Methylene blue-containing compounds for treatment of methemoglobinemia

Abstract

The present invention provides novel methods of oral treatment of methemoglobinemia in a subject comprising orally administering to the subject methylthionine ("MT") comprising a salt of the formula: # imgabs0 # wherein each HnA and HnB, if present, are protonic acids that may be the same or different, and wherein p = 1 or 2; q = 0 or 1; n = 1 or 2; (p + q) * n = 2, or a hydrate or solvate thereof. The administration should provide the subject with a daily total oral dose of 4 mg to 60 mg MT per day.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the priority benefit of GB 2204185.9 filed on March 24, 2022, the contents of which are incorporated herein in their entirety. Technical Field

[0003] The present invention generally relates to methods and materials for reducing methemoglobinemia or treating methemoglobinemia in a subject. Background Art

[0004] Methemoglobinemia is a rare condition associated with the oxidation of the ferrous iron of heme to the ferric iron of methemoglobin (see “Iolascon, Achille et al. “Recommendations for diagnosis and treatment of methemoglobinemia.” American Journal of Hematology (2021) 96:1666-1678.”).

[0005] Iron is ferric iron [Fe 3+ ] state leads to an allosteric change, which causes oxygen to bind irreversibly. The corresponding iron-globulin in the tetramer shifts the oxygen dissociation curve of Hb to the left. This shift leads to an increase in the affinity of ferrous iron for oxygen, thereby weakening the release of oxygen into the tissues, leading to hypoxia, the so-called "functional anemia", but Hb will not decrease.

[0006] Methemoglobinemia can be caused by either inherited or acquired processes. The acquired form is the most common and is primarily due to exposure to substances that directly or indirectly cause Hb oxidation. The inherited form is due to autosomal recessive variants in the CYB5R3 gene or autosomal dominant variants in the globin genes, collectively referred to as hemexia.

[0007] Intravenous injection of 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 can exist in an oxidized MT+ form (in MTC) and a reduced or "leuco" form, methylthioninium hydrochloride (LMT) (see "Harrington, CR et al. "Cellular models of aggregation-dependent template-directed proteolysis to characterize tau aggregationinhibitors for treatment of Alzheimer disease." Journal of BiologicalChemistry 2015; 290: 10862-10875."). MT + It needs to be converted to LMT first to allow absorption, distribution and uptake into cells (see "Baddeley, TC et al. "Complex disposition of methylthioninium redoxforms 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 action site, where it promotes electron transfer from LMT to Fe 3+ , which is reduced to Fe 2+ And oxidize LMT to MT +. LMT is continuously regenerated via ongoing erythrocyte glycolysis, allowing 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).

[0008] However, the role of MTC in treating methemoglobinemia is contradictory. For example, MTC can actually cause acquired methemoglobinemia (see Iolascon 2021, supra).

[0009] Methylene blue-vitamin CN-acetyl cysteine ​​(MCN) has been reported to provide benefit to patients with severe COVID-19, with one putative mechanism for this agent and dose being via a reduction in 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.

[0010] WO2021 / 224146 is about the use of hydromethylthionine and related salts (referred to therein as "LMTX") as therapeutic agents for alleviating hypoxemia in a subject. It is therefore suggested that these salts can be used to alleviate hypoxia and treat pathological or other causes of hypoxia. The effects disclosed therein are said to be independent of any known effects on metHb, and it is noted in WO2021 / 224146 that high concentrations of LMT (associated with oral doses in the range of 150-250 mg / day) can cause a measurable increase in metHb. The results described in WO2021 / 224146 indicate that high doses of LMTX compounds systematically increase metHb levels over a period of time (see Figure 3 of WO2021 / 224146) while still alleviating hypoxia.

[0011] From the foregoing it can be seen that providing compounds that can be safely and conveniently used to treat methemoglobinemia (ie, to reduce metHb levels in a subject) would provide a useful contribution to the art. Summary of the invention

[0012] The present invention relates to the use of "LMTX" compounds (including LMT) delivered orally at appropriate doses for treating methemoglobinemia (i.e., reducing methemoglobin levels) in a subject. The treatment is independent of and does not depend on the presence or absence of hypoxia. This provides a more convenient way to treat methemoglobinemia than using intravenous MTC.

[0013] There is no prior disclosure of oral LMTX for the treatment of methemoglobinemia. However, the inventors believe that LMT and the Fe 3+ The nature of the binding interaction is such that oral treatment, at appropriate doses, may provide benefit in terms of conversion to ferroglobulin.

[0014] Oral treatments may be applicable to either inherited or acquired methemoglobinemias. The availability of oral treatments with a good safety profile for use in these conditions would represent a valuable addition to currently available treatment options.

[0015] Thus, in one aspect, a method of treating (or reducing) methemoglobinemia in a subject is disclosed, the method comprising orally administering to the subject a compound containing methylthioninium (MT),

[0016] wherein the administration provides the subject with a total daily oral dose of 4 mg to 60 mg MT per day, optionally divided into 2 or more doses,

[0017] The compound containing MT is a LMTX compound of the following formula:

[0018]

[0019] wherein each HnA and HnB (if present) are protonic acids which may be the same or different,

[0020] Wherein p=1 or 2; q=0 or 1; n=1 or 2; (p+q)*n=2,

[0021] or a hydrate or a solvate thereof.

