TECHNICAL FIELS

The present invention discloses a new formulation and composition which use reduced methylene blue (Leucomethylene) for treatment of COVID-19 and patients with sepsis or diabetes or hypoxemia or etcetera.

BACKGROUND

COVID-19 is a global catastrophic event that causes the severe acute respiratory syndrome. [1] There is no currently approved protocol for treatment of COVID-19 patients. The mechanism of the disease remains unclear, and hypoxemia is one of the main complications. The microbial threat as induced by COVID-19 causes the activation of macrophages to produce a huge amount of inflammatory molecules and nitric oxide (NO). Activation of macrophages population into a pro-inflammatory phenotype induces a self-reinforcing cycle. Oxidative stress is documented in COVID-19 patients. Oxidative stress and NO contribute to this cycle, establishing a cascade inflammatory state that can kill the patient. Interrupting this vicious cycle by a simple remedy may save critical patients' lives. Nitrite, nitrate (the metabolites of NO), methemoglobin (met- Hb), and prooxidant-antioxidant balance (PAB) were estimated as involving factors to intensify hypoxemia in ICU patients and COVID-19 patients.

A probable reason for hypoxemia in COVID-19 patients is methemoglobinemia which results from oxidation of the iron contained in hemoglobin from the ferrous to the ferric form. The oxidation is associated with a decrement in the capacity of hemoglobin to carry oxygen.

The FDA approved the treatment of methemoglobinemia is the intravenous infusion of oxidized methylene blue (1-2 mg/kg body weight). After entering methylene blue into the cell of body, it is reduced by NADH to LMB which in turn reduces the methemoglobin to hemoglobin.

Other rationales for considering LMB for treatment is the following proven mechanism. 1) MB has direct inhibitory effects on nitric oxide synthases (produce nitric oxide that cooperate in generating reactive nitrogen species which damage the cells and biomolecules) and guanylate cyclase enzyme,; 2) MB increases the activity of normally slow NADPH-methemoglobin reductase pathway, which decreasing hypoxemia through reducing methemoglobin; 3) MB could prohibit the cytopathic effect and reduce the propagation of RNA virus (2).; 4) MB has formed the basis of antimicrobial chemotherapy,- particularly in the area of antimalarials. It is used in an antibacterial foam dressing for the management of chronic wounds with local infection (3).; 5) MB is a powerful oxygen superoxide scavenger that eliminates rapidly this ion not to damage the tissue; 6) MB inhibits xanthine oxidase which prevent to produce ROS (4).; 7) MB prevents platelet activation, adhesion, and aggregation. 8) Reduced form of MB (Leucomethylene: LMB) quench ROS as a reducing agent.

In the human body, MB first induces oxidative stress through absorbing electron (like a free radical) from other molecules, then decreases the oxidative stress through other mechanisms. After injection, 65-85% of MB (oxidized form) is reduced to LMB. The oxidative effect of MB is documented in culture media, dead cells and senescent cells cannot reduce it to leukomethylene blue (LMB, colorless), therefore the cells remain blue (5).

In all published papers, the missing point is that they use MB (oxidized form) which causes the increasing the oxidative stress, since at first MB should be reduced to LMB (a lipophilic substance), then it could penetrate into cell through the cell membrane. Since MB exacerbates oxidative stress, diseases such as glucose-6-phosphate dehydrogenase deficiency, which has deficiency in antioxidant defense system cannot tolerate the increment of oxidative stress and there is the risk of hemolytic anemia after administration of MB. Also it is mentioned that methylene blue doses over 7 mg/kg paradoxically can exhibit an oxidizing effect in patients, resulting in hemolysis and methemoglobin production (4). This phenomena can be attributed to oxidizing effect of MB.

SUMMARY

Several therapeutic procedures with controversial results have been suggested which included antiviral drugs, antibiotics, anticoagulants, corticosteroids, fluid therapy, H2 blockers and oxygen support for decreasing hypoxemia. In severe cases, patients continue to have increased respiratory distress and hypoxemia despite a high percentage of oxygen therapy. For decreasing hypoxemia, all attentions are focused on using only oxygen support by a nasal cannula or mask or non-invasive ventilation, but no one has paid attention to pharmacological treatment that alleviates hypoxemia, which is very important in improving patients. In this invention, we describe a new composition of methylene blue for pharmacological intervention for the treatment of hypoxemia in COVID-19 patients. This composition is consisting of reduced form of methylene blue. In COVID-19 patients, since there is high oxidative stress in them, if the oxidized form of methylene blue is given to patients, it causes more oxidative stress and consequently more inflammation, which in turn may worsen the situation of patients. Also, the LMB could quench inflammation.

BRIEF DESCRIPTION DETAILED DESCRIPTION

Oxidized form MB was converted to LMB (reduced form) by chemical reducing substances or electrochemical methods.

