CN118922191A - Organic compound - Google Patents

Abstract

The present disclosure relates to the use of phosphodiesterase 1 (PDE1) inhibitors alone or in combination with immune checkpoint inhibitor therapy for the treatment of breast cancer, including promoting anti-tumor immunity and reducing side effects associated with checkpoint inhibitor therapy (i.e., inflammation-related adverse events).

Description

Organic compounds

Related Applications

This patent application claims the benefit of U.S. Provisional Application No. 63/269,209, filed on March 11, 2022, and U.S. Provisional Application No. 63/479,938, filed on January 13, 2023. The entire contents of these applications are incorporated herein by reference.

Public domain

The present invention relates to the use of phosphodiesterase 1 (PDE1) inhibitors alone or in combination with immune checkpoint inhibitor therapy for the treatment of breast cancer, including for promoting anti-tumor immunity and reducing side effects associated with checkpoint inhibitor therapy (i.e., inflammation-related adverse events).

BACKGROUND OF THE DISCLOSURE

Breast cancer is a cancer that develops from breast tissue. About 10% to 15% of breast cancers are classified as triple-negative breast cancer (TNBC). TNBC is a complex and aggressive subtype of breast cancer that lacks estrogen receptors, progesterone receptors, and HER2. TNBC is one of the most challenging breast cancers to treat because TNBC does not respond to drugs that target estrogen receptors, progesterone receptors, or HER2. Therefore, better treatment options for TNBC are greatly needed.

Immunotherapy, including checkpoint inhibitors, has revolutionized cancer treatment and is effective for many breast cancer patients. Immunotherapy helps the patient's immune system to prevent the growth of cancer. As part of its normal function, the immune system detects and destroys abnormal cells and can also prevent or control the growth of many cancers. However, cancer cells have ways to evade immune responses. Immune activation is primarily mediated by T cells and is regulated by stimulatory, co-stimulatory, and inhibitory (checkpoint) signals. When T cells encounter self cells, there are important receptor-ligand interactions that provide a check on activation so the immune cells do not attack normal cells of the body. Cancer cells have genetic and epigenetic alterations that result in antigen expression, which triggers immune activation, but cancer cells also exploit immune checkpoint interactions such as PD-1/PD-L1 and CTLA4/B7-1/B7-2 to inactivate immune cells, rendering the immune system unable to effectively destroy the cancer. Immune checkpoint inhibitors are effective for many patients with various types of cancer because they allow the patient's own immune system to eliminate the cancer. Unfortunately, some patients do not benefit from these therapies, and the development of resistance can lead to cancer progression in patients with major clinical responses. Therefore, resistance to checkpoint inhibitors (e.g., PD-1/PD-L) blockade remains a major challenge and hinders their wider application.

In addition, immunotherapy-related adverse events can limit the use of checkpoint blockade therapy and may lead to serious adverse consequences. Blocking immune checkpoints can cause the immune system to attack normal tissues. This can lead to inflammatory conditions such as dermatitis, colitis, arthritis, nephritis, myositis, polymyalgia-like syndrome, and cytokine release syndrome (CRS) caused by the rapid release of cytokines in large quantities into the blood by immune cells affected by immunotherapy. These side effects can be very serious and sometimes fatal. Therefore, although immune checkpoint blockade has considerable benefits for cancer patients, immune checkpoint blockade may be limited by the occurrence of adverse events related to immunotherapy.

Eleven families of phosphodiesterases (PDEs) have been identified, but only family I PDEs, Ca2+/calmodulin-dependent phosphodiesterases (CaM-PDEs) activated by Ca2+/calmodulin, have been shown to mediate calcium-dependent cyclic nucleotide (e.g., cGMP and cAMP) signaling pathways. The three known CaM-PDE genes, PDE1A, PDE1B, and PDE1C, are all expressed in central nervous system tissues. PDE1A is expressed in the brain, lungs, and heart. PDE1B is primarily expressed in the central nervous system, but has also been detected in monocytes and neutrophils and has been shown to be involved in inflammatory responses in these cells. PDE1C is expressed in the olfactory epithelium, cerebellar granule cells, striatum, heart, vascular smooth muscle, and tumor cells. PDE1C has been shown to be a master regulator of smooth muscle proliferation in human smooth muscle. Cyclic nucleotide phosphodiesterases downregulate intracellular cAMP and cGMP signaling by hydrolyzing these cyclic nucleotides into their respective 5'-monophosphates (5'AMP and 5'GMP), which are inactive in terms of intracellular signaling pathways. Both cAMP and cGMP are central intracellular second messengers, and they play a role in regulating a variety of cellular functions. PDE1A and PDE1B preferentially hydrolyze cGMP relative to cAMP, while PDE1C shows approximately equal hydrolysis of cGMP and cAMP.

There is a substantial need for new therapies that complement and augment checkpoint inhibitor therapy, and safe and selective strategies are needed to mitigate the severe side effects associated with checkpoint inhibitor therapy (i.e., inflammation-related adverse events).

Overview of the Disclosure

In one aspect, the present disclosure provides a method for treating breast cancer, the method comprising administering a pharmaceutically acceptable amount of a PDE1 inhibitor alone or in combination with a pharmaceutically acceptable amount of an immune checkpoint inhibitor to an individual in need. In some embodiments, breast cancer is triple negative breast cancer (TNBR) with negative estrogen receptors, negative progesterone receptors and negative HER2. In certain embodiments, TNBC is a high-risk early TNBC. In another embodiment, treatment is adjuvant therapy after surgical removal of TNBC. In another embodiment, the individual suffers from locally recurrent unresectable or metastatic TNBC, and the individual's tumor expresses PD-L1, for example, a combined positive score (Combined Positive Score, CPS) ≥ 1 is determined by an FDA-approved test, wherein CPS is the number of PD-L1 stained cells (tumor cells, lymphocytes, macrophages) divided by the total number of live tumor cells, multiplied by 100.

In some embodiments, the immune checkpoint inhibitor is selected from one or more CTLA-4, PD-1 and/or PD-L1 inhibitors. In certain embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, for example, an anti-PD-1 antibody. In some embodiments, the PDE1 inhibitor is a PDE1 inhibitor of Formula I, Ia, II, III, IV, V, and/or VI in free form or in a pharmaceutically acceptable salt form as described below. In some embodiments, the PDE1 inhibitor is Compound A in free form or in a pharmaceutically acceptable salt form or Compound B in free form or in a pharmaceutically acceptable salt form.

In another aspect, the present disclosure provides a method for preventing or alleviating a disease, disorder or adverse reaction caused by administering an immune checkpoint inhibitor therapy to an individual, the method comprising reducing the amount of the checkpoint inhibitor administered to the individual and administering a pharmaceutically acceptable amount of a PDE1 inhibitor in combination with the immune checkpoint inhibitor therapy to the individual.

In another aspect, the present disclosure provides a drug combination therapy comprising a pharmaceutically acceptable amount of a PDE1 inhibitor and a pharmaceutically acceptable amount of an immune checkpoint inhibitor for administration in a method for treating breast cancer or for preventing or alleviating a disease, disorder or adverse reaction caused by administration of a checkpoint inhibitor therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the mean volume of E0771 tumors from mice treated with 900 ppm Compound A, 10 mg/kg anti-PD1, or combination therapy of Compound A (900 ppm) + anti-PD1 (10 mg/kg) in the diet. n=6-7/group. *p<0.05, ns indicates no statistical difference.

Figure 2 shows individual growth curves (volume) of E0771 tumors from mice treated with 900 ppm Compound A, Anti-PD1 10 mg/kg, or combination therapy of Compound A (900 ppm) + Anti-PD1 (10 mg/kg) in the diet.

Figure 3 shows the mean tumor weights at harvest day of E0771 tumors from mice treated with 900 ppm Compound A, Anti-PD1 10 mg/kg, or combination therapy of Compound A (900 ppm) + Anti-PD1 (10 mg/kg) in the diet. n=6-7/group. *p<0.05.

Figures 4A and B show flow cytometric analysis of E0771 tumors from mice treated with 900 ppm Compound A, 10 mg/kg anti-PD1, or a combination of Compound A (900 ppm) + anti-PD1 (10 mg/kg) in the diet. Figure 4A shows the relative proportions of CD45 cells and macrophages in E0771 tumors. Figure 4B shows the M1/M2 ratio of macrophages in E0771 tumors. n=6-7/group. t-test; *P<0.05.

Figure 5 shows flow cytometry analysis of E0771 tumors from mice treated with 900 ppm Compound A, 10 mg/kg anti-PD1, or a combination of Compound A (900 ppm) + anti-PD1 (10 mg/kg) in the diet. Figure 5 shows the relative proportions of T cells, CD8+ cells, CD4+ cells, and NK cells in E0771 tumors. n=6-7/group. t-test; *P<0.05.

Figure 6 shows a volcano plot comparing gene expression in compound A (900ppm) + anti-PD1 (10mg/kg) tumors and (control) groups. The volcano plot shows that 48 genes were downregulated and 136 genes were upregulated in compound A (900ppm) + anti-PD1 (10mg/kg) tumors (fold change <-1.5 or >1.5; P<0.05).

Figure 7 shows pathways enriched for genes differentially expressed in Compound A (900 ppm) + anti-PD1 (10 mg/kg) tumors (top), and transcriptional regulators associated with up- or down-regulated genes in Compound A (900 ppm) + anti-PD1 (10 mg/kg) tumors (bottom).

Figure 8 shows the mean volume of 4T1 tumors from mice treated with 300ppm or 900ppm Compound A, anti-PD1 10mg/kg, or combination therapy of Compound A (300ppm or 900ppm) + anti-PD1 (10mg/kg) in the diet. n=7-8/group. *p<0.05; **p<0.01; and ***p<0.001, ns indicates no statistical difference.

Figure 9 shows individual growth curves (volume) of 4T1 tumors from mice treated with 300 ppm or 900 ppm Compound A, Anti-PD1 10 mg/kg, or combination therapy of Compound A (300 ppm or 900 ppm) + Anti-PD1 (10 mg/kg) in the diet.

Figure 10 shows the average tumor weights at harvest day from 4T1 tumors of mice treated with 300ppm or 900ppm Compound A, anti-PD1 10mg/kg, or combination therapy of Compound A (300ppm or 900ppm) + anti-PD1 (10mg/kg) in the diet. n=7-8/group. *p<0.05; **p<0.01; and ***p<0.001, ns indicates no statistical difference.

Figure 11 shows the survival curves of mice treated with 300 ppm or 900 ppm Compound A, Anti-PD1 10 mg/kg, or combination therapy of Compound A (300 ppm or 900 ppm) + Anti-PD1 (10 mg/kg) in the diet. n=7-8/group.