[0022] ***

[0023] The total daily dose of MT may be between 8 or 10 or 20 or 20.5 or 21 and 50 or 60 mg.

[0024] The total daily dose can be any of about 4, 8, 12, 16, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24 mg to about 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.

[0025] The total daily dose may 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.

[0026] An exemplary dosage is 10 to 50 mg.

[0027] Another exemplary dosage is 20 or 20.5 to 50 mg.

[0028] Another exemplary dosage is 30 to 50 mg.

[0029] Another exemplary dosage is 40 to 50 mg.

[0030] ***

[0031] The total daily dosage of the compound may be administered in divided doses two or three times daily.

[0032] As explained below, when administering a dose of MT divided into larger doses per day, it may be necessary to use smaller total amounts within the stated ranges compared to single daily dosing or fewer doses per day.

[0033] The patient may be an adult, and the population-based doses described herein are based on this premise (typical body weight is 50 to 70 kg). If desired, the corresponding dose may be used for subjects falling outside this range by using the subject's body weight factor, wherein the subject's body weight is divided by 60 kg to provide a multiplication factor for an individual subject.

[0034] ***

[0035] Subjects for treatment may be characterized or selected by certain criteria.

[0036] Selected signs, symptoms, and causes of methemoglobinemia may include any of those in Table 7 (see Iolascon 2021, supra):

[0037] In one embodiment, the subject is not hypoxic or suffers from hypoxemia.

[0038] In one embodiment, the subject has a blood oxygen saturation level (SpO2) greater than 95% on room air.

[0039] For the present invention, the subject must be able to breathe and swallow, as the treatment is to be administered orally.

[0040] The method of the present invention may comprise the step of selecting a subject according to one or more of the above criteria.

[0041] Thus, in some embodiments, the subject may be a human who has been diagnosed with methemoglobinemia, or wherein the method comprises making such a diagnosis.

[0042] The present invention can be used to treat acquired or inherited forms of methemoglobinemia. Examples of acquired and inherited forms of methemoglobinemia are shown in Table 6. Thus, treatment can be directed to methemoglobinemia caused by drug exposure (e.g., benzopyridine (pyridine), sulfamethoxazole, diaminophenylsulfone, aniline, monolinuron, nitrates, nitroglycerin, amyl nitrite, isobutyl nitrite, sodium nitrite, benzocaine, lidocaine, prilocaine or chloramine).

[0043] In some embodiments, the inherited disease is methemoglobinemia type I, II, III, or IV, or HbM disease, or unstable hemoglobin.

[0044] In some embodiments, the subject has a metHb level greater than 20%.

[0045] Symptoms of methemoglobinemia may vary depending on how quickly metHb is formed. For example, people who have had methemoglobinemia their whole life may have symptoms, but people who have been exposed to drugs or toxins and suddenly develop the same degree of methemoglobinemia may have severe symptoms.

[0046] In some embodiments, the subject has a metHb level as described in Table 7. For example, the subject may be symptomatic, have a metHb level greater than 30%, 50%, or 70%.

[0047] In some embodiments, the subject has acute methemoglobinemia.

[0048] In some embodiments, the subject has chronic methemoglobinemia.

[0049] In some embodiments, the genetic disease is autosomal recessive congenital methemoglobinemia.

[0050] ***

[0051] WO2007 / 110627 discloses certain 3,7-diamino-10H-phenothiazine salts that are effective as drugs or prodrugs for treating diseases including Alzheimer's disease and other diseases such as frontotemporal dementia (FTD), as well as general viral diseases. When referring to MTC, these compounds are also in "reduced" or "colorless" form. These colorless-methylthioninium compounds are referred to therein as "LMTX" salts.

[0052] WO2012 / 107706 describes other LMTX salts with superior properties to those listed above, including colorless-methylthioninium bis(hydrogen methanesulfonate) (LMTM) (WHO INN name: Hydromethylthioninium):

[0053]

[0054] Preferably, the LMT compound is a "LMTX" compound of the type described in WO2007 / 110627 or WO2012 / 107706.

[0055] Therefore, the compound can be selected from the compounds of the following formulae or their hydrates or solvates:

[0056]

[0057] Each HnA and HnB (if present) is a protic acid which may be the same or different.

[0058] "Protic acid" means a proton (H + ) donor. Therefore, in the proton acid, A - or B- Therefore, the pH value of protonic acid in water is less than 7 (that is, the concentration of hydronium ions is greater than 10 -7 mol / L).

[0059] In one embodiment, the salt is a mixed salt having the formula wherein HA and HB are different monoprotic acids:

[0060]

[0061] However, the salt is preferably not a mixed salt and has the formula:

[0062]

[0063] wherein each HnX is a protic acid, such as a diprotic acid or a monoprotic acid.

[0064] In one embodiment, the salt has the formula wherein H 2 A is a diprotic acid:

[0065]

[0066]

[0067] Preferably, the salt has the formula, which is a dimonoprotic acid:

[0068]

[0069] Examples of protonic acids that may be present in the LMTX compounds used herein include:

[0070] Inorganic acids: halogen acids (e.g. HCl, HBr), nitric acid (HNO 3 ), sulfuric acid (H 2 SO 4 )

[0071] Organic acid: Carbonic acid (H 2 CO 3 ), acetic acid (CH 3 COOH), methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, p-toluenesulfonic acid,

[0072] Preferably the acid is a monoprotic acid and the salt is a bis(monoprotic acid) salt.

