USE OF BILIVERDIN REDUCTASE B INHIBITORS TO ALLOW MALARIA ERADICATION IN PATIENTS WITH G6PD DEFICIENCY

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Serial No. 63/147,438, filed on February 9, 2021, the content of which is incorporated by reference herein in entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCII text file (Name: 3062_107_2_PCT_ST25.txt; Size: 29 kilobytes; and Date of Creation: February 9, 2022) filed with the instant application is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The presently disclosed subject matter relates in some embodiments to uses of Biliverdin Reductase B inhibitors to enhance the safety profile of 8-Aminoquinoline drugs such as primaquine and tafenoquine used as treatments for the dormant liver stage of the malaria-causing pathogens, P. vivax and P. ovale , especially in patients deficient in G6PD activity.

BACKGROUND

Malaria is an infectious disease afflicting hundreds of millions people annually and causing 1 to 3 million deaths every year, most of which are children under the age of 5. Malaria is caused by protozoan parasites of the Plasmodium genus, five species of which, P. falciparum , P. ovale , P. vivax , P. malariae , and P. knowlesi , are able to cause infection in humans. Upon infection, the parasites (sporozoites) travel to the liver where they mature and release another form of parasites called merozoites. The parasites enter the bloodstream and multiply inside red blood cells, which then break open and infect more red blood cells. P. vivax and I’ ovale form hypnozoites that manifest in a dormant liver stage. This dormancy causes patients who have recovered from an initial infection to have a subsequent relapse, even up to years after the original infection.

Primaquine and tafenoquine are currently the only approved drugs in the United States that can eradicate the liver phase of P. vivax or P. ovale. Unfortunately, primaquine and tafenoquine have significant and potentially lethal side effects in patients with glucose- e-phosphate dehydrogenase enzyme deficiency, a genetic trait common in regions where malaria is endemic. Therefore, it is urgent to identify new treatments capable of eliminating the parasite during the liver phase of its life cycle.

Herein are described compositions and methods to treat patients receiving primaquine or tafenoquine with a Biliverdin Reductase B inhibitor to enhance the safety profile of these drugs, e.g ., by reducing or preventing dangerous side effects (e.g, hemolysis) that may occur in patients using 8-Aminoquinoline containing compounds such as primaquine and/or tafenoquine, especially in patients deficient in G6PD activity.

SUMMARY

This Summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments of the presently disclosed subject matter. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

In some embodiments, the presently disclosed subject matter relates to methods for using BlvrB inhibitors to permit malaria eradication in patients with G6PD deficiency when treated with 8-Aminoquinoline-containing compounds such as but not limited to primaquine and/or tafenoquine.

In some embodiments, there is a method of reducing the risk of drug-related hemolysis toxicity in a subject receiving a drug selected from the group consisting of primaquine and tafenoquine, comprising co-administering to the subject an effective amount of a BlvrB inhibitor.

In some embodiments, there is a method of improving the therapeutic index of a drug selected from the group consisting of primaquine and tafenoquine in a subject, comprising co-administering to the subject an effective amount of a BlvrB inhibitor.

In some embodiments, there is a method of reducing the production of reactive oxygen species in a subject receiving a drug selected from the group consisting of primaquine and tafenoquine comprising co-administering to the subject an effective amount of a BlvrB inhibitor.

In some embodiments, the subject has a G6PD deficiency. In some embodiments, the subject’s red blood cells are deficient in G6PD activity. In some embodiments, the G6PD deficiency is established by measurement of enzymatic activity, level of enzyme expression, RBC hemolysis to phenylhydrazine, reduction of methylene blue, or genotyping.

In some embodiments, the BlvrB inhibitor is selected from the group consisting of phloxine B, erythrosin B, NSC130813, NSC12516, PH001924, lumichrome, PH006888, ZINC4366439/NSC 12516, xanthene, proflavine, alizarin red S, NSC ID 371876, NSC ID 179187, NSC ID 53396, NSC ID10936, NSC ID 169534, NSC ID 117269, NSC ID 143491, NSC ID 305821, NSC ID 130813, ZINC ID ZINC0977089, ZINC ID ZINC27528243, ZINC ID ZINC09330686, ZINC ID ZINC71767103, ZINC ID ZINC04160108, ZINC ID ZINC09777107, ZINC ID ZINC21093196, ZINC ID ZINC71767097, asunaprevir (BMS- 650032), micafungin, tamibarotene, TSU-68 (SU6668, Orantinib), sulfasalazine, febuxostat, crenolanib (CP-868596), olsalazine, PTC 124 (ataluren), deferasirox, flunixin in combination with meglumin, azelastine, benzbromarone, triclabendazole, nifedipine, nisoldipine, zafirlukast, pyrantel in combination with pamoate, candesartan cilexetil, and azilsartan medoxomil, prodrugs thereof, metabolites thereof, and/or pharmaceutically acceptable salts thereof, or any combination thereof.

In some embodiments, the BlvrB inhibitor is selected from the group consisting of phloxine B, erythrosin B, NSC130813, NSC12516, and PH001924.

In some embodiments, the BlvrB inhibitor is selected from the group consisting of phloxine B and erythrosin B.

In some embodiments, the co-administration reduces the risk or severity of anemia in the subject as compared to the risk or severity of anemia in the subject not receiving the inhibitor or a control subject not receiving the inhibitor.

In some embodiments, the risk or severity of anemia is assayed by measuring hematocrit levels, reticulocyte counts, LDH levels, or haptoglobin levels in the subject or the control subject.

In some embodiments, intravascular hemolysis is prevented or reduced in the subject receiving the inhibitor as compared to the subject not receiving the inhibitor or a control subject not receiving the inhibitor.

In some embodiments, extravascular hemolysis is prevented or reduced in the subject receiving the inhibitor as compared to the subject not receiving the inhibitor or a control subject not receiving the inhibitor. In some embodiments, methemoglobinemia is prevented or reduced in the subject receiving the inhibitor as compared to the subject not receiving the inhibitor or a control subject not receiving the inhibitor.

In some embodiments, intravascular hemolysis, extravascular hemolysis, and methemoglobinemia are prevented or reduced in the subject receiving the inhibitor as compared to the subject not receiving the inhibitor or a control subject not receiving the inhibitor.

In some embodiments, the subject is undergoing treatment for malaria. In some embodiments, the treatment results in eradication of malaria in the subject’s liver.

In some embodiments, 15 mg of primaquine is administered to the subject once a day for 14 days. In some embodiments, 45 mg of primaquine is administered to the subject once a week for 8 weeks. In some embodiments, 200 mg of tafenoquine is administered to the subject once a day for 3 days, followed by 200 mg once a week for up to 6 months of continuous dosing.

In some embodiments, the BlvrB inhibitor is administered 6 times/day, 4 times/day, 3 times/day, 2 times/day, once per day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once per week.

In some embodiments, the BlvrB inhibitor is selected from the group consisting of micafungin, febuxostat, deferasirox, benzbromarone, triclabendazole, nifedipine, zafirlukast, candesartan and/or candesartan cilexetil, and azilsartan medoxomil. In some embodiments, the BlvrB inhibitor is micafungin and the dose administered is 50 mg/day. In some embodiments, the BlvrB inhibitor is micafungin and the dose administered is 150 mg/day. In some embodiments, the BlvrB inhibitor is febuxostat and the dose administered is 40 mg/day. In some embodiments, the BlvrB inhibitor is deferasirox and the dose administered is 5 mg/ kg/day. In some embodiments, the BlvrB inhibitor is deferasirox and the dose administered is 10 mg/ kg/day. In some embodiments, BlvrB inhibitor is benzbromarone and the dose administered is 5 mg/day. In some embodiments, the BlvrB inhibitor is benzbromarone and the dose administered is 10 mg/day.

In some embodiments, the BlvrB inhibitor is triclabendazole and the dose administered is 20 mg/ kg/day. In some embodiments, the dose is divided into two separate doses.

In some embodiments, the BlvrB inhibitor is nifedipine and the dose administered is 30 mg/day. In some embodiments, the BlvrB inhibitor is zafirlukast and the dose administered is 20 mg/day. In some embodiments, the dose is divided into two separate doses.

In some embodiments, the BlvrB inhibitor is zafirlukast and the dose administered is 40 mg/day. In some embodiments, the dose is divided into two separate doses.

In some embodiments, the BlvrB inhibitor is candesartan cilexetil and the dose administered is between 8- 32 mg/day. In some embodiments, the daily dose is divided into two separate doses.

In some embodiments, the BlvrB inhibitor is azilsartan medoxomil and the dose administered is 40 mg/day. In some embodiments, the BlvrB inhibitor is azilsartan medoxomil and the dose administered is 80 mg/day.

In some embodiments, the BlvrB inhibitor is administered before, with, or after the administration of primaquine or tafenoquine. In some embodiments, wherein the BlvrB inhibitor is administered for up to 6 months. In some embodiments, the BlvrB inhibitor is administered for 1 day, 2, days, 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the BlvrB inhibitor is a tablet or pill.

BRIEF DESCRIPTION OF THE FIGURES

Figure l is a schematic depiction of an exemplary mechanism for preventing toxicity of 8-AQs in RBCs but allowing anti-malarial activity in liver cells using inhibitors of BlvrB.

Figure 2 is a graph of electron paramagnetic resonance (EPR) using a spin probe specific for superoxide (O2Q of 5,6-Orthoquinone (5,6-POQ) showing that BlvrB catalyzes transfer of electrons from NADPH to 5,6-POQ. 5,6-POQ (POQ) was incubated with NADPH and recombinant BlvrB in the presence of 1 -hydroxy-3 -methoxycarbonyl-2, 2,5,5- tetramethylpyrrolidine (CMH), which is a spin probe that rapidly reacts with superoxide and forms a stable nitroxide radical. The nitroxide radical was measured by electron paramagnetic resonance (EPR). NADPH was incubated with 5,6-POQ (no BlvrB) as a negative control to establish background signal. The lines represent a regression from the multiple data points from multiple runs.

Figure 3 is a graph showing that BlvrB KO RBCs were resistant to treatment with the active primaquine metabolite (5,6-POQ). Wild-type (WT; squares) or BlvrB knockout (KO) RBCs (circles) were treated with 500 mM 5,6-POQ dissolved in methanol (MeOH). Also shown are negative control untreated wild-type or BlvrB knockout RBCs (triangles) treated with vehicle only (methanol; MeOH). RBCs were then transfused into mice and circulation was monitored over time by flow cytometry. Error bars (± standard deviation) were calculated for each timepoint, but were smaller than the symbols (triangles, circles, and squares) and are thus not visible in the Figure.

Figures 4A-4C present the results of hG6PD activity in humanized G6PD deficient (G6PDd) mice. Figure 4Ais a schematic representation of the changes made in the murine locus to generate humanized G6PDd and nondeficient control mice. The wild-type locus of the mouse G6PD gene was modified to introduce the non-deficient human genomic sequence (hG6PD(n)) or mutations V68M and N126D that constitute the (hG6PD(A-)) variant. Figure 4B is a graph of the percent of hG6PD activity in humanized G6PDd and nondeficient control mice. G6PD activity was measured in RBCs from wild-type (WT), hG6PD(n), and hG6PD(A-) RBCs. Activity was normalized to hG6PD(n) activity. **** indicates a difference of hG6PD(A-) compared to hG6PD(n) with a p value < 0.0001. Figure 4C is a western blot analysis carried out with an antibody to human G6PD that was non reactive with mouse G6PD. hG6PD was easily detectable in hG6PD(n) but not hG6PD(A-) RBCs (top blot) - overexposure allowed detection of the trace amounts of hG6PD in hG6PD(A-) samples (middle blot). Parallel blots were stained with an antibody to beta-actin to rule out differences in loading (lower blot). Samples with the same label represent replicates.

Figure 5 is two bar graphs showing that hG6PD(A-) RBCs were sensitive to primaquine-induced hemolysis. The percent changes in hematocrit levels (left panel) and reticulocytes (right panel) were measured in normal mice and G6PD deficient mice after 5 days of primaquine injections.

Figure 6 is two bar graphs showing that erythrosine and phloxine prevented primaquine-induced hemolysis in hG6PD(A-) mice. The percent changes in hematocrit levels (left panel) and reticulocytes (right panel) are shown in G6PD deficient mice 5 days after the receiving the indicated treatment for 5 days.

Figure 7 is a graph showing that hG6PD(A-) RBCs were sensitive to an active primaquine metabolite (5,6-POQ). RBCs from hG6PD(n) or hG6PD(A-) mice were treated with 5,6-POQ and then transfused. RBC circulation was determined by flow cytometry.

DETAILED DESCRIPTION T General Considerations and Medical Significance

Malaria is a major health problem worldwide. According to the World Health Organization (WHO), there were an estimated 219 million cases of malaria in 87 countries in 2017. Of these cases, approximately 435,000 deaths were recorded. In addition to the cost to human life, the equivalent of over 3 billion U.S. dollars were spent on malaria control in 2017. Moreover, the WHO 2018 annual report indicated that efforts to control malaria are making little progress overall.

Of the five different species of Plasmodium that cause malarial disease in humans, two, P. vivax and P. ovale , are particularly problematic from both a treatment and also eradication standpoint. The reason for this is that they form hypnozoites that manifest in a dormant liver stage. Due to this dormancy, patients who have convalesced from an initial infection can have a subsequent relapse, even up to years after the original infection. It is estimated that 66%-95% of acute episodes of P. vivax are due to relapse rather than primary infection. This is not only a problem for afflicted patients, but also represents a problem for general eradication efforts because relapsed patients can serve as a source of infection for whole populations through mosquito-based transmission.

The only currently approved drugs in the United States that can eradicate the liver phase of P. vivax or P. ovale (i.e., radical cure) are primaquine and tafenoquine, both which are 8-aminoquinolines (8-AQs).

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme in the pentose phosphate pathway (PPP). It oxidizes glucose-6-phosphate into 6- phosphogluconate and converts NADP ⁺ to NADPH, the latter being the major electron donor for antioxidant pathways. Insufficient G6PD activity (i.e., G6PD deficiency (G6PDd)) is the most common enzymopathy known in humans, affecting between 400 and 500 million individuals worldwide. G6PD deficiency can be assayed by enzymatic activity, level of enzyme expression, RBC hemolysis to phenylhydrazine or similar oxidant stresses, reduction of methylene blue, and/or genotyping. Affected individuals can present with acute hemolysis when exposed to sources of oxidant stress, including illnesses and consumption of certain foods or drugs. Primaquine and tafenoquine are contraindicated in patients with G6PD deficiency, due to hemolytic toxicity.

Many countries where G6PD deficiency is common do not have the resources to adequately determine if a patient presents with G6PD deficiency. Therefore, in areas where populations where G6PD deficiency is common, primaquine and tafenoquine treatment are not used as treatments for malaria.

Primaquine, also known as N-(6-methoxyquinolin-8-yl)pentane- 1,4-diamine phosphate, is known to cause hemolytic anemia in persons deficient in the enzyme glucose- 6-phosphate dehydrogenase (G6PD; Beutler, 1969; Capote et al., 1997). Patients with hemolysis may present with acute anemia, pallor, jaundice, hematuria, dyspnea, fatigue, lightheadedness, dizziness, tachycardia, and possibly hypotension. Laboratory test results that may confirm hemolysis include reticulocytosis, as well as increased lactate dehydrogenase, increased unconjugated bilirubin, and decreased haptoglobin levels. A peripheral blood smear should be performed when hemolysis is present to identify abnormal red blood cell morphologies. Treatment for hemolytic anemia include a red blood cell transfusion or intravenously administered immunoglobulin.