Figure 12 shows the mean volume of E0771 tumors from mice treated with 100 ppm, 300 ppm or 900 ppm Compound B, anti-PD1 1 mg/kg, or combination therapy of Compound B (100 ppm, 300 ppm or 900 ppm) + anti-PD1 (1 mg/kg) in the diet. n=5-6/group. *p<0.05, ns indicates no statistical difference.

Figure 13 shows individual growth curves (volume) of E0771 tumors from mice treated with 100 ppm, 300 ppm or 900 ppm Compound B, Anti-PD1 1 mg/kg, or combination therapy of Compound B (100 ppm, 300 ppm or 900 ppm) + Anti-PD1 (1 mg/kg) in the diet.

Figure 14 shows the mean tumor weight at harvest day from E0771 tumors of mice treated with 100 ppm, 300 ppm or 900 ppm Compound B, Anti-PD1 1 mg/kg, or combination therapy of Compound B (100 ppm, 300 ppm or 900 ppm) + Anti-PD1 (1 mg/kg) in the diet. n=5-6/group. *p<0.05; **p<0.01.

Figure 15 shows the mean volume of 4T1 tumors from mice treated with 100ppm, 300ppm or 900ppm Compound B, anti-PD1 10mg/kg, or combination therapy of Compound B (100ppm, 300ppm or 900ppm) + anti-PD1 (10mg/kg) in the diet. n=7-9/group. *p<0.05; **p<0.01; and ***p<0.001, ns indicates no statistical difference.

16 shows individual growth curves (volume) of 4T1 tumors from mice treated with 100 ppm, 300 ppm or 900 ppm Compound B, Anti-PD1 10 mg/kg, or combination therapy of Compound B (100 ppm, 300 ppm or 900 ppm) + Anti-PD1 (10 mg/kg) in the diet.

Figure 17 shows the average tumor weight at harvest day of 4T1 tumors from mice treated with 100 ppm, 300 ppm or 900 ppm Compound B, anti-PD1 10 mg/kg, or combination therapy of Compound B (100 ppm, 300 ppm or 900 ppm) + anti-PD1 (10 mg/kg) in the diet. n=7-9/group. *p<0.05; **p<0.01; and ***p<0.001, ns indicates no statistical difference.

Figures 18A and B show flow cytometry analysis of 4T1 tumors in mice treated with 900 ppm Compound B, 10 mg/kg anti-PD1, or a combination of Compound B (900 ppm) + anti-PD1 (10 mg/kg) in the diet. Figure 18A shows the relative proportions of CD45 cells and macrophages in 4T1 tumors. Figure 18B shows the M1/M2 ratio of macrophages in 4T1 tumors. n = 6-7/group. t-test, *p < 0.05.

Figure 19 shows flow cytometry analysis of 4T1 tumors in mice treated with 900 ppm Compound B, 10 mg/kg anti-PD1, or a combination of Compound B (900 ppm) + anti-PD1 (10 mg/kg) in the diet. Figure 19 shows the relative proportions of various T cells in 4T1 tumors. n=6-7/group. t-test, *p<0.05.

Figure 20 shows a volcano plot of gene expression comparison of compound B (900ppm) + anti-PD1 (10mg/kg) tumors and (control) groups. The volcano plot shows that 281 genes were downregulated and 708 genes were upregulated in compound B (900ppm) + anti-PD1 (10mg/kg) tumors (fold change <-1.5 or >1.5; P<0.05).

Figure 21 shows pathways enriched for genes differentially expressed in Compound B (900 ppm) + anti-PD1 (10 mg/kg) tumors (Figure 21A), and transcriptional regulators associated with up-regulated or down-regulated genes in Compound B (900 ppm) + anti-PD1 (10 mg/kg) tumors (Figure 21B).

DETAILED DESCRIPTION OF THE DISCLOSURE

The inventors have previously shown that inhibition of PDE1 activity using the presently disclosed compounds can safely restore cAMP function in a wide range of pathological conditions, including neurodegeneration and neuroinflammation, heart failure, pulmonary hypertension, and peripheral inflammation models, as well as in humans with certain diseases. More recently, the inventors have shown that PDE1 inhibitors modulate immune cell function (microglia and macrophages) by altering cell migration and levels of key cytokines, primarily CCL2 and TNF-α. Recent evidence suggests that PDE1, and in particular the PDE1C isoform, is overexpressed in experimental tumor models such as melanoma, neuroblastoma, renal cell carcinoma, and colon cancer, as well as osteosarcoma. In addition, focal genomic overexpression of PDE1C in glioblastoma multiforme (GBM) cells has been demonstrated. Genomic amplification of PDE1C is associated with increased expression in GBM-derived cell cultures and is critical for driving cell proliferation, migration, and invasion in cancer cells.

Many types of cancer cells overexpress PDE1 activity, which can be identified by various biomarkers, such as increased RNA expression, DNA copy number, PDE1 binding (PET or radioisotope retention of PDE1 inhibitor molecules), or enzyme activity. These cancer cells also exhibit low levels of cAMP, which is increased by PDE1 inhibitors.

In the present invention, it has been found that PDE-1 inhibitors promote anti-tumor immunity when administered alone or in combination with immune checkpoint inhibitor therapy, resulting in growth inhibition of breast cancer. It has been found that in a triple-negative breast cancer (TNBC) mouse model, PDE-1 inhibitors alone or in combination with sub-effective amounts of anti-PD-1 antibodies can inhibit the growth of breast cancer. In contrast, the tumor growth of mice treated with anti-PD-1 antibodies alone was similar to that of isotype controls. It was further found that combined therapy shifted the polarization of macrophages in the tumor microenvironment to a more inflammatory phenotype. Without being bound by any theory, it is believed that PDE-1 inhibitors affect macrophage infiltration and polarization, thereby promoting anti-tumor immunity. The synergistic ability of PDE-1 inhibitors and immune checkpoint inhibitors to change the tumor microenvironment and inhibit tumor growth can provide a method to expand the utility of immune checkpoint inhibitors in the treatment of refractory tumors such as TNBC. Furthermore, this synergistic ability of PDE-1 inhibitors in combination with less potent immune checkpoint inhibitors may provide a means to mitigate the adverse effects caused by checkpoint inhibitor therapy administered to breast cancer patients by reducing the dose of checkpoint inhibitors administered to patients.

Compounds for use in the methods of the present disclosure

In one embodiment, the PDEl inhibitors used in the treatment and prevention methods described herein are selective PDEl inhibitors.

PDE1 inhibitors

In one embodiment, the PDE1 inhibitor used in the methods of treatment and prevention described herein is a compound of formula I in free form, salt form, or prodrug form:

in

(i) R ₁ is H or C _1-4 alkyl (e.g., methyl);

(ii) R ₄ is H or C _1-4 alkyl and R ₂ and R ₃ are independently H or C _1-4 alkyl (e.g., R ₂ and R ₃ are both methyl, or R ₂ is H and R ₃ is isopropyl), aryl, heteroaryl, (optionally hetero)arylalkoxy, or (optionally hetero)arylalkyl; or

_R2 is H and _R3 and _R4 together form a di-, tri- or tetramethylene bridge (preferably, wherein _R3 and _R4 together have a cis configuration, e.g., wherein the carbons carrying _R3 and _R4 have the R and S configuration, respectively);

(iii) R ₅ is substituted heteroarylalkyl, for example, substituted with haloalkyl;

or R ₅ is attached to a nitrogen on the pyrazolo portion of formula I,

and R ₅ is a moiety of formula A

wherein X, Y, and Z are independently N or C, and R ₈ , R ₉ , R ₁₁ , and R ₁₂ are independently H or halogen (e.g., Cl or F), and R ₁₀ is halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluoromethyl), aryl (e.g., phenyl), heteroaryl optionally substituted with halogen (e.g., pyridyl (e.g., pyridin-2-yl), or thiadiazolyl (e.g., 1,2,3-thiadiazol-4-yl)), diazolyl, triazolyl, tetrazolyl, arylcarbonyl (e.g., benzoyl), alkylsulfonyl (e.g., methylsulfonyl), heteroarylcarbonyl, or alkoxycarbonyl; provided that when X, Y, or Z is nitrogen, R ₈ , R ₉ , or R ₁₀ , respectively, is absent; and

(iv) R ₆ is H, alkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), arylamino (e.g., phenylamino), heteroarylamino, N,N-dialkylamino, N,N-diarylamino, or N-aryl-N-(arylalkyl)amino (e.g., N-phenyl-N-(1,1'-biphenyl-4-ylmethyl)amino); and

(v) n = 0 or 1;

(vi) When n=1, A is -C(R ₁₃ R ₁₄ )-

wherein R ₁₃ and R ₁₄ are independently H or C _1-4 alkyl, aryl, heteroaryl, (optionally hetero)arylalkoxy or (optionally hetero)arylalkyl;

It includes their enantiomers, diastereomers and racemates.

In another embodiment, the PDEl inhibitor used in the methods of treatment and prevention described herein is Formula Ia in free form, in pharmaceutically acceptable salt form, or in prodrug form:

in

(i) _R2 and _R5 are independently H or hydroxy and _R3 and _R4 together form a trimethylene or tetramethylene bridge [preferably, wherein the carbons carrying _R3 and _R4 have the R and S configuration respectively]; or _R2 and _R3 are each methyl and _R4 and _R5 are each H; or _R2 , _R4 and _R5 are H and _R3 is isopropyl [preferably, the carbon carrying _R3 has the R configuration];

(ii) R ₆ is phenylamino (optionally substituted by halogen atoms), benzylamino (optionally substituted by halogen atoms), C _1-4 alkyl, or C _1-4 alkylthio; for example, phenylamino or 4-fluorophenylamino;

(iii) R ₁₀ is C _1-4 alkyl, methylcarbonyl, hydroxyethyl, carboxylic acid, sulfonyl, phenyl (optionally substituted by halogen or hydroxy), pyridyl (optionally substituted by halogen or hydroxy) (e.g. 6-fluoropyridin-2-yl), or thiadiazolyl (e.g. 1,2,3-thiadiazol-4-yl); and

(iv) X and Y are independently C or N,

It includes their enantiomers, diastereomers and racemates.

