherwise requires, the word "comprise", as well as variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not to the exclusion of any other integer or step or group of integers or steps.

[0105] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0106] Ranges are often expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the individual value forms another embodiment.

[0107] Any subheadings herein are included for convenience only and should not be construed as limiting the disclosure in any way.

[0108] The invention will now be further described with reference to the following non-limiting figures and examples, in the light of which other embodiments of the invention will occur to those skilled in the art.

[0109] The disclosures of all references cited in this specification, to the extent that they can be used by those skilled in the art to practice the invention, are individually incorporated into this specification by cross-reference. [Brief description of the drawings]

[0110] drawing [Figure 1] Change in oxygen saturation in patients undergoing LMTM comparing pre-dose and 4 hours (post-dose) administration of a single dose of LMT at 4 mg and higher doses (75 / 100 / 125 mg shown as mean 100 mg). Data represent mean (%) ± standard error. [Diagram 2] Change in oxygen saturation in patients undergoing LMTM over 6 weeks. Pooled data for all treatments represent mean (%) ± standard error. [Diagram 3] Change in methemoglobin levels in patients undergoing LMTM comparing pre-dose and 4 hours (post-dose) administration of a single dose of LMT at 4 mg and higher doses (75 / 100 / 125 mg shown as mean 100 mg). Data represent mean (%) ± standard error. [Figure 4] Changes in methemoglobin levels in patients receiving low (8 mg / day) and high (150 mg, 200 mg, and 250 mg) doses of HMTM over a 6-week period. Data represent mean (%) ± standard error. [Diagram 5]Putative effect of LMTM treatment on oxygen-hemoglobin dissociation curves as described in the text. Data for patients with mild hypoxemia are from subjects treated with LMTM. Data for patients with severe hypoxemia are from results reported by Hamidi-Alamdari, D., et al. “Methylene blue for treatment of hospitalized COVID-19 patients, randomized, controlled, open-label clinical trial, phase 2.” Revista de Investigacion Clinica. 2021; 73:190-198 (where a reconstituted MTC preparation was used to deliver LMT orally). [Figure 6] Computational chemical modeling of the high affinity LMT / MT+-heme interaction. (A and B) Nonplanar, unbound T-state heme showing protrusion of the iron atom above the plane of the porphyrin ring. (C and D) Flat, oxygen-bound R-state heme. (E and F) LMT binding leads to the heme adopting the R-state conformation. EXAMPLES

[0111] Working Example Example 1 - Methylthioninium chloride (MTC) and LMTX MTC (methylthioninium chloride, methylene blue) has been available as a drug since 1876. It is on the World Health Organization's list of essential medicines, a list of the safest and most effective medicines in the health care system.

[0112] MTC has been previously applied in many areas of clinical medicine, including the treatment of methemoglobinemia, malaria, nephrolithiasis, bipolar disorder, ifosfamide encephalopathy, and more recently in Alzheimer's disease (AD) (see Wischik, CM, et al. "Tau aggregation inhibitor therapy: an exploratory phase 2 study in mild or moderate Alzheimer's disease" Journal of Alzheimer's Disease 2015;44:705-720 and Nedu, ME, et al. Comparative study regarding the properties of methylene blue and proflavine and their optimal concentrations for in vitro and in vivo applications. Diagnostics 2020;10:223).

[0113] The MT portion is composed of oxidized MT + form and in the reduced LMT form (Harrington et al., 2015; see above). [ka]

[0114] MTC is oxidized MT + It is a chloride salt in the form of leuco-MT (LMT; International Generic Name: hydromethylthionine) which must be converted in the intestine by thiazine dye reductase activity to the reduced leuco-MT (LMT; International Generic Name: hydromethylthionine) form for absorption and distribution to deep compartments, including erythrocytes and the brain (Baddeley et al., 2015; see above). Similarly, in isolated erythrocyte preparations, MT +must be converted to LMT to allow uptake into both erythrocytes (see May, JM, et al., "Reduction and uptake of methylene blue by human erythrocytes." American Journal of Physiology - Cell Physiology 2004; 286:C1390-C1398) and pulmonary endothelial cells (Merker et al., 1997; supra).

[0115] Since MTC is actually a prodrug of LMT (the predominant form in the body), TauRx developed a stabilized reduced form of MT as LMTM (leuco-methylthioninium bis(hydromethanesulfonate); hydromethylthionine mesylate) to allow for direct administration of the LMT form.

[0116] The synthesis of LMTX and LMTM compounds can be carried out according to methods described in the art (see, for example, WO 2007 / 110627 and WO 2012 / 107706).