Moreover, methemoglobin toxicity is another predictable dose-related adverse effect associated with primaquine. Methemoglobin toxicity, or methemoglobinemia, can be assayed by a complete blood count, an enzyme test, examination of blood color, examination of blood levels or other drugs, pulse oximetry to check the saturation of oxygen, and/or genotyping. Methemoglobinemia can be treated either with methylene blue, which is contraindicated for people with a congenital type of methemoglobinemia, or a blood transfusion.

The mechanism of 8-AQ induced hemolysis in G6PDd humans has long been appreciated to consist of redox cycling. Two active metabolites, 5-hydroxy primaquine and primaquine-5, 6-ortho-quinone, are produced by hepatic metabolism of primaquine. Redox cycling occurs when primaquine metabolites (PMs) catalyze the transfer of electrons (e-) from NADPH to molecular oxygen (O2) to generate superoxide (Ch-; see Figure 1).

Early on, it was recognized that redox cycling does not happen spontaneously and requires an enzymatic reductase to transfer an e- from NADPH to the PMs (Baird. Front Cell Infect Microbiol 2021; 11;696598). Characteristics of the reductase (called Diaphorase II) included the ability to reduce methylene blue 8 and an approximate molecular weight of 18 kiloDaltons (kDa; Fisher et al., 1977). In separate fields, a reductase (named either

Biliverdin reductase B (BlvrB) or Flavin reductase) was also shown to reduce methylene blue and have a similar molecular weight as Diaphorase II (Komoro et al., 1996; Shalloe, 1996). Finally, BlvrB is known to be able to reduce other quinoline orthoquinones (i.e., pyrroloquinoline quinone; Xu et al., 1993. BlvrB has now been cloned and characterized (Komoro et al., 1996).

Tafenoquine is disclosed in U.S. Patent No. 4,617,394. Though more effective than primaquine, the drug was found to cause methemoglobin toxicity almost three times more than that of primaquine (Anders et al., 1988), hence has drawbacks in terms of safety. Because 8-AQs are the only known drugs that can eradicate the liver phase of P vivax and/! ovale , but also cause hemolysis in patients with G6PD deficiency, 8-AQs cannot safely be used to cure P. vivax or P ovale in patients with G6PD deficiency, without the ability to transfuse during therapy and hospitalize if necessary. Because hemolysis can have negative sequelae in addition to anemia ( e.g ., kidney damage), even with transfusion support, 8-AQ therapy may not be entirely safe in patients with G6PD deficiency. Ironically, because G6PD deficiency evolved as a natural resistance to malaria, it is particularly prevalent in populations indigenous to malaria endemic regions, increasing the percentage of malaria infected people in whom 8-AQs are toxic.

Primaquine and tafenoquine cause hemolysis in G6PD deficient subjects because they induce oxidative stress; G6PD RBCs have diminished antioxidant capacity. The mechanisms by which primaquine and tafenoquine generate oxidative stress is through generation of reactive oxygen species (ROS) by redox cycling. In particular, an electron is transferred to primaquine and tafenoquine (i.e., chemical reduction) by an enzymatic reductase to give rise to primaquine and tafenoquine radicals, which then transfer electrons to O2 to generate superoxide (O2-). Up until now, the identity of the cellular reductase has not been known.

IT Definitions

In describing and claiming the presently disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the presently disclosed and claimed subject matter.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. The term “about”, as used herein when referring to a measurable value such as an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±9%, in some embodiments ±8%, in some embodiments ±7%, in some embodiments ±6%, in some embodiments ±5%, in some embodiments ±4%, in some embodiments ±3%, in some embodiments ±2%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods and/or employ the disclosed compositions. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

As used herein, the term “and/or” when used in the context of a list of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

As use herein, the terms “administration of’ and/or “administering” a compound should be understood to refer to providing a compound of the presently disclosed subject matter to a subject in need of treatment.

As used herein, the phrase “biological sample” refers to a sample isolated from a subject ( e.g ., a biopsy, blood, serum, etc.) or from a cell or tissue from a subject (e.g, RNA and/or DNA and/or a protein or polypeptide isolated therefrom). Biological samples can be of any biological tissue or fluid or cells from any organism as well as cells cultured in vitro, such as cell lines and tissue culture cells. Frequently the sample will be a “clinical sample” which is a sample derived from a subject (i.e., a subject undergoing a diagnostic procedure and/or a treatment). Typical clinical samples include, but are not limited to cerebrospinal fluid, serum, plasma, blood, saliva, skin, muscle, olfactory tissue, lacrimal fluid, synovial fluid, nail tissue, hair, feces, urine, a tissue or cell type, and combinations thereof, tissue or fine needle biopsy samples, and cells therefrom. Biological samples can also include sections of tissues, such as frozen sections or formalin fixed sections taken for histological purposes.

As used herein, term “comprising”, which is synonymous with “including”, “containing”, or “characterized by”, is inclusive or open-ended and does not exclude additional, unrecited elements and/or method steps. “Comprising” is a term of art used in claim language which means that the named elements are present, but other elements can be added and still form a composition or method within the scope of the presently disclosed subject matter. By way of example and not limitation, a pharmaceutical composition comprising a particular active agent and a pharmaceutically acceptable carrier can also contain other components including, but not limited to other active agents, other carriers and excipients, and any other molecule that might be appropriate for inclusion in the pharmaceutical composition without any limitation.

As used herein, the phrase “consisting of’ excludes any element, step, or ingredient that is not particularly recited in the claim. When the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. By way of example and not limitation, a pharmaceutical composition consisting of an active agent and a pharmaceutically acceptable carrier contains no other components besides the particular active agent and the pharmaceutically acceptable carrier. It is understood that any molecule that is below a reasonable level of detection is considered to be absent.

As used herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. By way of example and not limitation, a pharmaceutical composition consisting essentially of an active agent and a pharmaceutically acceptable carrier contains active agent and the pharmaceutically acceptable carrier, but can also include any additional elements that might be present but that do not materially affect the biological functions of the composition in vitro or in vivo.

With respect to the terms “comprising”, “consisting essentially of’, and “consisting of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter encompasses the use of either of the other two terms. For example, “comprising” is a transitional term that is broader than both “consisting essentially of’ and “consisting of’, and thus the term “comprising” implicitly encompasses both “consisting essentially of’ and “consisting of’. Likewise, the transitional phrase “consisting essentially of’ is broader than “consisting of’, and thus the phrase “consisting essentially of’ implicitly encompasses “consisting of’.

As used herein the term “BlvrB” refers to a Biliverdin reductase B (also called Flavin reductase) gene and its transcriptional and/or translational products. The human BlvrB genetic locus is present on chromosome 19 and has a nucleotide sequence that is the reverse complement of nucleotides 40,447,768-40,465,745 of Accession No. NC_000019.10 of the GENBANK® biosequence database (SEQ ID NO: 3). The human cDNA sequence is disclosed as Accession No. NM_000713.3 of the GENBANK® biosequence database (SEQ ID NO: 1), and encodes a protein having the amino acid sequence disclosed as Accession No. NP_000704.1 of the GENBANK® biosequence database (SEQ ID NO: 2).

As used herein, the term “BlvrB inhibitor” refers to a compound that inhibits or reduces a biological activity of a Biliverdin reductase B (BlvrB; also called Flavin reductase) gene product, including but not limited to a human BlvrB polypeptide. Exemplary BlvrB inhibitors that can be employed in the compositions and methods of the presently disclosed subject matter are disclosed herein below.

The term “biocompatible”, as used herein, refers to a material that does not elicit a substantial detrimental response in the host.

The term “effective amount,” including related terms “prophylaxis-effective amount,” “treatment effective amount,” and “therapeutically effective amount,” refers to a concentration of a compound that is effective in inhibiting, decreasing the likelihood of the disease by malarial parasites, or preventing malarial infection or preventing the delayed onset of the disease by malarial parasites, when administered before infection, i.e. before, during and/or slightly after the exposure period to malarial parasites.

The term “malarial parasites,” including the related term “malaria parasite,” refers to protozoan parasites of the Plasmodium genus, including the species of P. falciparum , P. ovale , P. vivax, P. malariae and P knowlesi. The term “parasite” includes the related term sporozoites.

As used herein, the terms “treat’ or “treatment” are used interchangeably and are meant to indicate administering one or more compounds in accordance with the methods of the invention to assist in malaria treatment to obtain a desired therapeutic objective. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom, or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom, or adverse effect attributed to the disease. The terms “treatment” and “treating” as used herein covers any treatment of a disease in a mammal, both therapeutic treatment and prophylactic or preventative measures, particularly in a human, wherein in some embodiments the objects are: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and/or (c) relieving the disease, i.e., causing regression of the disease and/or its symptoms or conditions, even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have or predisposed to having a condition, disease, or disorder, or those in whom the condition is to be prevented.

As used herein, the term “prevention” including the related terms such as “prevent” or “preventing,” is meant to refer to provide a subject not yet affected by the condition with a benefit that serves to avoid, delay, forestall, minimize, or reduce the recurrence/onset of the condition to be prevented and/or its attendant symptoms. Such preventative benefits include, for example, delaying development and/or recurrence of the condition, or reducing the duration, severity, or intensity of one or more unwanted features associated with the condition if it eventually develops.

As used herein, the term “small molecule” is meant to indicate a chemical compound having a molecular weight of less than about 500 daltons. Small molecules do not include biologic polymers such as polypeptides and polynucleotides.

As used herein, the term “pharmaceutically acceptable” or “pharmacologically acceptable” refers to a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. As used herein, the term “physiological pH” or a “pH in the physiological range” refers to a pH in the range of approximately 7.2 to 8.0 inclusive, more typically in the range of approximately 7.2 to 7.6 inclusive.

The term “malaria” includes disease and conditions related to an infection by Plasmodium species such as P. vivax and P. ovale.

The term “co-administer”, including the related terms “co-administering” and co administered”, is used to include any dosing regimen that results in levels of BlvrB effective to reduce ROS production resulting from administration of 8-AQ drugs.

A “compound”, as used herein, refers to a polypeptide, an isolated nucleic acid, or other agent used in the method of the presently disclosed subject matter.

A “control” cell, tissue, sample, or subject is a cell, tissue, sample, or subject of the same type as a test cell, tissue, sample, or subject. The control may, for example, be examined at precisely or nearly the same time the test cell, tissue, sample, or subject is examined. The control may also, for example, be examined at a time distant from the time at which the test cell, tissue, sample, or subject is examined, and the results of the examination of the control may be recorded so that the recorded results may be compared with results obtained by examination of a test cell, tissue, sample, or subject. The control may also be obtained from another source or similar source other than the test group or a test subject, where the test sample is obtained from a subject suspected of having a condition, disease, or disorder for which the test is being performed.

A “test” cell is a cell being examined.

As used herein, the terms “condition”, “disease condition”, “disease”, “disease state”, and “disorder” refer to physiological states in which diseased cells or cells of interest can be targeted with the compositions of the presently disclosed subject matter.

As used herein, the term “diagnosis” refers to detecting a risk or propensity to a condition, disease, or disorder. In any method of diagnosis exist false positives and false negatives. Any one method of diagnosis does not provide 100% accuracy.

A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

As used herein “injecting”, “applying”, and administering” include administration of a compound of the presently disclosed subject matter by any number of routes and modes including, but not limited to, topical, oral, buccal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ophthalmic, pulmonary, vaginal, and rectal approaches.

The term “otherwise identical sample”, as used herein, refers to a sample similar to a first sample, that is, it is obtained in the same manner from the same subject from the same tissue or fluid, or it refers a similar sample obtained from a different subject. The term “otherwise identical sample from an unaffected subject” refers to a sample obtained from a subject not known to have the disease or disorder being examined. The sample may of course be a standard sample. By analogy, the term “otherwise identical” can also be used regarding regions or tissues in a subject or in an unaffected subject.

As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue- penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, intratumoral, and kidney dialytic infusion techniques.

The term “pharmaceutical composition” refers to a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

“Pharmaceutically acceptable” means physiologically tolerable, for either human or veterinary application. Similarly, “pharmaceutical compositions” include formulations for human and veterinary use. As used herein, the term “pharmaceutically acceptable carrier” means a chemical composition with which an appropriate compound or derivative can be combined and which, following the combination, can be used to administer the appropriate compound to a subject.

As used herein, the term “physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

“Plurality” means at least two.

The term “prevent”, as used herein, means to stop something from happening, or taking advance measures against something possible or probable from happening. In the context of medicine, “prevention” generally refers to action taken to decrease the chance of getting a disease or condition. It is noted that “prevention” need not be absolute, and thus can occur as a matter of degree.

A “preventive” or “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs, or exhibits only early signs, of a condition, disease, or disorder. A prophylactic or preventative treatment is administered for the purpose of decreasing the risk of developing pathology associated with developing the condition, disease, or disorder.

As used herein, the term “purified” and like terms relate to an enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in a native environment. The term “purified” does not necessarily indicate that complete purity of the particular molecule has been achieved during the process.

A “highly purified” compound as used herein refers to a compound that is in some embodiments greater than 90% pure, that is in some embodiments greater than 95% pure, and that is in some embodiments greater than 98% pure.

As used herein, the term “mammal” refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

The term “subject” as used herein refers to a member of species for which analysis, diagnosis, and/or treatment and/or prevention of a disease or disorder using the compositions and methods of the presently disclosed subject matter might be desirable. Accordingly, the term “subject” is intended to encompass in some embodiments any member of the Kingdom Animalia including, but not limited to the phylum Chordata ( e.g ., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals), and all Orders and Families encompassed therein.

The compositions and methods of the presently disclosed subject matter are particularly useful for warm-blooded vertebrates. Thus, in some embodiments the presently disclosed subject matter concerns mammals and birds. More particularly provided are compositions and methods derived from and/or for use in mammals such as humans and other primates, such as chimpanzees, and other apes and monkey species, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), rodents (such as mice, rats, guinea pigs, and rabbits), marsupials, and horses. Also provided is the use of the disclosed methods and compositions on birds, including those kinds of birds that are endangered, kept in zoos, as well as fowl, and more particularly domesticated fowl, e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the use of the disclosed methods and compositions on livestock, including but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like. It is noted that the term does not denote a particular age or gender.

A “sample”, as used herein, refers in some embodiments to a biological sample from a subject, including, but not limited to, normal tissue samples, diseased tissue samples, biopsies, blood, saliva, feces, semen, tears, and urine. A sample can also be any other source of material obtained from a subject which contains cells, tissues, or fluid of interest. A sample can also be obtained from cell or tissue culture.

The term “standard”, as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered and used for comparing results when administering a test compound, or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or compound on a parameter or function. Standard can also refer to an “internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured. Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker.

The term “substantially pure” describes a compound, e.g ., a protein or polypeptide, which has been separated from components which naturally accompany it. Typically, a compound is substantially pure when in some embodiments at least 10%, in some embodiments at least 20%, in some embodiments at least 50%, in some embodiments at least 60%, in some embodiments at least 75%, in some embodiments at least 90%, and in some embodiments at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g. , in the case of polypeptides by column chromatography, gel electrophoresis, or HPLC analysis. A compound, e.g. , a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.

The term “symptom”, as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a “sign” is objective evidence of disease. For example, a bloody nose is a sign. It is evident to the patient, doctor, nurse, and other observers.

A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

As used herein, the phrase “therapeutic agent” refers to an agent that is used to, for example, treat, inhibit, prevent, mitigate the effects of, reduce the severity of, reduce the likelihood of developing, slow the progression of, and/or cure, a disease or disorder.