In another embodiment, the PDE1 inhibitor used in the methods of treatment and prevention described herein is a compound of Formula II in free form, salt form, or prodrug form:

(i) X is C _1-6 alkylene (e.g., methylene, ethylene or prop-2-yn-1-yl);

(ii) Y is a single bond, an alkynylene group (e.g., —C≡C—), an arylene group (e.g., phenylene) or a heteroarylene group (e.g., pyridylene);

(iii) Z is H, aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, e.g., pyridin-2-yl), halogen (e.g., F, Br, Cl), halogenated C _1-6 alkyl (e.g., trifluoromethyl), —C(O)—R ¹ , —N(R ² )(R ³ ), or C _3-7 cycloalkyl optionally containing at least one atom selected from N or O (e.g., cyclopentyl, cyclohexyl, tetrahydro-2H-pyran-4-yl, or morpholinyl);

(iv) R ¹ is C _1-6 alkyl, halogenated C _1-6 alkyl, —OH or —OC _1-6 alkyl (e.g., —OCH ₃ );

(v) ^R2 and ^R3 are independently H or _C1-6 alkyl;

(vi) ^R4 and ^R5 are independently H, _C1-6 alkyl, or aryl (e.g., phenyl) (the aryl is optionally substituted with one or more halogen atoms (e.g., fluorophenyl, e.g., 4-fluorophenyl), hydroxy (e.g., hydroxyphenyl, e.g., 4-hydroxyphenyl or 2-hydroxyphenyl)) or _C1-6 alkoxy;

(vii) wherein X, Y and Z are independently and optionally substituted by one or more halogen atoms (e.g., F, Cl or Br), Ci _-6 alkyl (e.g., methyl), halo-Ci _-6 alkyl (e.g., trifluoromethyl), for example, Z is heteroaryl, for example, pyridyl, which is substituted by one or more halogen atoms (e.g., 6-fluoropyridin-2-yl, 5-fluoropyridin-2-yl, 6-fluoropyridin-2-yl, 3-fluoropyridin-2-yl, 4-fluoropyridin-2-yl, 4,6-dichloropyridin-2-yl), halo-Ci _-6 alkyl (e.g., 5-trifluoromethylpyridin-2-yl) or Ci- ₆ -alkyl (e.g., 5-methylpyridin-2-yl), or Z is aryl, for example, phenyl, which is substituted by one or more halogen atoms (e.g., 4-fluorophenyl).

In yet another embodiment, the PDE1 inhibitor used in the methods of treatment and prevention described herein is of Formula III in free form or in salt form:

in

(i) R1 is H or _C1-4 alkyl (e.g., methyl or ethyl);

(ii) R ₂ and R ₃ are independently H or C _1-6 alkyl (e.g., methyl or ethyl);

(iii) R ₄ is H or C _1-4 alkyl (e.g., methyl or ethyl);

(iv) R ₅ is aryl (eg, phenyl), which is optionally substituted with one or more groups independently selected from -C(=O)-C _1-6 alkyl (eg, -C(=O)-CH ₃ ) and C _1-6 -hydroxyalkyl (eg, 1-hydroxyethyl);

(v) _R and _R are independently H or aryl (e.g., phenyl) optionally substituted by one or more groups independently selected from C _1-6 alkyl (e.g., methyl or ethyl) and halogen (e.g., F or Cl), such as unsubstituted phenyl, or phenyl substituted by one or more halogen (e.g., F), or phenyl substituted by one or more C _1-6 alkyl and one or more halogen, or phenyl substituted by one C _1-6 alkyl and one halogen, such as 4-fluorophenyl or 3,4-difluorophenyl or 4-fluoro-3-methylphenyl; and

(vi) n is 1, 2, 3, or 4.

In yet another embodiment, the PDE1 inhibitor used in the treatment and prevention methods described herein is a free form or salt form of Formula IV

in

(i) R ₁ is C _1-4 alkyl (eg, methyl or ethyl), or -NH(R ₂ ), wherein R ₂ is phenyl optionally substituted by a halogen atom (eg, fluorine), for example, 4-fluorophenyl;

(ii) X, Y and Z are independently N or C;

(iii) R ₃ , R ₄ and R ₅ are independently H or C _1-4 alkyl (e.g. methyl); or R ₃ is H and R ₄ and R ₅ together form a trimethylene bridge (preferably, wherein R ₄ and R ₅ together have a cis configuration, e.g., wherein the carbons carrying R ₄ and R ₅ have an R and S configuration, respectively),

(iv) R ₆ , R ₇ and R ₈ are independently:

H,

C _1-4 alkyl (e.g., methyl),

pyridin-2-yl substituted by hydroxy, or

–S(O) ₂ -NH ₂ ;

(v) provided that when X, Y and/or Z is N, then _R6 , _R7 and/or _R8, respectively, are absent; and when X, Y and Z are all C, then at least one of _R6 , _R7 or _R8 is -S(O) ₂ - _NH2 or pyridin-2-yl substituted with hydroxy.

In another embodiment, the PDEl inhibitor used in the methods described herein is of Formula V in free form or in salt form:

in

(i) R ₁ is -NH(R ₄ ), wherein R ₄ is phenyl optionally substituted by a halogen atom (eg, fluorine), such as 4-fluorophenyl;

(ii) R ₂ is H or C _1-6 alkyl (e.g., methyl, isobutyl or neopentyl);

(iii) R ₃ is -SO ₂ NH ₂ or -COOH.

In another embodiment, the PDEl inhibitor used in the methods described herein is of Formula VI in free form or in salt form:

in

(i) R ₁ is -NH(R ₄ ), wherein R ₄ is phenyl optionally substituted by a halogen atom (eg, fluorine), such as 4-fluorophenyl;

(ii) R ₂ is H or C _1-6 alkyl (e.g., methyl or ethyl);

(iii) R ₃ is H, halogen (e.g., bromine), C _1-6 alkyl (e.g., methyl), aryl optionally substituted by halogen (e.g., 4-fluorophenyl), heteroaryl optionally substituted by halogen (e.g., 6-fluoropyridin-2-yl or pyridin-2-yl), or acyl (e.g., acetyl).

In one embodiment, the disclosure provides for administration of a PDE1 inhibitor (e.g., a compound according to Formula I, Ia, II, III, IV, V, and/or VI) for use in the methods described herein, wherein the inhibitor is a compound according to:

In one embodiment, the invention provides for the administration of a PDEl inhibitor for treatment or prevention as described herein, wherein the inhibitor is a compound according to:

In yet another embodiment, the present invention provides for the administration of a PDEl inhibitor for treatment or prevention as described herein, wherein the inhibitor is a compound according to:

In yet another embodiment, the present invention provides for the administration of a PDEl inhibitor for treatment or prevention as described herein, wherein the inhibitor is a compound according to:

In yet another embodiment, the present invention provides for the administration of a PDEl inhibitor for treatment or prevention as described herein, wherein the inhibitor is a compound according to:

In one embodiment, the selective PDE1 inhibitor of any of the foregoing formulae (e.g., Formula I, Ia, II, III, IV, V, and/or VI) in free form or salt form is a compound that inhibits phosphodiesterase-mediated (e.g., PDE1-mediated, particularly PDE1B-mediated) hydrolysis of cGMP, e.g., preferred compounds have an IC50 of less than 1 μM, preferably less than 500 nM, preferably less than 50 nM, and preferably less than ₅ nM in an immobilized metal affinity microparticle reagent PDE assay.

In other embodiments, the present invention provides for the administration of a PDEl inhibitor for the treatment or prevention described herein, wherein the inhibitor is a compound according to:

Other examples of PDE1 inhibitors suitable for use in the methods and treatments discussed herein can be found in the following documents: International Publication WO2006133261A2; U.S. Patent 8,273,750; U.S. Patent 9,000,001; U.S. Patent 9,624,230; International Publication WO2009075784A1; U.S. Patent 8,273,751; U.S. Patent 8,829,008; U.S. Patent 9,403,836; International Publication WO2014151409A1, U.S. Patent 9,073,936; U.S. Patent 9,598,426; U.S. Patent 9,556,186; U.S. Publication 2017/0231994A1, International Publication WO2016022893A1 and U.S. Publication 2017/0226117A1, each of which is incorporated by reference in its entirety.

Other examples of PDE1 inhibitors suitable for use in the methods and treatments discussed herein can be found in the following documents: International Publication WO2018007249A1; U.S. Publication 2018/0000786; International Publication WO2015118097A1; U.S. Patent 9,718,832; International Publication WO2015091805A1; U.S. Patent 9,701,665; U.S. Publication 2015/0175584A1; U.S. Publication 2017/0267664A1; International Publication W O2016055618A1; U.S. Publication 2017/0298072A1; International Publication WO2016170064A1; U.S. Publication 2016/0311831A1; International Publication WO2015150254A1; U.S. Publication 2017/0022186A1; International Publication WO2016174188A1; U.S. Publication 2016/0318939A1; U.S. Publication 2017/0291903A1; International Publication WO2018073251 A1; International Publication WO2017178350A1; U.S. Publication 2017/0291901A1; International Publication WO2018/115067; U.S. Publication 2018/0179200A; U.S. Publication US20160318910A1; U.S. Patent 9,868,741; International Publication WO2017/139186A1; International Application WO2016/040083; U.S. Publication 2017/0240532; International Publication WO2016033776 A1; U.S. Publication 2017/0233373; International Publication WO2015130568; International Publication WO2014159012; U.S. Patent 9,034,864; U.S. Patent 9,266,859; International Publication WO2009085917; U.S. Patent 8,084,261; International Publication WO2018039052; U.S. Publication US20180062729; and International Publication WO2019027783, each of which is incorporated by reference in its entirety. In any case where the statements of any reference document incorporated by reference are contradictory or incompatible with any statement in the present disclosure, the statement of the present disclosure should be understood to be controlling.

Unless otherwise specified or clear from the context, the following terms in this document have the following meanings:

(a) As used herein, "selective PDEl inhibitor" refers to a PDEl inhibitor that has at least 100-fold selectivity for inhibition of PDEl relative to inhibition of any other PDE isoform.

(b) As used herein, "alkyl" is a saturated or unsaturated hydrocarbon moiety, preferably saturated, preferably having 1-6 carbon atoms, which may be straight chain or branched and may be optionally mono-, di- or tri-substituted, for example by halogen (e.g. chlorine or fluorine), hydroxyl or carboxyl.

(c) As used herein, "cycloalkyl" is a saturated or unsaturated non-aromatic hydrocarbon moiety, preferably saturated, preferably containing 3-9 carbon atoms, at least some of which form non-aromatic mono- or bicyclic or bridged ring structures, and may be optionally substituted, for example, by halogen (e.g., chlorine or fluorine), hydroxyl or carboxyl. Wherein the cycloalkyl optionally contains one or more atoms selected from N and O and/or S, the cycloalkyl may also be a heterocycloalkyl.