Methylene Blue + 2H+ + 2e ====== Leucomethylene Blue ( E0= 0.53V)

The treatment of severe COVID-19 with reduced form of MB is safe and feasible. The reduced MB has rapid and delayed effects. The rapid effect increases the Sp02% (All patients have been received 100 % oxygen) by reducing met-Hb. Delayed effects are through the acceleration of normally slow NADPH-methemoglobin reductase, the improvement of inflammatory markers such as CRP level and LDH, decreasing severity of disease that may be also due to antimicrobial effect. The hospital stay was significantly shortened in the MB-group and the mortality rate was decreased in MB-group. No serious adverse effects were observed after consuming LMB. We suggest the optimal time of reduced methylene blue (LMB) administration should be before entering the patient to a very severe stage of the disease and multi-organ involvement and failure.

The addition of LMB to the standard treatment protocols for severe COVID-19 patients was associated with statistically significant clinical benefits resulted in decreasing hospital stay and 28-day mortality.

The chemical compositions are:

1- Active chemical substance: Lecomethylene blue (reduced form of methylene blue)

2- Supplementary materials for reducing: carboxyl compound (like C6 to C8), carbamate, fatty acid (saturated and non-saturated fatty acid in milk), vitamin C, N-actyl cysteine, alpha-lipoic acid, Glutathione, Vitamin N hexanoic acid, caprilic, Oleic, linoleic, Trizomal Glutathione, stabilizer compound such as dextrose, alcoholic sugar such as sorbitol and mannitol and maltitol, thiosulfate sodium, mercaptoethanol.

3- Substance increasing the absorption of LMB: ethanol, urea

This composition is prepared in form of syrup and powder. We called it Corona blue.

Also LMB can be prepared in from of ampule for intravenous injection.

MB aggregates (dimer, trimer or larger) in concentration more than 10 mM, which limits its diffusion into cells. It is proved that the addition of urea to an aqueous solution of MB at pH 7.4 causes to dissociate the molecules of MB to monomer by forming a weak complex with MB (1:1) and also prevent the destruction of MB (21,22). The apparent binding constant (K=1.2x0.4 M-l) is small, ensuring that high concentrations of urea are required to saturate the complexation event. Urea has been used in pharmacology to enhance the penetration of other drugs into tissue and also has an anti-inflammatory effect (6). Three covid-19 cases which were treated by lecumethylene blue in Mashhad University of Medical Sciences (Trial registration number: NCT04370288). The formulation was patented (IR- 139950140003002083) in Iran.

Case 1

1. On May 13, 2020, a 75-year-old female had an emergency operation for coronary artery bypass grafting and two days later she showed a decrease in SP02 and respiratory distress.

2. Based on the criteria of Yang et al. (Yang et al., 2020), the patient had a severe form of the disease due to the presence of fever, respiratory symptoms, and radiological signs of pneumonia.

3. Lung HRCT revealed ground-glass opacities (GGOs). She had a combination of consolidation and GGOs. The distribution of abnormalities was bilateral in the subpleural lung regions. Both upper and lower lobes were involved.

4. She had past medical history of diabetes and hypertension.

5. WBC: 8.9 xl03 / mI with 80% neutrophils and 12.6% lymphocytes, platelets count: 276 ^c 103/mI, LDH: 1151 IU/1, CRP: 145 mg/dl, D-Dimer: 1374 ng/ml, total bilirubin: 1.5 mg/dl, AST: 115 IU/1 and ALT: 90 IU/1. RT- PCR was negative for SARS-CoV-2.

6. On May 15, 2020, she transferred to ICU, she had a stable hemodynamic. SP02 was 65-68% without oxygen therapy and 78-80% with oxygen therapy via a reserve bag.

7. After stating non-invasive ventilation (NIV) SP02 reached 87-88%.

8. Treatment was initiated with azithromycin (500mg/day), lopinavir/ritonavir (200/50 mg, 2 tablets xb.i.d), hydroxychloroquine (400 mg stat), heparin 5000 IU (q.i.dintravenously)., hydrocortisone(starting dose 100 mg TDS, lately tapered) and continued for five days.

9. Nitrite and nitrate, met-Hb, and PAB were 13.5 mitioI/I, 104.3 pmol/l, 17%, and 97, respectively.

10. On May 16, 2020, MB (lmg/ kg) vitamin C (1500mg), N-acetyl Cysteine (2gr) were added in 100 ml dextrose and prescribed orally.

11. There was no side effect and an allergic reaction. After 8-12 h the color of urine became blue or green.

12. Due to heart operation and leukocytosis, the prophylactic antibiotic was administrated.

13. At the first day, after MB therapy, there was no significant change in SP02

14. On the second day of MB therapy SP02 increased and the duration of noninvasive ventilation (NIV) was decreased.

15. On the fourth day of MB therapy, the patient did not need NIV, and SP02 was 80-82% without oxygen therapy and reached 97-99% with oxygen therapy. 16. On the fifth day of MB therapy, the patient did not need oxygen therapy and SP02 was 90- 92% and she was completely awake and oral feeding was started.