As used herein, the phrase “therapeutic index” refers a comparison of the amount of a therapeutic agent that causes the desired therapeutic effect to the amount that causes toxicity. In animal studies, the therapeutic index is a ratio of the median lethal dose (LDso) to the median effective dose (EDso), whereas in humans it is frequently expressed as a ratio of the median toxic dose (TDso) to the EDso.

All genes, gene names, and gene products disclosed herein are intended to correspond to homologs and/or orthologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. It is noted that it is customary to present gene names that correspond to human genes and their products in all capital letters and to present gene names that correspond to human genes and their products in a combination of capital and lower case letters. In the instant disclosure, this convention is not strictly adhered to and gene names presented in all capital letters or in a combination of capital and lower case letters can refer to any gene or gene product of any subject including, but not limited to mammals, particular humans. III Description of Exemplary Embodiments

TTT A Generally

Herein is described the identification of Biliverdin Reductase B (BlvrB) as a reductase in red blood cells (RBCs) that transfers electrons to 8-AQ metabolites and is directly involved in reactive oxygen species (ROS) generation. Biliverdin reductase B, also known as Diaphorase II or Flavin reductase, is an enzyme found in all tissues under normal conditions. There are two isozymes, in humans, each encoded by its own gene, biliverdin reductase A (BlvrA) and biliverdin reductase B (BlvrB). Biliverdin reductase converts biliverdin to bilirubin which is a chain-breaking intracellular antioxidant and a scavenger of free radicals. Bilirubin is converted back into biliverdin through the actions of reactive oxygen species (ROS). This cycle allows therefore the neutralization of ROS and the reductase function of biliverdin reductase is therefore considered to be cytoprotective (Bai et ah, 2015) showed that biliverdin plays a role in prevention of UVB irradiation-induced skin photo-damage mediated by its antioxidant mechanism and cell signal regulatory action.

Having identified BlvrB as an 8-AQ reductase, described herein is the discovery that inhibiting BlvrB mitigates the toxicity of 8-AQ to G6PD deficient RBCs. Thus, in some embodiments, the presently disclosed subject matter comprises, consists essentially of, or consists of co-administering a BlvrB inhibitor with primaquine or tafenoquine to allow safe and effective eradication of P. vivax and P. ovale in patients with G6PD deficiency. In addition, the use of BlvrB inhibitors to mitigate 8-AQ toxicity in RBCs is not limited to use in G6PD deficient patients, as ROS induced hemolysis and/or methemoglobinemia are dose limiting toxicities even in patients with normal G6PD activity. As used herein, the term “co administering” refers to any dosing regimen of at least two active agents. In some embodiments, co-administering a BlvrB inhibitor with an 8-AQ including but not limited to primaquine and/or tafenoquine results in a level of BlvrB biological activity that is effective to reduce ROS production that would otherwise have resulted from administration of the 8- AQ.

Most previous attempts to solve the G6PD malaria problem focus on the development of new 8-AQ analogs that can maintain anti-malarial activity, but without hemolysis. In contrast, the presently disclosed subject matter relates to compounds that can inhibit the redox cycling of 8-AQs, i.e., by inhibiting the BlvrB-mediated transfer of electrons from NADPH to 8-AQs, thus preventing formation of 8-AQ radicals and the subsequent transfer of an electron by the newly formed 8-AQ radical to O2 to generate superoxide (O2Q. Thus, in some embodiments, the presently disclosed subject matter is based on the identification of Biliverdin Reductase B (BlvrB) as the NADPH diaphorase that transfers electrons to 8-AQs, thereby chemically reducing the 8-AQ, allowing redox cycling to occur with the resulting generation of Ch- and depletion of NADPH.

Herein demonstrated is that BlvrB generates reactive oxygen species when incubated with primaquine 5,6-Orthoquinone (5,6-POQ), an active metabolite of primaquine. BlvrB KO cells are also shown to be resistant to hemolysis caused by incubation with 5,6-POQ.

Based on this identification, known BlvrB inhibitors were tested for their ability to prevent the redox cycling of oxidized 8-AQ. Mice with BlvrB deleted in their RBCs were also assessed to support the identification of BlvrB as the NADPH diaphorase. Mice with modified G6PD have been previously described in PCT International Patent Application Publication No. WO 2016/196666, which is incorporated herein by reference in its entirety.

IILB Screening for BlyrB Inhibitors

A small molecule screen identified a series of BlvrB inhibitors, most having a tricyclic hydrocarbon core structure, that have potency in the nanomolar range (Nesbitt et al., 2018; Kim et al., 2021). For example, lumichrome was identified as a lead BlvrB inhibitor compound. Lumichrome is a natural photoproduct of riboflavin (vitamin B2). Other BlvrB inhibitors include, but are not limited to phloxine B (disodium 2',4',5',7'- tetrabromo-4,5,6,7-tetrachloro-3-oxo-3H-spiro[[2]benzofuran- l,9'-xanthene]-3',6'- bis(olate); CAS Number 18472-87-2), erythrosin B (3’,6 ^, -dihydroxy-2 ^, ,4 ^, ,5 ^, ,7’- tetraiodospiro[2-benzofuran-3,9’-xanthene]-l-one; CAS Number 15905-32-5), NSC130813 (4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-methylpiper azin- 1 - yl)methyl)phenol; CAS Number 500565-15-1), NSC12516 (4-(6-chloro-2-methoxyacridin- 9-ylamino)-2-(pyrrolidin-l-ylmethyl)phenol, hydrochloride), PH001924 (4-[(6-chloro-2- methoxy-9-acridinyl)amino]-2-[(diethylamino)methyl]phenol; also called ZINC4366439), lumichrome (7,8-dimethyl-benzo[g]pteridine-2,4(lH,3H)-dione; CAS Number 1086-80-2), PH006888 (N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulfonamide) , xanthene, proflavine, alizarin red S, NSC371876 (4-(2-amino-6-naphthalen-l-yl-pyrimidin-4-yl)-6- naphthalen- 1 -yl-pyrimidin-2-amine), NSC179187 (5 ’ ,7, 9, 13 -tetramethylspiro[5- oxapentacyclo[10.8.0.02,9.04,8.013,18]icos-18-ene-6,2’-pip eridine]-16-ol; also called purapuridine), NSC53396 (4-[(3S,5R,8R,9S,10S,13R,14S,17S)-14-hydroxy-10,13- dimethyl-3-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy- l,2,3,4,5,6,7,8,9,ll,12,15, 16,17-tetradecahydrocyclopenta[a]phenanthren-17-yl]-2H-furan -5-one ; also called actodigin), NSC 10936 (2-(benzylamino)-6-methoxy-2, 3 -dihydro- lH-inden- 1 - ol;hydrochloride), NSC 169534 (7-(4-amino-5-hydroxy-6-methyloxan-2-yl)oxy-6,9,ll- trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tet racene-5,12-dione),

NSC 117269 (4-[4,6-diamino-2-(4-chlorophenyl)-2H-l,3,5-triazin-l-yl]-N- pyrimidin-2- ylbenzenesulfonamide), NSC 143491 (7-(4-amino-5-hydroxy-6-methyloxan-2-yl)oxy- 6,9,ll-trihydroxy-9-(N-hydroxy-C-methylcarbonimidoyl)-4-meth oxy-8,10-dihydro-7H- tetracene-5,12-dione), NSC305821 ([(3E)-3-[(4-chlorophenyl)methylidene]-5,7-dimethyl- l,2-dihydrocyclopenta[b]quinolin-9-yl]-piperidin-2-ylmethano l), NSC 130813 (5- naphthal en- 1 -y 1 - 1 -pheny ltetrazol e), ZINC ID ZINC0977089, ZINC27528243 (N-[4- (acridin-9-ylamino)phenyl]-4-methylbenzenesulfonamide), ZINC ID ZINC09330686, ZINC71767103 (1- [ [5 - [(7-chloro- 1 -hydroxy quinolin-4-ylidene)amino] -2- hydroxyphenyl]methyl]piperidin-4-ol), ZINC ID ZINC04160108, ZINC ID ZINC09777107, ZINC21093196 (l-[(2S)-5-anthracen-9-yl-2-(5-bromo-2-ethoxyphenyl)- 2H-l,3,4-oxadiazol-3-yl]ethenone), ZINC71767097 (l-[[5-[(7-chloroquinolin-4- yl)amino]-2-hydroxyphenyl]methyl]piperidin-4-ol), Asunaprevir (BMS-650032; tert-butyl N-[(2S)-l-[(2S,4R)-4-(7-chloro-4-methoxyisoquinolin-l-yl)oxy -2-[[(lR,2S)-l- (cyclopropylsulfonylcarbamoyl)-2-ethenylcyclopropyl]carbamoy l]pyrrolidin-l-yl]-3,3- dimethyl-l-oxobutan-2-yl]carbamate), micafungin (5-[(lS,2S)-2-[(3S,6S,

9S,llR,15S,18S,20R,21R,24S,25S,26S)-3-[(lR)-3-amino-l-hyd roxy-3-oxopropyl]-ll,20, 21,25-tetrahydroxy-15-[(lR)-l-hydroxyethyl]-26-methyl-2,5,8, 14,17,23-hexaoxo-18-[[4- [5-(4-pentoxyphenyl)- 1 ,2-oxazol-3 -yl]benzoyl]amino]- 1,4,7,13,16,22- hexazatricyclo[22.3.0.09, 13]heptacosan-6-yl]-l, 2-dihydroxy ethyl]-2-hydroxyphenyl] hydrogen sulfate), tamibarotene (4-[(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2- yl)carbamoyl]benzoic acid), TSU-68 (SU6668, Orantinib; 3-[2,4-dimethyl-5-[(Z)-(2-oxo- lH-indol-3-ylidene)methyl]-lH-pyrrol-3-yl]propanoic acid), sulfasalazine (2-hydroxy-5- [[4-(pyridin-2-ylsulfamoyl)phenyl]diazenyl]benzoic acid), febuxostat (2-[3-cyano-4-(2- methylpropoxy)phenyl]-4-methyl-l,3-thiazole-5-carboxylic acid), crenolanib (CP-868596; l-[2-[5-[(3-methyloxetan-3-yl)methoxy]benzimidazol-l-yl]quin olin-8-yl]piperi din-4- amine), olsalazine (5-[(3-carboxy-4-hydroxyphenyl)diazenyl]-2-hydroxybenzoic acid), PTC124 (ataluren; 3-[5-(2-fluorophenyl)-l,2,4-oxadiazol-3-yl]benzoic acid), deferasirox (- [3,5-bis(2-hydroxyphenyl)-l,2,4-triazol-l-yl]benzoic acid), flunixin in combination with meglumine ((2R,3R,4R,5S)-6-(methylamino)hexane-l,2,3,4,5-pentol;2-[2-m ethyl-3- (trifluoromethyl)anilino]pyridine-3-carboxylic acid), azelastine (4-[(4- chlorophenyl)methyl]-2-(l-methylazepan-4-yl)phthalazin-l-one ), benzbromarone ((3,5- dibromo-4-hydroxyphenyl)-(2-ethyl-l-benzofuran-3-yl)methanon e), triclabendazole (6- chloro-5-(2,3-dichlorophenoxy)-2-methylsulfanyl-lH-benzimida zole), nifedipine

(dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-l,4-dihydropyridine-3,5-dicar boxylate), nisoldipine (3-O-methyl 5-0-(2-methylpropyl) 2,6-dimethyl-4-(2-nitrophenyl)-l,4- dihydropyridine-3,5-dicarboxylate), zafirlukast (cyclopentyl N-[3-[[2-methoxy-4-[(2- methylphenyl)sulfonylcarbamoyl]phenyl]methyl]-l-methylindol- 5-yl]carbamate), pyrantel in combination with pamoate (4-[(3-carboxy-2-hydroxynaphthalen-l-yl)methyl]-3- hydroxynaphthalene-2-carboxylic acid;l-methyl-2-[(E)-2-thiophen-2-ylethenyl]-5,6- dihydro-4H-pyrimidine), candesartan cilexetil (1-cy cl ohexyloxycarbonyloxy ethyl 2- ethoxy-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]benzi midazole-4-carboxylate), and azilsartan medoxomil ((5-methyl-2-oxo-l,3-dioxol-4-yl)methyl 2-ethoxy-3-[[4-[2-(5- oxo-4H-l,2,4-oxadiazol-3-yl)phenyl]phenyl]methyl]benzimidazo le-4-carboxylate). Phloxine B, for example, is also a dye used in cosmetics and as a food coloring. Erythrosin B (also called erythrosin extra bluish) is a dye used in cosmetics, hair dyes, and color filters. Many of these compounds have been tested for their ability to inhibit BlvrB (Nesbitt et al., 2018), such as dye compounds (see Table 2), NSC inhibitors (see Table 3), and ZINC compounds (see Table 4).

In particular, phloxine B and erythrosin B inhibit BlvrB with an ICso of 0.7 and 0.41 mM, respectively (Nesbitt et al., 2018). Of note, this ICso was determined regarding reduction of BlvrB’s natural substrate (i.e., flavins), but the ICso dropped to as low as 20 nM for non-naturally occurring substrates (i.e., 2,6-Dichlorophenolindophenol; DCPIP). Importantly, phloxine B is rapidly taken up by cells and accumulates to an intracellular concentration of 10 times the extracellular concentration. Both phloxine B and erythrosin B have low toxicity, with LDso values of 8500 mg/Kg and 1300 mg/Kg, respectively. It is for this reason that phloxine B and erythrosin B are approved for human consumption as food coloring and also as a dental dye. Thus, in aggregate, phloxine B and erythrosin B are non toxic and bioavailable inhibitors of BlvrB that are potent in the nanomolar range in vitro.

Importantly, to be an effective additive to 8-AQs, BlvrB should preferably not prevent anti-malarial activity of 8-AQs in the liver. However, a different reductase (cytochrome p450s) causes redox cycling of 8-AQ in the liver (killing the malarial parasite). Thus, while it is not desired to be bound by any particular mechanism of action, in some embodiments a selective inhibitor of BlvrB (and which does not inhibit cytochrome p450) reduces and/or prevents RBC toxicity while preserving anti-malarial activity (as shown in Figure 1).

In some embodiments, an effective small molecule BlvrB inhibitor for the purposes of the presently disclosed subject matter will substantially decrease both the reduction in hematocrit and the increase in reticulocytes usually observed in the blood of patients with a G6PD deficiency. In some embodiments, this can be assayed by observing the circulation of both wild type or G6PD deficient red blood cells that have been incubated with primaquine-5, 6-ortho-quinone (5,6-POQ), an active metabolite of primaquine to induce hemolysis, and with and without the BlvrB inhibitor. Alternatively, G6PD or wild-type mice can be treated with primaquine, with or without the BlvrB inhibitor, and hemolysis can be monitored by measuring circulating blood for hematocrit, hemoglobin levels, reticulocytes, and/or morphological changes on peripheral blood smear by microscopic analysis including but not limited to formation of Heinz bodies ( e.g ., evaluation for Heinz bodies on blood smears stained with Wright-Giemsa Stain).

III.C. Formulations

The compositions of the presently disclosed subject matter can be administered in any formulation or route that would be expected to deliver the compositions to whatever target site might be appropriate.

The compositions of the presently disclosed subject matter comprise in some embodiments a composition that includes a carrier, particularly a pharmaceutically acceptable carrier, such as but not limited to a carrier pharmaceutically acceptable in humans. Any suitable pharmaceutical formulation can be used to prepare the compositions for administration to a subject. For example, suitable formulations can include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostatics, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of the presently disclosed subject matter can include other agents conventional in the art with regard to the type of formulation in question. For example, sterile pyrogen-free aqueous and non-aqueous solutions can be used.