(d) Unless otherwise indicated, "heterocycloalkyl" is a saturated or unsaturated non-aromatic hydrocarbon moiety, preferably saturated, preferably containing 3-9 carbon atoms, at least some of which form a non-aromatic mono- or bicyclic or bridged ring structure, in which at least one carbon atom is substituted by N, O or S, which heterocycloalkyl may be optionally substituted, for example, by halogen (e.g. chlorine or fluorine), hydroxy or carboxyl.

(e) As used herein, "aryl" is a mono- or bicyclic aromatic hydrocarbon, preferably phenyl, which is optionally substituted, for example, by alkyl (e.g., methyl), halogen (e.g., chloro or fluoro), haloalkyl (e.g., trifluoromethyl), hydroxy, carboxyl, or by another aryl or heteroaryl (e.g., biphenyl or pyridylphenyl).

(f) As used herein, "heteroaryl" is an aromatic moiety in which one or more of the atoms constituting the aromatic ring is sulfur or nitrogen rather than carbon, such as pyridyl or thiadiazolyl, which may be optionally substituted, for example, with alkyl, halogen, haloalkyl, hydroxyl or carboxyl.

The compounds of the present disclosure, such as the PDE1 inhibitors described herein, may exist in free form or in salt form, such as in the form of an acid addition salt. In this specification, unless otherwise indicated, phrases such as "compounds of the present disclosure" should be understood to include compounds in any form, such as free form or acid addition salt form, or if the compound contains an acidic substituent, a base addition salt form. The compounds of the present disclosure are intended for use as drugs, and therefore, pharmaceutically acceptable salts are preferred. Salts that are not suitable for pharmaceutical use may be useful, for example, for isolating or purifying free compounds of the present disclosure or their pharmaceutically acceptable salts, and are therefore also included.

Compounds of the present disclosure may also exist in prodrug form in some cases. Prodrug form is a compound that is converted into a compound of the present disclosure in vivo. For example, when a compound of the present disclosure contains a hydroxyl or carboxyl substituent, these substituents can form physiologically hydrolyzable and acceptable esters. As used herein, "physiologically hydrolyzable and acceptable esters" refer to esters of compounds of the present disclosure, which can be hydrolyzed to generate acids (in the case of compounds of the present disclosure with hydroxyl substituents) or alcohols (in the case of compounds of the present disclosure with carboxyl substituents) under physiological conditions, which themselves are physiologically tolerable at the administered dose. Therefore, if a compound of the present disclosure contains a hydroxyl group, such as compound-OH, the acyl ester prodrug of such compound, i.e., compound-O-C (O)-C1-4 alkyl, can be hydrolyzed into physiologically hydrolyzable alcohols (compound-OH) on the one hand in vivo, and can be hydrolyzed into physiologically hydrolyzable acids (such as HOC (O)-C1-4 alkyl) on the other hand in vivo. Alternatively, if a compound of the present disclosure contains a carboxylic acid, such as compound-C(O)OH, then an acidic ester prodrug of such compound, i.e., compound-C(O)O-C1-4 alkyl, can be hydrolyzed to compound-C(O)OH and HO-C1-4 alkyl. As can be appreciated, the term therefore includes conventional pharmaceutically acceptable prodrug forms.

In another embodiment, the present disclosure also provides a pharmaceutical composition comprising a combination of a PDE1 inhibitor and an immune checkpoint inhibitor (each of which is in the form of a free form or a pharmaceutically acceptable salt) and a pharmaceutically acceptable carrier. As used herein, the term "combination" includes simultaneous, sequential or contemporaneous administration of a PDE1 inhibitor and an immune checkpoint inhibitor. In some embodiments, the combination of a PDE1 inhibitor and an immune checkpoint inhibitor allows the immune checkpoint inhibitor to be administered at a dose lower than the dose that would be effective if administered as the only monotherapy.

Methods of using the disclosed compounds

In one embodiment, the application provides a method for treating breast cancer (Method 1), comprising administering a pharmaceutically acceptable amount of a PDE1 inhibitor (e.g., a PDE1 inhibitor according to Formula I, Ia, II, III, IV, V, and/or VI) to an individual in need thereof, alone or in combination with a pharmaceutically acceptable amount of an immune checkpoint inhibitor.

1.1 Method 1, wherein PDE1 expression is elevated in breast cancer.

1.2 Any of the foregoing methods, wherein the breast cancer expresses PD-L1, such as a combined positive score (CPS) ≥ 1 as determined by an FDA-approved test, wherein the CPS is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100.

1.3 Any of the preceding methods, wherein the breast cancer is triple-negative breast cancer (TNBC), which is estrogen receptor negative, progesterone receptor negative, and HER2 negative.

1.4 Method 1.3, wherein the TNBC is high-risk early TNBC.

1.5 Method 1.3, wherein the treatment is adjuvant therapy after surgery.

1.6 Method 1.3, wherein the individual has locally recurrent unresectable or metastatic TNBC, wherein the individual's tumor expresses PD-L1, e.g., as determined by an FDA-approved test, with a combined positive score (CPS) ≥ 1, wherein CPS is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100.

1.7 Any of the preceding methods, wherein administration of a pharmaceutically acceptable amount of a PDEl inhibitor in combination with a pharmaceutically acceptable amount of an immune checkpoint inhibitor to the individual increases the M1/M2 ratio of macrophages in the tumor microenvironment.

1.8 Any of the foregoing methods, wherein administration of a pharmaceutically acceptable amount of an immune checkpoint inhibitor to the individual alone (ie, not in combination with a PDE1 inhibitor) is ineffective for treating breast cancer, eg, it does not inhibit the growth of breast cancer.

1.9 Any of the foregoing methods, wherein administering a pharmaceutically acceptable amount of a PDEl inhibitor to the individual alone (ie, not in combination with a checkpoint inhibitor) is ineffective for treating breast cancer, eg, it does not inhibit the growth of breast cancer.

1.10 Any of the preceding methods, wherein the immune checkpoint inhibitor is selected from one or more CTLA-4, PD-1 and/or PD-L1 inhibitors.

1.11 Any of the preceding methods, wherein the immune checkpoint inhibitor is a PD-1 inhibitor, optionally wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, further optionally wherein the antibody is monoclonal or polyclonal.

1.12 Any of the preceding methods, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor, optionally wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody, further optionally wherein the antibody is monoclonal or polyclonal.

1.13 Any of the preceding methods, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor, optionally wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody, further optionally wherein the antibody is monoclonal or polyclonal.

1.14 Any of the preceding methods, wherein the immune checkpoint inhibitor comprises one or more members selected from the group consisting of nivolumab, pembrolizumab, cemiplizumab, ipilimumab, avelumab, durvalumab, atezolizumab, and spartalizumab.

1.15 Any of the foregoing methods, wherein the individual suffers from a systemic inflammatory response, a gastrointestinal inflammatory disorder, an endocrine inflammatory disorder, a skin inflammatory disorder, an ophthalmic inflammatory disorder, a neurological inflammatory disorder, a blood inflammatory disorder, a genitourinary inflammatory disorder, a respiratory inflammatory disorder, a musculoskeletal inflammatory disorder, a cardiac inflammatory disorder, or a defined systemic inflammatory disorder.

1.16 Any of the foregoing methods, wherein the individual in need thereof has previously been administered an immune checkpoint inhibitor therapy, optionally wherein the pharmaceutically acceptable amount of the immune checkpoint inhibitor administered to the individual is lower than the amount of the immune checkpoint inhibitor in the previous immune checkpoint inhibitor therapy.

1.17 Any of the foregoing methods, wherein the individual in need thereof suffers from a disease, disorder, or adverse effect of immune checkpoint inhibitor therapy.

1.18 Any of the foregoing methods, wherein the individual suffers from an inflammation-related disorder following immune checkpoint inhibitor therapy.

1.19 Any of the foregoing methods, wherein the individual suffers from, or is at risk of suffering from, a side effect of immune checkpoint inhibitor therapy, for example, wherein the side effect is selected from: a systemic inflammatory response, a gastrointestinal inflammation-related disorder, an endocrine inflammation-related disorder, a skin inflammation-related disorder, an ophthalmic inflammation-related disorder, a neurological inflammation-related disorder, a blood inflammation-related disorder, a genitourinary inflammation-related disorder, a respiratory system inflammation-related disorder, a musculoskeletal inflammation-related disorder, a cardiac inflammation-related disorder, or a well-defined systemic inflammation-related disorder.

1.20 Any of the foregoing methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a gastrointestinal inflammatory-related disorder, e.g., selected from colitis, enterocolitis, colitis complicated by intestinal perforation, hepatitis, pancreatitis.

1.21 Any of the foregoing methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is an endocrine inflammation-related disorder, e.g., selected from hypophysitis (e.g., manifesting as panhypopituitarism), thyrotoxicosis, hypothyroidism, syndrome of inappropriate antidiuretic hormone secretion, central adrenal insufficiency, primary adrenal insufficiency, and diabetes mellitus.

1.22 Any of the foregoing methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a skin inflammation-related disorder, e.g., selected from rash, pruritus, vitiligo, dermatitis, Sweet's syndrome, drug eruption, gray hair, delayed hypersensitivity reaction, alopecia universalis, grower's disease, pyoderma gangrenosum, toxic epidermal necrolysis, chronic non-caseation granuloma, bullous pemphigoid, and psoriasis.

1.23 Any of the foregoing methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is an ophthalmic inflammation-related disorder, e.g., selected from uveitis, conjunctivitis, orbital inflammation, Graves' ophthalmopathy, choroidal neovascularization, optic neuropathy, keratitis, and retinopathy.

1.24 Any of the foregoing methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a neuroinflammation-related disorder, e.g., selected from encephalopathy, Guillain-Barré syndrome, polyradiculoneuropathy, symmetric multifocal neuropathy, transverse myelitis, necrotizing myelopathy, myasthenia gravis, phrenic nerve palsy, immune-related meningitis, meningoradiculoneuritis, peripheral neuropathy, autoimmune inner ear disease, multiple sclerosis, and inflammatory enteric neuropathy.

1.25 Any of the foregoing methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a blood inflammation-related disorder, e.g., selected from thrombocytopenia, pancytopenia, neutropenia, eosinophilia, pure red cell aplasia, acquired hemophilia A, and disseminated intravascular coagulopathy.

1.26 Any of the foregoing methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a genitourinary inflammation-related disorder, e.g., selected from renal failure, acute/granulomatous interstitial nephritis, acute tubular necrosis, and lymphocytic vasculitis (e.g., uterine lymphocytic vasculitis).

1.27 Any of the preceding methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a respiratory inflammation-related disorder, e.g., selected from pneumonia and acute respiratory distress.