[0117] Example 2 - Study Overview Objective: To determine the effect of oral LMTM on SpO2 and metHb levels in patients with mild hypoxemia not due to COVID-19, and to explore the structural interactions between the LMT moiety and the heme group.

[0118] Methods: Eighteen AD trial participants randomized to 4 / 75 / 100 / 125 mg unit doses of LMTM had SpO2 levels below 94% at baseline. Patients were routinely monitored by pulse oximetry 4 hours after twice daily dosing and at 2 and 6 weeks. Computational chemistry was used to understand LMT-heme binding.

[0119] Example 3 - Clinical Trial Methods and Results method patient The study design and results of two phase III double-blind, controlled, randomized studies of LMTM as a potential treatment for AD have been described elsewhere (Gauthier, S., et al. “Efficacy and safety of tau-aggregation inhibitor therapy in patients with mild or moderate Alzheimer's disease: a randomised, controlled, double-blind, parallel-arm, phase 3 trial.” Lancet 2016;388:2873-2884 and Wilcock, GK, et al. “Potential of low dose leuco-methylthioninium bis(hydromethanesulphonate) (LMTM) monotherapy for treatment of mild Alzheimer's disease: cohort analysis as modified primary outcome in a Phase III clinical trial.” Journal of Alzheimer's Disease 2017;61:435-457). In summary, 890 patients with mild / moderate AD were randomly assigned (3:3:4) to 150 mg / day, 250 mg / day, or 8 mg / day (intended as control) for 15 months (clinicaltrials.gov NCT01689246). Similarly, 800 patients with mild AD were randomly assigned to 200 mg / day or 8 mg / day LMTM for 18 months (clinicaltrials.gov NCT01689233). In both trials, MetHb and SpO2 levels were measured by pulse oximetry (Massimo Corporation rad 57) at screening, baseline (within 1 hour prior to dosing), during the 4-hour post-dose observation period, and at subsequent visits at 2 and 6 weeks.

[0120] Estimating the effect of LMT treatment on the oxygen-hemoglobin dissociation curve We use standard clinical SpO2 to PaO2 conversion tables (e.g., https: / / www.acphospitalist.org / archives / 2013 / 11 / acph-201311-codingt1.pdf) to compare the oxygen-hemoglobin dissociation curves in patients undergoing LMTM or reduced forms of MTC to normal (see “Hamidi-Alamdari, D., et al. “Methylene blue for treatment of hospitalized COVID-19 patients, randomized, controlled, open-label clinical trial, phase 2,” Revista de Investigacion Clinica. 2021; 73:190-198”). Estimated baseline PaO2 values ​​and corresponding SpO2 values ​​were plotted for patients treated with LMTM. After treatment with LMTM, SpO2 values ​​were plotted against the same PaO2 values ​​to estimate the putative shift of the dissociation curve. To compare these treatment effects with those reported by Hamidi-Alamdari et al. (2021) (above), the SpO2 values ​​observed in patients receiving standard treatment were used to calculate estimated non-treatment PaO2 values. SpO2 values ​​observed after 5 days of treatment with the reduced MTC preparation were plotted against the same PaO2 values ​​to infer a putative shift in oxygen-hemoglobin dissociation.

[0121] statistics Statistical analysis was performed using R-version 3.5.1 using paired samples t-tests to compare mean SpO2 and metHb levels.

[0122] Computer modeling Quantum chemical calculations were performed using the self-consistent field (SCF) method within the MOE software suite (Chemical Computing Group, University College London). SCF calculations used the Austin Model-1 basis set, and geometry optimizations were initially performed using the AMBER 10 force field using the Extended Hueckel Theory. The crystal structure of human hemoglobin in its oxy form (2DN1) was used to align the LMT-heme complex (see "Park, S.-Y., et al. "1.25 Å resolution crystal structures of human haemoglobin in the oxy, deoxy and carbonmonoxy forms." Journal of Molecular Biology 2006;360:690-701."). The crystal structure of human hemoglobin in the deoxy form (PDB ID 2DN2; Park et al., 2006) and the crystal structure of human hemoglobin in the carbonmonoxide form (PDB ID 3DN2; Park et al. 2006) were aligned using Pymol.

[0123] result Baseline population characteristics Data were available for 18 subjects from both AD studies who had oxygen saturation <94% at baseline (the lower limit of the normal range is 95%), whose demographic characteristics are summarized in Table 1. They ranged from 70 to 80 years, with a mean age of 76.1 years, and there were more women (61%) than men (39%).