HI D. Dosages

An effective dose of a composition of the presently disclosed subject matter is administered to a subject in need thereof. A “treatment effective amount” or a “therapeutic amount” is an amount of a therapeutic composition sufficient to produce a measurable response ( e.g ., a biologically or clinically relevant response in a subject being treated, such as but not limited to a reduction in seizure activity and/or in the incidence of death, particularly as compared to the same subject had the subject not received the composition). Actual dosage levels of active ingredients in the compositions of the presently disclosed subject matter can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend upon the activity of the composition, the route of administration, combination with other drugs or treatments, the severity of the disease, disorder, and/or condition being treated, and the condition and prior medical history of the subject being treated. However, it is within the skill of the art to start doses of the compositions of the presently disclosed subject matter at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The potency of a composition can vary, and therefore a “treatment effective amount” can vary. However, using the methods described herein, one skilled in the art can readily assess the potency and efficacy of a composition of the presently disclosed subject matter and adjust the therapeutic regimen accordingly.

After review of the disclosure of the presently disclosed subject matter presented herein, one of ordinary skill in the art can tailor the dosages to an individual subject, taking into account the particular formulation, method of administration to be used with the composition, and particular disease, disorder, and/or condition treated. Further calculations of dose can consider subject height and weight, severity and stage of symptoms, and the presence of additional deleterious physical conditions. Such adjustments or variations, as well as evaluation of when and how to make such adjustments or variations, are well known to those of ordinary skill in the art of medicine.

In some embodiments, a pharmaceutically or therapeutically effective amount of primaquine and tafenoquine and a BlvrB inhibitor are delivered to the subject.

In some embodiments, the present invention provides a method for preventing infection with a malaria parasite, preventing development of malaria disease after infection with a malaria parasite, or treating malaria disease, wherein the method comprising administering the combination an effective amount of primaquine and tafenoquine and a BlvrB inhibitor to a subject.

In some embodiments, the administration of primaquine and tafenoquine have been established. In some embodiments, a therapeutically effective amount of primaquine is administered once a day for 14 days. In some embodiments, 15 mg of primaquine are administered once a day for 14 days. In some embodiments, 45 mg of primaquine are administered once a week for 8 weeks when the patient has a G6PD deficiency.

In some embodiments, a therapeutically effective amount of tafenoquine is administered. In some embodiments, 200 mg of tafenoquine are administered once a day for 3 days, followed my 200 mg once a week for up to 6 months of continuous dosing. In some embodiments, the same doses are employed when the patient has a G6PD deficiency, although in the absence of the BlvrB inhibitor, such a dose would be strictly contraindicated.

In some embodiments, the BlvrB inhibitor is administered prior to, with, or after the primaquine or tafenoquine dosage. In some embodiments, the BlvrB inhibitor is phloxine B or erythrosin B. Any of these administration regimens can be considered “co administration” regimens provided the BlvrB inhibitor plasma level present in a subject is effective to reduce the incidence, likelihood, or severity of toxicity arising from dosing of the subject with primaquine or tafenoquine.

In some embodiments, a therapeutically effective amount of the BlvrB inhibitor is orally administered.

In some embodiments, the therapeutically effective amount of the BlvrB inhibitor is between about 10 mg/day and about 8500 mg/day ( e.g ., between about 10 mg/day and about 8400 mg/day, between about 10 mg/day and about 8300 mg/day, between about 10 mg/day and about 8200 mg/day, between about 10 mg/day and about 8100 mg/day, between about 10 mg/day and about 8000 mg/day, between about 10 mg/day and about 7900 mg/day, between about 10 mg/day and about 7800 mg/day, between about 10 mg/day and about 7700 mg/day, between about 10 mg/day and about 7600 mg/day).

In some embodiments, the therapeutically effective amount of the BlvrB inhibitor is between about 10 mg/day and about 7500 mg/day ( e.g ., between about 10 mg/day and about 7400 mg/day, between about 10 mg/day and about 7300 mg/day, between about 10 mg/day and about 7200 mg/day, between about 10 mg/day and about 7100 mg/day, between about 10 mg/day and about 7000 mg/day, between about 10 mg/day and about 6900 mg/day, between about 10 mg/day and about 6800 mg/day, between about 10 mg/day and about 6700 mg/day, between about 10 mg/day and about 6600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 6400 mg/day).

In some embodiments, the therapeutically effective amount of the BlvrB inhibitor is between about 10 mg/day and about 6500 mg/day (e.g., between about 10 mg/day and about 6400 mg/day, between about 10 mg/day and about 6300 mg/day, between about 10 mg/day and about 6200 mg/day, between about 10 mg/day and about 6100 mg/day, between about 10 mg/day and about 6000 mg/day, between about 10 mg/day and about 5900 mg/day, between about 10 mg/day and about 5800 mg/day, between about 10 mg/day and about 5700 mg/day, between about 10 mg/day and about 5600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 5400 mg/day).

In some embodiments, the therapeutically effective amount of the BlvrB inhibitor is between about 10 mg/day and about 5500 mg/day (e.g, between about 10 mg/day and about 5400 mg/day, between about 10 mg/day and about 5300 mg/day, between about 10 mg/day and about 5200 mg/day, between about 10 mg/day and about 5100 mg/day, between about 10 mg/day and about 5000 mg/day, between about 10 mg/day and about 4900 mg/day, between about 10 mg/day and about 4800 mg/day, between about 10 mg/day and about 4700 mg/day, between about 10 mg/day and about 4600 mg/day, between about 10 mg/day and about 4500 mg/day or about 10 mg/day and about 4400 mg/day).

In some embodiments, the therapeutically effective amount of the BlvrB inhibitor is between about 10 mg/day and about 4500 mg/day (e.g, between about 10 mg/day and about 4400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 4200 mg/day, between about 10 mg/day and about 4100 mg/day, between about 10 mg/day and about 4000 mg/day, between about 10 mg/day and about 3900 mg/day, between about 10 mg/day and about 3800 mg/day, between about 10 mg/day and about 3700 mg/day, between about 10 mg/day and about 3600 mg/day, between about 10 mg/day and about 3500 mg/day or about 10 mg/day and about 3400 mg/day).

In some embodiments, the therapeutically effective amount of the BlvrB inhibitor is between about 10 mg/day and about 3500 mg/day ( e.g ., between about 10 mg/day and about 3400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 3200 mg/day, between about 10 mg/day and about 3100 mg/day, between about 10 mg/day and about 3000 mg/day, between about 10 mg/day and about 2900 mg/day, between about 10 mg/day and about 2800 mg/day, between about 10 mg/day and about 2700 mg/day, between about 10 mg/day and about 2600 mg/day, between about 10 mg/day and about 2500 mg/day or about 10 mg/day and about 2400 mg/day).

In some embodiments, the therapeutically effective amount of the BlvrB inhibitor is between about 10 mg/day and about 2500 mg/day (e.g., between about 10 mg/day and about 2400 mg/day, between about 10 mg/day and about 2300 mg/day, between about 10 mg/day and about 2200 mg/day, between about 10 mg/day and about 2100 mg/day, between about 10 mg/day and about 2000 mg/day, between about 10 mg/day and about 1900 mg/day, between about 10 mg/day and about 1800 mg/day, between about 10 mg/day and about 1700 mg/day, between about 10 mg/day and about 1600 mg/day, between about 10 mg/day and about 1500 mg/day or about 10 mg/day and about 1400 mg/day).

In some embodiments, the therapeutically effective amount of the BlvrB inhibitor is between about 10 mg/day and about 1500 mg/day (e.g, between about 10 mg/day and about 1400 mg/day, between about 10 mg/day and about 1300 mg/day, between about 10 mg/day and about 1200 mg/day, between about 10 mg/day and about 1100 mg/day, between about 10 mg/day and about 1000 mg/day, between about 10 mg/day and about 900 mg/day, between about 10 mg/day and about 800 mg/day, between about 10 mg/day and about 700 mg/day, between about 10 mg/day and about 600 mg/day, between about 10 mg/day and about 500 mg/day or about 10 mg/day and about 400 mg/day).

In some embodiments, the therapeutically effective amount of the BlvrB inhibitor is between about 10 mg/day and about 500 mg/day (e.g, between about 10 mg/day and about 400 mg/day, between about 10 mg/day and about 300 mg/day, between about 10 mg/day and about 200 mg/day, between about 10 mg/day and about 100 mg/day, between about 10 mg/day and about 90 mg/day, between about 10 mg/day and about 80 mg/day, between about 10 mg/day and about 70 mg/day, between about 10 mg/day and about 60 mg/day, between about 10 mg/day and about 50 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 30 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 20 mg/day).

In some embodiments, the therapeutically effective amount of the BlvrB inhibitor is about 8500 mg/day, about 8400 mg/day, about 8300 mg/day, about 8200 mg/day, about 8100 mg/day, about 8000 mg/day, about 7900 mg/day, about 7800 mg/day, about 7700 mg/day, about 7600 mg/day, about 7500 mg/day, about 7400 mg/day, about 7300 mg/day, about 7200 mg/day, about 7100 mg/day, about 7000 mg/day, about 6900 mg/day, about 6800 mg/day, about 6700 mg/day, about 6600 mg/day, about 6500 mg/day, about 6400 mg/day, about 6300 mg/day, about 6200 mg/day, about 6100 mg/day, about 6000 mg/day, about 5900 mg/day, about 5800 mg/day, about 5700 mg/day, about 5600 mg/day, about 5500 mg/day, about 5400 mg/day, about 5300 mg/day, about 5200 mg/day, about 5100 mg/day, about 5000 mg/day, about 4900 mg/day, about 4800 mg/day, about 4700 mg/day, about 4600 mg/day, about 4500 mg/day, about 4400 mg/day, about 4300 mg/day, about 4200 mg/day, about 4100 mg/day, about 4000 mg/day, about 3900 mg/day, about 3800 mg/day, about 3700 mg/day, about 3600 mg/day, about 3500 mg/day, about 3400 mg/day, about 3300 mg/day, about 3200 mg/day, about 3100 mg/day, about 3000 mg/day, about 2900 mg/day, about 2800 mg/day, about 2700 mg/day, about 2600 mg/day, about 2500 mg/day, about 2400 mg/day, about 2300 mg/day, about 2200 mg/day, about 2100 mg/day, about 2000 mg/day, about 1900 mg/day, about 1800 mg/day, about 1700 mg/day, about 1600 mg/day, about 1500 mg/day, about 1400 mg/day, about 1300 mg/day, about 1200 mg/day, about 1100 mg/day, about 1000 mg/day, about 900 mg/day, about 800 mg/day, about 700 mg/day, about 600 mg/day, about 500 mg/day, about 400 mg/day, about 300 mg/day, about 200 mg/day, about 100 mg/day, about 90 mg/day, about 80 mg/day, about 70 mg/day, about 60 mg/day, about 50 mg/day, about 40 mg/day, about 30 mg/day, about 20 mg/day, about 10 mg/day.

In some embodiments, the BlvrB inhibitor is administered 6 times/day ( e.g . every 4 hours).

In some embodiments, the method comprises administering to the subject 6 times/day (e.g., every 4 hours) a dose between about 10 mg/day and about 8500 mg/day (e.g, between about 10 mg/day and about 8400 mg/day, between about 10 mg/day and about 8300 mg/day, between about 10 mg/day and about 8200 mg/day, between about 10 mg/day and about 8100 mg/day, between about 10 mg/day and about 8000 mg/day, between about 10 mg/day and about 7900 mg/day, between about 10 mg/day and about 7800 mg/day, between about 10 mg/day and about 7700 mg/day, between about 10 mg/day and about 7600 mg/day).

In some embodiments, the method comprises administering to the subject 6 times/day ( e.g ., every 4 hours) a dose between about 10 mg/day and about 7500 mg/day (e.g, between about 10 mg/day and about 7400 mg/day, between about 10 mg/day and about 7300 mg/day, between about 10 mg/day and about 7200 mg/day, between about 10 mg/day and about 7100 mg/day, between about 10 mg/day and about 7000 mg/day, between about 10 mg/day and about 6900 mg/day, between about 10 mg/day and about 6800 mg/day, between about 10 mg/day and about 6700 mg/day, between about 10 mg/day and about 6600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 6400 mg/day).

In some embodiments, the method comprises administering to the subject 6 times/day (e.g, every 4 hours) a dose between about 10 mg/day and about 6500 mg/day (e.g, between about 10 mg/day and about 6400 mg/day, between about 10 mg/day and about 6300 mg/day, between about 10 mg/day and about 6200 mg/day, between about 10 mg/day and about 6100 mg/day, between about 10 mg/day and about 6000 mg/day, between about 10 mg/day and about 5900 mg/day, between about 10 mg/day and about 5800 mg/day, between about 10 mg/day and about 5700 mg/day, between about 10 mg/day and about 5600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 5400 mg/day).

In some embodiments, the method comprises administering to the subject 6 times/day (e.g, every 4 hours) a dose between about 10 mg/day and about 5500 mg/day (e.g, between about 10 mg/day and about 5400 mg/day, between about 10 mg/day and about 5300 mg/day, between about 10 mg/day and about 5200 mg/day, between about 10 mg/day and about 5100 mg/day, between about 10 mg/day and about 5000 mg/day, between about 10 mg/day and about 4900 mg/day, between about 10 mg/day and about 4800 mg/day, between about 10 mg/day and about 4700 mg/day, between about 10 mg/day and about 4600 mg/day, between about 10 mg/day and about 4500 mg/day or about 10 mg/day and about 4400 mg/day).

In some embodiments, the method comprises administering to the subject 6 times/day (e.g, every 4 hours) a dose between about 10 mg/day and about 4500 mg/day (e.g, between about 10 mg/day and about 4400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 4200 mg/day, between about 10 mg/day and about 4100 mg/day, between about 10 mg/day and about 4000 mg/day, between about 10 mg/day and about 3900 mg/day, between about 10 mg/day and about 3800 mg/day, between about 10 mg/day and about 3700 mg/day, between about 10 mg/day and about 3600 mg/day, between about 10 mg/day and about 3500 mg/day or about 10 mg/day and about 3400 mg/day).

In some embodiments, the method comprises administering to the subject 6 times/day ( e.g ., every 4 hours) a dose between about 10 mg/day and about 3500 mg/day (e.g, between about 10 mg/day and about 3400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 3200 mg/day, between about 10 mg/day and about 3100 mg/day, between about 10 mg/day and about 3000 mg/day, between about 10 mg/day and about 2900 mg/day, between about 10 mg/day and about 2800 mg/day, between about 10 mg/day and about 2700 mg/day, between about 10 mg/day and about 2600 mg/day, between about 10 mg/day and about 2500 mg/day or about 10 mg/day and about 2400 mg/day).

In some embodiments, the method comprises administering to the subject 6 times/day (e.g, every 4 hours) a dose between about 10 mg/day and about 2500 mg/day (e.g, between about 10 mg/day and about 2400 mg/day, between about 10 mg/day and about 2300 mg/day, between about 10 mg/day and about 2200 mg/day, between about 10 mg/day and about 2100 mg/day, between about 10 mg/day and about 2000 mg/day, between about 10 mg/day and about 1900 mg/day, between about 10 mg/day and about 1800 mg/day, between about 10 mg/day and about 1700 mg/day, between about 10 mg/day and about 1600 mg/day, between about 10 mg/day and about 1500 mg/day or about 10 mg/day and about 1400 mg/day).

In some embodiments, the method comprises administering to the subject 6 times/day (e.g, every 4 hours) a dose between about 10 mg/day and about 1500 mg/day (e.g, between about 10 mg/day and about 1400 mg/day, between about 10 mg/day and about 1300 mg/day, between about 10 mg/day and about 1200 mg/day, between about 10 mg/day and about 1100 mg/day, between about 10 mg/day and about 1000 mg/day, between about 10 mg/day and about 900 mg/day, between about 10 mg/day and about 800 mg/day, between about 10 mg/day and about 700 mg/day, between about 10 mg/day and about 600 mg/day, between about 10 mg/day and about 500 mg/day or about 10 mg/day and about 400 mg/day).