[0073] The preferred MT polymer is LMTM:

[0074]

[0075] Weight Factor

[0076] The anhydrous salt has a molecular weight of approximately 477.6. Based on the molecular weight of the LMT core of 285.1, the weight factor for using this MT compound in the present invention is 1.67. "Weight factor" means the relative weight of a pure MT-containing compound compared to the weight of the MT it contains.

[0077] Other weighting factors, such as the MT compounds herein, can be calculated, and corresponding dosage ranges can be calculated therefrom.

[0078] Other exemplary LMTX compounds are as follows. Their molecular weights (anhydrous) and weighting factors are also shown:

[0079]

[0080]

[0081] The dosages described herein for MT therefore apply mutatis mutandis to these MT-containing compounds, as adjusted for their molecular weight.

[0082] Accumulation Factor

[0083] As will be appreciated by those skilled in the art, for a given daily dose, more frequent dosing may result in greater accumulation of the drug.

[0084] Thus, in certain embodiments of the claimed invention, the total daily dose of the MT compound may be relatively lower when administered more frequently (eg, twice a day [bid] or three times a day [tid]), or higher when administered once a day [qd].

[0085] Treatment and prevention

[0086] As used herein in the context of treating a condition, the term "treat" generally refers to treatment and therapy, whether in humans or animals (e.g., in veterinary applications), in which some desired therapeutic effect (e.g., inhibition of progression of the condition) is achieved, and includes a reduction in the rate of progression, a halt in the rate of progression, regression of the condition, amelioration of the condition, and cure of the condition.

[0087] As used herein, the term "therapeutically effective amount" refers to that amount of a compound of the invention, or a material, composition or dosage form comprising the compound, which, when administered according to the desired treatment regimen, is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio. The inventors have demonstrated that, for the diseases of the invention, the therapeutically effective amount of the MT compound can be much lower than what has been known in the art to date.

[0088] The present invention also encompasses treatment as a prophylactic measure.

[0089] As used herein, the term "prophylactically effective amount" refers to that amount of a compound of the invention, or a material, composition, or dosage form containing the compound, which, when administered according to the desired treatment regimen, is effective for producing some desired prophylactic effect, commensurate with a reasonable benefit / risk ratio.

[0090] In the context of this specification, "prevention" should not be understood as limiting complete success, i.e., complete protection or complete prevention. Instead, prevention in this context refers to measures taken before a symptom appears or before the symptom worsens, with the goal of maintaining health by helping to delay, alleviate or avoid the particular symptom.

[0091] ***

[0092] The term "treatment" includes "combination" treatments and therapies in which two or more treatments or therapies are combined, e.g. sequentially or simultaneously. They may be symptomatic or disease-modifying treatments.

[0093] The specific combination will be determined by the physician.

[0094] In combination therapy, the agents (i.e., MT compounds as described herein plus one or more other agents) can be administered simultaneously or sequentially, and can be administered at individually different dosage regimens and via different routes. For example, when administered sequentially, the agents can be administered closely spaced (e.g., within a 5-10 minute period) or at longer intervals (e.g., 1, 2, 3, 4, or more hours apart, or, if desired, even longer periods of time apart), the precise dosage regimen being commensurate with the properties of the therapeutic agents.

[0095] In other embodiments, the treatment is "monotherapy," that is, the MT-containing compound is not used in combination (within the meaning discussed above) with another active agent.

[0096] ***

[0097] For the treatment of methemoglobinemia, the treatment regimen based on the MT compounds described herein will preferably be extended for a duration appropriate to the disease and symptoms. The specific duration will be determined by the physician.

[0098] For example, the duration of treatment may be:

[0099] 1 to 14, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days.

[0100] 1 to 4, for example 1, 2, 3 or 4 weeks.

[0101] In all cases, the duration of treatment is usually based on the advice and review of your physician.

[0102] ***

[0103] The MT compound of the present invention or a pharmaceutical composition comprising the same can be administered orally (or via a nasogastric tube) to the stomach of a subject / patient.

[0104] Generally, in the practice of the present invention, the compound will be administered as a composition comprising the compound and a pharmaceutically acceptable carrier or diluent.

[0105] In some embodiments, the composition is a pharmaceutical composition (eg, formulation, preparation, medicament) comprising a compound described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0106] As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc. that are suitable for use in contact with the tissues of relevant subjects (e.g., humans) within the scope of sound medical judgment without excessive toxicity, irritation, allergic response, or other problems or complications, 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.

[0107] In some embodiments, the composition is a pharmaceutical composition comprising at least one compound as described herein and one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents and sweeteners.

[0108] In some embodiments, the composition further comprises other active agents, for example, other therapeutic or prophylactic agents.

[0109] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical textbooks. See, for example, Handbook of Pharmaceutical Additives, 2nd edition (M. Ash and I. Ash eds.), 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.

[0110] 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 pharmaceutically acceptable carrier, diluent, or excipient.

[0111] The "MT compound" is the active agent of the dosage unit, although it may be present in relatively low amounts, in other words, is intended to have a therapeutic or preventive effect on methemoglobinemia. In contrast, other ingredients in the dosage unit will not be therapeutically active, such as carriers, diluents or excipients.

[0112] Thus, preferably, there will be no other active ingredients in the dosage unit, and no other agents other than those relevant to the combination therapy described herein, intended to have a therapeutic or prophylactic effect on the condition for which the dosage unit is intended.

