WO2022036111A1

WO2022036111A1 - Methods and compositions for treating sickle cell disease

Info

Publication number: WO2022036111A1
Application number: PCT/US2021/045765
Authority: WO
Inventors: Rahul Dilip BALLAL
Original assignee: Imara Inc.
Priority date: 2020-08-13
Filing date: 2021-08-12
Publication date: 2022-02-17

Abstract

The present disclosure relates to methods for decreasing or increasing one or more biomarkers by administering PDE9 pharmaceutical compositions comprising 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-a]pyrazin-8-one (Compound 1), or a pharmaceutically acceptable salt, solvate, or polymorph thereof, in a patient suffering from sickle cell disease (SCD) and/or sickle cell anemia (SCA).

Description

METHODS AND COMPOSITIONS FOR TREATING SICKLE CELL DISEASE

CROSS-REFERENCE

[001] This application claims the benefit of U.S. Application No. 63/065,389, filed August 13, 2020; U.S. Application No. 63/133,697, filed January 4, 2021; U.S. Application No. 63/134,113, filed January 5, 2021; U.S. Application No. 63/152,208, filed February 22, 2021; and U.S. Application No. 63/209,211, filed June 10, 2021, which are hereby incorporated by reference in their entirety.

FIELD OF THE DISCLOSURE

[002] The present disclosure relates to methods of using pharmaceutical compositions comprising cyclic guanylate monophosphate (cGMP)-specific phosphodiesterase type 9 inhibitors (hereinafter referred to as PDE9 inhibitors), namely 6-[(3S,4S)-4-methyl-l- (pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8- one (Compound 1), or a pharmaceutically acceptable salt, solvate, or polymorph thereof.

BACKGROUND

[003] Sickle Cell Disease (SCD, also called sickle cell anemia (SCA)) is a genetic disorder leading to vaso-occlusive processes responsible for much of the mortality in SCD patients. SCD disease results from a point mutation in the hemoglobin (HBB) gene producing abnormal sickle hemoglobin (HbS or HbSS), which polymerizes and creates rigid and sticky sickled red blood cells. Sickled red blood cells result in chronic inflammation, elevated cell adhesion, oxidative stress, and endothelial dysfunction culminating in vaso-occlusive processes. [004] There is to date no cure for SCD. Treatment options include blood transfusion and treatment with the anti-cancer agent hydroxyurea. Blood transfusions correct anemia by increasing the number of normal, non-sickled red blood cells in circulation. Regular transfusion therapy can help prevent recurring strokes in children at high risk. Hydroxyurea (HU) has been approved for the treatment of SCD and shown to reduce the frequency of painful crisis and hospitalization. Unfortunately, HU is often poorly tolerated and its widespread use is limited by concerns about its potential impact on fertility and reproduction; challenges achieving and maintaining an efficacious dose due to its hematologic toxicities; and requirements for monthly monitoring (Heeney et al., Pediatr Clin North Am., 2008, 55(2):483). In fact, it is estimated that only 1 out of 4 adult patients, and possibly even fewer, are treated with this drug (Stettler et al., JAMA, 2015, 313:1671). In addition, many patients are dosed with sub-efficacious doses of HU due to these challenges. Thus, novel, safe, and effective treatments that can be safely employed globally to prevent the morbid complications of SCD in patients of all ages are urgently needed. [005] There remains a need for treating sickle cell disease.

SUMMARY OF THE DISCLOSURE

[006] The present disclosure provides methods of making and using Compound 1 and/or pharmaceutical compositions comprising Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, to treat sickle cell disease.

[007] One aspect of the present invention provides methods of using PDE9 inhibitors of the present invention and pharmaceutical compositions comprising PDE9 inhibitors of the present invention.

[008] PDE9 inhibitors of the present invention may be used to treat sickle cell disease or any disease and/or symptom related to sickle cell disease, such as anemia, sickle-hemoglobin C disease (SC), beta thalassemia (beta-plus thalassemia and beta- zero thalassemia), vasoocclusive crisis, attacks of pain (sickle cell crisis), splenic sequestration crisis, acute chest syndrome, aplastic crisis, hemolytic crisis, long-term pain, bacterial infections, and stroke.

[009] One aspect, disclosed herein is a method of decreasing vaso-occlusive crisis (VOC) in a patient suffering therefrom, the method comprising administering a therapeutically effective amount of 6-[(3S,4S)-4-methyl-l-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3- tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8-one (Compound 1), or a pharmaceutically acceptable salt, solvate, or polymorph thereof. In some embodiments, the patient suffering therefrom is diagnosed with SCD, sickle cell anemia (SCA), thalassemia, or a combination thereof. In some embodiments, VOC is decrease by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% in number prior to treatment. In some embodiments, the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount of about at least 100 mg per day. In some embodiments, the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount of from about 100 mg to about 200 mg per day, from about 200 mg to about 500 mg per day, or from about 300 mg to about 800 mg per day. In some embodiments, the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount of about 100 mg per day, 200 mg per day, 300 mg per day, 400 mg per day, 500 mg per day , 600 mg per day, 700 mg per day, or 800 mg per day. In some embodiments, the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered orally once per day. In some embodiments, the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered for at least 4 weeks, 12 weeks, 16 weeks, or 24 weeks, 1 year, or 1.5 years. In some embodiments, the method further comprises administering hydroxyurea (HU).

[0010] In another aspect, disclosed herein is a method of modulating one or more biomarkers over baseline levels prior to treatment in a patient suffering from sickle cell disease (SCD), wherein said biomarkers are selected from absolute F-cell percentage, absolute fetal hemoglobin (HbF) percentage, mean corpuscular volume (MCV), and hemoglobin (Hb) level; the method comprising administering a therapeutically effective amount of 6-[(3S,4S)-4- methyl- 1 -(pyrimidin-2-ylmethyl)pyrrolidin-3 -y 1 ] -3 -tetrahydropyran-4-yl-7H-imidazo[ 1,5- a]pyrazin-8-one (Compound 1), or a pharmaceutically acceptable salt, solvate, or polymorph thereof.

[0011] In some embodiments, said absolute F-cell percentage is increased by at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30%. In some embodiments, said absolute HbF percentage is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%. In some embodiments, said MCV is increased by at least at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, at least 5.0 units, at least 6.0 units, at least 7.0 units, at least 8.0 units, at least 9.0 units, or at least 10.0 units. In some embodiments, said Hb level is increased by at least about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, or about 2.0 g/dL. In some embodiments, said method further comprises modulating one or more of a reticulocyte percentage, absolute reticulocyte count (ARC), bilirubin level, and LDH level. In some embodiments, said reticulocyte percentage is decreased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40% or about 50%. In some embodiments, said absolute reticulocyte count (ARC) is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, or at least 5.0 units. In some embodiments, said bilirubin level is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, or at least 5.0 units. In some embodiments, said LDH level is decreased is decreased by at least at least 5.0 units, at least 6.0 units, at least 7.0 units, at least 8.0 units, at least 9.0 units, at least 10 units, or at least 15 units. In some embodiments, Compound 1 is administered to the patient in an amount from about 200 mg to about 500 mg. In some embodiments, Compound 1 is administered to the patient in an amount of 200 mg or 300 mg. In some embodiments, the Compound 1 is administered orally once per day. In some embodiments, the Compound 1 is administered for at least 4 weeks, 12 weeks, 16 weeks, or 24 weeks. In some embodiments, the method further comprises administering hydroxyurea (HU).

[0012] Another aspect, disclosed herein is a method of modulating one or more biomarkers over baseline levels prior to treatment in a patient suffering from sickle cell disease (SCD), wherein said biomarkers are selected from reticulocyte percentage, absolute reticulocyte count (ARC), bilirubin level, and LDH level; the method comprising administering a therapeutically effective amount of 6-[(3S,4S)-4-methyl-l-(pyrimidin-2-ylmethyl)pyrrolidin-3- yl]-3-tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8-one (Compound 1), or a pharmaceutically acceptable salt, solvate, or polymorph thereof. In some embodiments, said reticulocyte percentage is decreased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40% or about 50%. In some embodiments, said absolute reticulocyte count (ARC) is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, or at least 5.0 units. In some embodiments, said bilirubin level is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, or at least 5.0 units. In some embodiments, said LDH level is decreased is decreased by at least at least 5.0 units, at least 6.0 units, at least 7.0 units, at least 8.0 units, at least 9.0 units, at least 10 units, or at least 15 units. In some embodiments, Compound 1 is administered to the patient in an amount from about 200 mg to about 500 mg. In some embodiments, Compound 1 is administered to the patient in an amount of 200 mg or 300 mg. In some embodiments, the Compound 1 is administered orally once per day. In some embodiments, the Compound 1 is administered for at least 4 weeks, 12 weeks, 16 weeks, or 24 weeks. In some embodiments, the method further comprises administering hydroxyurea (HU).

BRIEF DESCRIPTION OF DRAWINGS

[0013] Fig. 1 shows that single dose mean concentrations of 100 mg and 200 mg are above the IC90 for 16 to 18 hrs respectively. Predicted concentrations for phase 2b at 400 mg dose suggests a mean concentration above IC90 for full 24 hr.

[0014] Fig. 2 shows schematic of a SAD/MAD 800 mg QD or BID study.

[0015] Fig. 3 Shows a schematic summary of Phase 2a study with monotherapy of

Compound 1 and a combination therapy of Compound 1 and HU. [0016] Fig. 4 shows that F-cell % increases in Phase 2a. Mean absolute increase in F- cells of 5.9% in treatment interrupted patients after 4 months (left) and mean absolute increase in F-cells of 10.2% in roll -over patients after 4 months (right).

[0017] Fig. 5 shows that HbF increases in Phase 2a. Mean absolute increase in HbF of 1.7% in treatment interrupted patients after 4 months (left) and mean absolute increase in HbF of 2.3% in roll-over patients after 4 months (right).

[0018] Fig. 6 shows that Hb has minimal changes in Phase 2a. Small changes in Hb were seen across both interrupted and roll-over patients.

[0019] Fig. 7 shows that MCV increases in Phase 2a. Small changes in MCV were seen across both interrupted and roll over patients.

[0020] Fig. 8 shows trends in improvement in biomarkers of hemolysis across interrupted patient cohort at 4 months.

[0021] Fig. 9 shows trends in exploratory markers NTproBNP and CRP. NTproBNP levels show 55% mean decrease across interrupted cohort at 4 months.

[0022] Fig. 10 shows trends for markers of hemolysis for roll-over cohort.

[0023] Fig. 11 shows VOC events for representative Patient #1 treated with Compound 1 in Phase 2a direct-rollover monotherapy arm.

[0024] Fig. 12 shows results for representative Patient #1 in F-cells, HbF, Hb and MCV.

[0025] Fig. 13 shows results for representative Patient #1 in reties and reties %.

[0026] Fig. 14 shows VOC events for representative Patient #2 treated with Compound 1 in Phase 2a interrupted cohort of the combo therapy arm.

[0027] Fig. 15 shows results for representative Patient #2 in F-cells, HbF, Hb and MCV.

[0028] Fig. 16 shows results for representative Patient #2 in hemolysis biomarkers.

[0029] Fig. 17 shows that the annualized VOC rate decreased by 40% in the Compound

1 (IMR-687) treated groups versus the placebo groups in the Phase 2a study. The left side shows the mean annualized VOC rate while the right side shows the distribution of annualized VOC rates.

[0030] Fig. 18 shows the individual distribution of annualized VOC rates in the Compound 1 (IMR-687) treated groups versus the placebo groups in the Phase 2a study. The distribution shows that 52% of patients in the Compound 1 treated groups had zero VOCs versus 30% in the placebo groups.

[0031] Fig. 19 shows the Kaplan-Meier analysis of time to first VOC. The median time to first VOC for the pooled Compound 1 (IMR-687) treated groups was significantly longer than the placebo groups, 169 days versus 87 days respectively (p=0.029), in the Phase 2a study. [0032] Fig. 20 shows that individual distribution of annualized VOC rates in the Compound 1 (IMR-687) treated groups versus the placebo groups with or without HU in the Phase 2a study. The mean annualized VOC rate is shown on the left while the individual annualized VOC rate is shown on the right. The distribution shows that 60% of patients in the Compound 1 + HU treated groups had zero VOCs versus 30% in the placebo and HU groups and that the mean annualized VOC rate was 68% lower in the Compound 1+ HU groups versus the placebo + HU groups.

[0033] Fig. 21 shows the annualized VOC rate versus AUC with Compound 1. The trend demonstrates that VOC rate is reduced with increased Compound 1 (IMR-687) exposure (AUCo-24h), p=0.1192, in the Phase 2a study.

[0034] Fig. 22 shows that the ASCQ-Me pain episode frequency and severity improved with increased dosage of Compound 1 (IMR-687) with or without HU, in the Phase 2a study. [0035] Fig. 23 shows that Compound 1 (IMR-687) monotherapy results in overall increase in F-cell (%) over time. Both high dose and low dose groups showed increase in % cells containing HbF (F-cells) compared to placebo. LS mean (SEM) differences between the 100/200 mg group and placebo groups at week 24 was +11.66 (4.72), p=0.019, in the Phase 2a study.

[0036] Figs. 24A and 24B show correlation of F-cells % with HbF and AUC following Compound 1 (IMR-687) monotherapy up to Week 24. (24A) shows change in F cell % correlates with absolute change in HbF (%0, p=0.002. (24B) shows F-cell % correlates with Compound 1 exposure (AUCo-24h), p=0.043, in the Phase 2a study.