In some embodiments, the method comprises administering to the subject 6 times/day (e.g, every 4 hours) a dose between about 10 mg/day and about 500 mg/day (e.g, between about 10 mg/day and about 400 mg/day, between about 10 mg/day and about 300 mg/day, between about 10 mg/day and about 200 mg/day, between about 10 mg/day and about 100 mg/day, between about 10 mg/day and about 90 mg/day, between about 10 mg/day and about 80 mg/day, between about 10 mg/day and about 70 mg/day, between about 10 mg/day and about 60 mg/day, between about 10 mg/day and about 50 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 30 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 20 mg/day).

In some embodiments, the method comprises administering to the subject 6 times/day ( e.g ., every 4 hours) a dose of about 8500 mg/day, about 8400 mg/day, about 8300 mg/day, about 8200 mg/day, about 8100 mg/day, about 8000 mg/day, about 7900 mg/day, about 7800 mg/day, about 7700 mg/day, about 7600 mg/day, about 7500 mg/day, about 7400 mg/day, about 7300 mg/day, about 7200 mg/day, about 7100 mg/day, about 7000 mg/day, about 6900 mg/day, about 6800 mg/day, about 6700 mg/day, about 6600 mg/day, about 6500 mg/day, about 6400 mg/day, about 6300 mg/day, about 6200 mg/day, about 6100 mg/day, about 6000 mg/day, about 5900 mg/day, about 5800 mg/day, about 5700 mg/day, about 5600 mg/day, about 5500 mg/day, about 5400 mg/day, about 5300 mg/day, about 5200 mg/day, about 5100 mg/day, about 5000 mg/day, about 4900 mg/day, about 4800 mg/day, about 4700 mg/day, about 4600 mg/day, about 4500 mg/day, about 4400 mg/day, about 4300 mg/day, about 4200 mg/day, about 4100 mg/day, about 4000 mg/day, about 3900 mg/day, about 3800 mg/day, about 3700 mg/day, about 3600 mg/day, about 3500 mg/day, about 3400 mg/day, about 3300 mg/day, about 3200 mg/day, about 3100 mg/day, about 3000 mg/day, about 2900 mg/day, about 2800 mg/day, about 2700 mg/day, about 2600 mg/day, about 2500 mg/day, about 2400 mg/day, about 2300 mg/day, about 2200 mg/day, about 2100 mg/day, about 2000 mg/day, about 1900 mg/day, about 1800 mg/day, about 1700 mg/day, about 1600 mg/day, about 1500 mg/day, about 1400 mg/day, about 1300 mg/day, about 1200 mg/day, about 1100 mg/day, about 1000 mg/day, about 900 mg/day, about 800 mg/day, about 700 mg/day, about 600 mg/day, about 500 mg/day, about 400 mg/day, about 300 mg/day, about 200 mg/day, about 100 mg/day, about 90 mg/day, about 80 mg/day, about 70 mg/day, about 60 mg/day, about 50 mg/day, about 40 mg/day, about 30 mg/day, about 20 mg/day, about 10 mg/day.

In some embodiments, the BlvrB inhibitor is administered 4 times/day (e.g. every 6 hours). In some embodiments, the method comprises administering to the subject 4 times/day ( e.g ., every 6 hours) a dose between about 10 mg/day and about 8500 mg/day (e.g, between about 10 mg/day and about 8400 mg/day, between about 10 mg/day and about 8300 mg/day, between about 10 mg/day and about 8200 mg/day, between about 10 mg/day and about 8100 mg/day, between about 10 mg/day and about 8000 mg/day, between about 10 mg/day and about 7900 mg/day, between about 10 mg/day and about 7800 mg/day, between about 10 mg/day and about 7700 mg/day, between about 10 mg/day and about 7600 mg/day).

In some embodiments, the method comprises administering to the subject 4 times/day (e.g, every 6 hours) a dose between about 10 mg/day and about 7500 mg/day (e.g, between about 10 mg/day and about 7400 mg/day, between about 10 mg/day and about 7300 mg/day, between about 10 mg/day and about 7200 mg/day, between about 10 mg/day and about 7100 mg/day, between about 10 mg/day and about 7000 mg/day, between about 10 mg/day and about 6900 mg/day, between about 10 mg/day and about 6800 mg/day, between about 10 mg/day and about 6700 mg/day, between about 10 mg/day and about 6600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 6400 mg/day).

In some embodiments, the method comprises administering to the subject 4 times/day (e.g, every 6 hours) a dose between about 10 mg/day and about 6500 mg/day (e.g, between about 10 mg/day and about 6400 mg/day, between about 10 mg/day and about 6300 mg/day, between about 10 mg/day and about 6200 mg/day, between about 10 mg/day and about 6100 mg/day, between about 10 mg/day and about 6000 mg/day, between about 10 mg/day and about 5900 mg/day, between about 10 mg/day and about 5800 mg/day, between about 10 mg/day and about 5700 mg/day, between about 10 mg/day and about 5600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 5400 mg/day).

In some embodiments, the method comprises administering to the subject 4 times/day (e.g, every 6 hours) a dose between about 10 mg/day and about 5500 mg/day (e.g, between about 10 mg/day and about 5400 mg/day, between about 10 mg/day and about 5300 mg/day, between about 10 mg/day and about 5200 mg/day, between about 10 mg/day and about 5100 mg/day, between about 10 mg/day and about 5000 mg/day, between about 10 mg/day and about 4900 mg/day, between about 10 mg/day and about 4800 mg/day, between about 10 mg/day and about 4700 mg/day, between about 10 mg/day and about 4600 mg/day, between about 10 mg/day and about 4500 mg/day or about 10 mg/day and about 4400 mg/day).

In some embodiments, the method comprises administering to the subject 4 times/day ( e.g ., every 6 hours) a dose between about 10 mg/day and about 4500 mg/day (e.g, between about 10 mg/day and about 4400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 4200 mg/day, between about 10 mg/day and about 4100 mg/day, between about 10 mg/day and about 4000 mg/day, between about 10 mg/day and about 3900 mg/day, between about 10 mg/day and about 3800 mg/day, between about 10 mg/day and about 3700 mg/day, between about 10 mg/day and about 3600 mg/day, between about 10 mg/day and about 3500 mg/day or about 10 mg/day and about 3400 mg/day).

In some embodiments, the method comprises administering to the subject 4 times/day (e.g, every 6 hours) a dose between about 10 mg/day and about 3500 mg/day (e.g, between about 10 mg/day and about 3400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 3200 mg/day, between about 10 mg/day and about 3100 mg/day, between about 10 mg/day and about 3000 mg/day, between about 10 mg/day and about 2900 mg/day, between about 10 mg/day and about 2800 mg/day, between about 10 mg/day and about 2700 mg/day, between about 10 mg/day and about 2600 mg/day, between about 10 mg/day and about 2500 mg/day or about 10 mg/day and about 2400 mg/day).

In some embodiments, the method comprises administering to the subject 4 times/day (e.g, every 6 hours) a dose between about 10 mg/day and about 2500 mg/day (e.g, between about 10 mg/day and about 2400 mg/day, between about 10 mg/day and about 2300 mg/day, between about 10 mg/day and about 2200 mg/day, between about 10 mg/day and about 2100 mg/day, between about 10 mg/day and about 2000 mg/day, between about 10 mg/day and about 1900 mg/day, between about 10 mg/day and about 1800 mg/day, between about 10 mg/day and about 1700 mg/day, between about 10 mg/day and about 1600 mg/day, between about 10 mg/day and about 1500 mg/day or about 10 mg/day and about 1400 mg/day).

In some embodiments, the method comprises administering to the subject 4 times/day (e.g, every 6 hours) a dose between about 10 mg/day and about 1500 mg/day (e.g, between about 10 mg/day and about 1400 mg/day, between about 10 mg/day and about 1300 mg/day, between about 10 mg/day and about 1200 mg/day, between about 10 mg/day and about 1100 mg/day, between about 10 mg/day and about 1000 mg/day, between about 10 mg/day and about 900 mg/day, between about 10 mg/day and about 800 mg/day, between about 10 mg/day and about 700 mg/day, between about 10 mg/day and about 600 mg/day, between about 10 mg/day and about 500 mg/day or about 10 mg/day and about 400 mg/day).

In some embodiments, the method comprises administering to the subject 4 times/day ( e.g ., every 6 hours) a dose between about 10 mg/day and about 500 mg/day (e.g, between about 10 mg/day and about 400 mg/day, between about 10 mg/day and about 300 mg/day, between about 10 mg/day and about 200 mg/day, between about 10 mg/day and about 100 mg/day, between about 10 mg/day and about 90 mg/day, between about 10 mg/day and about 80 mg/day, between about 10 mg/day and about 70 mg/day, between about 10 mg/day and about 60 mg/day, between about 10 mg/day and about 50 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 30 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 20 mg/day).

In some embodiments, the method comprises administering to the subject 4 times/day (e.g, every 6 hours) a dose of about 8500 mg/day, about 8400 mg/day, about 8300 mg/day, about 8200 mg/day, about 8100 mg/day, about 8000 mg/day, about 7900 mg/day, about 7800 mg/day, about 7700 mg/day, about 7600 mg/day, about 7500 mg/day, about 7400 mg/day, about 7300 mg/day, about 7200 mg/day, about 7100 mg/day, about 7000 mg/day, about 6900 mg/day, about 6800 mg/day, about 6700 mg/day, about 6600 mg/day, about 6500 mg/day, about 6400 mg/day, about 6300 mg/day, about 6200 mg/day, about 6100 mg/day, about 6000 mg/day, about 5900 mg/day, about 5800 mg/day, about 5700 mg/day, about 5600 mg/day, about 5500 mg/day, about 5400 mg/day, about 5300 mg/day, about 5200 mg/day, about 5100 mg/day, about 5000 mg/day, about 4900 mg/day, about 4800 mg/day, about 4700 mg/day, about 4600 mg/day, about 4500 mg/day, about 4400 mg/day, about 4300 mg/day, about 4200 mg/day, about 4100 mg/day, about 4000 mg/day, about 3900 mg/day, about 3800 mg/day, about 3700 mg/day, about 3600 mg/day, about 3500 mg/day, about 3400 mg/day, about 3300 mg/day, about 3200 mg/day, about 3100 mg/day, about 3000 mg/day, about 2900 mg/day, about 2800 mg/day, about 2700 mg/day, about 2600 mg/day, about 2500 mg/day, about 2400 mg/day, about 2300 mg/day, about 2200 mg/day, about 2100 mg/day, about 2000 mg/day, about 1900 mg/day, about 1800 mg/day, about 1700 mg/day, about 1600 mg/day, about 1500 mg/day, about 1400 mg/day, about 1300 mg/day, about 1200 mg/day, about 1100 mg/day, about 1000 mg/day, about 900 mg/day, about 800 mg/day, about 700 mg/day, about 600 mg/day, about 500 mg/day, about 400 mg/day, about 300 mg/day, about 200 mg/day, about 100 mg/day, about 90 mg/day, about 80 mg/day, about 70 mg/day, about 60 mg/day, about 50 mg/day, about 40 mg/day, about 30 mg/day, about 20 mg/day, about 10 mg/day.

In some embodiments, the BlvrB inhibitor is administered 3 times/day (e.g. every 8 hours).

In some embodiments, the method comprises administering to the subject 3 times/day (e.g, every 8 hours) a dose between about 10 mg/day and about 8500 mg/day (e.g, between about 10 mg/day and about 8400 mg/day, between about 10 mg/day and about 8300 mg/day, between about 10 mg/day and about 8200 mg/day, between about 10 mg/day and about 8100 mg/day, between about 10 mg/day and about 8000 mg/day, between about 10 mg/day and about 7900 mg/day, between about 10 mg/day and about 7800 mg/day, between about 10 mg/day and about 7700 mg/day, between about 10 mg/day and about 7600 mg/day).

In some embodiments, the method comprises administering to the subject 3 times/day (e.g, every 8 hours) a dose between about 10 mg/day and about 7500 mg/day (e.g, between about 10 mg/day and about 7400 mg/day, between about 10 mg/day and about 7300 mg/day, between about 10 mg/day and about 7200 mg/day, between about 10 mg/day and about 7100 mg/day, between about 10 mg/day and about 7000 mg/day, between about 10 mg/day and about 6900 mg/day, between about 10 mg/day and about 6800 mg/day, between about 10 mg/day and about 6700 mg/day, between about 10 mg/day and about 6600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 6400 mg/day).

In some embodiments, the method comprises administering to the subject 3 times/day (e.g, every 8 hours) a dose between about 10 mg/day and about 6500 mg/day (e.g, between about 10 mg/day and about 6400 mg/day, between about 10 mg/day and about 6300 mg/day, between about 10 mg/day and about 6200 mg/day, between about 10 mg/day and about 6100 mg/day, between about 10 mg/day and about 6000 mg/day, between about 10 mg/day and about 5900 mg/day, between about 10 mg/day and about 5800 mg/day, between about 10 mg/day and about 5700 mg/day, between about 10 mg/day and about 5600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 5400 mg/day).

In some embodiments, the method comprises administering to the subject 3 times/day (e.g, every 8 hours) a dose between about 10 mg/day and about 5500 mg/day (e.g., between about 10 mg/day and about 5400 mg/day, between about 10 mg/day and about 5300 mg/day, between about 10 mg/day and about 5200 mg/day, between about 10 mg/day and about 5100 mg/day, between about 10 mg/day and about 5000 mg/day, between about 10 mg/day and about 4900 mg/day, between about 10 mg/day and about 4800 mg/day, between about 10 mg/day and about 4700 mg/day, between about 10 mg/day and about 4600 mg/day, between about 10 mg/day and about 4500 mg/day or about 10 mg/day and about 4400 mg/day).

In some embodiments, the method comprises administering to the subject 3 times/day (e.g, every 8 hours) a dose between about 10 mg/day and about 4500 mg/day (e.g, between about 10 mg/day and about 4400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 4200 mg/day, between about 10 mg/day and about 4100 mg/day, between about 10 mg/day and about 4000 mg/day, between about 10 mg/day and about 3900 mg/day, between about 10 mg/day and about 3800 mg/day, between about 10 mg/day and about 3700 mg/day, between about 10 mg/day and about 3600 mg/day, between about 10 mg/day and about 3500 mg/day or about 10 mg/day and about 3400 mg/day).

In some embodiments, the method comprises administering to the subject 3 times/day (e.g, every 8 hours) a dose between about 10 mg/day and about 3500 mg/day (e.g, between about 10 mg/day and about 3400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 3200 mg/day, between about 10 mg/day and about 3100 mg/day, between about 10 mg/day and about 3000 mg/day, between about 10 mg/day and about 2900 mg/day, between about 10 mg/day and about 2800 mg/day, between about 10 mg/day and about 2700 mg/day, between about 10 mg/day and about 2600 mg/day, between about 10 mg/day and about 2500 mg/day or about 10 mg/day and about 2400 mg/day).

In some embodiments, the method comprises administering to the subject 3 times/day (e.g, every 8 hours) a dose between about 10 mg/day and about 2500 mg/day (e.g, between about 10 mg/day and about 2400 mg/day, between about 10 mg/day and about 2300 mg/day, between about 10 mg/day and about 2200 mg/day, between about 10 mg/day and about 2100 mg/day, between about 10 mg/day and about 2000 mg/day, between about 10 mg/day and about 1900 mg/day, between about 10 mg/day and about 1800 mg/day, between about 10 mg/day and about 1700 mg/day, between about 10 mg/day and about 1600 mg/day, between about 10 mg/day and about 1500 mg/day or about 10 mg/day and about 1400 mg/day).