[0113] In some embodiments, the dosage unit is a tablet.

[0114] In some embodiments, the dosage unit is a capsule.

[0115] In some embodiments, the capsule is a gelatin capsule.

[0116] In some embodiments, the capsule is a HPMC (hydroxypropyl methylcellulose) capsule.

[0117] The appropriate amount of MT in the composition will depend on how often the subject takes it per day or how many units are taken at one time. Thus, a dosage unit may contain less than the total daily dose individually.

[0118] Exemplary dosage units may contain 10, 20, 30, 40, 50 or 60 mg of MT.

[0119] Using the weighting factors described or explained herein, one skilled in the art can select the appropriate amount of MT-containing compound for use in an oral formulation.

[0120] As noted above, the MT weighting factor for LMTM is 1.67. Because of the convenience of using a single or simple portion of the active ingredient, a non-limiting exemplary LMTM dosage unit may include 17 mg to provide 10 mg of LMT or MT, etc.

[0121] In one embodiment, a dosage unit pharmaceutical composition is provided comprising about 17, 34, 51 mg, etc. of LMTM.

[0122] ***

[0123] The unit dose compositions described herein (LMTX compound plus optional other ingredients) can be provided in labeled packages together with instructions for their use.

[0124] In one embodiment, the package is a bottle, such as is well known in the pharmaceutical art. A typical bottle may be made of pharmacopoeial grade HDPE (high density polyethylene), with a child-resistant HDPE push-lock closure, and contain a silica gel desiccant, in the form of a pouch or can. The bottle itself may include a label and be packaged in a cardboard container with instructions for use and optionally a further copy of the optional label.

[0125] In one embodiment, the package or packet is a blister package (preferably a blister package with an aluminum cavity and aluminum foil), so it is substantially impermeable to water. In this case, the package can be packaged in a cardboard container with instructions for use and a label on the container.

[0126] The label or instructions may provide information regarding the treatment of methemoglobinemia.

[0127] Treatment

[0128] As noted above, another aspect of the present invention relates to a method for treating methemoglobinemia 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.

[0129] Use in therapeutic approaches

[0130] Another aspect of the present invention relates to a compound or composition as described herein for use in a method of treating methemoglobinemia in the human or animal body by therapy.

[0131] Use in pharmaceutical manufacturing

[0132] Another aspect of the present invention relates to the use of a MT compound or composition as described herein in the manufacture of a medicament for the treatment of methemoglobinemia.

[0133] In some embodiments, the medicament is a composition, eg, a dosage composition as described herein.

[0134] Mixture of oxidized and reduced MT compounds

[0135] The LMT-containing compounds used in the present invention may include oxidized (MT + ) compounds as "impurities" during synthesis may also be oxidized (e.g., auto-oxidized) after synthesis to produce the corresponding oxidized form. Thus, even if it is not inevitable, compositions containing compounds of the invention will contain at least some of the corresponding oxidized compounds as impurities. For example, a "LMT" salt may include up to 15%, e.g., 10 to 15%, of MT+ Salt.

[0136] When mixed MT compounds are used, the MT dosage can be easily calculated using factors for the molecular weight of the compounds present.

[0137] Salts and solvates

[0138] Although the MT-containing compounds described herein are themselves salts, they may also be provided in the form of mixed salts (i.e., a compound of the invention in combination with another salt). Such mixed salts are intended to be encompassed by the term "and pharmaceutically acceptable salts thereof." Unless otherwise specified, reference to a particular compound also includes its salts.

[0139] The compounds of the present invention may also be provided in the form of solvates or hydrates. The term "solvate" is used herein in a conventional 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 is preferably referred to as a hydrate, such as a monohydrate, a dihydrate, a trihydrate, a pentahydrate, etc. Unless otherwise indicated, any reference to a compound also includes a solvate and any hydrate form thereof.

[0140] Of course, solvates or hydrates of the salts of the compounds are also included in the present invention.

[0141] ***

[0142] In order to more fully describe and disclose the present invention and the state of the art to which the present invention pertains, a number of patents and publications are cited herein. Each of these references incorporated herein is hereby incorporated by reference in its entirety to the same extent as if each individual reference was specifically and individually incorporated by reference.

[0143] Unless the context requires otherwise, throughout this specification, including the appended claims, the word "comprise" and 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 the exclusion of any other integer or step or group of integers or steps.

[0144] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents 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.

[0145] 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 a value is expressed as an approximation by using the antecedent "about," it is understood that the particular value forms another embodiment.

[0146] Any sub-headings included herein are for convenience only and should not be construed as limiting the disclosure in any way.

[0147] The present invention will now be further described with reference to the following non-limiting figures and examples. Other embodiments will occur to those skilled in the art in view of this.