1.28 Any of the foregoing methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a musculoskeletal inflammation-related disorder, e.g., selected from polyarthritis, arthralgia, myalgia, chronic granulomatous inflammation of the rectus abdominis muscle, and rhabdomyolysis.

1.29 Any of the preceding methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a cardiac inflammation-related disorder, e.g., selected from precarditis and Takotsubo-like syndrome.

1.30 Any of the foregoing methods, wherein the individual suffers from a side effect of immune checkpoint inhibitor therapy, wherein the side effect is a systemic inflammation-related disorder, e.g., selected from pulmonary nodules, skin and pulmonary nodules, polymyalgia rheumatica, giant cell arteritis, muscular sarcoidosis, neural and pulmonary nodules, celiac disease, lupus nephritis, dermatomyositis, autoimmune inflammatory myopathy, and Vogt-Koyanagi like syndrome.

1.31 Any of the preceding methods, wherein the PDE1 inhibitor and the immune checkpoint inhibitor are administered before, after, or together with radiotherapy or chemotherapy.

1.32 Any of the preceding methods, wherein the PDE1 inhibitor and the immune checkpoint inhibitor are administered concurrently with radiation therapy or chemotherapy.

1.33 Any of the preceding methods, wherein the PDE1 inhibitor and the immune checkpoint inhibitor are administered prior to radiotherapy or chemotherapy.

1.34 Any of the preceding methods, wherein the PDE1 inhibitor and the immune checkpoint inhibitor are administered after radiation therapy or chemotherapy.

1.35 Any of the preceding methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered together with an additional anti-tumor agent, chemotherapeutic, gene therapeutic and/or immunotherapeutic.

1.36 Any of the preceding methods, wherein the PDE1 inhibitor is a PDE1 inhibitor according to Formula I, Ia, II, III, IV, V, and/or VI in free form or in the form of a pharmaceutically acceptable salt or a compound according to:

1.37 Any of the preceding methods, wherein the PDEl inhibitor is a PDEl inhibitor according to Formula Ia.

1.38 Any of the preceding methods, wherein the PDE1 inhibitor is a compound in free form or in the form of a pharmaceutically acceptable salt:

1.39 Any of the preceding methods, wherein the PDE1 inhibitor is a compound in free form or in a pharmaceutically acceptable salt form (eg, monophosphate form):

1.40 Any of the preceding methods, further comprising administering to the individual a pharmaceutically acceptable amount of a beta blocker.

1.41 Any of the preceding methods wherein the subject is a human.

In another embodiment, the present disclosure provides a PDE1 inhibitor for use alone or in combination with an immune checkpoint inhibitor in the treatment of breast cancer, for example, for use in any one of Method 1, etc.

In another embodiment, the present application provides a method for preventing or alleviating a disease, disorder, or adverse reaction following administration of an immune checkpoint inhibitor to an individual with breast cancer (Method 2), the method comprising reducing the amount of the checkpoint inhibitor administered to the individual and administering to the individual a pharmaceutically acceptable amount of a PDE1 inhibitor (i.e., a PDE1 inhibitor according to Formula I, Ia, II, III, IV, V, and/or VI) and an immune checkpoint inhibitor therapy in combination.

2.1 Method 2, wherein PDE1 expression is elevated in breast cancer.

2.2 Any of the foregoing methods, wherein the breast cancer expresses PD-L1, e.g., as determined by an FDA-approved test, with a combined positive score (CPS) ≥ 1, wherein CPS is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100.

2.3 Any of the preceding methods, wherein the breast cancer is triple negative breast cancer (TNBC).

2.4 Method 2.3, wherein the TNBC is high-risk early TNBC.

2.5 Method 2.3, wherein immune checkpoint inhibitor therapy is an adjuvant therapy after surgery.

2.6 Method 2.3, wherein the individual has locally recurrent unresectable or metastatic TNBC, wherein the individual's tumor expresses PD-L1, e.g., as determined by an FDA-approved test, with a combined positive score (CPS) ≥ 1, wherein CPS is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100.

2.7 Any of the foregoing methods, wherein administration of a reduced amount of an immune checkpoint inhibitor to an individual is ineffective for treating breast cancer when administered alone (i.e., not in combination with a PDE1 inhibitor), for example, administration of a reduced amount of an immune checkpoint inhibitor to an individual alone (i.e., not in combination with a PDE1 inhibitor) does not inhibit the growth of breast cancer.

2.8 Any of the preceding methods, wherein administration of a pharmaceutically acceptable amount of a PDEl inhibitor in combination with a pharmaceutically acceptable amount of an immune checkpoint inhibitor to the individual increases the M1/M2 ratio of macrophages in the tumor microenvironment.

2.9 Any of the preceding methods, wherein the immune checkpoint inhibitor is selected from one or more CTLA-4, PD-1 and/or PD-L1 inhibitors.

2.10 Any of the preceding methods, wherein the immune checkpoint inhibitor is a PD-1 inhibitor, optionally wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, further optionally wherein the antibody is monoclonal or polyclonal.

2.11 Any of the preceding methods, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor, optionally wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody, further optionally wherein the antibody is monoclonal or polyclonal.

2.12 Any of the preceding methods, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor, optionally wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody, further optionally wherein the antibody is monoclonal or polyclonal.

2.13 Any of the preceding methods, wherein the immune checkpoint inhibitor comprises one or more members selected from the group consisting of nivolumab, pembrolizumab, cemiplizumab, ipilimumab, avelumab, durvalumab, atezolizumab, and spartalizumab.

2.14 Any of the foregoing methods, wherein the individual suffers from an inflammation-related disorder following immune checkpoint inhibitor therapy.

2.15 Any of the foregoing methods, wherein the individual suffers from a systemic inflammatory response, a gastrointestinal inflammatory disorder, an endocrine inflammatory disorder, a skin inflammatory disorder, an ophthalmic inflammatory disorder, a neurological inflammatory disorder, a blood inflammatory disorder, a genitourinary inflammatory disorder, a respiratory inflammatory disorder, a musculoskeletal inflammatory disorder, a cardiac inflammatory disorder, or a defined systemic inflammatory disorder.

2.16 Method 2.15 wherein the disorder associated with gastrointestinal inflammation is selected from colitis, enterocolitis, colitis complicated by intestinal perforation, hepatitis, and pancreatitis.

2.17 Method 2.15 wherein the endocrine inflammation-related disorder is selected from hypophysitis (e.g., manifesting as panhypopituitarism), thyrotoxicosis, hypothyroidism, syndrome of inappropriate antidiuretic hormone secretion, central adrenal insufficiency, primary adrenal insufficiency, and diabetes mellitus.

2.18 Method 2.15 wherein the disorder associated with skin inflammation is selected from the group consisting of rash, pruritus, vitiligo, dermatitis, Sweet's syndrome, drug eruption, graying of hair, delayed hypersensitivity reaction, alopecia universalis, transient acantholytic dermatosis, pyoderma gangrenosum, toxic epidermal necrolysis, chronic noncaseating granulomatosis, bullous pemphigoid, and psoriasis.

2.19 Method 2.15 wherein the ophthalmic inflammation-related disorder is selected from the group consisting of uveitis, conjunctivitis, orbital inflammation, Graves' ophthalmopathy, choroidal neovascularization, optic neuropathy, keratitis, and retinopathy.

2.20 Method 2.15, wherein the neuroinflammation-related disorder is selected from encephalopathy, Guillain-Barré syndrome, polyradiculoneuropathy, symmetric multifocal neuropathy, transverse myelitis, necrotizing myelopathy, myasthenia gravis, phrenic nerve palsy, immune-related meningitis, meningoradiculoneuritis, peripheral neuropathy, autoimmune inner ear disease, multiple sclerosis, and inflammatory enteric neuropathy.

2.21 Method 2.15 wherein the blood inflammation-related disorder is selected from thrombocytopenia, pancytopenia, neutropenia, eosinophilia, pure red cell aplasia, acquired hemophilia A, and disseminated intravascular coagulopathy.

2.22 Method 2.15 wherein the disorder associated with genitourinary inflammation is selected from renal failure, acute/granulomatous interstitial nephritis, acute tubular necrosis, and lymphocytic vasculitis (eg, uterine lymphocytic vasculitis).

2.23 Method 2.15, wherein the disorder associated with respiratory inflammation is selected from pneumonia and acute respiratory distress.

2.24 Method 2.15 wherein the musculoskeletal inflammation-related disorder is selected from polyarthritis, arthralgia, myalgia, chronic granulomatous inflammation of the rectus abdominis muscle, and rhabdomyolysis.

2.25 Method 2.15 wherein the disorder associated with cardiac inflammation is selected from pericarditis and Takotsubo-like syndrome.

2.26 Method 2.15, wherein the identified systemic inflammatory-related disorder is selected from the group consisting of pulmonary nodules, skin and pulmonary nodules, polymyalgia rheumatica, giant cell arteritis, muscular sarcoidosis, neural and pulmonary nodules, celiac disease, lupus nephritis, dermatomyositis, autoimmune inflammatory myopathy, and Voigt-Koyanagi syndrome.

2.27 Any of the preceding methods, wherein administration of the PDE1 inhibitor and the immune checkpoint inhibitor is concomitant with radiation therapy or chemotherapy.

2.28 Any of the preceding methods, wherein the PDE1 inhibitor and the immune checkpoint inhibitor are administered concurrently with radiation therapy or chemotherapy.

2.29 Any of the preceding methods, wherein the PDE1 inhibitor and the immune checkpoint inhibitor are administered prior to radiotherapy or chemotherapy.

2.30 Any of the preceding methods, wherein the PDE1 inhibitor and the immune checkpoint inhibitor are administered after radiation therapy or chemotherapy.

2.31 Any of the preceding methods, wherein the PDE1 inhibitor and immune checkpoint inhibitor are administered together with an additional anti-tumor agent, chemotherapeutic, gene therapeutic and/or immunotherapeutic.

2.32 Any of the preceding methods, wherein the PDE1 inhibitor is a PDE1 inhibitor according to Formula I, Ia, II, III, IV, V, and/or VI in free form or in the form of a pharmaceutically acceptable salt, or a compound according to:

2.33 Any of the preceding methods, wherein the PDEl inhibitor is a PDEl inhibitor according to Formula Ia.

2.34 Any of the preceding methods, wherein the PDE1 inhibitor is a compound in free form or in the form of a pharmaceutically acceptable salt:

2.35 Any of the preceding methods, wherein the PDE1 inhibitor is a compound in free form or in pharmaceutically acceptable salt form (eg, monophosphate form):

2.36 Any of the preceding methods, further comprising administering to the individual a pharmaceutically acceptable amount of a beta blocker.