[0037] Fig. 25 shows the annualized VOC rate comparison between the parent study and the OLE study (left side) and a comparison between the placebo and the Open Label Extension (“OLE”) study (right side). It can be seen that subjects previously treated (e.g. in the Phase 2a study/ “Parent Study”) with Compound 1 (IMR-687) maintained low VOC rates in the OLE study and that patients previously treated (e.g. in the Phase 2a study/ “Parent Study”) with placebo had a 39% reduction in VOC rate when switched to Compound 1 in the OLE study. [0038] Figs. 26A and 26B show the absolute change from baseline to month 8 in HbF (%) and F-cell % in the OLE study. (26 A) shows that 36% of subjects had a > 3% response in HbF % at the Month 8 date. (26B) shows that 47% of subjects had a > 6% response in F-cell % at the 8 Month date, in the OLE study.

[0039] Fig. 27 shows reduction in VOC events in Patient #2 treated with Compound 1 (IMF-687) and HU. VOC rate was reduced by 75% during 12 months of OLE study relative to the 12 months prior to treatment (e.g. Phase 2a/Parent Study). [0040] Figs. 28A and 28B show an increase in both HbF and F-cells for OLE patients #1 and #2. (28A) shows an absolute increase in HbF of 3.7% and 8.7% for patients #1 and #2 respectively. (28B) shows an absolute increase in F-cell of 35.9% and 20.9% for patients #1 and #2 respectively.

[0041] Figs. 29A and 29B show a reduction biomarkers of hemolysis for OLE patients #1 and #2. (29A) shows a reduction in indirect bilirubin of -38% (month 24) for patient #1 and -33% (month 12) for patient #2. (29B) shows a reduction in absolute reticulocytes of -63% (month 18) for patient #1 and -12% (month 8) for patient #2.

DETAILED DESCRIPTION

[0042] Phosphodiesterases (PDEs) are a family of enzymes degrading cyclic nucleotides and thereby regulating the cellular levels of second messengers throughout the entire body. PDEs represent attractive drug targets, as proven by many compounds that have been introduced to clinical testing and the market, respectively. PDEs are encoded by 21 genes that are functionally separated into 11 families differing with respect to kinetic properties, substrate selectivity, expression, localization pattern, activation, regulation factors and inhibitor sensitivity. The function of PDEs is the degradation of the cyclic nucleotide monophosphates cyclic Adenosine Monophosphate (cAMP) and/or Guanosine Monophosphate (cGMP), which are important intracellular mediators involved in numerous vital processes including the control of neurotransmission and smooth muscle contraction and relaxation.

[0043] PDE9 is cGMP specific (K_m cAMP is >1000x for cGMP) and is hypothesized to be a key player in regulating cGMP levels as it has the lowest K_m among the PDEs for this nucleotide. PDE9 is expressed throughout the brain at low levels with the potential for regulating basal cGMP.

[0044] In the periphery, PDE9 expression is highest in prostate, intestine, kidney and haematopoietic cells, enabling therapeutic potential in various non-CNS indications.

[0045] In the present disclosure, pharmaceutical compositions comprising PDE9 inhibitors are designed for treatment for Sickle Cell Disease (SCD).

Compounds of the Disclosure

[0046] In the context of the present disclosure, a compound is considered to be a PDE9 inhibitor if the amount required to reach the 50% inhibition level PDE9 is 10 micromolar or less, preferably less than 9 micromolar, such as 8 micromolar or less, such as 7 micromolar or less, such as 6 micromolar or less, such as 5 micromolar or less, such as 4 micromolar or less, such as 3 micromolar or less, more preferably 2 micromolar or less, such as 1 micromolar or less, in particular 500 nM or less. In preferred embodiments the required amount of PDE9 inhibitor required to reach the IC50 level of PDE9 is 400nM or less, such as 300 nM or less, 200nM or less, 100 nM or less, or even 80 nM or less, such as 50 nM or less, for example 25 nM or less.

[0047] Throughout this application the notations IC50 and IC50 are used interchangeably. [0048] In some embodiments, the PDE9 inhibitor of the present disclosure has low or no blood brain barrier penetration. For example, the ratio of the concentration of a PDE9 inhibitor of the present disclosure in the brain to the concentration of it in the plasma (brain/plasma ratio) may be less than about 0.50, about 0.40, about 0.30, about 0.20, about 0.10, about 0.05, about 0.04, about 0.03, about 0.02, or about 0.01. In some embodiments, the brain/plasma ration is measured 30 min or 120 min after administration of the PDE9 inhibitor.

[0049] In some embodiments, the PDE9 inhibitor may be any imidazo pyrazinone PDE9 inhibitor disclosed in WO 2013/053690, the contents of which is incorporated herein by reference in its entirety. In some embodiments, the PDE9 inhibitor is Compound 1 or a pharmaceutically acceptable salt, cocrystal, solvate, or polymorph thereof. A racemate form of Compound 1 and an anhydrous form of Compound 1 have been described in WO 2013/053690 and WO 2017/005786, the contents of which are incorporated herein by reference in their entirety. In some embodiments, Compound 1 is IMR-687.

[0050] In some embodiments, the PDE9 inhibitor is 6-[(3S,4S)-4-methyl-l-(pyrimidin-2- ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8-one (Compound 1), or a pharmaceutically acceptable salt, solvate, or polymorph thereof. In some Compound 1 has the following structure:

6-[(3S,4S)-4-methyl-l-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H- imidazo[l,5-a]pyrazin-8-one; Formula C21H26N6O2; calculated molecular weight about 394 g/mol. In some embodiments, Compound 1 is enantiopure or substantially enantiopure.

Pharmaceutical compositions

[0051] The present disclosure further provides a pharmaceutical composition comprising a therapeutically effective amount of any of the PDE9 inhibitors and a pharmaceutically acceptable carrier or diluent. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, and a pharmaceutically acceptable carrier or diluent or excipient.

Pharmaceutically Acceptable Salts

[0052] The present disclosure also comprises salts of the PDE9 inhibitors, typically, pharmaceutically acceptable salts. Such salts include pharmaceutically acceptable acid addition salts. Acid addition salts include salts of inorganic acids as well as organic acids.

[0053] Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, sulfamic, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, itaconic, lactic, methanesulfonic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methane sulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids, theophylline acetic acids, as well as the 8- halotheophyllines, for example 8-bromotheophylline and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in Berge, S.M. et al., J. Pharm. Sci. 1977, 66, 2, the contents of which are hereby incorporated by reference.

[0054] Furthermore, the compounds of this disclosure may exist in unsolvated as well as in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of this disclosure.

[0055] In some embodiments, the pharmaceutical composition comprises Compound 1 as the solvated, unsolvated, or crystalline form. In some embodiments, Compound 1 is present as the unsolvated form. In some embodiments, Compound 1 is present as the present as the crystalline form. In some embodiments, Compound 1 is present as a monohydrate crystalline form.

Formulations

[0056] The compounds of the disclosure may be administered alone or in combination with pharmaceutically acceptable carriers, diluents or excipients, in either single or multiple doses. The pharmaceutical compositions according to the disclosure may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2013.

[0057] The pharmaceutical compositions may be specifically formulated for administration by any suitable route, such as oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intracisternal, intraperitoneal, vaginal, and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous, and intradermal) routes. It will be appreciated that the route will depend on the general health and age of the subject to be treated, the nature of the condition to be treated, and the active ingredient. In some embodiments, the pharmaceutical composition is formulated for oral administration to a subject. In some embodiments, the pharmaceutical composition is formulated as a tablet or pill. In some embodiments, the pharmaceutical composition is formulated as a solid tablet suitable for oral administration to a subject.

[0058] Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders, and granules. Where appropriate, the compositions may be prepared with coatings, such as enteric coatings or they may be formulated so as to provide controlled release of the active ingredient such as sustained or prolonged release according to methods well known in the art. Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups, and elixirs, either manufactured as such, or as a solid form for reconstitution prior to use.

[0059] Pharmaceutical compositions for parenteral administration include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions, or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. Other suitable administration forms include, but are not limited to, suppositories, sprays, ointments, creams, gels, inhalants, dermal patches, and implants.

[0060] The present disclosure also provides a process for making a pharmaceutical composition comprising admixing a therapeutically effective amount of a compound of the present disclosure and at least one pharmaceutically acceptable carrier or diluent.

[0061] The compounds of this disclosure are generally utilized as the free substance or as a pharmaceutically acceptable salt thereof. Such salts are prepared in a conventional manner by treating a solution or suspension of a compound of the present disclosure with a pharmaceutically acceptable acid. Representative examples of suitable organic and inorganic acids are described above.

[0062] For parenteral administration, solutions of the compounds of the present disclosure in sterile aqueous solution, aqueous propylene glycol, aqueous vitamin E or sesame or peanut oil may be employed. The aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The compounds of the present disclosure may be readily incorporated into known sterile aqueous media using standard techniques known to those skilled in the art.

[0063] Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers include lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers include, but are not limited to, syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. The pharmaceutical compositions formed by combining the compounds of the present disclosure and a pharmaceutically acceptable carrier are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration. The formulations may conveniently be presented in unit dosage form by methods known in the art of pharmacy.

[0064] Pharmaceutical compositions of the present disclosure suitable for oral administration may be presented as discrete units such as capsules or tablets, each containing a predetermined amount of the active ingredient, and optionally a suitable excipient.

Furthermore, the orally available formulations may be in the form of a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion.

[0065] The pharmaceutical composition comprises PDE9 inhibitors such as Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof. The pharmaceutical composition comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight of Compound 1. The pharmaceutical composition comprises at least about 1 % to about 90 % by weight of Compound 1. The pharmaceutical compositions comprises at least about 1 % to about 10 %, about 1 % to about 20 %, about 1 % to about 30 %, about 1 % to about 40 %, about 1 % to about 50 %, about 1 % to about 60 %, about 1 % to about 70 %, about 1 % to about 80 %, about 1 % to about 90 %, about 10 % to about 20 %, about 10 % to about 30 %, about 10 % to about 40 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 70 % to about 80 %, about 70 % to about 90 %, or about 80 % to about 90 % by weight of Compound 1. The pharmaceutical compositions comprise at least about 1 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, or about 90 % by weight of Compound 1. The pharmaceutical compositions comprise at least at least about 1 %, about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, or about 80 % by weight of Compound 1. The pharmaceutical compositions comprise at least at most about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, or about 90 % by weight of Compound 1. The pharmaceutical composition comprises at least about 90 % to about 99.9 % by weight of Compound 1. The pharmaceutical composition comprise at least about 90 % to about 91 %, about 90 % to about 92 %, about 90 % to about 93 %, about 90 % to about 94 %, about 90 % to about 95 %, about 90 % to about 96 %, about 90 % to about 97 %, about 90 % to about 98 %, about 90 % to about 99 %, about 90 % to about

99.9 %, about 91 % to about 92 %, about 91 % to about 93 %, about 91 % to about 94 %, about

91 % to about 95 %, about 91 % to about 96 %, about 91 % to about 97 %, about 91 % to about 98 %, about 91 % to about 99 %, about 91 % to about 99.9 %, about 92 % to about 93 %, about

92 % to about 94 %, about 92 % to about 95 %, about 92 % to about 96 %, about 92 % to about 97 %, about 92 % to about 98 %, about 92 % to about 99 %, about 92 % to about 99.9 %, about

93 % to about 94 %, about 93 % to about 95 %, about 93 % to about 96 %, about 93 % to about

97 %, about 93 % to about 98 %, about 93 % to about 99 %, about 93 % to about 99.9 %, about

94 % to about 95 %, about 94 % to about 96 %, about 94 % to about 97 %, about 94 % to about

98 %, about 94 % to about 99 %, about 94 % to about 99.9 %, about 95 % to about 96 %, about

95 % to about 97 %, about 95 % to about 98 %, about 95 % to about 99 %, about 95 % to about

99.9 %, about 96 % to about 97 %, about 96 % to about 98 %, about 96 % to about 99 %, about

96 % to about 99.9 %, about 97 % to about 98 %, about 97 % to about 99 %, about 97 % to about 99.9 %, about 98 % to about 99 %, about 98 % to about 99.9 %, or about 99 % to about

99.9 % by weight of Compound 1. The pharmaceutical composition comprises at least about 90 %, about 91 %, about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 %, or about 99.9 % by weight of Compound 1. The pharmaceutical compositions comprise at least at least about 90 %, about 91 %, about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 %, or about 99 % by weight of Compound 1. The pharmaceutical composition comprises at least at most about 91 %, about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 %, or about 99.9 % by weight of Compound 1. The pharmaceutical composition comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weight of Compound 1.

[0066] In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is formulated as a pharmaceutical composition for oral administration. For example, it may be in a solid tablet form.

[0067] In some other embodiments, the composition comprising Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is suitable for pediatric uses and can be taken by pediatric sickle cell anemia patients.

[0068] In some embodiments, the pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is taken with food. In some embodiments, the pharmaceutical composition, is taken after a meal. In some embodiments, the pharmaceutical composition, is taken without food.