[0124] Using medical histories available from the patients' Case Report Forms, hypoxemia in these subjects was found to be associated with a variety of respiratory or other underlying conditions of varying degrees of severity that would plausibly contribute to chronic hypoxemia (summarized in Table 2). These included sleep apnea, insomnia, asbestosis, edema, asthma, bronchitis, allergies, angioedema, pneumonia, acute myocardial infarction / hypertension, coronary artery disease with angioplasty and stent insertion, transient ischemic attack (TIA), hypothyroidism, diabetes, syncope, tachycardia, and sepsis. In three of these subjects, no etiological clinical history factors were identified.

[0125] A prospective clinical study of peripheral oxygen saturation and methemoglobinemia in AD patients treated with LMTM SpO2 levels were compared before and after 4 hours in the clinic after administration of a single dose of LMTM at 4 mg, or at a dose of 75, 100, or 125 mg (summarized as an average of 100 mg for the high dose; FIG. 1). As can be seen in FIG. 1, the 4 mg dose of LMTM significantly increased the mean blood oxygen saturation. The mean oxygen saturation in the group with a baseline SpO2 level of less than 94% was 91.71%. Four hours after receiving the 4 mg dose of LMTM, the mean SpO2 level increased to 95.43% (+3.72%, p=0.0205; Table 3, FIG. 1). Similarly, after administration of the high dose of LMTM, oxygen saturation increased from 92.45% at baseline to 95.27% after 4 hours (+2.82%, p=0.0045; Table 3, FIG. 1). Thus, LMTM is capable of increasing blood oxygen saturation within 4 hours over a wide dose range without discernible dose-dependent differences. As can be seen from Figure 2 and Table 4, the effect was stable over 6 weeks at 8 mg / day and 150-250 mg / day, and the difference from baseline was statistically significant at 2 weeks (+3.17% ± SEM, p=0.0034) and 6 weeks (+3.23% ± SEM, p=0.0005). Thus, LMTM provides rapid improvement in oxygen saturation at a single dose in the 4-125 mg range, which is sustained over 6 weeks at doses in the 8-250 mg / day range.

[0126] The putative effect of treatment on the oxygen-hemoglobin dissociation curve is shown in FIG. 5. We use the standard dissociation curve to estimate the corresponding PaO2 value at baseline. It can be seen that the effect of LMTM treatment in increasing SpO2 values ​​corresponds to a putative shift of the oxygen-hemoglobin dissociation curve to the left. The cases in which LMTM treatment was used had relatively mild hypoxemia (FIG. 5, "Mild"). For the more severe cases (FIG. 5, "Severe"), we use the same approach to estimate the putative effect on oxygen-hemoglobin dissociation in patients receiving reduced MTC formulations reported by Hamidi-Alamdari et al. (2021) (above), using a non-treated population as a basis for comparison.

[0127] As can be seen, the putative effects of treatment with reduced MTC are similar.

[0128] We next investigated how the effect on hypoxemia relates to metHb levels measured simultaneously in the same patients. There was no consistent difference in metHb levels between baseline and 4 hours post-dose for both treatment groups (Figure 3, Table 3). Thus, the acute effect of LMTM on oxygen saturation is independent of any consistent corresponding effect on methemoglobin at low or high doses. Over a 6-week period, high doses (150, 200, and 250 mg / day) of LMTM consistently increased metHb levels, whereas the 8 mg / day dose did not (Figure 4, Table 5A), but none of the changes in metHb reached statistical significance in this small number of subjects. In the entire population, when the effect on metHb levels is considered, the small increases become statistically significant, including at the 8 mg / day dose (Table 5B). However, it should be understood that in terms of therapeutic efficacy, these small changes (approximately 0.7% to 1%) are not significant in terms of treating the levels of metHb seen in symptomatic patients (e.g., >20% or >30% metHb) (see Table 7).

[0129] Computational chemical modeling of the LMT-heme effect We have used computational modeling to study the minimum energy LMT-heme. This modeling suggests that the LMT can bind with high affinity to the heme iron of hemoglobin through the LMT nitrogen in an octahedral structure and within 2.1 Å of the iron atom (FIG. 6). The LMT is oriented toward the axial and equatorial angles of the octahedral complex, respectively, of the Fe 2+ Electronic dz 2 and dx 2 -y 2The LMT-heme complex strongly interacts with the orbital. We hypothesize that the formation of this LMT-heme complex acts in a manner similar to oxygen binding (FIGS. 6C and D) in helping to overcome the initial barrier to oxygen binding by promoting the conversion from the T state (FIGS. 6A and B) to the R state (FIGS. 6E and F). The resulting cooperativity would result in higher relative oxygen saturation occurring at lower PaO2 levels, which would be consistent with the putative leftward shift of the oxygen-hemoglobin dissociation curve suggested by our analysis.

