METHYLTHIONINIUM COMPOUNDS FOR USE IN THE TREATMENT OF COVID-19

Abstract

The present invention provides methods of treating COVID-19 in a subject using methylthioninium compounds.

Claims

1. A method of therapeutic treatment of COVID-19 in a subject, which method comprises administering to said subject a methylthioninium (MT)-containing compound, wherein said administration provides a total daily oral dose of between more than 30 to 250 mg of MT to the subject per day, optionally split into 2 or more doses, or wherein said administration provides a total daily intravenous (IV) dose of between 10 and 200 mg of MT to the subject per day, wherein the MT-containing compound is an LMTX compound of the following formula: ##STR00017## wherein each of H.sub.nA and H.sub.nB (where present) are protic acids which 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.

2. A method as claimed in claim 1 wherein the subject is a human who has been diagnosed with COVID-19, or wherein said method comprises making said diagnosis. 3 A method as claimed in claim 2 wherein the subject is characterised by having definite evidence of SARS-CoV-2 infection plus one, two or all of: (1) requiring medical care for COVID-19; (2) having an SpO2 less than 95% on room air; (3) having radiographic evidence of pulmonary infiltrates.

4. A method of prophylactic treatment of COVID-19 in a subject, which method comprises administering to said subject a methylthioninium (MT)-containing compound, wherein said administration provides a total daily oral dose of between more than 30 to 250mg of MT to the subject per day, optionally split into 2 or more doses, or wherein said administration provides a total daily intravenous (IV) dose of between 10 and 200 mg of MT to the subject per day, wherein the MT-containing compound is an LMTX compound of the following formula: ##STR00018## wherein each of H.sub.nA and H.sub.nB (where present) are protic acids which 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.

5. A method as claimed in claim 4 wherein the subject is a human who has been assessed as having suspected or probable COVID-19, and is optionally selected from: a subject who has been in close contact with one or more COVI D-19 cases; a subject who is at least 65 years old; a subject living in a nursing home, care home, or long-term care facility; a subject with an underlying medical condition which increases the likelihood of adverse effects from COVID-19.

6. A method as claimed in any one of claims 1 to 5 wherein the total daily oral dose is: (i) greater than 35, 40, 50, or 60 mg and less than or equal to 250 mg of MT to the subject per day; and/or (ii) greater than or equal to about 30.5, 30.6, 31, 35, 37.5, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 140, 150, 160, 170, 180, 190 or 200 mg MT.

7. A method as claimed in any one of claims 1 to 6 wherein the total daily oral dose is about 60, 75, 120, or 150 mg MT, which is optionally split twice a day or three times a day.

8. A method as claimed in any one of claims 1 to 5 wherein the total daily IV dose is between 26 and 200 mg of MT to the subject per day.

9. A method as claimed in any one of claims 1 to 5 wherein the total daily IV dose is: (i) about 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/day by continuous dosing. (ii) about, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 mg/day by bolus dosing.

10. A method as claimed in any one of claims 1 to 9 wherein the treatment is combined with a second agent.

11. A method as claimed in claim 10 wherein the second agent is selected from: chloroquine or hydroxychloroquine; lopinavir-ritonavir; arbidol; azithromycin, remdesivir, favipiravir, actemra; dexamethasone; convalescent plasma; a SARS-CoV-2-neutralizing antibody.

12. A method as claimed in claim 10 or claim 11 wherein the MT-containing compound and the second agent are administered sequentially within 12 hours of each other.

13. A method as claimed in any one of claims 10 to 12 wherein the subject is pre-treated with the second agent prior to commencement of the treatment with the MT-containing compound.

14. A method as claimed in claim 10 or claim 11 wherein the MT-containing compound and the second agent are administered simultaneously, optionally within a single dosage unit.

15. A method as claimed in any one of claims 1 to 14 wherein the MT-containing compound has the following formula, where HA and HB are different mono-protic acids: ##STR00019##

16. A method as claimed in any one of claims 1 to 14 wherein the MT-containing compound has the following formula: ##STR00020## wherein each of H.sub.nX is a protic acid.