Dosing

[0069] In some embodiments, the oral dosage ranges from about 0.001 to about 100 mg/kg body weight per day. In some embodiments, the oral dosage range is from about 0.01 to about 50 mg/kg body weight per day. In some embodiments, the oral dosage range is from about 0.05 to about 10 mg/kg body weight per day. Oral dosages are usually administered in one or more dosages, typically, one to three dosages per day. In some embodiments, the dose is administered once, twice, or three times a day. The exact dosage will depend upon the frequency and mode of administration, the gender, age, weight, and general health of the subject treated, the nature and severity of the condition treated and any concomitant diseases to be treated and other factors evident to those skilled in the art.

[0070] In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to a subject in need thereof, at a dose of less than 6.0 mg/kg or less than about 4.0 mg/kg per body weight of the subject. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered at a dose of from about 0.1 mg/kg to about 25.0 mg/kg per body weight of the subject. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered at a dose of from about 0.1 mg/kg to about 6.0 mg/kg per body weight of the subject. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered at a dose of from about 4.5 mg/kg to about 12.0 mg/kg per body weight of the subject. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered at a dose of from about 4.5 mg/kg to about 6.7 mg/kg per body weight of the subject. For example, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered at a dose of from about 0.3 to about 3.0 mg/kg, or from about 0.3 to about 1.0 mg/kg per body weight of the subject. The patient may have sickle cell disease. The patient may be an adult (>18 years old) or a child (<18 years old). In some embodiments, the patient receives Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof at a dose of around 0.3 mg/kg, around 0.2 mg/kg, around 0.1 mg/kg, or around 0.05 mg/kg per body weight of the subject. In some embodiments, the patient receives Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, at about 1 mg/kg per body weight of the subject. In some embodiments, the patient receives Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, at about 3 mg/kg per body weight of the subject. In some embodiments, the patient receives Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, at about 6 mg/kg per body weight of the subject.

[0071] In some embodiments, the patient receives Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, at about 1 mg/kg per body weight of the subject. [0072] In some embodiments, the patient receives Compound 1 or a pharmaceutically, acceptable salt, solvate, or polymorph thereof, at about 5 mg/kg per body weight of the subject. [0073] In some embodiments, the patient receives Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, at about 6 mg/kg per body weight of the subject. [0074] In some embodiments, the patient receives Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, at about 10 mg/kg per body weight of the subject.

[0075] In some embodiments, the patient receives Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, at about 12 mg/kg per body weight of the subject.

[0076] In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered to a patient in need thereof, at a flat dose of about 100 mg, about 200 mg, about 300 mg, about 400, about 500 mg, about 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg per day, 1,200 mg per day, 1,400 mg per day, or 1,600 mg per day. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to a patient at a dose about lOOmg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, or about 350 mg, about 400 mg, about, 450 mg, about 500, about 600 mg, about 700 mg, or about 800 mg per day. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 100 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered at a dose of about 150 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 200 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 250 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 300 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 350 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 400 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 450 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 500 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 600 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 800 mg. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a dose of about 1 g.

[0077] In some embodiments, of the pharmaceutical composition comprising Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered at a maximum dose per day or per dose. In some embodiments, a total combined dose of 1g of Compound 1, or a pharmaceutically acceptable salt, solvate or polymorph thereof, is administered per day or per dose. In some embodiments, a total combined dose of 800 mg Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered per day or per dose. In some embodiments, a total combined dose of 600 mg Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered per day or per dose. In some embodiments, a total combined dose of 500 mg Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered per day or per dose. In some embodiments, a total combined dose of 400 mg Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered per day or per dose. In some embodiments, a total combined dose of 300 mg Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered per day or per dose. In some embodiments, a total combined dose of 200 mg Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered per day or per dose. In some embodiments, Compound 1 or a pharmaceutically acceptable salt or polymorph thereof, is administered to a patient, wherein Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered once a day. In some embodiments, the pharmaceutical composition comprising Compound 1, is administered twice a day.

[0078] In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to a patient, wherein Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered once a day with food. It has been found that food can dramatically reduce the adverse event profile. The incidence and severity of the side effects, such as nausea, emesis and headache, can be reduced when Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is taken with food.

[0079] In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, is administered to a patient, wherein Compound 1 is administered once a day for at least 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, a year, 1.5 years, or 2 years. In some embodiments, the patient is treated for 3 months. In some embodiments, the patient is treated for 6 months. In some embodiments, the patient is treated for 1 year. In some embodiments, the patient is treated for 1.5 years. In some embodiments, the patient is treated for 2 years, 3 years, 4 years, 5 years, over 5 years, or the duration of life.

[0080] In some embodiments, the pharmaceutical compositions are presented in a unit dosage form by methods known to those skilled in the art. For illustrative purposes, a typical unit dosage form for oral administration may contain from about 0.01 to about 1000 mg, from about 0.05 to about 500 mg, or from about 0.5 mg to about 200 mg.

[0081] In some embodiments, Compound 1 can be administered at once-daily doses of 50 mg to 100 mg (lower dosage) through 12 weeks and then higher doses of 100 mg to 200 mg, respectively, through an additional 12 weeks. In some embodiments, Compound 1 can be administered at once-daily doses of 100 mg to 200 mg (lower dosage) through 12 weeks and then higher doses of 300 mg to 400 mg, respectively, through an additional 12 weeks. In some embodiments, Compound 1 can be administered at once-daily doses of 300 mg to 400 mg (lower dosage) through 12 weeks and then higher doses of 600 mg to 800 mg, respectively, through an additional 12 weeks. In some embodiments, Compound 1 can be administered at once-daily doses of about 3.4 to 5.0 mg/kg (lower dosage) through 12 weeks and then higher doses of about 5.0 to 6.7 mg/kg, respectively, through an additional 12 weeks. In some embodiments, Compound 1 can be administered at once-daily doses of about 5.0 to 6.7 mg/kg (lower dosage) through 12 weeks and then higher doses of about 6.7 to 12.3 mg/kg, respectively, through an additional 12 weeks. In some embodiments, the lower dosage is administered through about 1 week to about 12 weeks. In some embodiments, the lower dosage is administered through about 1 week to about 2 weeks, about 1 week to about 3 weeks, about 1 week to about 4 weeks, about 1 week to about 5 weeks, about 1 week to about 6 weeks, about 1 week to about 7 weeks, about 1 week to about 8 weeks, about 1 week to about 9 weeks, about 1 week to about 10 weeks, about 1 week to about 11 weeks, about 1 week to about 12 weeks, about 2 weeks to about 3 weeks, about 2 weeks to about 4 weeks, about 2 weeks to about 5 weeks, about 2 weeks to about 6 weeks, about 2 weeks to about 7 weeks, about 2 weeks to about 8 weeks, about 2 weeks to about 9 weeks, about 2 weeks to about 10 weeks, about 2 weeks to about 11 weeks, about 2 weeks to about 12 weeks, about 3 weeks to about 4 weeks, about 3 weeks to about 5 weeks, about 3 weeks to about 6 weeks, about 3 weeks to about 7 weeks, about 3 weeks to about 8 weeks, about 3 weeks to about 9 weeks, about 3 weeks to about 10 weeks, about 3 weeks to about 11 weeks, about 3 weeks to about 12 weeks, about 4 weeks to about 5 weeks, about 4 weeks to about 6 weeks, about 4 weeks to about 7 weeks, about 4 weeks to about 8 weeks, about 4 weeks to about 9 weeks, about 4 weeks to about 10 weeks, about 4 weeks to about 11 weeks, about 4 weeks to about 12 weeks, about 5 weeks to about 6 weeks, about 5 weeks to about 7 weeks, about 5 weeks to about 8 weeks, about 5 weeks to about 9 weeks, about 5 weeks to about 10 weeks, about 5 weeks to about 11 weeks, about 5 weeks to about 12 weeks, about 6 weeks to about 7 weeks, about 6 weeks to about 8 weeks, about 6 weeks to about 9 weeks, about 6 weeks to about 10 weeks, about 6 weeks to about 11 weeks, about 6 weeks to about 12 weeks, about 7 weeks to about 8 weeks, about 7 weeks to about 9 weeks, about 7 weeks to about 10 weeks, about 7 weeks to about 11 weeks, about 7 weeks to about 12 weeks, about 8 weeks to about 9 weeks, about 8 weeks to about 10 weeks, about 8 weeks to about 11 weeks, about 8 weeks to about 12 weeks, about 9 weeks to about 10 weeks, about 9 weeks to about 11 weeks, about 9 weeks to about 12 weeks, about 10 weeks to about 11 weeks, about 10 weeks to about 12 weeks, or about 11 weeks to about 12 weeks. In some embodiments, the lower dosage is administered through about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In some embodiments, the lower dosage is administered through at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or about 11 weeks. In some embodiments, the lower dosage is administered through at most about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In some embodiments, the higher dosage is administered through about 1 week to about 12 weeks. In some embodiments, the higher dosage is administered through about 1 week to about 2 weeks, about 1 week to about 3 weeks, about 1 week to about 4 weeks, about 1 week to about 5 weeks, about 1 week to about 6 weeks, about 1 week to about 7 weeks, about 1 week to about 8 weeks, about 1 week to about 9 weeks, about 1 week to about 10 weeks, about 1 week to about 11 weeks, about 1 week to about 12 weeks, about 2 weeks to about 3 weeks, about 2 weeks to about 4 weeks, about 2 weeks to about 5 weeks, about 2 weeks to about 6 weeks, about 2 weeks to about 7 weeks, about 2 weeks to about 8 weeks, about 2 weeks to about 9 weeks, about 2 weeks to about 10 weeks, about 2 weeks to about 11 weeks, about 2 weeks to about 12 weeks, about 3 weeks to about 4 weeks, about 3 weeks to about 5 weeks, about 3 weeks to about 6 weeks, about 3 weeks to about 7 weeks, about 3 weeks to about 8 weeks, about 3 weeks to about 9 weeks, about 3 weeks to about 10 weeks, about 3 weeks to about 11 weeks, about 3 weeks to about 12 weeks, about 4 weeks to about 5 weeks, about 4 weeks to about 6 weeks, about 4 weeks to about 7 weeks, about 4 weeks to about 8 weeks, about 4 weeks to about 9 weeks, about 4 weeks to about 10 weeks, about 4 weeks to about 11 weeks, about 4 weeks to about 12 weeks, about 5 weeks to about 6 weeks, about 5 weeks to about 7 weeks, about 5 weeks to about 8 weeks, about 5 weeks to about 9 weeks, about 5 weeks to about 10 weeks, about 5 weeks to about 11 weeks, about 5 weeks to about 12 weeks, about 6 weeks to about 7 weeks, about 6 weeks to about 8 weeks, about 6 weeks to about 9 weeks, about 6 weeks to about 10 weeks, about 6 weeks to about 11 weeks, about 6 weeks to about 12 weeks, about 7 weeks to about 8 weeks, about 7 weeks to about 9 weeks, about 7 weeks to about 10 weeks, about 7 weeks to about 11 weeks, about 7 weeks to about 12 weeks, about 8 weeks to about 9 weeks, about 8 weeks to about 10 weeks, about 8 weeks to about 11 weeks, about 8 weeks to about 12 weeks, about 9 weeks to about 10 weeks, about 9 weeks to about 11 weeks, about 9 weeks to about 12 weeks, about 10 weeks to about 11 weeks, about 10 weeks to about 12 weeks, or about 11 weeks to about 12 weeks. In some embodiments, the higher dosage is administered through about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In some embodiments, the higher dosage is administered through at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or about 11 weeks. In some embodiments, the higher dosage is administered through at most about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In some embodiments, the dosage of Compound 1 is escalated 4 weeks.

Combination Therapies

[0082] In another embodiment, the pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is used in combination with an additional active agent, such as Hydroxyurea (HU). Compound 1 and the additional active agent may be administered simultaneously, sequentially, or in any order. Compound 1 and the additional active agent may be administered at different dosages, with different dosing frequencies, or via different routes, whichever is suitable.

[0083] The term “administered simultaneously”, as used herein, is not specifically restricted and means that the compounds of the present disclosure and the additional active agent are substantially administered at the same time, e.g. as a mixture or in immediate subsequent sequence.

[0084] The term “administered sequentially”, as used herein, is not specifically restricted and means that the compounds of the present disclosure and the additional active agent are not administered at the same time but one after the other, or in groups, with a specific time interval between administrations. The time interval may be the same or different between the respective administrations of the compounds of the present disclosure and the additional active agent and may be selected, for example, from the range of 2 minutes to 96 hours, 1 to 7 days or one, two, or three weeks. Generally, the time interval between the administrations may be in the range of a few minutes to hours, such as in the range of 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 to 12 hours. Further examples include time intervals in the range of 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12 hours.

[0085] In some embodiments, HU is administered at the standard of care dose for treating SCD.

[0086] In some embodiments, HU is administered to the patient in need thereof at about 1 g to 5 g per day or per dose. In some embodiments, HU is administered at about 1,000 mg, about 1,500 mg, about 2,000 mg, about 2,500 mg, about 3,000 mg, about 3,500 mg, about 4,000 mg, about 4,500 mg, or about 5,000 mg per dose or per day. In some embodiments, HU is administered at about 1,000 mg per day. In some embodiments, HU is administered at about 1,500 mg per day. In some embodiments, HU is administered at about 2,000 mg per day. In some embodiments, HU is administered at about 2,500 mg per day. In some embodiments, HU is administered at about 3,000 mg per day. In some embodiments, HU is administered at about 3,500 mg per day. In some embodiments, HU is administered at about 4,000 mg per day. In some embodiments, HU is administered at about 5,000 mg per day.