[0148] The disclosures of all references cited herein, insofar as they may be used by one skilled in the art to practice the present invention, are hereby specifically incorporated herein by cross-reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1 : Changes in blood oxygen saturation levels in patients receiving LMTM, compared before administration and four hours after administration of a single dose of 4 mg LMT and a high dose (75 / 100 / 125 mg expressed as mean, 100 mg) (post-administration). Data represent mean (%) ± SE

[0150] Figure 2 : Changes in blood oxygen saturation levels in patients receiving LMTM over 6 weeks. Pooled data for all doses represent mean (%) ± SE

[0151] Figure 3 : Changes in methemoglobin levels in patients receiving LMTM, compared before administration and four hours after administration of a single dose of 4 mg LMT and a high dose (75 / 100 / 125 mg expressed as mean, 100 mg) (post-dose). Data represent mean (%) ± SE

[0152] Figure 4 : Changes in methemoglobin levels in patients receiving low-dose (8 mg / day) and high-dose (150 mg, 200 mg, and 250 mg) HMTM over a 6-week period. Data represent mean (%) ± SE

[0153] Figure 5: As described in the text, the implicit effect of LMTM treatment on the oxygen-heme dissociation curve. The data for patients with mild hypoxemia came from subjects treated with LMTM. The data for patients with severe hypoxemia came from the results reported in the following literature: Hamidi-Alamdari, D. et al. "Methylene blue for treatment of hospitalized COVID-19 patients, randomized, controlled, open-label clinical trial, phase 2." Revistade Investigacion Clinica. 2021; 73: 190-198., in which a reduced MTC formulation was used to deliver LMT orally.

[0154] Figure 6 :High affinity LMT / MT + Computational chemical modeling of the heme-HMT interaction. (A&B) Heme in the non-planar unbound T state showing the protrusion of the iron atom above the plane of the porphyrin ring. (C&D) Heme in the flat oxygen-bound R state. (E&F) LMT binding causes heme to adopt the R state. Example

[0155] Example 1 - Methylthioninium chloride (MTC) and LMTX

[0156] MTC (methylthioninium chloride) has been used as a medicine since 1876. It is on the World Health Organization's Essential Medicines List, a list of the safest and most effective medicines for a health system.

[0157] MTC has previously been applied in many areas of clinical medicine, including the treatment of methemoglobinemia, malaria, kidney stones, bipolar disorder, efavirenz encephalopathy, and most recently, 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.").

[0158] MT can be partially oxidized + The α-LMT form and the reduced LMT form exist (see Harrington et al., 2015; supra).

[0159]

[0160] MTC is oxidized MT + It is a chloride salt in the form of leuco-MT (LMT; International Nonproprietary Name: methylthionine) by thiazine dye reductase activity in the intestine to allow absorption and distribution to deep compartments including erythrocytes and the brain (see Baddeley et al., 2015; supra). Similarly, in isolated erythrocyte preparations, MT + Conversion to LMT is required 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 into lung endothelial cells (Merker et al., 1997; supra).

[0161] Because MTC is actually a prodrug of LMT, ie, the major form in the body, TauRx developed a stable reduced form of MT, LMTM (leuco-methylthioninium bis(hydrogen methanesulfonate); hydromethylthioninium methanesulfonate), to allow for direct administration of the LMT form.

[0162] The synthesis of LMTX and LMTM compounds can be carried out according to methods described in the art (see, for example, WO2007 / 110627 and WO2012 / 107706)

[0163] Example 2 - Study Overview

[0164] Objective: To determine the effect of oral LMTM on SpO in patients with mild hypoxemia not caused by COVID-19. 2 and metHb levels, and explored the structural interactions between the LMT part and the heme group.

[0165] Methods: Eighteen AD trial participants with baseline SpO less than 94% were randomized to LMTM at 4 / 75 / 100 / 125 mg per unit. 2 Patients were routinely monitored by pulse oximetry 4 hours after twice daily dosing, 2 weeks, and 6 weeks later. Computational chemistry was used to understand LMT-heme binding.

[0166] Example 3 - Test Methods and Results

[0167] method

[0168] patient

[0169] The study design and results of two phase 3, double-blind, controlled, randomized studies of LMTM as a potential treatment for AD are described elsewhere (see “Gauthier, S. et al. “Efficacy and safety of tau-aggregation inhibitortherapy 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.). Overall, 890 patients with mild / moderate AD were randomized (3:3:4) to 150 mg / day, 250 mg / day, or 8 mg / day (intended as a control) for 15 months (clinicaltrials.gov NCT01689246). Similarly, 800 patients with mild AD were randomized to 200 mg / day or 8 mg / day LMTM for 18 months (clinicaltrials.gov NCT01689233). In both trials, MetHb and SpO 2 Levels were measured by pulse oximetry (Massimo Corporation rad 57) at screening, baseline (within 1 hour before dosing), during a 4-hour observation period after dosing, and at subsequent 2-week and 6-week clinic visits.

[0170] Estimation of the effect of LMT treatment on the oxygen-heme dissociation curve

[0171] Use standard clinical SpO 2 To PaO 2A conversion table (e.g., https: / / www.acphospitalist.org / archives / 2013 / 11 / acph-201311-coding_t1.pdf) was used to compare patients who received LMTM or the reduced form of MTC (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.”) with the normal oxygen-heme dissociation curve. For patients treated with LMTM, this means plotting the PaO 2 Baseline values ​​and corresponding SpO 2 After treatment with LMTM, SpO 2 At the same PaO 2 To compare these treatment effects with those reported by Hamidi-Alamdari et al. (2021) supra, the SpO values ​​observed in patients receiving standard care were plotted at 1:10. 2 Values ​​used to calculate the implied untreated PaO 2 SpO observed after 5 days of treatment with the reduced MTC formulation 2 At the same PaO 2 The values ​​are plotted below to infer the implied shift of oxygen-heme dissociation.