2.37 Any of the preceding methods, wherein the individual is a human.

In another embodiment, the present disclosure provides a PDE1 inhibitor (e.g., a PDE1 inhibitor according to Formula I, Ia, II, III, IV, V, and/or VI) for use in preventing or alleviating a disease, disorder or adverse reaction caused by administration of a checkpoint inhibitor therapy, e.g., for use in any one of method 2, etc.

Combination therapy with PDE1 inhibitors.

In the present invention, PDE1 inhibitors can be administered in combination with immune checkpoint inhibitors. Combination therapy can be achieved by administering a single composition or pharmaceutical preparation including a PDE1 inhibitor and an immune checkpoint inhibitor, or by administering two different compositions or preparations separately, simultaneously or sequentially, wherein one composition includes a PDE1 inhibitor and another composition includes an immune checkpoint inhibitor. Therapy using inhibitors can be before or after the administration of other inhibitors, with intervals of several minutes to several weeks. In embodiments where other inhibitors are applied separately to cells, it is generally ensured that a significant period of time (asignificant period of time) between each delivery time does not exceed the deadline, so that PDE1 inhibitors and immune checkpoint inhibitors can still play a favorable combined effect on cells. In some embodiments, it is generally considered to contact two inhibitors with cells within about 12-24 hours apart from each other, and more preferably, within about 6-12 hours apart from each other, the most preferred delay time is only about 12 hours. However, in some cases, it may be necessary to significantly extend the time period of treatment, wherein between each administration, several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) are passed.

It is contemplated that more than one administration of a PDE1 inhibitor or immune checkpoint inhibitor may be required. In this regard, various combinations may be employed. By way of illustration, when the PDE1 inhibitor is "A" and the immune checkpoint inhibitor is "B", the following arrangements based on 3 and 4 total administrations are exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Therefore, in various embodiments, the present disclosure also provides a drug combination [combination 1] therapy, the therapy comprising a pharmaceutically acceptable amount of a PDE1 inhibitor (e.g., a compound according to Formula I, Ia, II, III, IV, V and/or VI) and a pharmaceutically acceptable amount of an immune checkpoint inhibitor, the therapy is used for administration in a method for treating breast cancer, such as according to any one of method 1, etc., or the therapy is used to prevent or alleviate a disease, disorder or adverse reaction caused by the administration of a checkpoint inhibitor therapy, such as according to any one of method 2, etc. For example, the present disclosure provides the following combinations:

1.1 Combination 1, wherein the PDE1 inhibitor and the checkpoint inhibitor are combined or associated with a pharmaceutically acceptable diluent or carrier in a single dosage form, for example, a tablet or capsule.

1.2 Combination 1 wherein the PDE1 inhibitor and the checkpoint inhibitor are in a single package, e.g. together with instructions for simultaneous or sequential administration.

1.3 Any of the preceding combinations, wherein the PDE1 inhibitor is a PDE1 inhibitor according to Formula I, Ia, II, III, IV, V, and/or VI in free form or in the form of a pharmaceutically acceptable salt or a compound according to:

1.4 Any of the preceding combinations, wherein the PDEl inhibitor is a PDEl inhibitor according to Formula Ia.

1.5 Any of the preceding combinations, wherein the PDE1 inhibitor is the following compound in free form or in the form of a pharmaceutically acceptable salt:

1.6 Any of the preceding combinations, wherein the PDE1 inhibitor is the following compound in free form or in the form of a pharmaceutically acceptable salt:

1.7 Any of the foregoing combinations, wherein the immune checkpoint inhibitor is selected from one or more CTLA-4, PD-1 and/or PD-L1 inhibitors.

1.8 Any of the foregoing combinations, wherein the immune checkpoint inhibitor is a PD-1 inhibitor, optionally wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, further optionally wherein the antibody is monoclonal or polyclonal.

1.9 Any of the foregoing combinations, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor, optionally wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody, further optionally wherein the antibody is monoclonal or polyclonal.

1.10 Any of the foregoing combinations, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor, optionally wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody, further optionally wherein the antibody is monoclonal or polyclonal.

1.11 Any of the foregoing combinations, wherein the immune checkpoint inhibitor comprises one or more members selected from the group consisting of nivolumab, pembrolizumab, cemiplizumab, ipilimumab, avelumab, durvalumab, atezolizumab, and spartalizumab.

1.12 Any of the preceding combinations, wherein the checkpoint inhibitor is administered concurrently with the PDE1 inhibitor.

1.13 Any of the preceding combinations, wherein the checkpoint inhibitor is administered prior to administration of the PDE1 inhibitor.

1.14 Any of the preceding combinations, wherein the checkpoint inhibitor is administered after administration of the PDEl inhibitor.

1.15 Any of the foregoing combinations, wherein the individual in need thereof has been previously or contemporaneously administered a checkpoint inhibitor therapy.

1.16 Any of the foregoing combinations, further comprising a pharmaceutically acceptable amount of a beta blocker.

In some embodiments, the pharmaceutical composition is administered in combination with one or more additional anti-tumor drugs, for example, drugs known to be effective in treating or eliminating cancer and/or tumors. Non-limiting examples of anti-tumor drugs are Abemaciclib, Abiraterone Acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, Acalabrutinib, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Doxorubicin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride/Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection (Melphalan Hydrochloride), Melphalan Tablets (Melphalan), Aloxi (Palocisetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Amifostine, Aminolevulinic Acid (Aminolevulinic Acid), Acid), Anastrozole, Aprepitant, Aredia, Pamidronate, Arimidex, Aromasin, Arranon, Arsenic Trioxide, Arzerra, Asparaginase, Erwiniachrysanthemi), Atezolizumab, Avastin (bevacizumab), Avelumab, Axicabtagene Ciloleucel, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto, Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantin-S-malate) ib-S-Malate), Cabozantinib-S-malate, CAF, Calquence (Acalabrutinib), Campath (Alemtuzumab), Camptosar (Irinotecan hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant, Casodex (Bicalutamide), CEM, Ceritinib (Ce ritinib), Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), Cetuximab, CEV, Chlorambucil, Chlorambucil-prednisone, CHOP, Cisplatin, Cladribine, Clafen (cyclophosphamide), Clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, Cobimetinib, Cometriq (cabozantinib-S-malate), Copanisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetini b), Crizotinib, CVP, Cyclophosphamide, Cyfos (ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposomal, Cytosar-U (Cytarabine), Cytoxan (Cytoxan), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposomal, Decitabine, Defibrotide Sodium Sodium), Defitelio (defibrotide sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (liposomal cytarabine), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (liposomal doxorubicin hydrochloride), Doxorubicin hydrochloride, Dox-SL (liposomal doxorubicin hydrochloride), DTIC-Dome (dacarbazine), Durvalumab, Efudex (topical fluorouracil), Elitek (rasburicase), Ellence (epirubicin hydrochloride), Elotuzumab, Eloxatin (oxaliplatin), Eltrombopag Olamine), Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge, Vismodegib, Erlotinib Hydrochloride, Erwinaze (Erwinia chrysanthemi Asparaginase), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Liposomal Hydrochloride), Everolimus, Evista (Raloxifene Hydrochloride) Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Lerozole), Filgrastim (Filgrastim), Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil Topical), Fluorouracil Injection, Fluorouracil Topical, Flutamide, Folex (Methotrexate), Folex PFS (methotrexate), irinotecan (Folfiri), irinotecan-bevacizumab, irinotecan-cetuximab, Folfirinox, oxaliplatin (Folfox), Folotyn (Pralatrexate), FU-LV, Fulvestane, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nine-valent vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine hydrochloride, Gemcitabine-cisplatin, Gemcitabine-oxaliplatin, Gemtuzumab Ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), HPV Bivalent Vaccine, Recombinant HPV Nine-valent Vaccine, Recombinant HPV Four-valent Vaccine, Recombinant (Recombinant), Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride Hydrochloride), Idarubicin hydrochloride, Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Gemtuzumab Ozogamicin (Inotuzumab Ozogamicin), Interferon α-2b, Recombinant Interleukin-2 (Aldesleukin), Intron A (recombinant interferon alpha-2b), ipilimumab, Iressa (gefitinib), Irinotecan hydrochloride, Irinotecan hydrochloride liposome, Istodax (romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate, Ruxolitinib Phosphate), JEB, Jevtana (cabazitaxel), Kadcyla (trastuzumab-emtansine conjugate), Raloxifene/Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Kyprolis (carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima (lenvatinib mesylate), Letrozole, Leucovorin Calcium (leucovorin Calcium), Leukeran (chlorambucil), Leuprolide Acetate, Leustatin (Cladribine, Chlorambucil), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (liposomal doxorubicin hydrochloride), Lomustine (Lomustine), Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lynparza (Olaparib), Marqibo (liposomal vincristine sulfate), Matulane (procarbazine hydrochloride), nitrogen mustard hydrochloride, megestrol acetate (Megestrol Acetate), Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex, Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone bromide (Methylnaltrexone Bromide), Mexate (methotrexate), Mexate-AQ (methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (nitrogen mustard hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), Paclitaxel Nanoparticles (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), Necitumumab, Nelarabine, Neosar (cyclophosphamide), Neratinib Maleate, Nerlynx (neratinib maleate), Netupitant and Palonosetron Hydrochloride), Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Methylsuccinate Mepesuccinate), Oncaspar (Pegaspagase), Ondansetron Hydrochloride, Onivyde (Irinotecan liposome), Ontak (denileukin), Opdivo (nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel albumin-stabilized nanoparticle formulation, PAD, Palbociclib, Palifermin, Palosetron Hydrochloride, Palosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), Pazopanib Hydrochloride Hydrochloride), PCV, PEB, Pegaspargase, Pegylated filgrastim, Peginterferon alfa-2b, PEG-Intron (pegylated interferon alfa-2b), Pembrolizumab, Pemetrexed disodium, Perjeta (pertuzumab), Pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), Plerixafor, Pomalidomide, Pomalyst (pomalidomide), Ponatinib hydrochloride, Portrazza (necitumumab), Pralatrexate, Prednisone, Procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nine-Valent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon α-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (rituximab and human hyaluronidase), Rituximab, Rituximab and human hyaluronidase, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib camsylate), Rucaparib camsylate, Ruxolitinib phosphate, Rydapt (midostaurin), Sclerosol intrapleural aerosol (talc), Siltuximab, Sipuleucel-T, Somatuline Depot (lanreotide acetate), Solidegi, Sorafenib p-toluenesulfonate (Sorafenib Tosylate), Sprycel (Dasatinib), STANFORD V, Sterile Talc (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate (Sunitinib Malate), Sutent (Sunitinib Malate), Sylatron (Peginterferon alpha-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab), Temodar (temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid, Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (topical fluorouracil), Topotecan hydrochloride, Toremifene, Torisel (temsirolimus), Totect (dexrazoxane hydrochloride, Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tripiracic Hydrochloride, Trisenox (Arsenic Trioxide), TykerB (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Valrubicin, Valstar (Valrubicin), Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride, Hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (leuprorelin acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, liposomal vincristine sulfate, vinorelbine tartrate, VIP, Vismodegib, Vistogard (uridine triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposomal), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium 223 chloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yescarta (axicillin), Ciloleucel), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filagrastim), Zejula (Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib), and Zytiga (Abiraterone Acetate).