[0087] The molar ratio of the compounds of the present disclosure and the additional active agent is not particularly restricted. For example, when the compounds of the present disclosure and one additional active agent are combined in a composition, the molar ratio of them may be in the range of 1 :500 to 500: 1, or of 1 :100 to 100: 1, or of 1 :50 to 50: 1, or of 1 :20 to 20: 1, or of 1 :5 to 5: 1, or 1 : 1. Similar molar ratios apply when the compounds of the present disclosure and two or more other active agents are combined in a composition. The compounds of the present disclosure compounds of the present disclosure may comprise a predetermined molar weight percentage from about 1% to 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to 40%, or about 40% to 50%, or about 50% to 60%, or about 60% to 70%, or about 70% to 80%, or about 80% to 90%, or about 90% to 99% of the composition.

Methods of Using Compounds of the Disclosure

[0088] PDE9 is expressed specifically in the human haematopoietic system including neutrophils, reticulocytes erythroid and erythrol eukaemic cells. Furthermore, SCD patients exhibit a marked and significant elevation of PDE9 expression in reticulocytes and neutrophils compared to healthy individuals (Almeida et al., Br J Haematol. 2008 Sep; 142(5), 836). Evidence additionally demonstrates a link between PDE9 and cell adhesion since pharmacologic PDE9 inhibition ameliorates the increased adhesive properties of SCD neutrophils (Miguel et al., Inflamm Res. 2011 Jul; 60(7), 633). The mechanism by which PDE9 inhibition decreases cell adhesion has been shown to be mediated by increased cGMP and decreased endothelial adhesion molecule expression. Importantly, in an animal model of SCD, the PDE9 inhibitor-mediated decrease in cell adhesion had the functional effect of increased cell survival. In addition to demonstrating decreased cell adhesion comparable to HU, PDE9 inhibition resulted in increased fetal non-sickled haemoglobin (HbF) production, which reduced the cellular concentration of abnormal haemoglobin (HbS) within red blood cells (RBCs) resulting in less polymerization of the abnormal haemoglobin and its associated sequelae. The importance of increasing HbF in treating SCD is evidenced by results of large studies like the Cooperative Study of Sickle Cell Disease, as well as studies in a variety of patient cohorts outside of the United States, showing that HbF is among the most important modifiers of this disease (Alsultan et al., Am JHematoL 2013, 88(6), 531) as well as data showing that modifiers of HbF improve other hematological parameters (Akinsheye, Blood, 2011, 118(1): 19). Finally, Almeida and colleagues demonstrated that treatment with HU combined with PDE9 inhibition in a mouse model of SCD leads to an additional beneficial amplification of the cGMP elevating effects of HU (Almeida et al., Blood. 2012 Oct ; 120(14), 2879). In conclusion, PDE9 inhibition can modulate both the expression of fetal haemoglobin production as well as decrease cell adhesion, both mechanisms key for the treatment of SCD.

[0089] PDE9 inhibitors, e.g. Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof, and hydroxyurea (HU) act through different mechanisms. HU increases nitric oxide (NO) levels, which activate soluble guanylyl cyclase (sGC) to generate cGMP. PDE9 inhibitors of the present disclosure block the degradation of cGMP by inhibiting PDE9 enzymatic activity, thus elevating cGMP levels. In erythroid lineages, cGMP binds to protein kinase G (PKG) and signals synthesis of fetal gamma globin and ultimately production of HbF. In hematopoietic cells where PDE9 expression is high, the direct inhibition of PDE9 activity increases cGMP levels, which promotes decreased leucocyte adhesion.

[0090] One aspect, of the present disclosure provides methods of using PDE9 inhibitors and pharmaceutical compositions comprising PDE9 inhibitors, e.g. Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof.

[0091] Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof may be used to treat sickle cell disease or any disease and/or symptom related to sickle cell disease (SCD), such as anemia, sickle-hemoglobin C disease (SC), vaso-occlusive crisis (VOC), attacks of pain (sickle cell crisis), beta thalassemia (beta-plus thalassemia and betazero thalassemia), splenic sequestration crisis, acute chest syndrome, aplastic crisis, hemolytic crisis, long-term pain, bacterial infections, and stroke.

[0092] The most common clinical manifestation of SCD is vaso-occlusive crisis. A vasoocclusive crisis occurs when the microcirculation is obstructed by sickled RBCs, causing ischemic injury to the organ supplied and resultant pain. Pain crises constitute the most distinguishing clinical feature of sickle cell disease and are the leading cause of emergency department visits and hospitalizations for affected patients. Approximately half the individuals with homozygous Hbs disease experience vaso-occlusive crisis. The frequency of crisis is extremely variable. Some have as many as 6 or more episodes annually, whereas others may have episodes only at great intervals or none at all. Each individual typically has a consistent pattern for crisis frequency. In some embodiments, Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof is used to treat the effects of sickle cell disease (SCD). Compound 1 may be used to decrease the number of vaso-occlusive crises and/or rate of developing a vaso-occlusive crisis (VOC). In some embodiments, the number of VOCs decrease by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the number or VOCs decrease by about 55%. In some instances, VOCs that result in hospitalization decrease by at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the number of VOCs decrease by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% in subjects suffering from SCD (or SC A) and/or thalassemia, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the thalassemia is betathalassemia.

[0093] In another embodiments, treatment with Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof; produces an effect on one or more SCD (or SCA) and/or thalassemia biomarkers, such as but not limited to fetal hemoglobin (HbF) and hemoglobin levels (Hb), compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the biomarkers are decreased or increase after the administration of Compound 1 to the subject in need thereof, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the biomarkers are decreased. In some embodiments, the biomarkers are increased.

[0094] In another embodiment, Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is used to increase cGMP levels in a cell or in the plasma of a subject, wherein the subject has SCD (or SCA) and/or thalassemia. The cell may be, but not limited to, red blood cells and/or white blood cells. The cGMP level may be increased by at least 50%, at least 100%, or at least 150%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the cGMP level may be increased at least 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, or 25 times, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1).

[0095] In another embodiment, Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, increases hemoglobin (Hb) levels in a subject, wherein the subject has SCD (or SCA) and/or thalassemia. In some embodiments, the Hb level is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 150%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the Hb level is increased by at least about 1 % to about 10 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the Hb level is increased by at least about 1 % to about 2 %, about 1 % to about 3 %, about 1 % to about 4 %, about 1 % to about 5 %, about 1 % to about 10 %, about 2 % to about 3 %, about 2 % to about 4 %, about 2 % to about 5 %, about 2 % to about 10 %, about 3 % to about 4 %, about 3 % to about 5 %, about 3 % to about 10 %, about 4 % to about 5 %, about 4 % to about 10 %, or about 5 % to about 10 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the Hb level is increased by at least about 1 %, about 2 %, about 3 %, about 4 %, about 5 %, or about 10 % compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the Hb level is increased by at least at least about 1 %, about 2 %, about 3 %, about 4 %, or about 5 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the Hb level is increased by at least at most about 2 %, about 3 %, about 4 %, about 5 %, or about 10 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the Hb level is increased by at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, or 25 times, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, Compound 1 increases the absolute level of hemoglobin (Hb) by about 0.1 g/dL to about 5 g/d or by about 0.5 g/dL to about 1.5 g/dL. In some embodiments, the absolute level of hemoglobin is increased by about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.2, about 1.3, about 1.4, about 1.5, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.5, about 3.0, about 4.0 or about 5.0 g/dL. In some embodiments, the absolute level of hemoglobin is increased by about 1.0 g/dL.

[0096] In another embodiment, Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, increases fetal hemoglobin (HbF) positive red blood cell number in a subject, wherein the subject has SCD (or SCA) and/or thalassemia. In some embodiments, the HbF positive red blood cell number is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 150%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the HbF positive red blood cell number is increased by at least about 1 % to about 10 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the HbF positive red blood cell number is increased by at least about 1 % to about 2 %, about 1 % to about 3 %, about 1 % to about 4 %, about 1 % to about 5 %, about 1 % to about 10 %, about 2 % to about 3 %, about 2 % to about 4 %, about 2 % to about 5 %, about 2 % to about 10 %, about 3 % to about 4 %, about 3 % to about 5 %, about 3 % to about 10 %, about 4 % to about 5 %, about 4 % to about 10 %, or about 5 % to about 10 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the HbF positive red blood cell number is increased by at least about 1 %, about 2 %, about 3 %, about 4 %, about 5 %, or about 10 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the HbF positive red blood cell number is increased by at least at least about 1 %, about 2 %, about 3 %, about 4 %, or about 5 %. In some embodiments, the HbF positive red blood cell number is increased by at least at most about 2 %, about 3 %, about 4 %, about 5 %, or about 10 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the HbF positive red blood cell number is increased by at least 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, or 25 times, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, HbF is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1).

[0097] In another embodiment, Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, increases the absolute F-cell percentage. In some embodiments, the absolute F-cell percentage is increased from at least about 5% to at least about 50%. In some embodiments, the absolute F-cell percentage is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 150%. In some embodiments, the absolute F-cell percentage is increased by at least about 1 % to about 10 %. In some embodiments, the absolute F-cell percentage is increased by at least about 1 % to about 2 %, about 1 % to about 3 %, about 1 % to about 4 %, about 1 % to about 5 %, about 1 % to about 10 %, about 2 % to about 3 %, about 2 % to about 4 %, about 2 % to about 5 %, about 2 % to about 10 %, about 3 % to about 4 %, about 3 % to about 5 %, about 3 % to about 10 %, about 4 % to about 5 %, about 4 % to about 10 %, or about 5 % to about 10 %. In some embodiments, the absolute F-cell percentage is increased by at least about 1 %, about 2 %, about 3 %, about 4 %, about 5 %, or about 10 %. In some embodiments, the absolute F-cell percentage is increased by at least at least about 1 %, about 2 %, about 3 %, about 4 %, or about 5 %. In some embodiments, the absolute F-cell percentage is increased by at least at most about 2 %, about 3 %, about 4 %, about 5 %, or about 10 %. In some embodiments, the absolute F-cell percentage is increase by at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least 31%, at least 32%, at least 33%, at least 34%, or at least 35%. [0098] In another embodiment, Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, increases in mean corpuscular volume (MCV). In some embodiments, MCV is increased by at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 150%. In some embodiments, MCV is increased by at least 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, or 25 times. In some embodiments, MCV is increased by at least 1.5 units, 2.0 units, 2.5 units, 3.0 units, 3.5 units, 4.0 units, 4.5 units, 5.0 units, 6.0 units, 7.0 units, 8.0 units, 9.0 units, 10.0 units, 12.5 units, or 15.0 units. In some embodiments, MCV is increased by about 3.5 units.

[0099] Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, produces improvements across markers of hemolysis, such as but not limited to. percentage reticulocytes, total leucocytes, and total bilirubin. In some embodiments, Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof; is used to reduce sickle red blood cell percentage (% sickle RBC), stasis percentage (% stasis), total bilirubin, or total leucocyte count in a subject, wherein the subject has SCD (or SC A) and/or thalassemia. The % sickle RBC, % stasis, total bilirubin, total leucocyte count or spleen weight is decreased by at least 10%, 20%, 30%, 40%, 50%, 60% or 70%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1).

[00100] In some embodiments, Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, decreases the percentage of reticulocytes and the absolute reticulocyte count (ARC). In some embodiments, Compound 1, decreases the percentage of reticulocytes. In some embodiments, the percentage of reticulocytes is decreased by at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 150%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the percentage reticulocytes is decreased by at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the percentage of reticulocytes and the absolute reticulocyte count is decreased by at least about 10 % to about 50 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the percentage of reticulocytes and the absolute reticulocyte count is decreased by at least about 10 % to about 15 %, about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 15 % to about 20 %, about 15 % to about 25 %, about 15 % to about 30 %, about 15 % to about 35 %, about 15 % to about 40 %, about 15 % to about 45 %, about 15 % to about 50 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 40 % to about 45 %, about 40 % to about 50 %, or about 45 % to about 50 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the percentage of reticulocytes and the absolute reticulocyte count is decreased by at least about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, or about 50 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the percentage of reticulocytes and the absolute reticulocyte count is decreased by at least at least about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, or about 45 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the percentage of reticulocytes and the absolute reticulocyte count is decreased by at least at most about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, or about 50 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, the ARC is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, at least 5.0 units, at least 6.0 units, at least 7.0 units, at least 8.0 units, at least 9.0 units, at least 10 units, or more.