[0172] statistics

[0173] Statistical analysis was performed using R version 3.5.1, and the mean SpO was compared using paired sample t test. 2 and metHb levels.

[0174] Computational modeling

[0175] Quantum chemical calculations were performed using the self-consistent field (SCF) method in the MOE software suite, Chemical Computing Group, University College London. The SCF calculations used the Austin Model-1 basis set, and geometry optimization was initially performed using the AMBER 10 force field and Extended Hückel Theory. The crystal structure of human heme in the oxygen form (2DN1) was used to align the LMT-heme complex (see "Park, S.-Y. et al." The crystal structure of the deoxy form of human haemoglobin (PDB ID 2DN2; Park et al., 2006) was aligned with the crystal structure of the carbon monoxide form of human haemoglobin (PDB ID 3DN2; (Park et al., 2006) using Pymol.

[0176] result

[0177] Baseline population characteristics

[0178] Data were available for 18 subjects in both AD studies with baseline oxygen saturation <94% (lower limit of normal range 95%). Their demographic characteristics are summarized in Table 1. The mean age was 76.1 years, ranging from 70 to 80 years, and there were more females (61%) than males (39%).

[0179] Using the medical history available from the patient case report forms, the hypoxemia in these subjects was found to be associated with a variety of underlying respiratory conditions or other conditions of varying severity that may contribute to chronic hypoxemia (summarized in Table 2). These conditions included sleep apnea, insomnia, asbestosis, edema, asthma, bronchitis, allergies, angioedema, pneumonia, acute myocardial infarction / hypertension, coronary artery disease with angioplasty and stenting, transient ischemic attack (TIA), hypothyroidism, diabetes, syncope, tachycardia, and sepsis. No predisposing clinical history factors were found in three of the subjects.

[0180] A prospective clinical study of peripheral blood oxygen saturation and methemoglobinemia in AD patients treated with LMTM

[0181] Comparison of SpO before and 4 hours after a single 4 mg dose of LMTM or 75, 100, or 125 mg doses (summarized as the mean of the high dose, 100 mg; Figure 1) 2 As can be seen in Figure 1, the 4 mg dose of LMTM substantially increased the mean blood oxygen saturation level. With a baseline SpO below 94% 2 The mean oxygen saturation in the group with 4 mg of LMTM was 91.71%. Four hours after receiving the 4 mg dose of LMTM, the mean SpO 2Levels increased to 95.43% (+3.72%, p=0.0205; Table 3, Figure 1). Similarly, after administration of high-dose LMTM, oxygen saturation levels increased from 92.45% at baseline to 95.27% after 4 hours (+2.82%, p=0.0045; Table 3, Figure 1). Thus, LMTM was able to increase blood oxygen saturation within 4 hours over a wide range of doses, with no significant dose-dependent differences. As can be seen in 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 week 2 (+3.17%±SE, p=0.0034) and week 6 (+3.23%±SE, p=0.0005). Thus, LMTM produced rapid improvements in oxygen saturation at single doses in the 4-125 mg range and sustained improvements for more than 6 weeks at doses in the 8-250 mg / day range.

[0182] The implied effects of treatment on the oxygen-heme dissociation curve are shown in Figure 5. The standard dissociation curve was used to estimate the corresponding PaO 2 Baseline value. It can be seen that LMTM treatment improves SpO 2 The effect of the value corresponds to an implicit left shift in the oxygen-heme dissociation curve. Cases treated with LMTM had relatively mild hypoxemia (Figure 5, "mild"). For more severe cases (Figure 5, "severe"), the same method was used to infer the implicit effect on oxygen-heme dissociation in patients receiving the reduced MTC formulation reported by Hamidi-Alamdari et al. (2021) (supra), using the untreated population as a comparison basis.

[0183] It can be seen that the implicit effects of reduced MTC treatment are similar.

[0184] It was next investigated how the effect on hypoxemia related to metHb levels measured simultaneously in the same patients. There were no consistent differences in metHb levels between baseline and 4 hours after dosing for both treatment groups (Figure 3, Table 3). Thus, the short-term effects of LMTM on oxygen saturation were independent of any consistent corresponding effects on methemoglobin at low or high doses. Over 6 weeks, higher doses of LMTM (150, 200, and 250 mg / day) systematically increased metHb levels, but not at the 8 mg / day dose (Figure 4, Table 5A), although the changes in metHb did not reach statistical significance in this small number of subjects. When the effects on metHb levels were considered in the entire population, the small increases became statistically significant, including the 8 mg / day dose (Table 5B). However, it should be understood that these small changes (approximately 0.7% to 1%) are not relevant to the levels of metHb seen in symptomatic patients (e.g., >20%, or >30% metHb) in terms of therapeutic efficacy (see Table 7).

[0185] Computational chemistry model of LMT-heme effect

[0186] Computational modeling was used to investigate the minimum energy LMT-heme. The modeling suggested that LMT can bind with high affinity to the heme iron of heme via the LMT nitrogen in an octahedral geometry at a distance of 1.3 m from the iron atom. (Figure 6). LMT and Fe 2+ Electronic and The orbitals have strong interactions, and they are oriented at the axial ends and equatorial angles of the octahedral complex, respectively. It is hypothesized that the formation of this LMT-heme complex acts in a manner similar to oxygen binding (Figures 6C and D), thereby helping to overcome the initial barrier to oxygen binding by promoting the transition from the T state (Figures 6A and B) to the R state (Figures 6E and F). The resulting cooperativity would allow for the formation of the heme complex at lower PaO 2 The higher relative oxygen saturation at the lower level is consistent with the implied leftward shift of the oxygen-hemoglobin dissociation curve suggested by our analysis.