In some embodiments, the method comprises administering to the subject 3 times/day ( e.g ., every 8 hours) a dose between about 10 mg/day and about 1500 mg/day (e.g, between about 10 mg/day and about 1400 mg/day, between about 10 mg/day and about 1300 mg/day, between about 10 mg/day and about 1200 mg/day, between about 10 mg/day and about 1100 mg/day, between about 10 mg/day and about 1000 mg/day, between about 10 mg/day and about 900 mg/day, between about 10 mg/day and about 800 mg/day, between about 10 mg/day and about 700 mg/day, between about 10 mg/day and about 600 mg/day, between about 10 mg/day and about 500 mg/day or about 10 mg/day and about 400 mg/day).

In some embodiments, the method comprises administering to the subject 3 times/day (e.g, every 8 hours) a dose between about 10 mg/day and about 500 mg/day (e.g, between about 10 mg/day and about 400 mg/day, between about 10 mg/day and about 300 mg/day, between about 10 mg/day and about 200 mg/day, between about 10 mg/day and about 100 mg/day, between about 10 mg/day and about 90 mg/day, between about 10 mg/day and about 80 mg/day, between about 10 mg/day and about 70 mg/day, between about 10 mg/day and about 60 mg/day, between about 10 mg/day and about 50 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 30 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 20 mg/day).

In some embodiments, the method comprises administering to the subject 3 times/day (e.g, every 8 hours) a dose of about 8500 mg/day, about 8400 mg/day, about 8300 mg/day, about 8200 mg/day, about 8100 mg/day, about 8000 mg/day, about 7900 mg/day, about 7800 mg/day, about 7700 mg/day, about 7600 mg/day, about 7500 mg/day, about 7400 mg/day, about 7300 mg/day, about 7200 mg/day, about 7100 mg/day, about 7000 mg/day, about 6900 mg/day, about 6800 mg/day, about 6700 mg/day, about 6600 mg/day, about 6500 mg/day, about 6400 mg/day, about 6300 mg/day, about 6200 mg/day, about 6100 mg/day, about 6000 mg/day, about 5900 mg/day, about 5800 mg/day, about 5700 mg/day, about 5600 mg/day, about 5500 mg/day, about 5400 mg/day, about 5300 mg/day, about 5200 mg/day, about 5100 mg/day, about 5000 mg/day, about 4900 mg/day, about 4800 mg/day, about 4700 mg/day, about 4600 mg/day, about 4500 mg/day, about 4400 mg/day, about 4300 mg/day, about 4200 mg/day, about 4100 mg/day, about 4000 mg/day, about 3900 mg/day, about 3800 mg/day, about 3700 mg/day, about 3600 mg/day, about 3500 mg/day, about 3400 mg/day, about 3300 mg/day, about 3200 mg/day, about 3100 mg/day, about 3000 mg/day, about 2900 mg/day, about 2800 mg/day, about 2700 mg/day, about 2600 mg/day, about 2500 mg/day, about 2400 mg/day, about 2300 mg/day, about 2200 mg/day, about 2100 mg/day, about 2000 mg/day, about 1900 mg/day, about 1800 mg/day, about 1700 mg/day, about 1600 mg/day, about 1500 mg/day, about 1400 mg/day, about 1300 mg/day, about 1200 mg/day, about 1100 mg/day, about 1000 mg/day, about 900 mg/day, about 800 mg/day, about 700 mg/day, about 600 mg/day, about 500 mg/day, about 400 mg/day, about 300 mg/day, about 200 mg/day, about 100 mg/day, about 90 mg/day, about 80 mg/day, about 70 mg/day, about 60 mg/day, about 50 mg/day, about 40 mg/day, about 30 mg/day, about 20 mg/day, about 10 mg/day.

In some embodiments, the BlvrB inhibitor is administered 2 times/day (e.g. every 12 hours).

In some embodiments, the method comprises administering to the subject 2 times/day (e.g, every 12 hours) a dose between about 10 mg/day and about 8500 mg/day (e.g, between about 10 mg/day and about 8400 mg/day, between about 10 mg/day and about 8300 mg/day, between about 10 mg/day and about 8200 mg/day, between about 10 mg/day and about 8100 mg/day, between about 10 mg/day and about 8000 mg/day, between about 10 mg/day and about 7900 mg/day, between about 10 mg/day and about 7800 mg/day, between about 10 mg/day and about 7700 mg/day, between about 10 mg/day and about 7600 mg/day).

In some embodiments, the method comprises administering to the subject 2 times/day (e.g, every 12 hours) a dose between about 10 mg/day and about 7500 mg/day (e.g, between about 10 mg/day and about 7400 mg/day, between about 10 mg/day and about 7300 mg/day, between about 10 mg/day and about 7200 mg/day, between about 10 mg/day and about 7100 mg/day, between about 10 mg/day and about 7000 mg/day, between about 10 mg/day and about 6900 mg/day, between about 10 mg/day and about 6800 mg/day, between about 10 mg/day and about 6700 mg/day, between about 10 mg/day and about 6600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 6400 mg/day).

In some embodiments, the method comprises administering to the subject 2 times/day (e.g, every 12 hours) a dose between about 10 mg/day and about 6500 mg/day (e.g, between about 10 mg/day and about 6400 mg/day, between about 10 mg/day and about 6300 mg/day, between about 10 mg/day and about 6200 mg/day, between about 10 mg/day and about 6100 mg/day, between about 10 mg/day and about 6000 mg/day, between about 10 mg/day and about 5900 mg/day, between about 10 mg/day and about 5800 mg/day, between about 10 mg/day and about 5700 mg/day, between about 10 mg/day and about 5600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 5400 mg/day).

In some embodiments, the method comprises administering to the subject 2 times/day ( e.g ., every 12 hours) a dose between about 10 mg/day and about 5500 mg/day (e.g, between about 10 mg/day and about 5400 mg/day, between about 10 mg/day and about 5300 mg/day, between about 10 mg/day and about 5200 mg/day, between about 10 mg/day and about 5100 mg/day, between about 10 mg/day and about 5000 mg/day, between about 10 mg/day and about 4900 mg/day, between about 10 mg/day and about 4800 mg/day, between about 10 mg/day and about 4700 mg/day, between about 10 mg/day and about 4600 mg/day, between about 10 mg/day and about 4500 mg/day or about 10 mg/day and about 4400 mg/day).

In some embodiments, the method comprises administering to the subject 2 times/day (e.g, every 12 hours) a dose between about 10 mg/day and about 4500 mg/day (e.g, between about 10 mg/day and about 4400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 4200 mg/day, between about 10 mg/day and about 4100 mg/day, between about 10 mg/day and about 4000 mg/day, between about 10 mg/day and about 3900 mg/day, between about 10 mg/day and about 3800 mg/day, between about 10 mg/day and about 3700 mg/day, between about 10 mg/day and about 3600 mg/day, between about 10 mg/day and about 3500 mg/day or about 10 mg/day and about 3400 mg/day).

In some embodiments, the method comprises administering to the subject 2 times/day (e.g, every 12 hours) a dose between about 10 mg/day and about 3500 mg/day (e.g, between about 10 mg/day and about 3400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 3200 mg/day, between about 10 mg/day and about 3100 mg/day, between about 10 mg/day and about 3000 mg/day, between about 10 mg/day and about 2900 mg/day, between about 10 mg/day and about 2800 mg/day, between about 10 mg/day and about 2700 mg/day, between about 10 mg/day and about 2600 mg/day, between about 10 mg/day and about 2500 mg/day or about 10 mg/day and about 2400 mg/day).

In some embodiments, the method comprises administering to the subject 2 times/day (e.g, every 12 hours) a dose between about 10 mg/day and about 2500 mg/day (e.g., between about 10 mg/day and about 2400 mg/day, between about 10 mg/day and about 2300 mg/day, between about 10 mg/day and about 2200 mg/day, between about 10 mg/day and about 2100 mg/day, between about 10 mg/day and about 2000 mg/day, between about 10 mg/day and about 1900 mg/day, between about 10 mg/day and about 1800 mg/day, between about 10 mg/day and about 1700 mg/day, between about 10 mg/day and about 1600 mg/day, between about 10 mg/day and about 1500 mg/day or about 10 mg/day and about 1400 mg/day).

In some embodiments, the method comprises administering to the subject 2 times/day (e.g, every 12 hours) a dose between about 10 mg/day and about 1500 mg/day (e.g, between about 10 mg/day and about 1400 mg/day, between about 10 mg/day and about 1300 mg/day, between about 10 mg/day and about 1200 mg/day, between about 10 mg/day and about 1100 mg/day, between about 10 mg/day and about 1000 mg/day, between about 10 mg/day and about 900 mg/day, between about 10 mg/day and about 800 mg/day, between about 10 mg/day and about 700 mg/day, between about 10 mg/day and about 600 mg/day, between about 10 mg/day and about 500 mg/day or about 10 mg/day and about 400 mg/day).

In some embodiments, the method comprises administering to the subject 2 times/day (e.g, every 12 hours) a dose between about 10 mg/day and about 500 mg/day (e.g, between about 10 mg/day and about 400 mg/day, between about 10 mg/day and about 300 mg/day, between about 10 mg/day and about 200 mg/day, between about 10 mg/day and about 100 mg/day, between about 10 mg/day and about 90 mg/day, between about 10 mg/day and about 80 mg/day, between about 10 mg/day and about 70 mg/day, between about 10 mg/day and about 60 mg/day, between about 10 mg/day and about 50 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 30 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 20 mg/day).

In some embodiments, the method comprises administering to the subject 2 times/day (e.g, every 12 hours) a dose of about 8500 mg/day, about 8400 mg/day, about 8300 mg/day, about 8200 mg/day, about 8100 mg/day, about 8000 mg/day, about 7900 mg/day, about 7800 mg/day, about 7700 mg/day, about 7600 mg/day, about 7500 mg/day, about 7400 mg/day, about 7300 mg/day, about 7200 mg/day, about 7100 mg/day, about 7000 mg/day, about 6900 mg/day, about 6800 mg/day, about 6700 mg/day, about 6600 mg/day, about 6500 mg/day, about 6400 mg/day, about 6300 mg/day, about 6200 mg/day, about 6100 mg/day, about 6000 mg/day, about 5900 mg/day, about 5800 mg/day, about 5700 mg/day, about 5600 mg/day, about 5500 mg/day, about 5400 mg/day, about 5300 mg/day, about 5200 mg/day, about 5100 mg/day, about 5000 mg/day, about 4900 mg/day, about 4800 mg/day, about 4700 mg/day, about 4600 mg/day, about 4500 mg/day, about 4400 mg/day, about 4300 mg/day, about 4200 mg/day, about 4100 mg/day, about 4000 mg/day, about 3900 mg/day, about 3800 mg/day, about 3700 mg/day, about 3600 mg/day, about 3500 mg/day, about 3400 mg/day, about 3300 mg/day, about 3200 mg/day, about 3100 mg/day, about 3000 mg/day, about 2900 mg/day, about 2800 mg/day, about 2700 mg/day, about 2600 mg/day, about 2500 mg/day, about 2400 mg/day, about 2300 mg/day, about 2200 mg/day, about 2100 mg/day, about 2000 mg/day, about 1900 mg/day, about 1800 mg/day, about 1700 mg/day, about 1600 mg/day, about 1500 mg/day, about 1400 mg/day, about 1300 mg/day, about 1200 mg/day, about 1100 mg/day, about 1000 mg/day, about 900 mg/day, about 800 mg/day, about 700 mg/day, about 600 mg/day, about 500 mg/day, about 400 mg/day, about 300 mg/day, about 200 mg/day, about 100 mg/day, about 90 mg/day, about 80 mg/day, about 70 mg/day, about 60 mg/day, about 50 mg/day, about 40 mg/day, about 30 mg/day, about 20 mg/day, about 10 mg/day.

In some embodiments, the BlvrB inhibitor is administered 1 time/day (e.g. every 24 hours).