[00101] In some embodiments, Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof; indirectly reduces bilirubin and serum lactate dehydrogenase (LDH) levels in the plasma of a subject. In some embodiments, Compound 1, decreases bilirubin levels. In some embodiments, bilirubin is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 150%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, bilirubin is decreased by at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, bilirubin levels are decreased by at least about 10 % to about 50 %. In some embodiments, bilirubin levels are decreased by at least about 10 % to about 15 %, about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 15 % to about 20 %, about 15 % to about 25 %, about 15 % to about 30 %, about 15 % to about 35 %, about 15 % to about 40 %, about 15 % to about 45 %, about 15 % to about 50 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 40 % to about 45 %, about 40 % to about 50 %, or about 45 % to about 50 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, bilirubin levels are decreased by at least about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, or about 50 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, bilirubin levels are decreased by at least at least about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, or about 45 %. In some embodiments, bilirubin levels are decreased by at least at most about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, or about 50 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, bilirubin is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, at least 5.0 units, at least 6.0 units, at least 7.0 units, at least 8.0 units, at least 9.0 units, at least 10.0 units, or more, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, Compound 1 decreases LDH levels in the plasma of a subject. In some embodiments, LDH is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 150%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, LDH is decreased by at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, LDH levels are decreased by at least about 10 % to about 50 %. In some embodiments, LDH levels are decreased by at least about 10 % to about 15 %, about 10 % to about 20 %, about 10 % to about 25 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 15 % to about 20 %, about 15 % to about 25 %, about 15 % to about 30 %, about 15 % to about 35 %, about 15 % to about 40 %, about 15 % to about 45 %, about 15 % to about 50 %, about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 25 % to about 30 %, about 25 % to about 35 %, about 25 % to about 40 %, about 25 % to about 45 %, about 25 % to about 50 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 40 % to about 45 %, about 40 % to about 50 %, or about 45 % to about 50 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, LDH levels are decreased by at least about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, or about 50 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, LDH levels are decreased by at least at least about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, or about 45 %. In some embodiments, LDH levels are decreased by at least at most about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, or about 50 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, LDH is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, at least 5.0 units, at least 6.0 units, at least 7.0 units, at least 8.0 units, at least 9.0 units, at least 10 units, at least 15 units, at least 20 units, at least 20 units, at least 40 units, at least 50 units, at least 60 units, at least 70 units, at least 80 units, at least 90 units, at least 100 units, or more over pre-treatment levels. In some embodiments, LDH is decreased by at least 10 units.

[00102] In some embodiments, Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, indirectly reduces high-sensitivity C-reactive protein (“hsCRP”). In some embodiments, hsCRP is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 150%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, hsCRP is decreased by at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, hsCRP levels are decreased by at least about 10 % to about 100 %. In some embodiments, hsCRP levels are decreased by at least about 10 % to about 20 %, about 10 % to about 30 %, about 10 % to about 40 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 75 %, about 10 % to about 80 %, about 10 % to about 85 %, about 10 % to about 90 %, about 10 % to about 100 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 75 %, about 20 % to about 80 %, about 20 % to about 85 %, about 20 % to about 90 %, about 20 % to about 100 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 75 %, about 30 % to about 80 %, about 30 % to about 85 %, about 30 % to about 90 %, about 30 % to about 100 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 75 %, about 40 % to about 80 %, about 40 % to about 85 %, about 40 % to about 90 %, about 40 % to about 100 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 75 %, about 50 % to about 80 %, about 50 % to about 85 %, about 50 % to about 90 %, about 50 % to about 100 %, about 60 % to about 70 %, about 60 % to about 75 %, about 60 % to about 80 %, about 60 % to about 85 %, about 60 % to about 90 %, about 60 % to about 100 %, about 70 % to about 75 %, about 70 % to about 80 %, about 70 % to about 85 %, about 70 % to about 90 %, about 70 % to about 100 %, about 75 % to about 80 %, about 75 % to about 85 %, about 75 % to about 90 %, about 75 % to about 100 %, about 80 % to about 85 %, about 80 % to about 90 %, about 80 % to about 100 %, about 85 % to about 90 %, about 85 % to about 100 %, or about 90 % to about 100 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, hsCRP levels are decreased by at least about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 75 %, about 80 %, about 85 %, about 90 %, or about 100 %. In some embodiments, hsCRP levels are decreased by at least at least about 10 %, about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 75 %, about 80 %, about 85 %, or about 90 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, hsCRP levels are decreased by at least at most about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 75 %, about 80 %, about 85 %, about 90 %, or about 100 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1).

[00103] In some embodiments, Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, indirectly reduces N-terminal (NT)-pro hormone B-type natriuretic peptide (“NT-proBNP”). In some embodiments, NT-proBNP is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 150%, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, NT-proBNP is decreased by at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30%. In some embodiments, hsCRP levels are decreased by at least about 10 % to about 100 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, hsCRP levels are decreased by at least about 10 % to about 20 %, about 10 % to about 30 %, about 10 % to about 35 %, about 10 % to about 40 %, about 10 % to about 45 %, about 10 % to about 50 %, about 10 % to about 60 %, about 10 % to about 70 %, about 10 % to about 80 %, about 10 % to about 90 %, about 10 % to about 100 %, about 20 % to about 30 %, about 20 % to about 35 %, about 20 % to about 40 %, about 20 % to about 45 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 80 %, about 20 % to about 90 %, about 20 % to about 100 %, about 30 % to about 35 %, about 30 % to about 40 %, about 30 % to about 45 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 80 %, about 30 % to about 90 %, about 30 % to about 100 %, about 35 % to about 40 %, about 35 % to about 45 %, about 35 % to about 50 %, about 35 % to about 60 %, about 35 % to about 70 %, about 35 % to about 80 %, about 35 % to about 90 %, about 35 % to about 100 %, about 40 % to about 45 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 80 %, about 40 % to about 90 %, about 40 % to about 100 %, about 45 % to about 50 %, about 45 % to about 60 %, about 45 % to about 70 %, about 45 % to about 80 %, about 45 % to about 90 %, about 45 % to about 100 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 80 %, about 50 % to about 90 %, about 50 % to about 100 %, about 60 % to about 70 %, about 60 % to about 80 %, about 60 % to about 90 %, about 60 % to about 100 %, about 70 % to about 80 %, about 70 % to about 90 %, about 70 % to about 100 %, about 80 % to about 90 %, about 80 % to about 100 %, or about 90 % to about 100 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, hsCRP levels are decreased by at least about 10 %, about 20 %, about 30 %, about 35 %, about 40 %, about 45 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, or about 100 %. In some embodiments, hsCRP levels are decreased by at least at least about 10 %, about 20 %, about 30 %, about 35 %, about 40 %, about 45 %, about 50 %, about 60 %, about 70 %, about 80 %, or about 90 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1). In some embodiments, hsCRP levels are decreased by at least at most about 20 %, about 30 %, about 35 %, about 40 %, about 45 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, or about 100 %, compared to subjects that did not receive Compound 1 and/or compared to baseline (previous to receiving Compound 1).

[00104] cGMP level may be measured with any suitable method in the art, such as enzyme immunoassay.

[00105] HbF positive cells, as used herein, means red blood cells with HbF. HbF positive cells may be measured from a blood sample with any suitable method in the art, such as electrophoresis and/or colorimetric methods.

[00106] Sickle red blood cells, sickled red blood cells, as used herein, means red blood cells with a crescent or sickle shape. % sickle red blood cell may be measured from a blood sample with any suitable method in the art.

[00107] Stasis or microvascular stasis, as used herein, is serious slowing, or complete cessation, of blood or lymph flow through vessels. % stasis is the number of static (no flow) venules divided by the number of flowing venules times 100. Percent (%) stasis may be measured with any suitable method in the art.

[00108] Total bilirubin, as used herein, means both unconjugated and conjugated bilirubin. Total bilirubin levels may be measured from a blood sample with any suitable method in the art.

[00109] Total leucocyte count or total white blood cell count, as used herein, is a blood test that measures the number of white blood cells in the body. It may be measured from a blood sample with any suitable method in the art.

[00110] Another aspect of the present disclosure provides methods of administering Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, to a subject in need thereof, in combination with at least one other active agent. They may be administered simultaneously or sequentially. They may be present as a mixture for simultaneous administration, or may each be present in separate containers for sequential administration.

[00111] The term “simultaneous administration”, as used herein, is not specifically restricted and means that Compound 1 and the at least one other active agent are substantially administered at the same time, e.g. as a mixture or in immediate subsequent sequence.

[00112] The term “sequential administration”, as used herein, is not specifically restricted and means that Compound 1 and the at least one other active agent are not administered at the same time but one after the other, or in groups, with a specific time interval between administrations. The time interval may be the same or different between the respective administrations of Compound 1 and the at least one other active agent and may be selected, for example, from the range of 2 minutes to 96 hours, 1 to 7 days or one, two or three weeks. Generally, the time interval between the administrations may be in the range of a few minutes to hours, such as in the range of 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 to 12 hours. Further examples include time intervals in the range of 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12 hours.

[00113] The molar ratio of Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, and the at least one other active agent is not particularly restricted. For example, when Compound 1 and one other active agent are combined in a composition, the molar ratio of them may be in the range of 1 :500 to 500: 1, or of 1 : 100 to 100: 1, or of 1 :50 to 50: 1, or of 1 :20 to 20: 1, or of 1 :5 to 5: 1, or 1 : 1. Similar molar ratios apply when Compound 1 and two or more other active agents are combined in a composition. Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, may comprise a predetermined molar weight percentage from about 1% to 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to 40%, or about 40% to 50%, or about 50% to 60%, or about 60% to 70%, or about 70% to 80%, or about 80% to 90%, or about 90% to 99% of the composition. Without being bound by theory, amount of Compound 1 are usually calculated from the free base or unsolvated form.

[00114] The other active agent may be a different PDE9 inhibitor than Compound 1 or HU. The other active agent may also be an antibiotic agent such as penicillin, a nonsteroidal anti-inflammatory drug (NSAIDS) such as diclofenac or naproxen, a pain relief medication such as opioid, or folic acid.

[00115] Yet another aspect of the present disclosure provides methods of using Compound 1 or a pharmaceutically acceptable salt, solvate, or polymorph thereof in combination with at least one other therapy, such as but not limited to blood transfusion, bone marrow transplant, or gene therapy.

Kits and Devices

[00116] The disclosure provides a variety of kits and devices for conveniently and/or effectively carrying out methods of the present disclosure. Typically, kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.

[00117] In one embodiment, the present disclosure provides kits for treating sickle cell disease, comprising Compound 1, or a pharmaceutically acceptable salt or polymorph thereof, optionally in combination with any other active agents, such as HU, an antibiotic agent such as penicillin, a nonsteroidal anti-inflammatory drug (NSAIDS) such as diclofenac or naproxen, a pain relief medication such as opioid, or folic acid.

[00118] The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may comprise a saline, a buffered solution, or any delivery agent disclosed herein. The amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations. The components may also be varied in order to increase the stability of Compound 1 in the buffer solution over a period of time and/or under a variety of conditions.

[00119] The present disclosure provides for devices that may incorporate Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof. These devices contain in a stable pharmaceutical formulation available to be immediately delivered to a subject in need thereof, such as a human patient with sickle cell disease.

[00120] Non-limiting examples of the devices include a pump, a catheter, a needle, a transdermal patch, a pressurized olfactory delivery device, iontophoresis devices, multi-layered microfluidic devices. The devices may be employed to deliver Compound 1 according to single, multi- or split-dosing regiments. The devices may be employed to deliver Compound 1 across biological tissue, intradermal, subcutaneously, or intramuscularly. More examples of devices suitable for delivering Compound 1 include but not limited to a medical device for intravesical drug delivery disclosed in International Publication WO 2014036555, a glass bottle made of type I glass disclosed in US Publication No. 20080108697, a drug-eluting device comprising a film made of a degradable polymer and an active agent as disclosed in US Publication No. 20140308336, an infusion device having an injection micro-pump, or a container containing a pharmaceutically stable preparation of an active agent as disclosed in US Patent No. 5716988, an implantable device comprising a reservoir and a channeled member in fluid communication with the reservoir as disclosed in International Publication WO 2015023557, a hollow-fiber-based biocompatible drug delivery device with one or more layers as disclosed in US Publication No. 20090220612, an implantable device for drug delivery including an elongated, flexible device having a housing defining a reservoir that contains a drug in solid or semi-solid form as disclosed in International Publication WO 2013170069, a bioresorbable implant device disclosed in US Patent No. 7326421, contents of each of which are incorporated herein by reference in their entirety.

Definitions

[00121] The articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary.

[00122] The phrase “and/or,” as used herein, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements).

[00123] As used herein, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements.

[00124] In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[00125] As used herein, the phrase “at least one” in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.

[00126] Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. [00127] As used herein, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.

[00128] Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi -closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures.

[00129] As used herein, a “subject” or a “patient” refers to any mammal (e.g., a human), such as a mammal that may be susceptible to a disease or disorder, for example, tumorigenesis or cancer. Examples include a human, a non-human primate, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, or a rodent such as a mouse, a rat, a hamster, or a guinea pig. In various embodiments, a subject refers to one that has been or will be the object of treatment, observation, or experiment. For example, a subject can be a subject diagnosed with cancer or otherwise known to have cancer or one selected for treatment, observation, or experiment on the basis of a known cancer in the subject.

[00130] As used herein, “treatment” or “treating” refers to amelioration of a disease or disorder, or at least one sign or symptom thereof. “Treatment” or “treating” can refer to reducing the progression of a disease or disorder, as determined by, e.g., stabilization of at least one sign or symptom or a reduction in the rate of progression as determined by a reduction in the rate of progression of at least one sign or symptom. In another embodiment, “treatment” or “treating” refers to delaying the onset of a disease or disorder.

[00131] As used herein, “prevention” or “preventing” refers to a reduction of the risk of acquiring or having a sign or symptom a given disease or disorder, i.e., prophylactic treatment.

[00132] The phrase “therapeutically effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the present teachings that is effective for producing a desired therapeutic effect. Accordingly, a therapeutically effective amount treats or prevents a disease or a disorder, e.g., ameliorates at least one sign or symptom of the disorder. In various embodiments, the disease or disorder is a cancer.

[00133] A dash

that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -CONH2 is attached through the carbon atom (C). [00134] By “optional” or “optionally,” it is meant that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “optionally substituted aryl” encompasses both “aryl” and “substituted aryl” as defined herein. It will be understood by those ordinarily skilled in the art, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible, and/or inherently unstable.