In some embodiments, the PDE1 inhibitor and the immune checkpoint inhibitor are administered in combination with one or more beta blockers. A non-exhaustive list of such beta blockers includes various beta-adrenergic receptor antagonists, also known as beta-blockers, which are currently used in the clinic to eliminate harmful chronic myocardial stimulation caused by heart failure. Preferred beta-adrenergic receptor antagonists include metoprolol, metoprolol succinate, carvedilol, atenolol, propranolol, acebutolol, acebutolol hydrochloride, betaxolol, betaxolol HCL, nadolol, talinolol, bisoprolol, bisoprolol hemifumarate, carteolol, carteolol HCL, esmolol, esmolol HC L, labetalol, labetalol HCL, metoprolol, metoprolol succinate, metoprolol tartrate, nadolol, penbutolol, penbutolol sulfate, pindolol, propranolol, propranolol HCL, sotalol, sotalol HCL, timolol and timolol maleate salt or its pharmaceutically acceptable salt. According to the present invention, beta-adrenergic receptor antagonists can be used with the daily dose of this class of medicine accepted clinically. For example, according to the disease treated, route of administration, age, body weight and condition of the patient, the suitable daily dose of metoprolol in the form of tartrate or succinate is about 100-200mg, and the carvedilol daily dose is about 5-50mg.

As used herein, the term "antineoplastic agent" is understood to refer to any chemically active agent or drug that is effective in preventing or inhibiting the formation or growth of cancer or tumors. Antineoplastic agents described herein may include alkylating agents, antimetabolites, natural products, hormones and/or antibodies. The treatment of tumors or cancers may include limiting the proliferation, migration and/or invasion of cancer or tumor cells in the body or limiting symptoms associated with the cancer or tumor. As used herein, antineoplastic agents are understood to include anticancer agents, otherwise they are synonymous with anticancer agents.

Methods of preparing the disclosed compounds

The PDE1 inhibitors and pharmaceutically acceptable salts thereof of the present disclosure can be prepared using methods as described and exemplified in the following documents, and by methods analogous thereto and by methods well known in the chemical art: US 8,273,750, US2006/0173878, US 8,273,751, US2010/0273753, US 8,697,710, US 8,664,207, US 8,633,180, US 8,536,159, US2012/0136013, US2011/0281832, US2013/0085123, US2013/0324565, US2013/0338124, US2013/0331363, WO 2012/171016 and WO 2013/192556. Such methods include, but are not limited to, those described below. If not commercially available, the starting materials for these methods can be prepared by chemical methods selected using techniques similar or analogous to the synthesis of known compounds.

Various PDE1 inhibitors and their raw materials can be prepared using the methods described in the following documents: US 2008-0188492A1, US2010-0173878 A1, US2010-0273754 A1, US 2010-0273753A1, WO 2010/065153, WO 2010/065151, WO 2010/065151, WO 2010/065149, WO 2010/065147, WO 2010/065152, WO 2011/153129, WO 2011/133224, WO 2011/153135, WO 2011/153136, WO 2011/153138. All references cited herein are incorporated by reference in their entirety.

The compounds (PDE1 inhibitors) disclosed herein include enantiomers, diastereomers and racemates thereof and polymorphs, hydrates, solvates and complexes thereof. Some individual compounds within the scope of the disclosure may contain double bonds. The representation of double bonds in the disclosure is meant to include E and Z isomers of the double bonds. In addition, some compounds within the scope of the disclosure may contain one or more asymmetric centers. The disclosure includes the use of any one of optically pure stereoisomers and any combination of stereoisomers.

It is also expected that the disclosed compounds (PDE1 inhibitors) include stable and unstable isotopes thereof. Stable isotopes are non-radioactive isotopes that contain an extra neutron compared to the abundant nuclides of the same kind (i.e., element). It is expected that the activity of compounds containing such isotopes can be retained, and such compounds can also have the utility of determining the pharmacokinetics of non-isotopic analogs. For example, a hydrogen atom at a certain position on the compounds of the present disclosure can be replaced by deuterium (a non-radioactive stable isotope). Examples of known stable isotopes include, but are not limited to, deuterium, 13C, 15N, 18O. Alternatively, unstable isotopes such as 123I, 131I, 125I, 11C, 18F, which are radioactive isotopes containing an extra neutron compared to the abundant nuclides of the same kind (i.e., element), can replace the corresponding abundant species of I, C and F. Another example of a useful isotope of the disclosed compounds is the 11C isotope. These radioactive isotopes can be used for radioimaging and/or pharmacokinetic studies of the disclosed compounds.

The term "treatment" is thus to be understood to include treating or alleviating the symptoms of a disease as well as treating the cause of the disease.

For treatment methods, the term "effective amount" is intended to include a therapeutically effective amount for treating a particular breast cancer, such as inhibiting the growth (volume or weight) of breast cancer when a PDE-1 inhibitor and an immune checkpoint inhibitor are administered in combination. The effective amount of a PDE-1 inhibitor or an immune checkpoint inhibitor may be lower than the amount of a PDE-1 inhibitor or an immune checkpoint inhibitor administered as a monotherapy.

The terms "patient" and "subject" include human or non-human (i.e., animal) patients, and it is understood that "patient" and "subject" are interchangeable in the context of the present disclosure. In certain embodiments, the present disclosure includes humans and non-humans. In another embodiment, the present disclosure includes non-humans. In additional embodiments, the term includes humans.

The term "comprising" as used in this disclosure is intended to be open ended and does not exclude additional, undescribed elements or method steps.

The dosage used in the implementation of the present disclosure will of course vary according to, for example, the type of breast cancer being treated, a specific PDE-1 inhibitor or immune checkpoint inhibitor, the mode of administration, and the desired therapy. The PDE1 inhibitor can be administered by any suitable route, including oral, parenteral (intravenous, intramuscular or subcutaneous), transdermal or by inhalation, preferably oral administration. In certain embodiments, the PDE-1 inhibitor is preferably administered parenterally, for example, by injection in a depot formulation. The immune checkpoint inhibitor can be administered by any suitable route, including oral, parenteral (intravenous, intramuscular or subcutaneous), transdermal or by inhalation, preferably intravenous administration.

Typically, satisfactory results, for example for the treatment of breast cancer, are indicated to be obtained with oral administration of PDE-1 inhibitors in doses of the order of about 0.01-2.0 mg/kg. In larger mammals, such as humans, the indicated daily dosage for oral administration of PDE1 inhibitors is correspondingly in the range of about 0.50-300 mg, conveniently administered once daily, or in 2-4 divided doses, or in sustained release form. Unit dosage forms for oral administration may thus, for example, contain about 0.2-150 or 300 mg, for example about 0.2 or 2.0-10, 25, 50, 75, 100, 150 or 200 mg of the PDE-1 inhibitor together with a pharmaceutically acceptable diluent or carrier thereof.

PDE1 inhibitors and immune checkpoint inhibitors can be used in combination with one or more additional therapeutic agents, particularly at a dose lower than when a single active agent is used as a monotherapy, so as to enhance the therapeutic activity of the combined active agent without causing undesirable side effects that usually occur in conventional monotherapy. Therefore, PDE1 inhibitors and immune checkpoint inhibitors can be administered simultaneously, separately, sequentially or concurrently with other active agents for treating diseases. In another example, by combining the administration of PDE1 inhibitors and immune checkpoint inhibitors with one or more additional free forms or salt forms of therapeutic agents, side effects can be reduced or minimized, wherein the dose of (i) the additional therapeutic agent or (ii) the PDE1 inhibitor and the immune checkpoint inhibitor is lower than the dose if the active agent/compound is administered as a monotherapy.

The term "simultaneously" when referring to therapeutic use means that two or more active ingredients are administered at or about the same time and by the same route of administration.

The term "separately" when referring to therapeutic use means that two or more active ingredients are administered at or about the same time, by different routes of administration.

The pharmaceutical composition can be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus, oral dosage forms may include tablets, capsules, solutions, suspensions, and the like.

Example - Effects of PDE1 inhibitors alone and in combination with anti-PD-1 in a mouse model of breast cancer

Example 1

Materials and methods

In vivo tumor transplantation and treatment

E0771 murine breast cancer cells were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM medium containing 10% fetal bovine serum, 4mM glutamine, 20mM HEPES and 1% penicillin/streptomycin. Cells were cultured in a tissue culture flask at 37°C in a humidified incubator in 5% CO ₂ and 95% air. E0771 tumor cells for transplantation were harvested and resuspended in cold PBS at a concentration of 2.5×10 ⁶ cells/mL. 0.5×10 6 cells in 0.2 mL of cold PBS were injected subcutaneously on the right side of nine-week-old female C57Bl/6 mice (C57Bl/6J, Jackson Laboratory). After 10 days, the mice had tumors of palpable size and had a specific size (about 100 mm ^{3 )} for the start of treatment. After measuring the tumors in two dimensions with a vernier caliper to monitor the size, the mice were randomly divided into groups. Tumor size was calculated using the formula: Tumor volume ( ^mm3 ) = ( ^W2 x L)/2, where w = width and l = length in mm. Tumors were measured twice a week during the study. Mice were individually euthanized when they reached a tumor volume endpoint of 1500 ^mm3 , or if showing any type of discomfort, whichever came first.

Mice were treated with Compound A alone, anti-PD-1 antibody alone, or a combination of Compound A and anti-PD-1 antibody. Compound A was synthesized at Intra-Cellular Therapies Inc. Compound A diet was used to treat mice. A 900 ppm Compound A diet (900 mg Compound A/kg supplemented to Picolab rodent diet 5053) was produced by Envigo (Madison WI). Vehicle or Compound A was administered to mice 5 days a week.