[0187] Example 4 - Discussion of Example 3

[0188] Preliminary results of an exploratory analysis of the effect of LMTM treatment in patients with chronic mild hypoxemia at baseline are reported above.

[0189] Eighteen subjects were studied who were enrolled in either of two Phase 3 trials with mild to moderate AD and had oxygen saturation levels below 94% at baseline due to a range of incidental chronic cardiopulmonary conditions. A single dose of LMTM in the range of 4-125 mg was shown to be able to significantly improve oxygen saturation levels within 4 hours, and this effect was sustained over 6 weeks of treatment with the same dose given twice daily. This means that LMTM is able to bind to heme, thereby improving oxygen saturation by approximately 3% in patients with chronic hypoxemia.

[0190] The paradoxical interactions of MT with methemoglobinemia have been discussed above. 3+ Reduction to Fe 2+ (See “May, JM, et al. “Reduction and uptake of methylene blue by human erythrocytes.” American Journal of Physiology-Cell Physiology 2004; 286: 1390-1398.” and “Schirmer, RH et al. “Lest we forget you-methylene blue...” Neurobiology of Aging 2011; 32: 2325.e7-2325.e16.”), but both MT and LMT substances themselves can cause methemoglobinemia.

[0191] Computational chemistry was used to understand the anti-hypoxemic effect of LMT. Since the structures of heme and LMT are known, it is possible to calculate the minimum energy binding interaction between the two. It was shown that LMT can bind to the heme by transferring the central nitrogen of LMT to Fe 2+ Electronic and The orbital donates an electron pair to the iron atom of heme with high affinity According to crystal field theory, this type of interaction has an estimated field factor of 1.2-1.5 (see " CK "Absorption spectra and chemical bonding in complexes." Pergamon Press, Oxford, London, New York, Paris 1962.352pp." and " CK "Oxidation numbers and oxidation states. Springer Berlin Heidelberg; 1969."), suggesting that LMT can act as a strong displaceable field ligand. It is assumed that the formation of this complex promotes the transition from the T state to the R state, in which Fe 2+ have The ionic radius of Fe 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."). In the R state, Fe 2+ have The ionic radius of ferric and ferrous myoglobins is small, so that it can fit within the four nitrogen atoms coordinated to it. Compared with T-state hemoglobin, oxygen can bind to R-state hemoglobin with higher affinity (see "Lima, FA et al." "Probing the electronic and geometric structure of ferric and ferrous myoglobins in physiological solutions by Fe K-edge absorption spectroscopies." Physical Chemistry Chemical Physics 2014; 16: 1617-1631."), thereby overcoming the initial energy barrier, and subsequent oxygen binding is further promoted by cooperativity (see "Bohr, C. et al. "Ueber einen in biologischer Beziehung wichtigen Einfluss, den die des Blutes auf dessen Sauerstoffbindungübt.” Skandinavisches Archiv für Physiologie 1904; 16: 402-412.”. This is consistent with the leftward shift of the oxygen-hemoglobin dissociation curve (28).

[0192] However, LMT binding is not optimal compared to oxygen. The binding distance between LMT nitrogen and heme iron is The corresponding oxygen binding distance is This means that when oxygen is present at high pH or low pCO 2When available, it can replace LMT, allowing normal oxygen dissociation to occur while releasing bound oxygen to peripheral tissues. This is consistent with the reduced respiratory rate observed in critically ill patients treated with reduced MTC formulations of LMT (Hamidi-Alamdari, D. et al., 2021, supra). Respiratory rate is regulated by the response to hypoxia and elevated CO 2 Level (indicating tissue hypoxia) sensitive central and peripheral chemoreceptors drive (see "Davies, A. and Moores, C. "The Respiratory System: Basic science and clinical conditions." 2nd edition. Churchill Livingstone; 2010.").

[0193] The described formation of the heme-LMT complex provides a structural explanation for three different possible effects of the MT moiety on heme: Fe 3+ Reduction to Fe 2+ (13), Fe 2+ Converted to Fe 3+ (See "Bodansky, O. "Methemoglobinemia and methemoglobin-producing compounds." Pharmacological Reviews 1951; 3: 144-196."), and the effects on oxygen saturation described herein. In the first two, the formation of the heme-LMT complex allows the donation or removal of electrons, depending on the availability of sufficient levels of NADPH required to regenerate LMT. This is in the case of Fe 3+ MT generated from LMT during reduction + Required for conversion back to LMT. Under high concentrations of the MT fraction (or in the presence of G6PD deficiency), MT + Levels exceed the available reducing capacity of the cell. MT + The orientation of the nitrogen atom can form the same coordination with heme, thus 2+ Oxidized to Fe 3+ and form LMT. In the case of hypoxemia in the context of normal red blood cell physiology, low doses of LMTM were shown to improve oxygen saturation with minimal corresponding effect on metHb levels. At higher doses of LMTM, oxygen saturation was still improved, but in addition, Fe 2+ Also oxidized to Fe 3+ .