[00135] All numerical ranges herein include all numerical values and ranges of all numerical values within the recited range of numerical values. As a non-limiting example, (Ci- C₆) alkyls also include any one of Ci, C₂, C₃, C₄, C₅, C₆, (C1-C2), (C1-C3), (C1-C4), (Ci-C₅), (C₂- C₃), (C2-C4), (C₂-C₅), (C₂-C₆), (C₃-C₄), (C₃-C₅), (C₃-C₆), (C₄-C₅), (C₄-C₆), and (C₅-C₆) alkyls.

[00136] Further, while the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations as discussed above, the numerical values set forth in the Examples section are reported as precisely as possible. It should be understood, however, that such numerical values inherently contain certain errors resulting from the measurement equipment and/or measurement technique. LIST OF ABBREVIATIONS AND TERMS

^-NMR: Proton Nuclear Magnetic Resonance spectroscopy

ADME: Absorption, Distribution, Metabolism, and Excretion

AE: Adverse event

AUC0-24: area under the concentration -time curve from time 0 to 24 hours post dose

BBB: blood-brain barrier

Cmax: maximum plasma concentration cGMP: cyclic guanosine monophosphate

DMSO: dimethyl sulfoxide

F cells: blood cells with fetal hemoglobin

FIH: first in human

FTIR: Fourier transform infrared spectroscopy

GC: gas chromatography

HBB: hemoglobin subunit beta

Hb: hemoglobin

HbF : fetal hemoglobin

HBG: gamma-globin gene

HbSS: beta zero thalassemia

HbS: sickle hemoglobin hERG: human ether-a-go-go related gene

HPLC: high-performance liquid chromatography

HU: hydroxyurea

IC: inhibitory concentration

IC50: a half minimal inhibitory concentration

ICAM-1 : intercellular adhesion molecule- 1

ICH: International Conference on Harmonization

ICP-MS: inductively coupled plasma mass spectroscopy

IV: intravenous

LDH: lactate dehydrogenase

MAD: multiple-ascending dose

MTD: maximum tolerated dose

NO: nitric oxide

NOAEL: no-observed-adverse-effect level

PD: pharmacodynamic

PDE9: phosphodiester-9 PEG polyethylene glycol

PIC: Powder in capsule PK: pharmacokinetic(s) PKG: protein kinase G RBC: red blood cell RH: relative humidity SCD: sickle cell disease SD: standard deviation

SEM: standard error of the mean sGC: soluble guanylyl cyclase t>/₂: half-life TK: Toxicokinetic

Tmax: time of maximum concentration

VOC: vaso-occlusive crisis

WBC: white blood cell w/w%: weight/weight percent

EXAMPLES

[00137] It will be appreciated that the following examples are intended to illustrate but not to limit the present disclosure. Various other examples and modifications of the foregoing description and examples will be apparent to a person skilled in the art after reading the disclosure without departing from the spirit and scope of the disclosure, and it is intended that all such examples or modifications be included within the scope of the appended claims. All publications and patents referenced herein are hereby incorporated by reference in their entirety.

Example 1. Synthesis and formulation of Compound 1

[00138] Compound 1 is an enantiomer of 6-[4-methyl-l-(pyrimidin-2- ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8-one disclosed in WO 2013/053690., the contents of which are which are incorporated herein by reference in their entirety. Compound 1 may be prepared from chiral-selective purification from 6-[4- methyl- 1 -(pyrimidin-2-ylmethyl)pyrrolidin-3 -y 1 ] -3 -tetrahydropyran-4-yl-7H-imidazo[ 1,5- a]pyrazin-8-one according to the method disclosed in WO 2013/053690, the contents of which are incorporated herein by reference in their entirety. Compound 1 may also be prepared with the method disclosed in WO 2017/005786, the contents of which are incorporated herein by reference in their entirety. Compound 1 is 6-[(3S,4S)-4-methyl-l-(pyrimidin-2- ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8-one, the structure of which is disclosed below:

Compound 1.

[00139] Compound 1 drug product to be used in ongoing clinical development is an immediate release tablet. The coating is may be used to assure uniformity of appearance across different tablet strengths and with the placebo.

[00140] Earlier clinical studies were performed with Compound 1 drug substance directly filled into opaque white gelatin capsules (Powder in Capsule, PIC) with no excipients or processing aids. An excipient-blended tablet form of the drug product for oral administration has been developed, as this allowed for scale-up of the manufacturing process and assurance of content uniformity. These tablets were tested for defined limits for purity, potency, dissolution, total aerobic microbial count, as well as total yeast and mold count. In addition, tests for specified microorganisms were performed.

[00141] Each tablet comprises from 100 mg to 800 mg of Compound 1 drug substance (the monohydrate of the API) or placebo.

Example 2. Compound 1 Reduces White Cell Adhesion and Activation

[00142] Polymorphic mononuclear cells (PMN), particularly neutrophils, play a critical role in pathogenesis of sickle cell disease (SCD) and activated neutrophils have been shown to be more adhesive to each other, platelets and the vascular endothelium. Recently several drugs targeting white cell binding to endothelial cells, have been advancing in clinical studies in patients. Compound 1 is able to increase expression of fetal hemoglobin in patient derived cells and murine models of SCD and reduce vessel occlusion in SCD murine models. In this Example, the ability of Compound 1 to reduce the adhesive properties of neutrophils from SCD patients and reduce sE-Selectin (sE-Sel) and markers of PMN activation in murine SCD models was studied.

[00143] Endothelial E-selectin (E-Sel) slows leukocyte rolling, which is followed by stationary adhesion and transmigration of activated leukocytes. Plasma levels of sE-Sel, produced by the enzymatic cleavage of the extracellular domains of E-Sel, are increased in SCD patients and this may be mediated by its interaction with leukocytes. In the Townes mouse model, plasma sE-Sel is increased 144% (139 mg/ml) over levels seen in control mice (57 mg/ml). This was reduced significantly in Townes mice treated with Compound 1, where plasma sE-Sel levels were elevated by only 61% over control mice (92 mg/ml).

[00144] It was found that Compound 1 reduced circulating levels of PMNs in SCD models, but not in long term studies in healthy animals. This appears to be accompanied by a Compound 1- mediated reduction in disease specific cell activation including 67% lower levels of myeloid derived myeloperoxidase (MPO) and 26% lower levels of neutrophil derived arginase in the lung. Using a previously described in vitro adhesion assay mimicking blood flow, where activated endothelial cells HMEC-1 line the inner surface of microchannels, perfused whole blood samples from SS (the most common form of sickle cell) patients showed that neutrophils aggregate and bind to the endothelial monolayer. This was quantified by real time monitoring of the green florescent patches in the microchannel, as neutrophils are labeled by a specific Alexa Fluor® 488-conjugated antibody before the perfusion step. Untreated, patient neutrophils showed a significant amount of adhesion to activated HMEC-1. When added to blood samples prior to the perfusion step, Compound 1 reduced adhesions significantly and in a dose dependent manner. The inhibitory effect was initiated as early as 15 min of incubation, with the most potent inhibition of adhesion observed for 30 min incubation with 30 pM of Compound 1. Under these conditions, adhesion was reduced an average of 54 % (p=0.03). Mechanistically, not willing to be bound by any theory, Compound 1 may target the stationary adhesion step of neutrophils as it lowered expression levels of key neutrophil integrins including CD1 la [reduced 23 % (p=0.002)], CD1 lb [reduced 39 % (ns)) and CD18 [reduced 47 % (p=0.03)).

[00145] Together, these data indicate a role for Compound 1 in reducing PBMCs mediated pathology in SCD by targeting the abnormal adhesion of neutrophils independently from their cell count in the circulation.

Example 3. A Phase 2a, Randomized., Double-Blind, Placebo-Controlled Study of Compound 1 in Adult Patients with Sickle Cell Anemia (SCA) and Open Label Extension (OLE) Study

Objectives:

[00146] Primary Objectives: To assess the safety and tolerability of Compound 1 in adult patients with sickle cell anemia (SCA), defined as homozygous sickle hemoglobin (HbSS) or sickle-p⁰ thalassemia, who are not receiving hydroxyurea (HU) and in adult SCA patients who are receiving a stable dose of HU.

[00147] Secondary Objectives: To characterize the pharmacokinetic (PK) profile of Compound 1 in adult patients with SCA who are/are not receiving a stable dose of HU; to characterize the PK profile of HU in adult patients with SCA before and after receiving Compound 1 to determine if there is a clinically relevant PK interaction.

[00148] Exploratory Objectives: To assess the pharmacodynamic (PD) effects of Compound 1 in adult patients with SCA who are/are not receiving stable HU; to assess the potential efficacy of Compound 1 on SCA-related clinical outcome measures in adult patients with SCA who are/are not receiving stable HU.

Methodology:

[00149] This is a randomized, double-blind, placebo-controlled study to evaluate the safety, tolerability, PK, and exploratory PD and clinical outcomes of the phosphodiesterase 9 (PDE9) inhibitor Compound 1, administered once daily for 16 to 24 weeks in 2 populations of patients with SCA: (1) those who are not receiving HU (Population A) and (2) those who are currently receiving a stable dose of HU according to standard of care (Population B). Up to approximately 36 patients are enrolled in Population A and 18 patients are enrolled in Population B.

[00150] Population A: Following a Screening period of up to 4 weeks, eligible patients in Population A (i.e., those not receiving HU) receive either Compound 1 or placebo for a total of 24 weeks. On Day 1, patients are randomized 1 : 1 : 1 to receive oral Compound 1 dose of 50 mg or 100 mg, or placebo daily for the first 12 weeks; for the second 12 weeks (Weeks 13-24), each patient’s dose may be doubled (i.e., from 50 mg to 100 mg; from 100 mg to 200 mg; or placebo). Throughout the study, all available clinical data are reviewed approximately every 2 weeks, and dose escalation occurs on an individual patient basis on Day 85 only if approved based upon review of each patient’s individual clinical safety data.

[00151] Population B: Following a Screening period of up to 4 weeks, eligible patients in Population B (i.e., those receiving stable HU) enter a lead-in period and have blood samples drawn to characterize the PK profile of the patient’s prescribed dose of HU in the absence of Compound 1 (i.e., to characterize the patient’s baseline HU PK profile). Two full baseline HU PK profiles (with blood samples drawn over a 10-hour period at least 48 hours apart) are determined.

[00152] Compound 1 dosing in Population B do not begin until at least 4 weeks of safety data from 6 patients in Population A have been reviewed and determined that it is safe and appropriate to begin dosing in Population B. Following approval to initiate dosing in Population B and once the baseline HU PK blood draws are complete, patients are randomized 2: 1 on Day 1 to receive oral Compound 1 at 50 mg or placebo for 16 weeks. For the first 4 weeks (Weeks 1-4), patients receive study medication according to their randomized treatment assignment; for the following 12 weeks (Weeks 5-16), each patient’s dose may be doubled (e.g., from 50 mg to 100 mg; or placebo). As in Population A, dose escalation occurs on Day 29 only if approved based upon review of each patient’s individual clinical safety data.

[00153] Phase 2a and the OLE studies are outlined in Figure 3.

Study Design Rationale:

[00154] This is the first study in a patient population (patients with SCA), and as such, is designed to examine the safety, tolerability, and PK, as well as the potential PD effects and clinical efficacy, of Compound 1 across a range of doses in adult patients with SCA. Given the possibility that Compound 1, if approved, could be administered as a single agent or coadministered with HU, the effects of Compound 1 are evaluated in SCA patients who are not receiving HU or any other treatment known to modulate HbF levels (Population A) as well as in those who are currently receiving a stable dose of HU (Population B).

[00155] Available nonclinical and healthy volunteer clinical data suggest that Compound 1 is safe and well tolerated at once daily doses of 50, 100, and 200 mg and that a potentially clinical beneficial PD effect is likely to be observed when a dose of at least 100 mg is administered for at least 24 weeks. Therefore, Population A is designed to explore the PD dose response in patients as well as the tolerability of the 200 mg dose level in sickle cell patients who have tolerated the 100 mg dose well.

[00156] Results from Population B are intended to provide information on Compound 1 when administered concomitantly with HU, both of which increase HbF levels through alternative biochemical pathways that increase intracellular cGMP. Because there are no clinical data to support administration of Compound 1 concomitantly with HU, patients in Population B initiate Compound 1 dosing at the low dose (50 mg) used in Population A and only escalate to the 100 mg dose if the 50 mg dose has been safe and tolerated for 4 weeks. In addition, although available nonclinical data do not suggest that concomitant administration of HU with Compound 1 would increase Compound 1 exposure, dosing in Population B does not initiate until 4 weeks of safety data are available from Population A in 2 patients each at 50 mg (starting dose in Population B) and at 100 mg (2x the starting dose) as well as placebo.

[00157] A four-year open label extension (“OLE”) clinical trial which allows patients from Population A and Population B in the above to enroll in a long-term safety and tolerability study of Compound 1 following completion of the experiments described above. The OLE clinical trial was initially designed so that patients were administered a daily dose of 100 mg of Compound 1, and in the second quarter of 2020, a protocol amendment increased the daily dose to 200 mg. Patients from the combination sub-study continue to receive the same dose of HU that they received while on the Phase 2a clinical trial throughout the duration of the OLE clinical trial (Population B).