17. A method as claimed in any one of claims 1 to 14 wherein the MT-containing compound has the following formula and H.sub.2A is a di-protic acid: ##STR00021##

18. A method as claimed in claim 16 wherein the MT-containing compound has the following formula and is a bis-monoprotic acid: ##STR00022##

19. A method as claimed in any one of claims 1 to 18 wherein the or each protic acid is an inorganic acid.

20. A method as claimed in claim 19 wherein each protic acid is a hydrohalide acid.

21. A method as claimed in claim 19 wherein the or each protic acid is selected from HCl; HBr; HNO.sub.3; H.sub.2SO.sub.4.

22. A method as claimed in any one of claims 1 to 18 wherein the or each protic acid is an organic acid.

23. A method as claimed in claim 22 wherein the or each protic acid is selected from H.sub.2CO.sub.3; CH.sub.3COOH; methanesulfonic acid, 1,2-ethanedisulfonic acid, ethansulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid.

24. A method as claimed in any one of claims 1 to 18, or claim 23 wherein the MT-containing compound is LMTM: ##STR00023##

25. A method as claimed in claim 24 wherein the total daily dose of LMTM is at least 52 mg/day.

26. A method as claimed in claim 25 wherein the dose of LMTM is about 125 mg/once per day.

27. A method as claimed in any one of claims 1 to 18 wherein the MT-containing compound is selected from the list consisting of: ##STR00024##

28. A method as claimed in any one of claims 1 to 27 wherein the treatment with the MT compound is such as to achieve one or more of the following in the subject: (i) enhanced mitochondrial function; (ii) enhanced blood oxygen capacity; (iii) improved CNS sequelae arising from COVID-19.

29. An MT-containing compound as defined in any one of claims 1 to 28, for use in a method of treatment as defined in any one of claims 1 to 28.

30. Use of an MT-containing compound as defined in any one of claims 1 to 28, in the manufacture of a medicament for use in a method of treatment as defined in any one of claims 1 to 28.

Description

FIGURES

[0197] FIG. 1A: virucidal activity of MTC against SARS-CoV2 in vitro in the dark using Vero-E6 kidney cells (date from Cagno et al 2020).

[0198] FIG. 1B: data from FIG. 1A presented in terms of IC50 for antiviral activity.

[0199] FIGS. 2A and 2B: computational chemistry modelling of the high affinity LMT/M.sup.+-heme interaction.

[0200] FIG. 3A: calculation of human oral doses of MT provided as LMTX required to achieve the tissue concentrations required for inhibition of SARS-CoV-2 toxicity based on 20:1 tissue: plasma ratio determined from study in minipigs.

[0201] FIG. 3B: calculation as per FIG. 3A based on 40:1 tissue: plasma ratio inferred from minipig and rat autoradiography data.

[0202] FIG. 3C: calculation as per FIG. 3A based on 10:1 tissue: plasma ratio based on assumed lower lung penetration.

[0203] FIG. 3D: calculation as per FIG. 3A based on 80:1 tissue: plasma ratio, for reference.

[0204] FIG. 4: calculations for IV dosing based on 20:1 tissue: plasma ratio. The dose required as continuous infusion (mg/hr) is shown in FIG. 4A and for bolus doses given 6-hourly is shown in FIG. 4B

[0205] FIG. 5: calculations for IV dosing based on 40:1 tissue: plasma ratio. FIGS. 5A and 5B provide the corresponding estimates for continuous infusion or infusion over 5 minutes every 6 hrs.

[0206] FIG. 6: calculations for IV dosing based on 10:1 tissue: plasma ratio. FIGS. 6A and 6B provide the corresponding estimates for continuous infusion or infusion over 5 minutes every 6 hrs.