[0130] Example 4 - Discussion of Example 3 We report above preliminary results of an exploratory analysis of the effect of LMTM treatment on patients who were chronically mildly hypoxemic at baseline.

[0131] We investigated 18 subjects who entered either of two Phase III studies in mild-moderate AD and had oxygen saturation below 94% at baseline due to various chronic incident cardiopulmonary conditions. We show that a single dose of LMTM in the range of 4-125 mg was able to significantly increase oxygen saturation within 4 hours, and that the effect of treatment with the same dose given twice daily persisted for 6 weeks. This suggests that LMTM can bind to hemoglobin in such a way that it increases oxygen saturation by about 3% in patients with chronic hypoxemia.

[0132] The paradoxical interaction of the MT moieties with respect to methemoglobinemia has already been discussed above. LMT species are the most abundant species in methemoglobinemia. 3+ Fe 2+(May, JM, et al. "Reduction and uptake of methylene blue by human erythrocytes." American Journal of Physiology - Cell Physiology 2004;286:1390-1398; Schirmer, RH, et al. "Lest we forget you - methylene blue..." Neurobiology of Aging 2011;32:2325.e7-2325.e16), but both MT and LMT species can cause methemoglobinemia by themselves.

[0133] We have used computational chemistry to understand the antihypoxemic effect of LMT. Because the structures of both heme and LMT are known, it is possible to calculate the minimum energy binding interaction between the two. We show that LMT transfers a pair of electrons from the central nitrogen of LMT to the Fe 2+ Electronic dz 2 and dx 2 -y 2 orbitals, it has been shown that LMT can bind with high affinity to within 2.10 Å of the iron atom of hemoglobin. From crystal field theory, this type of interaction has an estimated field factor of 1.2-1.5 (see Jorgensen, CK "Absorption spectra and chemical bonding in complexes." Pergamon Press, Oxford, London, New York, Paris 1962. 352 pp. and Jorgensen, CK "Oxidation numbers and oxidation states. Springer Berlin Heidelberg; 1969)), suggesting that LMT can act as a strong displaceable ligand-field ligand. The inventors believe that the formation of this complex is due to the presence of Fe 2+However, the T state has an ionic radius of 2.06 Å, which is too large to fit into the cavity at the center of the porphyrin ring (see Perutz, MF “Proteins and nucleic acids-structure and function.” Amsterdam and New York: Elsevier Publishing Co.; 1962). 2+ We hypothesize that oxygen is able to bind with higher affinity to the R-state heme, which has an ionic radius of 1.96 Å that allows it to fit within the four nitrogen atoms that coordinate with it (see “Lima, FA, et al.” “Probing the electronic and geometric structure of ferric and ferrous myoglobins in physiological solutions by Fe K-edge absorption spectroscopy.” Physical Chemistry Chemical Physics 2014;16:1617-1631”), thereby overcoming the first energy barrier and the subsequent binding of oxygen is further promoted by cooperativity (see “Bohr, C., et al. “Ueber einen in biologischer Beziehung wichtigen Einfluss, den die Kohlensaeurespannung des Blutes auf dessen Sauerstoffbindung uebt.” Skandinavisches Archiv fuer Physiologie 1904;16:402-412). This is consistent with a leftward shift of the oxygen-hemoglobin dissociation curve (28).

[0134] However, LMT binding is suboptimal compared to oxygen. The bond distance between LMT nitrogen and heme iron is 2.10 Å, whereas the corresponding bond distance for oxygen is 1.98 Å. This suggests that oxygen, when available at high pH or low pCO2, can replace LMT, thereby resulting in normal oxygen dissociation with release of bound oxygen to peripheral tissues. This is consistent with the decreased respiratory rate observed in critically ill patients treated with reduced MTC preparations of LMT (Hamidi-Alamdari, D. et al., 2021, supra). Respiratory rate is driven by central and peripheral chemoreceptors that are sensitive to hypoxia, and increased CO2 levels that signal tissue hypoxia (see "Davies, A. and Moores, C. "The Respiratory System: Basic science and clinical conditions." 2nd Edition. Churchill Livingstone; 2010.").