In some embodiments, the method comprises administering to the subject 1 time/day (e.g, every 24 hours) a dose between about 10 mg/day and about 8500 mg/day (e.g, between about 10 mg/day and about 8400 mg/day, between about 10 mg/day and about 8300 mg/day, between about 10 mg/day and about 8200 mg/day, between about 10 mg/day and about 8100 mg/day, between about 10 mg/day and about 8000 mg/day, between about 10 mg/day and about 7900 mg/day, between about 10 mg/day and about 7800 mg/day, between about 10 mg/day and about 7700 mg/day, between about 10 mg/day and about 7600 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/day (e.g, every 24 hours) a dose between about 10 mg/day and about 7500 mg/day (e.g, between about 10 mg/day and about 7400 mg/day, between about 10 mg/day and about 7300 mg/day, between about 10 mg/day and about 7200 mg/day, between about 10 mg/day and about 7100 mg/day, between about 10 mg/day and about 7000 mg/day, between about 10 mg/day and about 6900 mg/day, between about 10 mg/day and about 6800 mg/day, between about 10 mg/day and about 6700 mg/day, between about 10 mg/day and about 6600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 6400 mg/day). In some embodiments, the method comprises administering to the subject 1 time/day ( e.g ., every 24 hours) a dose between about 10 mg/day and about 6500 mg/day (e.g, between about 10 mg/day and about 6400 mg/day, between about 10 mg/day and about 6300 mg/day, between about 10 mg/day and about 6200 mg/day, between about 10 mg/day and about 6100 mg/day, between about 10 mg/day and about 6000 mg/day, between about 10 mg/day and about 5900 mg/day, between about 10 mg/day and about 5800 mg/day, between about 10 mg/day and about 5700 mg/day, between about 10 mg/day and about 5600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 5400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/day (e.g, every 24 hours) a dose between about 10 mg/day and about 5500 mg/day (e.g, between about 10 mg/day and about 5400 mg/day, between about 10 mg/day and about 5300 mg/day, between about 10 mg/day and about 5200 mg/day, between about 10 mg/day and about 5100 mg/day, between about 10 mg/day and about 5000 mg/day, between about 10 mg/day and about 4900 mg/day, between about 10 mg/day and about 4800 mg/day, between about 10 mg/day and about 4700 mg/day, between about 10 mg/day and about 4600 mg/day, between about 10 mg/day and about 4500 mg/day or about 10 mg/day and about 4400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/day (e.g, every 24 hours) a dose between about 10 mg/day and about 4500 mg/day (e.g, between about 10 mg/day and about 4400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 4200 mg/day, between about 10 mg/day and about 4100 mg/day, between about 10 mg/day and about 4000 mg/day, between about 10 mg/day and about 3900 mg/day, between about 10 mg/day and about 3800 mg/day, between about 10 mg/day and about 3700 mg/day, between about 10 mg/day and about 3600 mg/day, between about 10 mg/day and about 3500 mg/day or about 10 mg/day and about 3400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/day (e.g, every 24 hours) a dose between about 10 mg/day and about 3500 mg/day (e.g, between about 10 mg/day and about 3400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 3200 mg/day, between about 10 mg/day and about 3100 mg/day, between about 10 mg/day and about 3000 mg/day, between about 10 mg/day and about 2900 mg/day, between about 10 mg/day and about 2800 mg/day, between about 10 mg/day and about 2700 mg/day, between about 10 mg/day and about 2600 mg/day, between about 10 mg/day and about 2500 mg/day or about 10 mg/day and about 2400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/day ( e.g ., every 24 hours) a dose between about 10 mg/day and about 2500 mg/day (e.g, between about 10 mg/day and about 2400 mg/day, between about 10 mg/day and about 2300 mg/day, between about 10 mg/day and about 2200 mg/day, between about 10 mg/day and about 2100 mg/day, between about 10 mg/day and about 2000 mg/day, between about 10 mg/day and about 1900 mg/day, between about 10 mg/day and about 1800 mg/day, between about 10 mg/day and about 1700 mg/day, between about 10 mg/day and about 1600 mg/day, between about 10 mg/day and about 1500 mg/day or about 10 mg/day and about 1400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/day (e.g, every 24 hours) a dose between about 10 mg/day and about 1500 mg/day (e.g, between about 10 mg/day and about 1400 mg/day, between about 10 mg/day and about 1300 mg/day, between about 10 mg/day and about 1200 mg/day, between about 10 mg/day and about 1100 mg/day, between about 10 mg/day and about 1000 mg/day, between about 10 mg/day and about 900 mg/day, between about 10 mg/day and about 800 mg/day, between about 10 mg/day and about 700 mg/day, between about 10 mg/day and about 600 mg/day, between about 10 mg/day and about 500 mg/day or about 10 mg/day and about 400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/day (e.g, every 24 hours) a dose between about 10 mg/day and about 500 mg/day (e.g, between about 10 mg/day and about 400 mg/day, between about 10 mg/day and about 300 mg/day, between about 10 mg/day and about 200 mg/day, between about 10 mg/day and about 100 mg/day, between about 10 mg/day and about 90 mg/day, between about 10 mg/day and about 80 mg/day, between about 10 mg/day and about 70 mg/day, between about 10 mg/day and about 60 mg/day, between about 10 mg/day and about 50 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 30 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 20 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/day (e.g, every 24 hours) a dose of about 8500 mg/day, about 8400 mg/day, about 8300 mg/day, about 8200 mg/day, about 8100 mg/day, about 8000 mg/day, about 7900 mg/day, about 7800 mg/day, about 7700 mg/day, about 7600 mg/day, about 7500 mg/day, about 7400 mg/day, about 7300 mg/day, about 7200 mg/day, about 7100 mg/day, about 7000 mg/day, about 6900 mg/day, about 6800 mg/day, about 6700 mg/day, about 6600 mg/day, about 6500 mg/day, about 6400 mg/day, about 6300 mg/day, about 6200 mg/day, about 6100 mg/day, about 6000 mg/day, about 5900 mg/day, about 5800 mg/day, about 5700 mg/day, about 5600 mg/day, about 5500 mg/day, about 5400 mg/day, about 5300 mg/day, about 5200 mg/day, about 5100 mg/day, about 5000 mg/day, about 4900 mg/day, about 4800 mg/day, about 4700 mg/day, about 4600 mg/day, about 4500 mg/day, about 4400 mg/day, about 4300 mg/day, about 4200 mg/day, about 4100 mg/day, about 4000 mg/day, about 3900 mg/day, about 3800 mg/day, about 3700 mg/day, about 3600 mg/day, about 3500 mg/day, about 3400 mg/day, about 3300 mg/day, about 3200 mg/day, about 3100 mg/day, about 3000 mg/day, about 2900 mg/day, about 2800 mg/day, about 2700 mg/day, about 2600 mg/day, about 2500 mg/day, about 2400 mg/day, about 2300 mg/day, about 2200 mg/day, about 2100 mg/day, about 2000 mg/day, about 1900 mg/day, about 1800 mg/day, about 1700 mg/day, about 1600 mg/day, about 1500 mg/day, about 1400 mg/day, about 1300 mg/day, about 1200 mg/day, about 1100 mg/day, about 1000 mg/day, about 900 mg/day, about 800 mg/day, about 700 mg/day, about 600 mg/day, about 500 mg/day, about 400 mg/day, about 300 mg/day, about 200 mg/day, about 100 mg/day, about 90 mg/day, about 80 mg/day, about 70 mg/day, about 60 mg/day, about 50 mg/day, about 40 mg/day, about 30 mg/day, about 20 mg/day, about 10 mg/day.

In some embodiments, the BlvrB inhibitor is administered 1 time/week.

In some embodiments, the method comprises administering to the subject 1 time/week a dose between about 10 mg/day and about 8500 mg/day ( e.g ., between about 10 mg/day and about 8400 mg/day, between about 10 mg/day and about 8300 mg/day, between about 10 mg/day and about 8200 mg/day, between about 10 mg/day and about 8100 mg/day, between about 10 mg/day and about 8000 mg/day, between about 10 mg/day and about 7900 mg/day, between about 10 mg/day and about 7800 mg/day, between about 10 mg/day and about 7700 mg/day, between about 10 mg/day and about 7600 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/week a dose between about 10 mg/day and about 7500 mg/day (e.g., between about 10 mg/day and about 7400 mg/day, between about 10 mg/day and about 7300 mg/day, between about 10 mg/day and about 7200 mg/day, between about 10 mg/day and about 7100 mg/day, between about 10 mg/day and about 7000 mg/day, between about 10 mg/day and about 6900 mg/day, between about 10 mg/day and about 6800 mg/day, between about 10 mg/day and about 6700 mg/day, between about 10 mg/day and about 6600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 6400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/week a dose between about 10 mg/day and about 6500 mg/day ( e.g ., between about 10 mg/day and about 6400 mg/day, between about 10 mg/day and about 6300 mg/day, between about 10 mg/day and about 6200 mg/day, between about 10 mg/day and about 6100 mg/day, between about 10 mg/day and about 6000 mg/day, between about 10 mg/day and about 5900 mg/day, between about 10 mg/day and about 5800 mg/day, between about 10 mg/day and about 5700 mg/day, between about 10 mg/day and about 5600 mg/day, between about 10 mg/day and about 6500 mg/day or about 10 mg/day and about 5400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/week a dose between about 10 mg/day and about 5500 mg/day (e.g., between about 10 mg/day and about 5400 mg/day, between about 10 mg/day and about 5300 mg/day, between about 10 mg/day and about 5200 mg/day, between about 10 mg/day and about 5100 mg/day, between about 10 mg/day and about 5000 mg/day, between about 10 mg/day and about 4900 mg/day, between about 10 mg/day and about 4800 mg/day, between about 10 mg/day and about 4700 mg/day, between about 10 mg/day and about 4600 mg/day, between about 10 mg/day and about 4500 mg/day or about 10 mg/day and about 4400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/week a dose between about 10 mg/day and about 4500 mg/day (e.g, between about 10 mg/day and about 4400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 4200 mg/day, between about 10 mg/day and about 4100 mg/day, between about 10 mg/day and about 4000 mg/day, between about 10 mg/day and about 3900 mg/day, between about 10 mg/day and about 3800 mg/day, between about 10 mg/day and about 3700 mg/day, between about 10 mg/day and about 3600 mg/day, between about 10 mg/day and about 3500 mg/day or about 10 mg/day and about 3400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/week a dose between about 10 mg/day and about 3500 mg/day (e.g, between about 10 mg/day and about 3400 mg/day, between about 10 mg/day and about 4300 mg/day, between about 10 mg/day and about 3200 mg/day, between about 10 mg/day and about 3100 mg/day, between about 10 mg/day and about 3000 mg/day, between about 10 mg/day and about 2900 mg/day, between about 10 mg/day and about 2800 mg/day, between about 10 mg/day and about 2700 mg/day, between about 10 mg/day and about 2600 mg/day, between about 10 mg/day and about 2500 mg/day or about 10 mg/day and about 2400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/week a dose between about 10 mg/day and about 2500 mg/day ( e.g ., between about 10 mg/day and about 2400 mg/day, between about 10 mg/day and about 2300 mg/day, between about 10 mg/day and about 2200 mg/day, between about 10 mg/day and about 2100 mg/day, between about 10 mg/day and about 2000 mg/day, between about 10 mg/day and about 1900 mg/day, between about 10 mg/day and about 1800 mg/day, between about 10 mg/day and about 1700 mg/day, between about 10 mg/day and about 1600 mg/day, between about 10 mg/day and about 1500 mg/day or about 10 mg/day and about 1400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/week a dose between about 10 mg/day and about 1500 mg/day (e.g., between about 10 mg/day and about 1400 mg/day, between about 10 mg/day and about 1300 mg/day, between about 10 mg/day and about 1200 mg/day, between about 10 mg/day and about 1100 mg/day, between about 10 mg/day and about 1000 mg/day, between about 10 mg/day and about 900 mg/day, between about 10 mg/day and about 800 mg/day, between about 10 mg/day and about 700 mg/day, between about 10 mg/day and about 600 mg/day, between about 10 mg/day and about 500 mg/day or about 10 mg/day and about 400 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/week a dose between about 10 mg/day and about 500 mg/day (e.g, between about 10 mg/day and about 400 mg/day, between about 10 mg/day and about 300 mg/day, between about 10 mg/day and about 200 mg/day, between about 10 mg/day and about 100 mg/day, between about 10 mg/day and about 90 mg/day, between about 10 mg/day and about 80 mg/day, between about 10 mg/day and about 70 mg/day, between about 10 mg/day and about 60 mg/day, between about 10 mg/day and about 50 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 30 mg/day, between about 10 mg/day and about 40 mg/day or about 10 mg/day and about 20 mg/day).

In some embodiments, the method comprises administering to the subject 1 time/week a dose of about 8500 mg/day, about 8400 mg/day, about 8300 mg/day, about 8200 mg/day, about 8100 mg/day, about 8000 mg/day, about 7900 mg/day, about 7800 mg/day, about 7700 mg/day, about 7600 mg/day, about 7500 mg/day, about 7400 mg/day, about 7300 mg/day, about 7200 mg/day, about 7100 mg/day, about 7000 mg/day, about 6900 mg/day, about 6800 mg/day, about 6700 mg/day, about 6600 mg/day, about 6500 mg/day, about 6400 mg/day, about 6300 mg/day, about 6200 mg/day, about 6100 mg/day, about 6000 mg/day, about 5900 mg/day, about 5800 mg/day, about 5700 mg/day, about 5600 mg/day, about 5500 mg/day, about 5400 mg/day, about 5300 mg/day, about 5200 mg/day, about 5100 mg/day, about 5000 mg/day, about 4900 mg/day, about 4800 mg/day, about 4700 mg/day, about 4600 mg/day, about 4500 mg/day, about 4400 mg/day, about 4300 mg/day, about 4200 mg/day, about 4100 mg/day, about 4000 mg/day, about 3900 mg/day, about 3800 mg/day, about 3700 mg/day, about 3600 mg/day, about 3500 mg/day, about 3400 mg/day, about 3300 mg/day, about 3200 mg/day, about 3100 mg/day, about 3000 mg/day, about 2900 mg/day, about 2800 mg/day, about 2700 mg/day, about 2600 mg/day, about 2500 mg/day, about 2400 mg/day, about 2300 mg/day, about 2200 mg/day, about 2100 mg/day, about 2000 mg/day, about 1900 mg/day, about 1800 mg/day, about 1700 mg/day, about 1600 mg/day, about 1500 mg/day, about 1400 mg/day, about 1300 mg/day, about 1200 mg/day, about 1100 mg/day, about 1000 mg/day, about 900 mg/day, about 800 mg/day, about 700 mg/day, about 600 mg/day, about 500 mg/day, about 400 mg/day, about 300 mg/day, about 200 mg/day, about 100 mg/day, about 90 mg/day, about 80 mg/day, about 70 mg/day, about 60 mg/day, about 50 mg/day, about 40 mg/day, about 30 mg/day, about 20 mg/day, about 10 mg/day.

In some embodiments, the BlvrB inhibitor is administered before, during, or after the administration of primaquine or tafenoquine. In some embodiments, the BlvrB inhibitor can be administered up to three days after the administration of primaquine or tafenoquine.

In some embodiments, the BlvrB inhibitor is phloxine B. In some embodiments, the BlvrB inhibitor is erythrosin B.

In some embodiments, the BlvrB inhibitor is micafungin (Mycamine). In some embodiments, micafungin is administered to a patient at a dose between 40-160 mg/day. In some embodiments, micafungin is administered to a patient at a dose between 50-150 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 160 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 150 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 140 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 130 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 120 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 110 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 100 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 90 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 80 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 70 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 60 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 50 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 40 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 30 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 20 mg/day. In some embodiments, micafungin is administered to a patient at a dose of about 10 mg/day.

In some embodiments, the BlvrB inhibitor is febuxostat (Uloric). In some embodiments, febuxostat is administered to a patient at a dose between 20-100 mg/day. In some embodiments, febuxostat is administered to a patient at a dose between 40-80 mg/day. In some embodiments, febuxostat is administered to a patient at a dose of about 100 mg/day. In some embodiments, febuxostat is administered to a patient at a dose of about 90 mg/day. In some embodiments, febuxostat is administered to a patient at a dose of about 80 mg/day. In some embodiments, febuxostat is administered to a patient at a dose of about 70 mg/day. In some embodiments, febuxostat is administered to a patient at a dose of about 60 mg/day. In some embodiments, febuxostat is administered to a patient at a dose of about 50 mg/day. In some embodiments, febuxostat is administered to a patient at a dose of about 40 mg/day. In some embodiments, febuxostat is administered to a patient at a dose of about 30 mg/day. In some embodiments, febuxostat is administered to a patient at a dose of about 20 mg/day.

In some embodiments, the BlvrB inhibitor is deferasirox (Exjade). In some embodiments, deferasirox is administered to a patient at a dose between 5 mg/kg/day and 25 mg/ kg/day. In some embodiments, deferasirox is administered to a patient at a dose between 10 mg/kg/day -20 mg/ kg/day. In some embodiments, deferasirox is administered to a patient at a dose of about 5 mg/kg/day. In some embodiments, deferasirox is administered to a patient at a dose of about 10 mg/kg/day. In some embodiments, deferasirox is administered to a patient at a dose of about 15 mg/kg/day. In some embodiments, deferasirox is administered to a patient at a dose of about 20 mg/kg/day. In some embodiments, deferasirox is administered to a patient at a dose of about 25 mg/kg/day.

In some embodiments, the BlvrB inhibitor is benzbromarone (Belsomra). In some embodiments, benzbromarone is administered to a patient at a dose between 2.5 mg/day - 25 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose between 5 mg/day - 20 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose of about 2.5 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose of about 5 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose of about 7.5 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose of about 10 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose of about 12.5 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose of about 15 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose of about 17.5 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose of about 20 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose of about 22.5 mg/day. In some embodiments, benzbromarone is administered to a patient at a dose of about 25 mg/day.

In some embodiments, the BlvrB inhibitor is triclabendazole (EGATEN®). In some embodiments, triclabendazole is administered to a patient at a dose between 5 mg/kg twice a day and 15 mg/ kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 4 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 5 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 6 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 7 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 8 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 9 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 10 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 11 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 12 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 13 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 14 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 15 mg/kg twice a day. In some embodiments, triclabendazole is administered to a patient at a dose of about 16 mg/kg twice a day.

In some embodiments, the BlvrB inhibitor is nifedipine (Procardia). In some embodiments, nifedipine is administered to a patient at a dose between 5 mg/day and 40 mg/day. In some embodiments, nifedipine is administered to a patient at a dose between 10 mg/day and 30 mg/day. In some embodiments, nifedipine is administered to a patient at a dose of about 5 mg/day. In some embodiments, nifedipine is administered to a patient at a dose of about 10 mg/day. In some embodiments, nifedipine is administered to a patient at a dose of about 15 mg/day. In some embodiments, nifedipine is administered to a patient at a dose of about 20 mg/day. In some embodiments, nifedipine is administered to a patient at a dose of about 25 mg/day. In some embodiments, nifedipine is administered to a patient at a dose of about 30 mg/day. In some embodiments, nifedipine is administered to a patient at a dose of about 35 mg/day. In some embodiments, nifedipine is administered to a patient at a dose of about 40 mg/day.