[0207] FIG. 7: calculations for IV dosing based on 80:1 tissue: plasma ratio for reference. FIGS. 7A and 7B provide the corresponding estimates for continuous infusion or infusion over 5 minutes every 6 hrs.

[0208] FIG. 8: oxygen saturation levels in patients receiving LMTX compared pre-dose and after 4 hrs in the clinic following administration of a single doses of LMT at 4 mg and ˜100mg (mean of 75 mg, 100 mg, 125 mg). Levels were measured pre-dose and 4 hrs after dosing (post-dose).

[0209] FIG. 9: the effects of LMTM on SpO2 levels over 4 hours was independent of any corresponding effect on metHb.

[0210] FIG. 10: LMTM at high dosages over a period of time systematically increases metHb levels.

REFERENCE EXAMPLE 1

Methylthioninium Chloride (MTC) as an Antiviral

[0211] MTC (methylthioninium chloride, methylene blue) has been available as a drug since 1876. It is on the world health organisation's list of essential medicines, which is a list of the safest and most effective medicines in a health system.

[0212] MTC has been applied previously in many areas of clinical medicine including treatment of methemoglobinemia, malaria, nephrolithiasis, bipolar disorder, ifosfamide encephalopathy and most recently in Alzheimer disease (AD; Wischik et al., 2015; Nedu et al 2020).

[0213] Several studies have investigated the antiviral activity of MTC. One such study reported a significant reduction in viral load in hepatitis C patients at a dose of 130 mg/MTC per day (i.e. 98 mg/MT-equivalent per day) for 50 days (Wood et al., 2006; Mehta et al., 2006). Photoactivated MTC is routinely used for viral sterilisation of blood products in vitro via a photo-oxidation mechanism whereby intercalated methylthioninium (MT) generates singlet oxygen following photo-activation which damages and breaks nucleic acids and inactivates viruses. Viruses susceptible to MTC treatment include HIV-1 and 2, herpes and hepatitis C (Muller-Breitkreutz 1998, Mohr, 1999).

[0214] Recently, there is increasing interest in MTC as a potential treatment for COVID-19. MTC inhibits binding of coronavirus spike protein to its main receptor, angiotensin-converting enzyme 2 (ACE2) through which the virus gains entry into cells (IC.sub.50 of 3.0 μM or 0.09 μg/ml; Bojadzic et al 2020).

[0215] A recent study published by Cagno and colleagues reported that MTC had virucidal activity against SARS-CoV2 in vitro in the dark using Vero-E6 kidney cells (Cagno et al 2020; FIG. 1A). The data have been replotted as percentage inhibition of SARS-CoV-2 toxicity to permit estimation of the IC.sub.50 for antiviral activity (FIG. 1B). The mechanism responsible for this viricidal effect is unknown, but, as explained further below, it is most likely mediated via the reduced form of the MT moiety (leuco-MT, LMT), since MT needs to be converted to the LMT form to gain entry into cells (Merker et al., 1997; May et al., 2004).

[0216] Using the Cagno et al. data, the calculated IC.sub.50 of the MT moiety for neutralising viral toxicity in a Vero cell assay is 0.032 μM at 20 hrs. The IC.sub.50 values for SARS-CoV-2 antiviral activity of two other compounds (hydroxychloroquine and remdesivir) in a similar Vero cell assay using viral replication as the endpoint has been reported (Yao et al., 2020; Wang et al., 2020). For hydroxychloroquine, the IC.sub.50 values are 6.25 μM at 24 hrs and 0.72 μM at 48 hrs. For remdesivir, the IC.sub.50 value is 0.77 μM at 48 hours. Therefore, assuming comparability of the assays, LMT appears to be approximately 23-fold more potent as a SARS-CoV-2 antiviral. The relatively low potency of hydroxychloroquine limits its clinical utility, since the upper limit of safe dosing is 400 mg/day, whereas the clinical dose required to achieve optimal antiviral activity is approximately 800 mg/day (Yao et al., 2020). Therefore, the typical dosing regimen for hydroxychloroquine is limited to 800 mg on day, followed by 400 mg per day on days 2-7.