[0194] It is proposed that oral doses of LMTM (and related compounds) above the control dose (4 mg) but below the higher dose tested here (75 mg) should provide Fe in methemoglobinemia (hereditary and acquired forms). 3+ Fe 2+ Such an effect would be consistent with the structural model and data described herein and would provide a valuable addition to currently available therapeutic options.

[0195] Table 1: Clinical characteristics of patients at baseline.

[0196]

[0197] Table 2: Clinical history of clinical trial subjects presenting with low oxygen saturation. 1 Aggravates clinical symptoms and, if severe or chronic enough, may also cause hypoxia. 2 Three patients had no listed predisposing clinical history factors.

[0198]

[0199]

[0200] Table 3: Changes in oxygen saturation and methemoglobin levels from before to after administration of LMT at a dose of 4 mg or a high dose (75 / 100 / 150 mg expressed as 100 mg). Data represent mean (%) ± SE

[0201]

[0202] Table 4: Changes in oxygen saturation levels in the summary data of patients receiving LMTM at baseline and over a 6-week period. Data represent mean (%) ± SE

[0203]

[0204] Table 5: (A) Changes in methemoglobin levels in the subgroup of patients with baseline oxygen saturation levels less than 94% who received low-dose (8 mg / day) or high-dose (150 / 200 / 250 mg / day) LMTM over a 6-week period. (B) Changes in methemoglobin levels in all patients with available data who received low-dose (8 mg / day) or high-dose (150 / 200 / 250 mg / day) LMTM over a 6-week period. Data represent mean (%) ± SE

[0205]

[0206] Table 5B (all patients):

[0207]

[0208] Table 6: (See Lolascon 2021, supra):

[0209]

[0210]

[0211] Abbreviation: DSD, disorder of sex differentiation.

[0212] Table 7: (See Lolascon 2021, supra):

[0213]

[0214]

Claims

1. A method of treating methemoglobinemia in a subject, The method comprises the steps of: orally administering to the subject a compound containing methylthioninium (MT), wherein the administration provides the subject with a total daily oral dose of 4 mg to 60 mg MT per day, optionally divided into 2 or more doses, The compound containing MT is a LMTX compound of the following formula: wherein each HnA and HnB (if present) are protonic acids which may be the same or different, Wherein p=1 or 2; q=0 or 1; n=1 or 2; (p+q)×n=2, or a hydrate or a solvate thereof.

2. The method of claim 1 , wherein the total daily dose is any of about 4, 8, 12, 16, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24 mg to about 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.

3. The method of claim 1 or claim 2, wherein the total daily dose is between 8 or 10 or 20 or 20.5 or 21 and 50 or 55 or 60 mg.

4. The method of claim 1 , wherein the total daily dose is 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, 50, 51, 52, 53, 54, or 55 mg.

5. The method of claim 1, wherein the total daily dose is 10 to 50 mg.

6. The method of claim 1, wherein the total daily dose is 20.5 to 50 mg.

7. The method of claim 1, wherein the total daily dose is 40 to 50 mg.

8. The method of claim 1, wherein the total daily dose is about 4, 8, 12 or 16 mg.

9. The method according to any one of claims 1 to 8, wherein the total daily dose of the compound is administered in divided doses twice a day or three times a day.

10. The method according to any one of claims 1 to 9, wherein the subject expresses the signs or symptoms shown in Table 7, but wherein the subject optionally (1) Not suffering from hypoxia or hypoxemia; (2) Blood oxygen saturation level (SpO2) greater than 95% under room air.

11. The method according to claim 10, comprising the step of selecting a subject based on the signs or symptoms and / or genetic testing of the genes shown in Table 6.

12. The method according to any one of claims 1 to 11, wherein the methemoglobinemia is acquired, and optionally the subject has a methemoglobin level of >30%, >50% or >70%.

13. The method according to any one of claims 1 to 11, wherein the methemoglobinemia is hereditary and optionally the subject has a methemoglobin level of >30%, >50% or >70%.

14. The method according to any one of claims 1 to 13, wherein the MT-containing compound has the following formula, wherein HA and HB are different monoprotic acids:

15. The method according to any one of claims 1 to 13, wherein the MT-containing compound has the formula: Each HnX is a protonic acid.

16. The method according to any one of claims 1 to 13, wherein the MT-containing compound has the formula and H2A is a diprotic acid:

17. The method of claim 15, wherein the MT-containing compound has the following formula and is a di-monoprotic acid:

18. A method according to any one of claims 1 to 17, wherein the or each protic acid is an inorganic acid.

19. The method of claim 18, wherein each protic acid is a hydrohalic acid.

20. A method according to claim 18, wherein the or each protic acid is selected from HCl; HBr; HNO 3 ; H 2 SO 4 。 21. A method according to any one of claims 1 to 17, wherein the or each protic acid is an organic acid.

22. The method of claim 21, wherein the protic acid or each protic acid is selected from H 2 CO 3 ; CH 3 COOH; Methanesulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, p-toluenesulfonic acid.

23. The method according to any one of claims 1 to 17 or claim 22, wherein the MT-containing compound is LMTM:

24. The method according to any one of claims 1 to 17, wherein the MT-containing compound is selected from the list consisting of:

25. The MT-containing compound according to any one of claims 1 to 24, for use in a method of treatment according to any one of claims 1 to 24.

26. Use of a MT-containing compound according to any one of claims 1 to 24 for the manufacture of a medicament for use in a method of treatment according to any one of claims 1 to 24.

Images