Diagnosis and main criteria for inclusion:

[00158] Inclusion Criteria: : Each patient must meet all of the following criteria to be enrolled in the study: (1). Male or female >18 or <50 years of age. (2). Confirmed diagnosis of SCA (HbSS or sickle- P° thalassemia). Note, if not already documented in the patient’s record, the diagnosis of SCA must be confirmed via electrophoresis, HPLC, and/or genotyping. (3). Use of HU: For patients in the Population A: Have not received HU within 90 days prior to Screening and are not planning to take HU within the next 6 months. For patients in Population B: Have received HU for at least 6 months, have been on a stable dose for at least 60 days prior to Screening, and are not planning to change the dose level, dose regimen, or discontinue HU within the next 6 months. 4. Female patients must not be pregnant and be highly unlikely to become pregnant. Male patients must be unlikely to impregnate a partner.

[00159] Exclusion Criteria: Patients who meet any of the following criteria are excluded from the study: (1). Total Hb at Screening >11.0 g/dL or < 6 g/dL. (2). Reticulocyte count <100 x 109/L. (3). >3 hospitalizations (for at least 24 hours) for vaso-occlusive crises (VOC), including acute chest syndrome (ACS) and priapism, within the prior year. (4). Receiving chronic outpatient opioid treatment (equivalent to >10 mg oral morphine daily) for any reason other than avascular necrosis (AVN). Note: chronic treatment is defined as continuous daily opioid use for >8 weeks. (5). Blood transfusion or donation of blood or any blood product within 60 days of Day 1 or on chronic transfusion therapy regimen. (6). Positive for human immunodeficiency virus (HIV), hepatitis C (HCV) antibodies (unless the patient has successfully completed drug therapy that results in cure/clearance of HCV), and hepatitis B surface antigen (HBsAg). (7). For female patients of childbearing potential, a positive serum human chorionic gonadotropin (hCG) test (Screening) or a positive urine hCG test on Day 1.

(8). Estimated glomerular filtration rate (eGFR) <50 mL/min as calculated by the equation from the Modification of Diet in Renal Disease (MDRD) Study using creatinine, age, sex, and ethnicity. (9). Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) >3x the upper limit of normal (ULN). (10). Body Mass Index (BMI) < 17.5 or > 35 kg/m2; a total body weight < 50 kg. (11). Use of PDE5 inhibitors (including but not limited to sildenafil, tadalafil, vardenafil) within 7 days prior to the first dose of study drug, or planning to use any time during study. (12). A history of drug or alcohol abuse as judged by the investigator within the past lyear, or a positive alcohol (breathalyzer) test (Screening or Day -1). (13). A cancer that has not been in complete remission for at least 5 years. Patients with squamous cell or basal cell carcinoma of the skin, localized cervical cancer, or localized prostate cancer are eligible if, in the opinion of the investigator, the condition has been adequately diagnosed, and is determined to be clinically in remission, and the patient’s participation in the study would not represent a safety concern. (14). A history of a clinically significant allergic reaction or hypersensitivity, as judged by the investigator, to any drug or any component of the study drug formulations used in the study. (15). On ECG, a corrected QT interval, Fridericia’s formula (QTcF) >450 ms in men and >470 ms in women or the presence of clinically significant abnormalities as determined by the investigator. (16). A history of major surgery within 4 weeks or minor surgery within 2 weeks of Day 1. (17). Any flu-like syndrome or other respiratory infection within 2 weeks of Day 1 or vaccination with attenuated live virus within 4 weeks of Day 1.

(18). Participation in an investigational drug or device study within 30 days prior to Day 1.

(19). Use within 30 days prior to Day 1, or planning to use during the study, of any drugs or substances that are known to strongly inhibit or induce cytochrome P450 enzymes (CYPs), including but not limited to cimetidine, cyclosporine, erythromycin, omeprazole, rifampin, ritonavir, and St. John’s wort. If there is any question as to whether a substance is permitted, please review the product labelling (if applicable) and consult the Sponsor. (20). Consumption of grapefruit, grapefruit juice, or grapefruit products within 24 hours prior to Day 1 or planning to consume grapefruit products during the study. (21). Use within 30 days prior to Day 1, or planning to use during the study, of any CYP3 A sensitive substrates, (excluding opioids), including but not limited to alfentanil, avanafil, budesonide, buspirone, conivaptan, darifenacin, darunavir, dasatinib, dronedarone, ebastine, eletriptan, eplerenone, everolimus, felodipine, ibrutinib, indinavir, lomitapide, lurasidone, maraviroc, midazolam, naloxegol, nisoldipine, quetiapine, saquinavir, sirolimus, tacrolimus, ticagrelor, tipranavir, tolvaptan, triazolam. (22). Use within 30 days prior to Day 1, or planning to use during the study, of any drugs or substances known to be significant substrates or inhibitors of P-gly coprotein (P-gp), including but not limited to cyclosporine, lovastatin, propranolol, quinidine, and simvastatin. If there is any question as to whether a substance is permitted, please review the product labelling (if applicable) and consult the Sponsor. (23). Other prior or ongoing medical condition, physical findings, or laboratory abnormality that, in the investigator's opinion, could adversely affect the safety of the patient, make it unlikely that the course of treatment or follow-up would be completed, or impair the assessment of study results.

Investigational product, dosage and mode of administration:

[00160] Compound 1 is supplied as a 100 mg or 200 mg white tablets and is administered orally with food. The different doses of Compound 1 are visually identical in tablet form. Reference therapy, dosage and mode of administration:

[00161] Placebo consists of tablets containing matrix absent Compound 1 and is identical in appearance to the Compound 1 tablets. Placebo is administered orally with food.

Duration of treatment:

[00162] The total duration of the study is approximately 32 weeks for Population A, including a Screening period of up to 4 weeks, a treatment period of 24 weeks, and a 4-week follow-up assessment after the last dose of study drug is administered.

[00163] The total duration of the study is approximately 32 weeks for Population B, including a Screening period of up to 4 weeks, a lead-in period of approximately 8 weeks, a treatment period of 16 weeks, and a 4-week follow-up assessment after the last dose of study drug is administered.

Endpoints:

[00164] The endpoints for Populations A and B are the same except where noted otherwise.

[00165] Primary Endpoints: Compound 1 safety and tolerability as measured by: Incidence and severity of adverse events (AEs) and serious adverse events (SAEs); Change from baseline in 12-lead electrocardiogram (ECG) parameters, clinical laboratory tests (chemistry, hematology, coagulation, urine), and vital signs; Physical examination findings. [00166] Secondary Endpoints: The plasma PK profile of Compound 1 after oral administration to adult patients with SC A (Populations A and B); The plasma PK profile of HU before and after oral administration of Compound 1 to adult patients with SCA (Population B only).

[00167] Exploratory Endpoints: Compound 1 PD as measured by the following (additional exploratory biomarkers may also be tested): Total hemoglobin (Hb) levels; HbF value (%); % F cells; Indices of red cell hemolysis (unconjugated bilirubin, reticulocyte count, lactase dehydrogenase [LDH], and haptoglobin levels); Soluble E-selectin (sE-Sel), Soluble P- selectin (sP-Sel) and soluble intercellular adhesion molecule 1 (sICAM-1); High sensitivity-C reactive protein (hs-CRP). Compound 1 clinical outcomes as measured by pain-related measures (frequency, severity, and duration of pain; impact of pain/fatigue on work/school and on activities of daily living; need for/use of pain medication; SCA-related events requiring professional medical or health care, including events requiring hospitalization or therapies, such as transfusions) and in the physical, social, and emotional impact of SCA as measured by the Adult Sickle Cell Quality-of-Life Measurement Information System (ASCQ-Me).

[00168] In addition, a separate blood sample is collected for confirmation of diagnosis by electrophoresis, high performance liquid chromatography (HPLC) and/or DNA sequencing (as needed) as well as for possible pharmacogenomic analyses of genes that may affect treatment response (including but not limited to alpha globin and BCL11 A).

Results:

[00169] A summary of the OLE safety results is displayed in Table 1. No related Serious Adverse Events (SAEs) were reported. The majority of AEs were mild or moderate. Grade 3 AEs were unrelated to treatment.

Table 1.

[00170] Summary of OLE study F-cells, HbF and biomarkers of hemolysis are shown in Figures 4-10, 26, and 28-29. Case narratives for two principle patients are summarized below. [00171] Patient #1 was originally randomized to the 100 mg/200 mg dose group in the monotherapy sub-study of the Phase 2a clinical trial, in which the patient received Compound 1 for six months, and then transitioned to an open label extension (“OLE”) clinical trial to test an escalated dose of Compound 1 along with HU at the standard of case dose, after a washout period of approximately one month. Patient 1 was transferred to the clinical trial for 18 months, either as part of the Phase 2a clinical trial (six months) or as part of the OLE clinical trial (additional 12 months).

[00172] A comparison of data for the 18-month period while the patient had been on Compound 1 versus information from a retrospective review of the patient’s medical records for the 18 months prior to initiation of Compound 1 indicate potential benefits of the compound for the patient with respect to reported vaso-occlusive crises, or VOCs, trends, healthcare facility use, and disease biomarkers. In the 18-month period on Compound 1, the patient had a 55% (38 to 17) reduction in reported VOCs, as compared to the 18 month period prior to Compound 1 administration. Furthermore, reported VOCs that resulted in emergency department, or ED, visits and out-patient visits decreased by 55% (22 to 10 visits) and 50% (14 to 7 visits) respectively, in the 18-month period on Compound 1 versus the 18-month period prior to initial Compound 1 administration. Reported VOCs that resulted in hospitalization decreased from two in the 18-month period prior to Compound 1 administration to zero, in the 18-month period on Compound 1.

[00173] A review of reported VOCs during the 18-month period on Compound 1 showed a decreased incidence of VOCs with increased time on Compound 1. In the first six months on Compound 1, the patient reported nine VOCs. In the next six months, the patients reported six VOCs, and in the last six months of the 18-month period, the patient reported two VOCs. Additionally, reported VOCs that resulted in ED visits dropped from five in the first and second six -month periods to zero in the most recent six-month period (100% reduction).

[00174] The patient was also administered the ASCQ-Me questionnaire, a National Institutes of Health validated SCD questionnaire, at the 12-month OLE visit. The ASCQ-ME questionnaire reports patient outcomes across seven domains (emotional, social functioning, pain, stiffness, sleep, pain episode frequency and pain episode severity). Examination of ASCQ-Me scores after 18 months of Compound 1 treatment showed improvement in five of the seven domains when compared to ASCQ-Me scores at the start of the Phase 2a clinical trial, including improvement in pain episode frequency and pain episode severity. The emotional domain score was the only domain score to regress when compared to the baseline Phase 2a clinical trial measurements.

[00175] Improvement across key SCD disease biomarkers were also observed. When comparing the patient’s lab values to the patient’s baseline lab values at the start of the Phase 2a clinical trial, an absolute increase in F-cell percentage of 20.2 percentage points (increasing from 26.1% to 46.3%), an absolute increase in HbF percentage of 3.9 percentage points (increasing from 12.3% to 16.2%), an increase in MCV of 3.5 units (increasing from 86.8 fL to 90.3 fL) and an increase in hemoglobin (Hb) of 1 g/dL (increasing from 7.6 g/dL to 8.6 g/dL) were observed. There were also improvements across markers of hemolysis, namely a five unit decrease in percent reticulocytes (decreasing from 15% to 10%), an 88 unit decrease in absolute reticulocyte count (ARCs) (decreasing from 408 xlO⁹/L to 320 xlO⁹/L), a 4 unit reduction in indirect bilirubin (decreasing from 29 pmol/L to 25 pmol/L) and a 10 units decrease in LDH (decreasing from 306 U/L to 296 U/L).

[00176] Biomarker results are summarized graphically in Figures 12 and 13. [00177] A summary of VOC events in shown in Figure 11. In 24 months prior to Compound 1 administration, the patient had 44 VOCs including 4 hospitalizations (average of 2.3 VOCs/month). In the 24 months during treatment, 20 VOCs were reported (average of 0.8 VOCs/month). VOC rate was reduced by 64% during a 24 month period relative to the 24 months prior to treatment.

[00178] Patient #2 entered the Phase 2a clinical trial as part of the HU combination substudy and was randomized to the placebo dose group, and therefore never received Compound 1. The patient started the OLE clinical trial 14 months after completing the Phase 2a clinical trial but remained on a stable HU dose (3,000 mg daily) during this period and while on the OLE clinical trial. A comparison of data for the six-month OLE clinical trial period versus information from a retrospective review of the patient’s medical records for the six month period prior to initiation of the OLE clinical trial indicate potential benefits of Compound 1 being administered in combination with HU. There were zero VOCs during the six-month period on the OLE clinical trial as compared to 15 reported VOCs in the previous six months while on HU alone.

[00179] Biomarkers were measured in Patient 2 and compared to the patient’s baseline lab values at the start of the OLE clinical trial. An absolute increase in F-cell percentage of 21.2 percentage points (increasing from 59.7% to 80.9%), an absolute increase in HbF percentage of 9 percentage points (increasing from 20.7% to 29.7%), an increase in MCV of 10.2 units (increasing from 111.9 fL to 122.1 fL) and an increase in hemoglobin (Hb) of 0.8 g/dL (increasing from 9.9 g/dL to 10.7 g/dL) were observed. There were also improvements across markers of hemolysis, namely a 0.4 unit decrease in percent reticulocytes (decreasing from 4.2% to 3.8%), a 12.7 unit decrease in absolute reticulocyte count (ARCs) (decreasing from 102.5 X10⁹/L to 89.8 xlO⁹/L), a 1 unit reduction in indirect bilirubin (decreasing from 12 pmol/L to 11 pmol/L) and a 87 unit decrease in LDH (decreasing from 332 U/L to 245 U/L).