EXAMPLE 2

Hydromethylthionine Salts as a Monotherapy for Covid 19

[0217] The MT moiety can exist in the oxidised MT.sup.+ form and in the reduced LMT form (Harrington et al., 2015;).

##STR00016##

[0218] MTC is the chloride salt of the oxidised MT.sup.+ form. It needs to be converted to the reduced leuco-MT (LMT; international non-proprietary name: hydromethylthionine) form by a thiazine dye reductase activity in the gut to permit absorption and distribution to deep compartments including red cells and brain (Baddeley et al., 2015). Likewise, in isolated red cell preparations, MT.sup.+ needs to be converted to LMT to permit uptake both into red cells (May et al., 2004) and into pulmonary endothelial cells (Merker et al., 1997).

[0219] Because MTC is actually a prodrug for LMT, the predominant form in the body, TauRx developed a stabilised reduced form of MT as LMTM (leuco-methylthioninium bis(hydromethanesulphonate); hydromethylthionine mesylate) in order to permit direct administration of the LMT form.

[0220] Synthesis of LMTX and LMTM compounds can be performed according to the methods described in the art (see e.g. WO2007/110627, and WO2012/107706)

[0221] Mitochondrial Dysfunction in COVID-19

[0222] There is mounting evidence to suggest a link between COVID-19 and mitochondrial dysfunction (Saleh et al 2020; Singh et al 2020). A significant number of COVID-19 patients develop severe consequences attributed to a surge of inflammatory events described as the “cytokine storm”. Mitochondria play a pivotal role in maintaining cellular oxidative homeostasis and a heightened inflammatory response is thought to lead to mitochondrial dysfunction in these patients. Mitochondria are the main source of reactive oxygen species (ROS) within the cells. Increased ROS generation causes both intra- and extracellular mitochondrial damage which in turn leads to microbiota dysbiosis and platelet dysfunction which plays a major role in blood clotting and coagulopathy events that further aggravate the inflammatory response in a vicious cycle of events contributing to COVID-19 disease progression (Melchinger et al 2019). A recent study aiming to determine which parts of the human interactome are most affected by SARS-CoV-2-infection demonstrated that a member of the mitochondrial complex I is downregulated by infection leading to apoptosis and ultimately cell death (Guzzi et al 2020). In addition, Singh and colleagues (2020) reported that SARS-CoV-2 upregulated genes in the interferon, cytokines, nuclear factor kappa B (NF-κB) and ROS processes, while downregulating mitochondrial organisation and respiratory processes, in a lung cell line.

[0223] The above studies indicate that mitochondrial dysfunction may represent an important mediator in the development of COVID-19 and could contribute to the dysregulated immune response of COVID-19 patients, resulting in accelerated progression of the disease and a hyper-inflammatory state.

[0224] LMTM has been shown to enhance mitochondrial function both in vitro (Atamna & Kumar 2010) and in vivo (Riedel et al., 2020). This is due to the fact that MT.sup.+/LMT has a redox potential close to zero which is mid-way between the potentials of Complex I and Complex IV in the mitochondrial electron transport chain and can therefore act as an electron shuttle. This activity translates into an anti-ischaemic activity which limits the extent of infarction in a unilaterally ligated rat-brain model of cerebral ischaemia (Rodriguez et al., 2014). Therefore, LMT has the ability to protect tissues in the context of hypoxia where oxygen delivery is limiting.