[0135] The formation of the heme-LMT complex that we describe has three different possible effects on hemoglobin of the MT moiety: Fe in methemoglobinemia; 3+ Fe 2+ (13), and at high concentrations of MT, 2+ Fe 3+ (see Bodansky, O. "Methemoglobinemia and methemoglobin-producing compounds." Pharmacological Reviews 1951;3:144-196.) and provides a structural explanation for the effect on oxygen saturation that we describe herein. In the first two, the formation of the heme-LMT coordination allows for the donation or removal of electrons, depending on the availability of adequate levels of NADPH required to regenerate LMT. This is followed by the conversion of the back-Fe 3+ During reduction of LMT, MTs were generated. +At high concentrations of MT fractions (or in the presence of G6PD deficiency), MT + The level of MT exceeds the available reducing capacity of the cell. + can form the same coordination as heme via the orientation of the nitrogen atom, and Fe 2+ Fe 3+ In the case of hypoxemia occurring in the context of normal red blood cell physiology, we show that low doses of LMTM are capable of improving oxygen saturation with minimal corresponding effects on metHb levels. At higher doses of LMTM, there is also an improvement in oxygen saturation, but in addition, there is a reduction in Fe 2+ Fe 3+ is oxidized to.

[0136] The use of oral doses of LMTM (and related compounds) greater than the control dose (4 mg) but less than the higher dose tested herein (75 mg) has been shown to reduce FeHb in patients with methemoglobinemia (hereditary and acquired forms). 3+ Fe 2+ It is proposed that these effects should provide a net reduction in . Such an effect is consistent with the structural model and data described herein and would provide a valuable addition to currently available treatment options.

[0137] [Table 1]

[0138] [Table 2]

[0139] [Table 3]

[0140] [Table 4]

[0141]

Table 5

[0142]

Table 6

[0143]

Table 7

[0144]

Table 8

[0145]

Table 9

Claims

1. A pharmaceutical composition comprising a methylthioninium (MT)-containing compound for use in a method of treating methemoglobinemia in a subject, The method includes orally administering the MT-containing compound to the subject, The aforementioned administration provides a total daily oral dose of 4 mg to 60 mg of MT per day to the subject, which may be optionally divided into two or more doses. The MT-containing compound is given by the following formula: 【Chemistry 1】 (In the formula, H ｎ A and H ｎ Each of B (if present) is a protonic acid, and these may be the same or different. p = 1 or 2; q = 0 or 1; n = 1 or 2; and (p + q) × n = 2) The compound, or its hydrate or solvate, Pharmaceutical composition.

2. The total daily dose mentioned above is (i) any of the following: approximately 4, 8, 12, 16, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24 mg to approximately 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 mg and / or (ii) 8 or 10 or 20 or 20.5 or 21 mg to 50 or 55 or 60 mg, The pharmaceutical composition according to claim 1.

3. The total daily dose mentioned above is (i) Approximately 8, 12, 16, 20, 20.5, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mg, or (ii) 10-50 mg, 20.5-50 mg, 40-50 mg, or approximately 4, 8, 12, or 16 mg. The pharmaceutical composition according to claim 1.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the total daily dose of the compound is administered in divided doses twice or three times a day.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the methemoglobinemia is acquired, and the subject optionally has a methemoglobin level of >30%, >50%, or >70%.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the methemoglobinemia is hereditary, and the subject is optionally selected to have a methemoglobin level of >30%, >50%, or >70%.

7. The MT-containing compound is given by the following formula: 【Chemistry 2】 (In the formula, HA and HB are different monoprotonic acids.) A pharmaceutical composition according to any one of claims 1 to 3, having the following characteristics.

8. The MT-containing compound is given by the following formula: 【Transformation 3】 (In the formula, H ｎ X is a protonate. A pharmaceutical composition according to any one of claims 1 to 3, having the following characteristics.

9. The MT-containing compound is given by the following formula: 【Chemistry 4】 It has H ２ The pharmaceutical composition according to any one of claims 1 to 3, wherein A is diprotonic acid.

10. The MT-containing compound is given by the following formula: 【Transformation 5】 The pharmaceutical composition according to claim 8, wherein it is a bis-mono-protonic acid.

11. The pharmaceutical composition according to any one of claims 1 to 3, wherein the above or each protonic acid is an inorganic acid.

12. The above or each proton acid is a hydrohalic acid, and / or HCl; HBr; HNO ３ ; H ２ SO ４ A pharmaceutical composition according to claim 11, selected from the above.

13. wherein the protonic acid or each protonic acid is an organic acid, and / or H ２ COOH ３ ; CH ３ COOH; methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid, the pharmaceutical composition according to any one of claims 1 to 3.

14. The MT-containing compound is 【Transformation 6】 The pharmaceutical composition according to any one of claims 1 to 3.

15. The MT-containing compound is 【Transformation 7】 【Transformation 8】 A pharmaceutical composition according to any one of claims 1 to 3, selected from the list comprising:

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