[00180] Biomarker results are summarized graphically in Figures 15 and 16.

[00181] A summary of VOC events in shown in Figure 14. In 8 months prior to treatment with Compound 1 and HU, the patient had 16 VOCs (all outpatient visits) were reported (average of 2.0 VOCs/month). In the 8 months during treatment, 5 VOCs (all outpatient visits) were reported (average of 0.6 VOCs/month). VOC rate was reduced by 69% during an 8 month period relative to the 8 months prior to treatment.

[00182] Compound 1 was well-tolerated as a monotherapy and in combination with HU. The mot frequent adverse events included headache and nausea. In the parent combination study, there as about a 40% lower mean annualized VOC rate in the Compound 1 treated groups versus the placebo groups. There was a significant difference between mean values, for example, a significant prolongation in time to first VOC in the Compound 1 treated groups versus the placebo groups, reduction in the mean annualized hospitalizations for VOCs were observed and improved patient-reported outcomes for pain episodes on the ASCQ-Me scale. Additionally, increases in F-cell (%) correlated with HbF (%) and with Compound 1 level of exposure.

[00183] In the open-label extension (OLE) study, low VOC rates were maintained in subject who remained on Compound 1 long term. 36% of subjects had increased HbF of greater than 3% at Month 8 date with minimal changes in total Hb.

Example 4. A Phase 2b, Randomized, Double-Blind, Placebo-Controlled Study of Compound 1 Monotherapy Dose Escalation in Adult Patients with Sickle Cell Anemia (SCA)

[00184] 40 patients received either placebo or Compound 1 at once-daily doses of 50 mg or 100 mg through 12 weeks and then higher doses of 100 mg or 200 mg, respectively, through an additional 12 weeks (24 weeks total). An additional 18 patients received either placebo or Compound 1 at a once-daily dose of 100 mg through 4 weeks and then 200 mg through an additional 20 weeks (24 weeks total).

[00185] With respect to the 18 patients that received either placebo or Compound 1 at a once-daily dose of 100 mg through 4 weeks and then 200 mg through an additional 20 weeks, biomarker results show no meaningful changes in F-cells, which are red blood cells containing fetal hemoglobin (HbF), HbF levels, and hemoglobin (Hb) levels from baseline through week 24. However, dose dependent trends in F-Cells and HbF levels were seen when patients dose escalated from lOOmg to 200mg, starting after 4 weeks and through 24 weeks. One of seven completers (14%) recorded an absolute increase in HbF percentage from baseline of greater than 1% (increase of 3.2%). Markers of hemolysis that include percent reticulocytes, absolute reticulocyte count, indirect bilirubin and LDH all improved from baseline in a dose dependent manner, with the greatest improvement occurring when patients were on the 200 mg dose. This similar trend occurred with high-sensitivity C-reactive protein (“hsCRP”) and N-terminal (NT)- pro hormone B-type natriuretic peptide (“NT -proBNP”) values. A summary of the mean results is found in Table 2. Finally, a decrease in vaso-occlusive crises/sickle cell-related pain crises (VOCs/SCPCs), as defined in the safety analysis, were observed in the treatment group when compared to placebo. 58% of patients (7 of 12) experienced at least one VOC/SCPC in the treatment group as compared to 83% (5 of 6) in the placebo population, a 25% decrease in the treatment group.

[00186] With respect to the 40 patients that received either placebo or Compound 1 at once-daily doses of 50 mg or 100 mg through 12 weeks and then higher doses of 100 mg or 200 mg, respectively, through an additional 12 weeks, the results comprising all patients in this group show that the high dose of Compound 1 (100mg/200mg) resulted in a relative increase in F-cell percentage of 13.3% from baseline (p=0.025) and a mean absolute increase in HbF percentage from baseline of 0.9%. Three of the nine completers (33%) recorded absolute HbF percentage increases of greater than 1%, with a mean absolute increase in HbF percentage of 3.1% in that subset. Decreases in VOCs/SCPCs, as defined in the safety analysis, were also observed in the higher dose of the treatment group when compared to placebo.

[00187] Results for dose escalation from 100 to 200 mg per day are above the IC90 for 16-18 hrs respectively (Figure 1). Predicted concentrations of 400 mg suggest mean concentration above IC90 for full 24 hrs. Higher doses are expected to result in improvements in key biomarkers and clinical outcomes. Patients will receive placebo or Compound 1 at once- daily doses of 200 mg or 300 mg through 12 weeks and then higher doses of 300 mg or 400 mg, respectively, through an additional 12 weeks (24 weeks total).

Example 5. A Phase 2, Randomized, Double-Blind., Placebo-Controlled Study of

Compound 1 Combination Therapy with Hydroxyurea (HU) in Adult Patients with Sickle Cell Anemia (SCA)

[00188] 21 patients received either placebo or Compound 1 once-daily at 50 mg on top of a stable dose of standard of care HU, with escalation after 4 weeks to 100 mg for an additional 12 weeks (16 weeks total). An additional 14 patients received either placebo or Compound 1 once-daily at 50 mg on top of a stable dose of standard of care HU, with escalation after 4 weeks to 100 mg for an additional 20 weeks (24 weeks total).

[00189] With respect to the 14 patients that received either placebo or Compound 1 once- daily at 50 mg on top of a stable dose of standard of care HU, with escalation after 4 weeks to 100 mg for an additional 20 weeks, the most frequent adverse events in the treatment group were generally consistent with those observed in groups that received the monotherapy of Compound 1 and included headache, sickle cell anemia with crisis and nausea. Biomarker results in treatment group showed an overall increase in F-cells and HbF levels from baseline to week 24. Three of the eight completers (33%) had absolute increases in HbF percentage of greater than 1%, with a mean absolute increase in HbF percentage of 4.3% in that subset. Similar to the groups that received the monotherapy of Compound 1, dose dependent improvements in markers of hemolysis that include percent reticulocytes, absolute reticulocyte count, and LDH were observed from baseline through week 24, with the greatest improvement occurring when patients were on the lOOmg dose. hsCRP and NT-proBNP values slightly increased in the treatment group. Indirect bilirubin values slightly worsened from baseline through week 24. A summary of the mean results in from the treatment group is found in

Table 3

[00190] Similar to the group treated with the monotherapy of Compound 1, the treatment group from the combination therapy had decreased VOCs/SCPCs, with approximately 38% of patients (5 of 13) experiencing at least one VOC/SCPC in the treatment group as compared to 71% (5 of 7) in the placebo group. Example 6. A Phase 2b, Randomized, Double-Blind., Placebo-Controlled Study of an Increase Dosage of Compound 1 Monotherapy in Adult Patients with Sickle Cell Anemia (SCA)

[00191] Patients receive either placebo or Compound 1 at once or twicedaily doses of 300 mg or 400 mg through 24 weeks (4.5 to 6.7 mg/kg). Higher doses are be assessed for tolerability. It is projected that biomarkers will change for the positive with the increased dosage. Higher dosages are proposed in Figure 2 in either a QD or BID administration strategy. [00192] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of decreasing vaso-occlusive crisis (VOC) in a patient suffering therefrom, the method comprising administering a therapeutically effective amount of 6-[(3S,4S)-4-methyl-l- (pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8- one (Compound 1), or a pharmaceutically acceptable salt, solvate, or polymorph thereof.

2. The method of claim 1, wherein the patient suffering therefrom is diagnosed with SCD, sickle cell anemia (SCA), thalassemia, or a combination thereof.

3. The method of claim 1 or claim 2, wherein VOC is decrease by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% in number prior to treatment.

4. The method of any one of claims 1 to 3, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount of about at least 100 mg per day.

5. The method of any one of claims 1 to 4, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount of from about 100 mg to about 200 mg per day, from about 200 mg to about 500 mg per day, or from about 300 mg to about 800 mg per day.

6. The method of any one of claims 1 to 5, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount of about 100 mg per day, 200 mg per day, 300 mg per day, 400 mg per day, 500 mg per day , 600 mg per day, 700 mg per day, or 800 mg per day.

7. The method of any one of claims 1 to 6, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered orally once per day.

8. The method of any one of claims 1 to 7, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered for at least 4 weeks, 12 weeks, 16 weeks, or 24 weeks, 1 year, or 1.5 years.

9. The method of any one of claims 1 to 8, further comprising administering hydroxyurea (HU).

10. A method of modulating one or more biomarkers over baseline levels prior to treatment in a patient suffering from sickle cell disease (SCD), wherein said biomarkers are selected from absolute F-cell percentage, absolute HbF percentage, MCV, and Hb level; the method comprising administering a therapeutically effective amount of 6-[(3S,4S)-4-methyl-l-

53 (pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8- one (Compound 1), or a pharmaceutically acceptable salt, solvate, or polymorph thereof.

11. The method of claim 10, wherein said absolute F-cell percentage is increased by at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30%.

12. The method of claim 10, wherein said absolute HbF percentage is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%.

13. The method of claim 10, wherein said MCV is increased by at least at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, at least 5.0 units, at least 6.0 units, at least 7.0 units, at least 8.0 units, at least 9.0 units, or at least 10.0 units.

14. The method of claim 10, wherein said Hb level is increased by at least about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, or about 2.0 g/dL.

15. The method of any one of claims 10 to 14, wherein said method further comprises modulating one or more of a reticulocyte percentage, absolute reticulocyte count (ARC), bilirubin level, and LDH level.

16. The method of claim 10, wherein said reticulocyte percentage is decreased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40% or about 50%.

17. The method of claim 10, wherein said absolute reticulocyte count (ARC) is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, or at least 5.0 units.

18. The method of claim 10, wherein said bilirubin level is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, or at least 5.0 units.

19. The method of claim 10, wherein said LDH level is decreased is decreased by at least at least 5.0 units, at least 6.0 units, at least 7.0 units, at least 8.0 units, at least 9.0 units, at least 10 units, or at least 15 units.

20. The method of any one of claims 10 to 19, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount from about 200 mg to about 500 mg or from about 300 mg to about 800 mg.

21. The method of any one of claims 10 to 20, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in

54 an amount of about 200 mg, about 300 mg, about 400 mg, about 500, about 600 mg, about 700 mg or about 800 mg

22. The method of claim 20 or claim 21, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered orally once per day.

23. The method of any one of claims 20 to 22, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered for at least 4 weeks, 12 weeks, 16 weeks, or 24 weeks, 1 year, or 1.5 years.

24. The method of any one of claims 10 to 23, further comprising administering hydroxyurea (HU).

25. A method of treating one or more symptoms associated with sickle cell disease (SCD) in a patient in need thereof; the method comprising administering a therapeutically effective amount of 6-[(3S,4S)-4-methyl-l-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4- yl-7H-imidazo[l,5-a]pyrazin-8-one (Compound 1), or a pharmaceutically acceptable salt, solvate, or polymorph thereof.

26. The method of claim 25, wherein said symptom of SCD is vaso-occlusive crisis (VOC).

27. The method of claim 25 or claim 26, wherein VOC is decrease by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% in number prior to treatment.

28. The method of any one of claims 25 to 27, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount from about 200 mg to about 500 mg or from about 300 mg to about 800 mg.

29. The method of any one of claims 25 or 28, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount of about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, or about 800 mg.

30. The method of claim 28 or claim 29, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered orally once per day.

31. The method of any one of claims 28 to 30, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered for at least 4 weeks, 12 weeks, 16 weeks, or 24 weeks, 1 year, or 1.5 years.

32. The method of any one of claims 25 to 31, further comprising administering hydroxyurea (HU).

33. A method of modulating one or more biomarkers over baseline levels prior to treatment in a patient suffering from sickle cell disease (SCD), wherein said biomarkers are selected from

55 reticulocyte percentage, absolute reticulocyte count (ARC), bilirubin level, and LDH level; the method comprising administering a therapeutically effective amount of 6-[(3S,4S)-4-methyl-l- (pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[l,5-a]pyrazin-8- one (Compound 1), or a pharmaceutically acceptable salt, solvate, or polymorph thereof.

34. The method of claim 33, wherein said reticulocyte percentage is decreased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40% or about 50%.

35. The method of claim 33, wherein said absolute reticulocyte count (ARC) is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, or at least 5.0 units.

36. The method of claim 33, wherein said bilirubin level is decreased by at least 1.5 units, at least 2.0 units, at least 2.5 units, at least 3.0 units, at least 3.5 units, at least 4.0 units, at least 4.5 units, or at least 5.0 units.

37. The method of claim 33, wherein said LDH level is decreased is decreased by at least at least 5.0 units, at least 6.0 units, at least 7.0 units, at least 8.0 units, at least 9.0 units, at least 10 units, or at least 15 units.

38. The method of any one of claims 33 to 37, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount from about 200 mg to about 500 mg or from about 300 mg to about 800 mg.

39. The method of any one of claims 33 to 37, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered to the patient in an amount of about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, or about 800 mg.

40. The method of claim 38 or 39, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered orally once per day.

41. The method of any one of claims 38 to 40, wherein the Compound 1, or a pharmaceutically acceptable salt, solvate, or polymorph thereof is administered for at least 4 weeks, 12 weeks, 16 weeks, or 24 weeks, 1 year, or 1.5 years.

42. The method of any one of claims 33 to 41, further comprising administering hydroxyurea (HU).

56