[0225] In addition to enhancing mitochondrial function, MT dosed orally as MTC has been shown to increase mitochondrial biogenesis (Stack et al., 2014). Enhancement of mitochondrial biogenesis is linked to cellular clearance mechanisms, such as macroautophagy, pathways related to scavenging of ROS as well as the ability to increase in Nrf2 levels (Gureev et al., 2016). De la Vega and colleagues (2016) argue in an extensive review that Nrf2 plays an important protective role with respect to oxidative and inflammatory lung damage in Acute Lung Injury/Acute Respiratory Distress Syndrome (ADI/ARDS). They present evidence to show that pharmacological activation of Nrf2 would be expected to ameliorate alveolar damage from the primary infection but also from mechanical and hyperoxic injury resulting from Ventilation Induced Lung Injury (VILI). Oral dosing with MTC at 30 mg/kg has been shown to increase Nrf2 levels in brain (Stack et al., 2014). As noted above, the oxidised MT+ needs to be reduced to LMT to permit uptake into pulmonary endothelial cells (Merker et al., 1997). It is therefore credible that LMTM would have similar ability to induce Nrf2 in ADI/ARDS.

[0226] Blood Oxygen Carrying Capacity

[0227] COVID-19 has been associated with the emergence of both methemoglobinemia and hypoxaemia in patients (Naymagon et al., 2020). Methemoglobinemia results from oxidation of the iron contained in haemoglobin from the ferrous (Fe.sup.2+) to the ferric (Fe.sup.3+) form. The oxidation is associated with a decrement in the capacity of haemoglobin to carry oxygen efficiently (Curry et al., 1982). MTC is the primary treatment for methemoglobinemia, and indeed represents the only approved indication for its clinical use. The oxidised MT.sup.+ form of methylthionine given as MTC is first reduced to LMT at the cell surface as a prerequisite for red cell entry (May et al., 2004). It is then LMT which is the active species at the heme site, binding to porphyrin and permitting the transfer of an electron which converts Fe.sup.3+ to Fe.sup.2+, thereby restoring normal oxygen-carrying capacity (Yubisui et al., 1980; Blank et al., 2012).

[0228] Computational chemistry modelling shown in FIGS. 2A and B provides a structural basis explaining the dynamics of the high affinity LMT-heme interaction. The LMT nitrogen coordinates with the heme iron atom by orientating itself towards the iron atom within 2.1 Å (dotted line in FIG. 2A). In methaemoglobinaemia, the iron atom is in the oxidised Fe.sup.3+ state.

[0229] In conditions associated with hypoxaemia where the iron the iron atom is in the Fe.sup.2+ state, the close formation of the LMT/heme coordinate facilitates oxygen carrying capacity via a process that does not require the transfer of an electron. When Hb is in the deoxygenated state, the heme is in the domed T state with Fe not fully accommodated in the tetrapyrrole ring, and is held by two histidines (His 87 in alpha subunit/His 92 in beta subunit and His 58 in alpha subunit/His 63 in beta submit). In this state, the ionic radius of the iron, which is in a high-spin Fe(II) state, is too large (radius 2.06 Å) to fit in the ring of nitrogens with which it coordinates; it is 0.6 Å out of the plane of the ring. When O.sub.2 binds to the heme group it assumes the R state, becomes planar and the iron ion lies in the plane of the ring, as it is in a low-spin Fe(II) state with a smaller radius (1.98 Å). All six coordination positions of the ion are occupied: the bound oxygen molecule accounts for the sixth. When O.sub.2 binds to Fe.sup.2+, it displaces the distal histidine and stabilises the heme moiety in the flat R-state. The binding of oxygen by haemoglobin is cooperative. As the haemoglobin tetramer units bind successive oxygens, the oxygen affinity of the subunits increases. The affinity for the fourth oxygen to bind is approximately 300 times that for the first. LMT is able to bind to the Fe of heme with an estimated field factor of 1.2-1.5. The field factor of LMT is sufficient to bind to Fe.sup.2+ (potentially f-factor of 1.2-1.5; C K Jorgensen, Oxidation numbers and oxidation states, Springer 1969 pp84-30 85). MT is therefore a strong field ligand and is able to bind to heme sufficiently to induce an R-state configuration within the protein. The LMT moiety is able to form a complex with Fe.sup.2+ by donation of lone pair electrons from the N atom to the d-orbitals of ferrous iron (Molecules 2013, 18(3), 3168-3182; https://doi.org/10.3390/molecules18033168). Therefore, binding of LMT overcomes the initial energy barrier for oxygen binding, which is thereafter able to bind and oxygenate all four heme groups of haemoglobin. Because O.sub.2 binds with higher affinity, it is able to displace LMT from the same binding site. This permits normal oxygen dissociation to occur with release of bound oxygen to peripheral tissues at low pH/high pCO.sub.2.