17. On the seventh day, she was discharged from ICU.

18. After treatment with MCN, nitrite and nitrate, met-Hb, and PAB were 5.1 pmol/l, 45.2 pmol/l, 4%, and 21, respectively.

Case 2

1. On April 13, 2020, a 49-year-old male was admitted to our ICU because of fever, low level of consciousness, decreased SP02, and highly purulent tracheal secretion. At admission, the patient had RASS -4 (Richmond Agitation Sedation Score), and under mechanical ventilation with fever, tachycardia, SP02 86-88%. His blood pressure was in the normal range.

2. He had no co-morbidity in his past medical history

3. The patient was admitted previously on February 15, 2020, to another ICU due to headache, cough, myalgia, fever, and dyspnea which are starting 14 days before admission.

4. Lung HRCT revealed diffuse bilateral ground-glass opacities (GGOs) and consolidation in the peripheral lung regions. Both upper and lower lobes were involved.

5. WBC: 16 xl03 /mI with 88% neutrophils and 3.6% lymphocytes, platelets count: 221 ^c 103/mI, LDH: 906 IU/1, CRP: 82 mg/dl, D-Dimer: 2078 ng/ml, total bilirubin: 2 mg/dl, AST: 134 IU/1 and ALT: 89 IU/1. RT- PCR was positive for SARS-CoV-2.

6. He was treated with azithromycin (500mg/day), hydroxychloroquine (400 mg stat and 200 mg BD), and meropenem 1 gr TDS.

7. Tracheostomy was done because of the long period of intubation.

8. The culture of tracheal secretion revealed multi-resistance microorganisms such as Acinetobacter and Pseudomonas.

9. After three days, because of progressive respiratory distress, Kaletra (lopinavir/ritonavir, 200/50 mg) and hydrocortisone were added to the treatment protocol.

10. Nitrite and nitrate, met-Hb, and PAB were 10.2 pmol/l, 35.1 pmol/l, 14%, and 95 HK, respectively.

11. On May 24, 2020, because of a weak response to antibiotics after 45 days and weaning failure, we started to administer oral MB (lmg/ kg) vitamin C (1500mg), N-acetyl Cysteine (2gr) in 100 ml dextrose for twice a day.

12. There was no side effect and an allergic reaction. After 8-12 h the color of urine became blue or green.

13. On the same day of MB administration, the patient was decannulated and tracheostomy was removed and oxygen therapy was started by a reserve bag mask.

14. Antibiotic therapy was continued and dexamethasone (8 mg QID) was started. 15. The day after starting MB therapy, the patient had a large volume of tracheal secretion from the tracheostomy site, but SP02 was 90-92% on high flow oxygen.

16. On the second and third days after starting MB therapy, the patient improved significantly and SP02 to 96 by a simple face mask.

17. From the fourth day after MB therapy, dexamethasone was tapered and patient consciousness was improved, the tracheal secretion was decreased but the patient had a low degree of fever.

18. On the sixth day after starting methylene blue, oxygen therapy was discontinued.

19. On the twenty-third day, he was discharged from ICU.

20. After treatment with MCN, nitrite and nitrate, met-Hb, and PAB were 6.6 pmol/l, 24.30 pmol/l, 4%, and 62 HK, respectively.

Case 3

1. On June 22, 2020, a 53-year-old male was admitted to our ICU because of high fever, low level of consciousness, decreased Sp02, and highly resistant blood glucose.

2. He had DM and HTN co-morbidity in his past medical history

3. Lung HRCT revealed diffuse bilateral ground-glass opacities (GGOs) and consolidation in the peripheral lung regions. Both upper and lower lobes were involved.

4. He was treated with azithromycin (500mg/day), Atazanavir (300 mg daily), and Meropenem 1 gr TDS and Ticoplanin (400mg/BID and then 200mg/BID).

5. Since the continuing of patient complications such as fever, hyperglycemia, and confusion, Interferon-Beta was started at 18 days later, according to the standard protocol in 3 doses for 5 days. But there was no response to this treatment and hyperglycemia and fever were continued for ten days after Interferon treatment.

6. The antibiotics were changed for sepsis control, but the fever was continued.

7. During the treatment, the patient was intubated and extubated two times in ICU and his hypoxemia (Sp02 78%) was continued and respiratory distress worsened.

8. 14 days after Interferon, as the last option of treatment, MB therapy was started and fortunately, two days later, fever and hyperglycemia along with hypoxemia started to improve.

9. After 5 days of MB therapy, Sp02 increased by 85 %, and respiratory distress significantly improved.

10. Duration MB therapy, no other new modality was added and supportive care therapy was continued.

12. After 8 days from the beginning of the MB therapy, Sp02 increased to 93% and he discharged from the hospital. 11. It should be noted that during the clinical improvement of patients, after the administration of MB, the change of the urine color from blue to green was observed. When the dark blue color started to change to green color, this means that the oxidative stress started to decrease.