In some embodiments, the BlvrB inhibitors is zafirlukast. In some embodiments, zafirlukast is administered to a patient at a dose between 5 mg/ twice a day and 25 mg/ twice a day. In some embodiments, zafirlukast is administered to a patient at a dose between 10 mg/ twice a day and 20 mg/ twice a day. In some embodiments, zafirlukast is administered to a patient at a dose of about 5 mg/ twice a day. In some embodiments, zafirlukast is administered to a patient at a dose of about 10 mg/ twice a day. In some embodiments, zafirlukast is administered to a patient at a dose of about 15 mg/ twice a day. In some embodiments, zafirlukast is administered to a patient at a dose of about 20 mg/ twice a day. In some embodiments, zafirlukast is administered to a patient at a dose of about 25 mg/ twice a day.

In some embodiments, the BlvrB inhibitor is candesartan and/or its prodrug candesartan cilexetil. In some embodiments, candesartan Cilexetil is administered to a patient at a dose between 2 mg/day and 36 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose between 4 mg/day and 32 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 2 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 4 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 6 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 8 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 10 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 12 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 14 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 16 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 18 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 20 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 24 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 28 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 32 mg/day. In some embodiments, candesartan cilexetil is administered to a patient at a dose of about 36 mg/day.

In some embodiments, the BlvrB inhibitors is azilsartan medoxomil (EDARBI®). In some embodiments, azilsartan medoxomil is administered to a patient at a dose between 36 mg/day and 88 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose between 40 mg/day and 80 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 32 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 36 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 40 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 44 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 48 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 52 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 56 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 60 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 64 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 68 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 72 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 76 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 80 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 84 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 86 mg/day. In some embodiments, azilsartan medoxomil is administered to a patient at a dose of about 90 mg/day.

III.E. Routes of Administration

Suitable methods for administration of the compositions of the presently disclosed subject matter include, but are not limited to intravenous administration and delivery directly to a target tissue or organ. Exemplary routes of administration include parenteral, enteral, intravenous, intraarterial, intracardiac, intrapericardial, intraosseal, intracutaneous, subcutaneous, intradermal, subdermal, transdermal, intrathecal, intramuscular, intraperitoneal, intrastemal, parenchymatous, oral, sublingual, buccal, inhalational, and intranasal. The selection of a particular route of administration can be made based at least in part on the nature of the formulation and the ultimate target site where the compositions of the presently disclosed subject matter are desired to act. In some embodiments, the method of administration encompasses features for regionalized delivery or accumulation of the compositions at the site in need of treatment. In some embodiments, the compositions are delivered directly into the site to be treated. By way of example and not limitation, in some embodiments a composition of the presently disclosed subject matter is administered to the subject via a route selected from the group consisting of intraperitoneal, intramuscular, intravenous, and intranasal, or any combination thereof.

In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the presently disclosed subject matter. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, United Kingdom), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Indiana, United States of America), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, New Jersey, United States of America), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, California, United States of America), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPI PEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park, Illinois, United States of America), to name only a few. See e.g., U.S. Patent Nos. 7,762,994; 8,409,149; 8,556,864; 8,579,869; 9,011,391; and 9,265,893, the disclosure of each of which is incorporated herein by reference in its entirety.

The methods described herein use pharmaceutical compositions comprising the molecules described above, together with one or more pharmaceutically acceptable excipients or vehicles, and optionally other therapeutic and/or prophylactic ingredients. Such excipients include liquids such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, cyclodextrins, modified cyclodextrins (i.e., sufobutyl ether cyclodextrins), etc. Suitable excipients for non-liquid formulations are also known to those of skill in the art. Pharmaceutically acceptable salts can be used in the compositions of the present invention and include, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, and salts is available in Remington’s Pharmaceutical Sciences, 1990.

Additionally, auxiliary substances, such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, may be present in such vehicles. A biological buffer can be virtually any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, i.e., a pH in the physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank’s buffered saline, and the like.

Depending on the intended mode of administration, the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, may include other pharmaceutical agents, adjuvants, diluents, buffers, etc.

In some embodiments, the mode of administration is a solid dosage form, such as tablets and pills that are orally administered.

EXAMPLES

The presently disclosed subject matter will be now be described more fully hereinafter with reference to the accompanying EXAMPLES, in which representative embodiments of the presently disclosed subject matter are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the presently disclosed subject matter to those skilled in the art.

EXAMPLE 1

BlyrB Promotes Redox Cycling by a Primaquine Metabolite One of the main active metabolites of primaquine is primaquine 5,6-Orthoquinone (5,6-POQ). 5,6-POQ was incubated with NADPH (with or without recombinant BlvrB) and O2- generation was monitored by electron paramagnetic resonance (EPR; see Suzen et al.. 2017) using a spin probe specific for 02 ^_ (see Figure 2). The presence of BlvrB significantly enhanced 02 ^_ generation by 5,6-POQ in the presence of NADPH. These data demonstrated that BlvrB could promote redox cycling by 5,6-POQ.

EXAMPLE 2

BlyrB is Required for Induction of Full RBC Hemolysis by a Primaquine Metabolite At high doses (about 500 micromolar for 1 hour), incubation with 5,6-POQ causes hemolysis of even RBCs with normal G6PD activity. 5,6-POQ was incubated with either wild-type RBCs or RBCs from BlvrB knockout mice. The treated RBCs were then transfused into wild-type recipients and RBC circulation was established by flow cytometry. BlvrB KO RBCs were resistant to 5,6-POQ induced hemolysis compared to wild-type RBCs (FIGURE 3). It was unclear from these data if the residual toxicity in BlvrB KO RBCs was due to an alternate reductase or spontaneous transfer of e from NADPH to 5,6-POQ. Since such high levels of 5,6-POQ were used, spontaneous transfer may occur in this study but not happen in vivo under normal primaquine treatment conditions. It is also important to note that even partial inhibition of hemolysis may be sufficient to solve the problem of primaquine toxicity in G6PDd patients, as patients can tolerate mild anemia and young RBCs from G6PDd patients have higher G6PD activity.

EXAMPLE 3

Generation of Novel Humanized G6PD Deficient Mice and Control Mice To generate humanized G6PDd and nondeficient control mice, which differ only in their G6PD sequence, the murine G6PD locus was replaced with either the canonical (nondeficient) human G6PD gene (hG6PD(n) mouse) or with the common A- variant [V68M/N126D] (hG6PD(A-) mouse) (Figure 4A). The presence of homologous recombination was confirmed (and random integration ruled out) in ES cell clones and then again in germline mice. ES cells from a C57BL/6 (B6) genetic background were used. Exon 2 was not replaced, so as to avoid disrupting other genetic elements in that region; however, human and mouse G6PD are identical in exon 2 except for 2 amino acids. The NEO cassette was flanked with FRT sites and removed with FLT recombinase (the FRT scar is 3’ of the last exon). Except for the nucleotides encoding amino acids 68 and 126, the targeting constructs were identical, giving rise to isogenic hG6PD(n) and hG6PD(A-) mice.

Red blood cells (RBCs) from hG6PD(n) mice had similar G6PD activity as wild- type B6 mice (Figure 4B), but a significant decrease was seen in hG6PD(A-) RBCs (about 12% of normal) - G6PD activity in RBCs of A- humans ranges from 10-60%3, so the mice were on the lower range of what is seen in humans. qPCR specific for human G6PD mRNA showed similar levels of expression in bone marrow between hG6PD(n) and hG6PD(A-) strains (data not shown). In contrast, G6PD protein was initially undetectable by Western blot of cytoplasm from hG6PD(A-) RBCs (Figure 4C, top panel), but faintly visible upon overexposure (Figure 4C, middle panel). This was not due to differences in protein loading as assessed by beta-actin (Figure 4C, bottom panel). The antibody used for Western blotting was specific to human G6PD and detected no band in RBCs from wild-type B6 mice (even upon overexposure).

EXAMPLE 4

Primaquine Induces Hemolysis in G6PD Deficient Mice To generate an in vivo model of primaquine induced hemolysis, wild type mice, G6PD(n), and G6PD deficient mice ,G6PD(d), were injected daily with primaquine bisphosphate (50 mg/kg/day) for 5 days. At the end of 5 days, peripheral blood was obtained and tested for hematocrit, hemoglobin, and reticulocytes. Whereas no significant change in hematocrit or reticulocytes were observed in G6PDn mice treated with primaquine, G6PDd mice had an approximately 14% decrease in hematocrit and a greater than 2-fold increase in reticulocytes (FIGURE 5). These findings are consistent with hemolysis of RBCs (decreased hematocrit) and compensatory bone marrow induction of increased RBC production (increased reticulocytes). This is precisely the pattern seen in human G6PD deficient patients receiving primaquine.

EXAMPLE 5

In Vivo Administration of BlyrB Inhibitors Prevents Primaquine-induced Anemia in G6PD Deficient Mice

To test the effects of inhibitors of BlvrB on primaquine induced hemolysis in G6PDd mice, either phloxine B or erythrosin B were administered daily, at the same time as primaquine injection (50 mg/kg/day) for 5 days. A negative control group received no injections, and a positive control group received only primaquine injections. Consistent with previous findings, primaquine induced a decrease in hematocrit and an increase in reticulocytes in G6PDd mice (FIGURE 6). However, the addition of either phloxine B or erythrosin B substantially decreased (erythrosin B) or eliminated (phloxine B) both the decrease in hematocrit and the increase in reticulocytes. Initial studies started with a relatively high dose of phloxine B or erythrosin B, seeking to achieve a 10 micromolar steady state blood concentration.

EXAMPLE 6 hG6PD(A-) RBCs Show Increased Sensitivity to PM Induced Hemolysis Primaquine is itself a prodrug, with neither hemolytic nor anti-malarial activities; however, primaquine metabolites (generated by hepatic metabolism) are pharmacologically active. The two most active metabolites are 5-hydroxy primaquine and primaquine-5, 6- ortho-quinone. To create a reductionist in vitro system to allow more controlled experimental manipulation, RBCs from hG6PD(n) or hG6PD(A-) mice were incubated for 1 hour at 37 °C with 150 micromolar 5,6-POQ. Treated RBCs were washed and infused into recipient mice expressing a fluorescent transgene in RBCs. Circulating treated RBCs were enumerated by gating on non-fluore scent RBCs. Prior to transfusion, RBCs expressing a red fluorescent protein (mCherry-RBCs) were mixed with the test cell. By normalizing the ratio of test RBCs/mCherry RBCs post-transfusion to the pre-transfusion ratio, one can control for any alterations due to differences in transfusion volume or phlebotomy. Exposure to 5,6- POQ caused in vivo hemolysis of hG6PD(A-) but not hG6PD(n) (FIGURE 7). No RBC clearance was seen in RBCs from either strain treated with the vehicle needed to dissolve 5,6-POQ (MeOH).

EXAMPLE 7

Assessing the Requirement for BlyrB in 5.6-POO-induced Hemolysis in G6PDd RBCs BlvrB KO mice are crossed with G6PD(n) or G6PD(A-) mice. The following experimental groups are tested: (1) G6PD(n); (2) G6PD(A-); (3) BlvrBKO.G6PD(n); and (4) BlrvBKO.G6PD(A-). RBCs from each group are incubated with the established dose of 5,6-POQ that induces clearance of G6PD(A-) and not G6PD(n) RBCS (i.e. 150 M). Treated RBCs are transfused to assess effect of treatment on ability to circulate in vivo. RBCs are also smeared on glass slides, stained with Wright-Giemsa Stain, and analyzed microscopically. No significant hemolysis is observed for Group 1 and Group 3. Hemolysis is observed for Group 2. Significantly less hemolysis is observed in Group 4 compared to Group 2. Group 2 (and no other group) has formation of Heinz bodies in RBCs.

EXAMPLE 8

Assessing the Ability of Phloxine B and/or Erythrosin B to Inhibit BlyrB in Whole RBCs RBCs from G6PD(n) and G6PD(A-) mice are incubated at 37°C with a range of concentrations of phloxine B or erythrosin B. RBCs are harvested each hour for 6 hours, supernatants and washed cell pellets are isolated and snap frozen at -80 °C. Frozen pellets are sent to Svalinn Labs where they are processed for mass spectrometry analysis. Specialized mass spec methods that can distinguish oxidized from reduced flavins (oxidized vs. reduced riboflavin, FMN vs. FMNH2, and FAD vs. FADH2) are performed. Mass spec methods to quantify phloxine B or erythrosin B in supernatants and inside RBCs are also utilized. In addition, RBCs are tested for BlvrB activity using a commercially available enzyme activity kit (Millipore Sigma CS1100). BlvrA is present in RBCs at 50-100 fold lower levels than BlvrB and does not interfere with assays. The concentrations of phloxine B or erythrosin B needed to inhibit BlvrB are established based on the generated standard curve.

EXAMPLE 9

Testing the Safety and Efficacy of In Vivo Administration of BlyrB to G6PDd Mice Exposed to Primaquine

The following mice strains are injected with primaquine (50 mg/kg/day) for 5 days: (1) hG6PD(n); (2) hG6PD(A-); (3) hG6PD(n) x BlvrB KO; and (4) hG6PD(A-) x BlvrB KO. In a separate set of studies, hG6PD(n) or hG6PD(A-) mice are given the same course of primaquine, either alone, or with simultaneous injection with either phloxine B or erythrosin B. In all cases, cohorts of mice (n = 5) are sacrificed at days 1, 3, 5, and 7 and peripheral blood is analyzed with regards to HCT, Hb, MetHb, reticulocyte count, morphology by peripheral smear and assessment of Heinz bodies. Initial doses of phloxine B and erythrosin B are chosen based upon the dose titrations carried out in Example 8 and adjusted for mouse blood volume (approximately 2 ml). Initial studies are performed to establish the distribution between vascular and extravascular spaces, half-life, amount in RBCs, and inhibition of BlvrB in RBCs by the same mass spec based methods used in Example 8. The final doses tested are a focused titration around the established minimum dose that provides stable inhibition of BlvrB in RBCs as measured by ratios of (oxidized/reduced riboflavin, FMN/FMNH2, and FAD/FADH2). In addition, at each time point tested, RBCs are tested for BlvrB activity using a commercially available enzyme activity kit (Millipore Sigma CS1100). BlvrA is present in RBCs at 50-100 fold lower levels than BlvrB and does not interfere with the assays. It is observed that Group 4 has significantly less hemolysis (decreased hematocrit and decreased hemoglobin) and also less reticulocytosis and formation of Heinz bodies than group 2 and that no hemolysis, reticulocytosis or formation of Heinz bodies is observed in groups 1 or 3. It is also observed that the administration of Phloxine B and Erythrosin B inhibits BlvrB in RBCs of both hG6PD(n) and hG6PD(A-) mice and also decreases primaquine induced hemolysis, reticulocytosis, and formation of Heinz bodies in hG6PD(A-) to a significant extent.

REFERENCES

All references listed below, as well as all references cited in the instant disclosure, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries ( e.g ., GENBANK® and UniProt biosequence database entries and all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

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Table 1

Properties of Top BlyrB Inhibitors

^# ISR: intrinsic specificity ratio; * Flavin reductase IC50 presented as mean ± S.D. of the fit of the experiment run in duplicate.

Table 2

Inhibition of the FR Activity of BlyrB by Dyes

^† Values represent the mean ± SEM of experiments run in duplicate. Table 3

BlyrB Inhibition by NSC Inhibitor Compound