[0230] Given that the LMT is the active form, the clinical evidence below showing that LMTM treatment enhances the oxygen carrying capacity of the blood confirms that this LMT-heme interaction facilitates oxygen uptake by haemoglobin.

[0231] Potential for LMTM to Improve CNS Sequelae of COVID-19

[0232] There are emerging clinical reports indicate that COVID-19 may have detrimental effects on the central nervous system (De Felice et al 2020; Baig et al 2020). It has been reported that SARS-CoV-2 preferentially targets soma of cortical neurons but not neural stem cells, the target cell type of ZIKA virus (Ramani et al 2020). Imaging analysis also revealed that SARS-CoV-2 co-localises with tau is associated with missorting tau and subsequent neuronal death.

[0233] LMTM was originally developed as a treatment for pathological tau protein aggregation in AD and other dementias. Therefore, LMTM may have a role to play in limiting the long-term functional disability and cognitive impairment that has been reported in some cases of COVID-19 infection (Zhou et al., 2020).

EXAMPLE 3

Estimation of Clinical Dose of LMTM Required for SAR-CoV-2 Antiviral Activity

[0234] TauRx originally focused on MTC as a potential treatment for AD because of its ability to block pathological aggregation of the microtubule associated protein tau which forms neurofibrillary tangles and is responsible for clinical dementia in Alzheimer's Disease (Wischik et al., 1996; Harrington et al., 2015).

[0235] Comparatively, LMTM shows better pharmacodynamic and pharmacokinetic properties than MTC (Harrington et al., 2015; Baddeley et al., 2015). Following oral administration, free plasma MT/LMT is subject to efficient first-pass metabolism which converts it to an inactive conjugate, and which is the predominant species in found plasma. The 20-fold better uptake into red cells is important for protection LMT from metabolic inactivation and permitting its efficient distribution to the brain and other tissue compartments (Baddeley et al., 2015).

[0236] An initial Phase 2 dose-finding study identified 138 mg/day as the minimum effective dose of MTC (Wischik et al., 2015). However, because LMT absorption from LMTM is much more efficient, the minimum effective dose required for anti-dementia effects was found to be 8 mg/day, and 16 mg/day was found to be the optimally effective dose (Schelter et al., 2019).

[0237] The reason for this has been elucidated in two unpublished preclinical studies which provide highly relevant insights into the use of LMTX for treating COVID-19:

[0238] A pharmacokinetic study in minipigs (nearest to humans in terms of pharmacokinetic properties) given LMTM orally at doses corresponding to human doses of 8, 24, 40, 71 and 155 mg/day found that the mean brain:plasma ratio at 2 and 4 hrs for the parent LMT moiety is ˜20:1 (compared to 0.3:1 for MTC).

[0239] A further whole body autoradiography study rats compared the distribution of LMT-associated radioactivity in brain, lung and heart following oral dosing at 10 mg/kg. This found that the ratio of heart and lung to brain is 2:1. However, this is for total MT, including the inactive conjugate. The ratio specific for LMT in lung is therefore unknown. It is possible to relate plasma levels determined in a large clinical population (Schelter et al., 2019) to expected tissue levels of LMT at steady state across a wide dosing range of 8-250 mg/day. However, this depends critically on the tissue:plasma ratio for specifically affected tissues such a lung.

[0240] Combining the human and animal PK data, it is possible to calculate the human doses required to achieve the tissue concentrations required for inhibition of SARS-CoV-2 toxicity as reported by Cagno et al. (2020). This is shown in FIG. 3A below using the 20:1 tissue: plasma ratio determined from study in minipigs. The dose required to achieve 99% reduction in toxicity in 95% population would be approximately 60 or 75 mg/day.

[0241] However, other scenarios should also be considered. If the tissue:plasma ratio is 40:1 (consistent with the minipig and rat autoradiography data), a dose of approximately 40 mg/day would be sufficient (FIG. 3B).

[0242] Furthermore if the plasma:lung ratio is closer to 10:1, then the dose required for 99% reduction in toxicity would be closer to 150 mg/da (FIG. 3C).

[0243] For reference FIG. 3D illustrates the corresponding estimates for the tissue:plasma ratio of 80:1. The dose required to achieve at least 99% inhibition of toxicity in at least 95% of population is approximately 20 mg/day.

[0244] Therefore, until further tissue-specific data are available for the tissue distribution of LMT (as distinct from total MT) in lung in particular, an appropriate dosing range would be at least 30 mg/day.

[0245] The safety of LMTM across doses ranging from 8-250 mg/day has been well established from three Phase 3 trials in over 2,000 patients with dementia. Therefore, doses up to 250 mg/day could be given safely for treatment of COVID-19 patients.

[0246] IV Dosing

[0247] The predicted tissue levels at IV doses depend on the bioavailability and the tissue:plasma ratio (study discussed above).

[0248] We investigated bioavailability (oral vs iv) of LMTM in minipigs, based on total radioactivity following dosing of .sup.14C-LMTM following dosing at 10 mg/kg oral and 5 mg/kg IV. Although absolute bioavailability adjusted for dose in this study was found to be ˜100%, we have assumed bioavailability of 75% for the purposes of dosage calculations.

[0249] For the reasons given above, a range of dosing regimes has been provided based on tissue:plasma ratios of 10:1, 20:1, 40:1, and 80:1 for reference.

[0250] The IV doses have been calculated for continuous infusion (mg/hr) or for IV bolus infusion administered 6-hourly. In each case, the infusion rates calculated from the population-PK model have been determined on the basis of the dose required for 95% of the population to have tissue levels above a given threshold required to achieve a given reduction in predicted SARS-CoV-2 tissue toxicity determined from the studies reported for Vero-E6 kidney cells for the MT moiety by Cagno et al. (2020).

[0251] For the 20:1 tissue:plasma ratio, the dose required as continuous infusion (mg/hr) is shown in FIG. 4A and for bolus doses given 6-hourly is shown in FIG. 4B. The doses required to achieve at least 99% inhibition of toxicity in at least 95% of the population are 2.8 mg/hr as continuous infusion or 20 mg as infusion over 5 minutes every 6 hrs or 27 mg as infusion over 5 minutes every 8 hrs, or 40 mg as infusion over 5 minutes every 12 hrs,

[0252] FIGS. 5A and 5B provide the corresponding estimates for the tissue:plasma ratio of 40:1. The doses required to achieve at least 99% inhibition of toxicity in at least 95% of the population are 1.2 mg/hr as continuous infusion or 12 mg as infusion over 5 minutes every 6 hrs or 16 mg as infusion over 5 minutes every 8 hrs, or 24 mg as infusion over 5 minutes every 12 hrs,

[0253] FIGS. 6A and 6B provide the corresponding estimates for the tissue:plasma ratio of 10:1. The doses required to achieve at least 99% inhibition of toxicity in at least 95% of the population are 6.8 mg/hr as continuous infusion or 50 mg as infusion over 5 minutes every 6 hrs or 67 mg as infusion over 5 minutes every 8 hrs, or 100 mg as infusion over 5 minutes every 12 hrs,

[0254] For reference, FIGS. 7A and 7B provide the corresponding estimates for the tissue:plasma ratio of 80:1. The doses required to achieve at least 99% inhibition of toxicity in at least 95% of the population are 0.7 mg/hr as continuous infusion or 5.3 mg as infusion over 5 minutes every 6 hrs or 7 mg as infusion over 5 minutes every 8 hrs, or 21 mg as infusion over 5 minutes every 12 hrs,

EXAMPLE 4

Preliminary Clinical Data

[0255] The present inventors have used data available for patients participating in clinical trials to determine whether LMT enhances oxygen saturation of blood. Data were available for 18 subjects with oxygen saturation <94% at baseline (lower limit of normal range is 95%). Oxygen saturation levels were compared pre-dose and after 4 hrs in the clinic following administration of a single doses of LMT at 4 mg and ˜100mg (mean of 75, 100, 125 mg; FIG. 8).

[0256] LMTM at both dosing ranges significantly increased oxygen saturation at 4 hours, again supporting multiple beneficial modes of action for LTMX for treatment of COVID-19 patients.

[0257] In order to understand this effect further the inventors investigated whether the low oxygen saturation in these patients is due to elevation in metHb levels. There was no difference in metHb levels at baseline between subjects with low SpO2 and those with SpO2 levels in the normal range. Furthermore, the effects on LMTM on SpO2 levels over 4 hours was independent of any corresponding effect on metHb (FIG. 9).

[0258] Therefore, LMTM is able to act on oxygen saturation in the blood by a novel mechanism unrelated to its known effects on metHb. Indeed LMTM at higher doses systematically increases metHb levels (FIG. 10).

EXAMPLE 5

A Clinical Trial for LMTM for Treatment of Covid-19

[0259] LMTM may be given at doses of 60 and 120 mg/day, or alternatively 75mg/day or 150mg/day (see Example 3 above), over 1 month to adult patients who are currently hospitalised and requiring medical care for COVID-19 with definite evidence of SARS-CoV-2 infection from nasal swab, who have an SpO2 less than 95% on room air at screening or PaO2/FiO2 <300 or respiratory rate≥20 per minute and have radiographic evidence of pulmonary infiltrates.

[0260] Patients already participating in any other clinical trial of an experimental agent treatment for COVI D-19, or in whom concurrent treatment or planned concurrent treatment with other agents with actual or possible direct acting antiviral activity against SARS-CoV-2, or who require mechanical ventilation at screening may be excluded, as will patients with a calculated creatinine clearance<30 ml/min.

[0261] The principal endpoints are change in clinical disease severity (7-point ordinal scale; Table 1), SpO2 change measured by Co-Oximeter, change in viral burden measured by PCR of nasal swabs, C-reactive protein levels in blood, percentage of lung involvement on lung CT scan and mortality.

TABLE-US-00009 TABLE 1 7-point ordinal scale 1: Not hospitalised with no limitations on activities 2: Not hospitalised but with limitations on activities 3: Hospitalised, not receiving supplemental oxygen 4: Hospitalised, receiving supplemental oxygen 5: Hospitalised, receiving non-invasive ventilation or high-flow nasal cannula 6: Hospitalised, receiving mechanical ventilation 7: Death

[0262] Based on publicly available data regarding the standard deviations on the key outcome measures, the number of subjects will be in the range of approximately 100 per arm.

EXAMPLE 6

Conclusion: Hydromethylthionine Salts as Treatment for COVID-19

[0263] For the foregoing rationale the LMTX class of compounds may provide benefits in the treatment (including prophylactic treatment) of COVID-19 patients both alone and in combination with other agents by reducing reducing viral toxicity at doses defined herein based on proprietary PK studies of LMTX in vivo. Also described herein are beneficial effects on blood increased oxygen saturation.

[0264] LMTX may also provide benefits to subjects in enhancing, mitochondrial function and improving CNS sequelae of COVID-19.

[0265] Furthermore, the LMTM does not have the cardiotoxicity that limits the dose and duration of certain other treatments.

REFERENCES

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