Anti-PD-1RMP1-14 (lot 800121F12A) and isotype (lot 749620N1) antibodies were purchased from BioXCell. The stock solution was diluted in PBS to produce a dosing solution of 0.1 or 1 mg/mL, which was delivered at a dosing volume of 0.2 mL (10 mL/kg for 20 mg mice) of 1 or 10 mg/kg, respectively. Mice were administered isotype or anti-PD-1RMP1-14 intraperitoneally at 0.2 mL (10 mL/kg for 20 mg mice) twice a week.

Animals were weighed twice weekly until completion of the study. Animals were frequently observed for any signs of toxicity, and notable clinical observations were recorded. Animal body weights were monitored, and any animal that lost more than 15% of body weight on any one measurement was euthanized.

Flow cytometry and cell sorting

Tumors were collected and minced in a digestion solution containing 2mg/mL collagenase D (Sigma-Aldrich) and 1mg/mL DNase I (Sigma-Aldrich). Samples were incubated at 37°C for 30-45min and passed through a 70-μm nylon cell strainer (Corning). The suspension was centrifuged at 1200rpm for 3min at 4°C. The precipitated cells were collected and resuspended in erythrocyte lysis buffer (Sigma-Aldrich) and incubated at room temperature for 5min, washed in PBS and centrifuged at 1200rpm for 3min at 4°C. The cells were resuspended in 2% Fc Blocker (Fc Block) (BD Bioscience) and blocked for 30min. Then, the primary antibody coupled to the fluorophore was incubated at RT for 30min in FACS buffer (1X HBSS (Thermo Fisher Scientific), 2% BSA and 0.5mMEDTA). The antibodies used for the macrophage panel were: PerCp-Cy5.5 anti-mouse CD45 (BD Bioscience), APC-Cy7 anti-mouse F4/80 (Invitrogen), PE anti-mouse CD11b (Biolegen), FITC anti-mouse INOS (BD Bioscience), BV510 anti-mouse CD80 (BD Bioscience), APC anti-mouse CD206 (Biolegen), and PE-Cy7 anti-mouse Arg1 (Invitrogen). The antibodies used for the T cell panel were: PerCp-Cy5.5 anti-mouse CD45 (BD Bioscience), APC-Cy7 anti-mouse CD3 (Biolegend), PE-Cy7 anti-mouse CD8 (BD Bioscience), BV610 anti-mouse CD4 (Biolegend), and BV786 anti-mouse NK1.1 (Biolegend). The antibodies used to sort macrophages were: PerCp-Cy5.5 anti-mouse CD45 (BD Bioscience), APC anti-mouse F4/80 (Invitrogen), PE anti-mouse CD11b (Biolegend), and BV785 anti-mouse CD11c (Invitrogen). All antibodies were used at 1:200. After washing in FACS buffer, cells were resuspended in FACS buffer and analyzed using an in-house CytoFLEX (Beckman Coulter) or using a FACSAria II cell sorter (BD Biosciences) to sort and collect macrophages (M1 and M2).

RNAseq

Tumors were extracted with the PureLINK RNA mini kit (Invitrogen), sorted macrophages were extracted with the PicoPure RNA Isolation kit (Thermo Fisher Scientific), and the quality and quantity were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies). RNAseq libraries were prepared using the TrueSeq strand mRNA kit for tumors or the Clontech SMARTer strand total RNA Seq kit-Pico Input Mammalian for sorted macrophages, respectively, according to the manufacturer's protocol. The library was purified using AMPure beads, equimolar amounts were combined, and run on HiSeq 6000, paired end reads. FASTQ files were obtained. The reads were mapped to the mouse MM10 genome, and gene level and differential expression analysis were performed using the Rosalind tool. Genes with p values <0.05 and FC (fold change) <-1.5 or >1.5 under different conditions were determined to be differentially expressed. Heat maps and pathway analysis were generated using Rosalind (https://www.rosalind.bio/).

LC/MS/MS quantification of compound A

Tumor, liver and fecal samples were diluted in phosphate buffer and homogenized using an ultrasonic processor. Plasma and homogenized tissue samples, together with a set of standards and quality control samples, were extracted on OstroPlate (Waters) using an acetonitrile solution of 1% formic acid with an internal standard by means of a protein precipitation technique. 2 μL of filtered samples were injected using an automatic sampler and chromatographic separation was performed using the following conditions: Phenomenex, Synergi TM, 2.5 μm, 50×3 mm, Polar-RP column, an isocratic mobile phase combination of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (40/60), flow rate 0.8 mL/min. A Sciex Qtrap 6500 mass spectrometer was used as a detector, positive electrospray was performed in MRM ionization mode, and the ion source temperature was 650°C. Compound A was quantified using a calibration curve established with standards of corresponding analyte concentrations, retention times and mass properties.

Data analysis

Statistical analysis was performed using GraphPad Prism 9.1.0. Data are reported as mean ± SD. Typically, a two-tailed unpaired Student's t test was used when comparing two groups, and p values < 0.05 were considered significant, and the significance level is indicated as *P < 0.05. For in vivo experiments, n = number of animals. Samples were randomly assigned to experimental groups, and data were not excluded in the analysis.

result

The effects of PDE1 inhibitors alone or in combination with sub-effective doses of programmed cell death-1 (PD-1) immune checkpoint inhibitors on tumor growth inhibition were studied in a syngeneic mouse model of triple-negative breast cancer. Compound A (900 ppm) delivered in the diet as a monotherapy or as a combination therapy in combination with a 10 mg/kg dose of an anti-PD1 antibody (RMP1-14) was evaluated for its effect on tumor growth.

Compound A (900ppm) alone and anti-PD-1 antibody (10mg/kg) alone did not show significant effects on tumor volume (Figures 1 and 2) or tumor weight (Figure 3) at any measurement time point (3rd, 7th, 10th, 14th and 17th day) at the end of the sacrifice (treatment day 17). However, the combination of compound A (900ppm) + anti-PD1 antibody (10mg/kg) resulted in a significant decrease in tumor volume and tumor weight compared with the isotype control (Figures 1-3). No significant differences were observed between the groups in terms of food intake or mouse weight. In all groups receiving compound A, the drug exposure of compound A in plasma, tissues, and feces was also comparable. These results demonstrate the synergistic ability of compound A and anti-PD-1 antibodies to inhibit tumor growth in a breast cancer mouse model.

Next, the drug-related changes of immune cells (macrophages, T cells and NK cells) in the tumor microenvironment from the isotype (control), compound A (900ppm) alone, anti-PD1 antibody (10mg/kg) and compound A (900ppm) + anti-PD1 antibody (10mg/kg) combination groups were analyzed by flow cytometry (Figures 4 and 5). The results showed that the combination treatment of compound A + anti-PD1 antibody did not change the total number of macrophages in the tumor microenvironment (Figure 4A), but the combination treatment had an effect on the M1/M2 ratio (Figure 4B). M1 macrophages are pro-inflammatory, while M2 macrophages are anti-inflammatory. The combination treatment of compound A + anti-PD1 antibody significantly increased the M1/M2 macrophage ratio compared to the isotype group (Figure 4B). As measured by flow cytometry, the combination treatment had no significant effect on T cell distribution (CD8+, CD4+) and NK cells (Figure 5).

RNAseq analysis was performed to explore changes in drug-related gene expression in Compound A + anti-PD1 (10 mg/kg) tumors compared to the isotype (control) group. Comparative volcano plots are shown in Figure 6. The volcano plot shows that 48 genes were downregulated and 136 genes were upregulated in Compound A (900ppm) + anti-PD1 (10 mg/kg) tumors (fold change <-1.5 or >1.5; P <0.05). In order to characterize the role of these genes differentially expressed in Compound A (900ppm) + anti-PD1 (10 mg/kg) tumors, pathway analysis was performed. Figure 7 shows pathways enriched in genes differentially expressed in Compound A (900ppm) + anti-PD1 (10 mg/kg) tumors. Figure 7 shows transcriptional regulators associated with genes upregulated or downregulated in Compound A + anti-PD1 (10 mg/kg) tumors. RNaseq analysis showed that the combination therapy significantly downregulated genes involved in cell growth, survival, and migration pathways, while upregulated genes involved in inflammatory pathways. These results suggest that the combination of PDE-1 inhibitors and anti-PD-1 antibodies promotes anti-tumor immunity, leading to tumor growth inhibition.

Example 2

Materials and methods

In vivo tumor transplantation and treatment

4T1 murine breast cancer cells were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI medium containing 10% fetal bovine serum, 4mM glutamine and 1% penicillin/streptomycin. Cells were cultured in a tissue culture bottle at 37°C in a humidified incubator in 5% CO ₂ and 95% air. 4T1 tumor cells for transplantation were harvested and resuspended in cold PBS at a concentration of 50×10 ³ cells/mL. 10×10 ³ cells in 0.2mL cold PBS were injected subcutaneously on the right side of nine-week-old female Balb/c mice (Balb/cJ, Jackson Laboratory). After 10 days, the mice had tumors of palpable size and had a specific size (about 100mm ³ ) for the start of treatment. After measuring the tumor in two dimensions with a vernier caliper to monitor the size, the mice were randomly divided into groups. Tumor size was calculated using the formula: Tumor volume ( ^mm3 ) = ( ^w2 xl)/2, where w = width and l = length in mm. Tumors were measured twice a week during the study. Mice were individually euthanized when they reached a tumor volume endpoint of 1500 ^mm3 , or if they showed any type of discomfort, whichever came first.

Mice were treated with Compound A alone, anti-PD-1 antibody alone, or a combination of Compound A and anti-PD-1 antibody. Compound A was synthesized at Intra-Cellular Therapies Inc. Compound A diet was used to treat mice. 300 ppm or 900 ppm Compound A diet (300 mg or 900 mg of Compound A/kg supplemented to Picolab rodent diet 5053) was produced by Envigo (Madison WI). Vehicle or Compound A was administered to mice 5 days a week.

Anti-PD-1RMP1-14 (lot 800121F12A) and isotype (lot 749620N1) antibodies were purchased from BioXCell. The stock solution was diluted in PBS to produce a dosing solution of 0.1 or 1 mg/mL, which was delivered at a dosing volume of 0.2 mL (10 mL/kg for 20 mg mice) of 1 or 10 mg/kg, respectively. Mice were intraperitoneally administered 0.2 mL (10 mL/kg for 20 mg mice) of isotype or anti-PD-1RMP1-14 twice a week.

Animals were weighed twice weekly until completion of the study. Animals were frequently observed for any signs of toxicity, and notable clinical observations were recorded. Animal body weights were monitored, and any animal that lost more than 15% of body weight on any one measurement was euthanized.

LC/MS/MS quantification of compound A

Tumor, liver and fecal samples were diluted in phosphate buffer and homogenized using an ultrasonic processor. Plasma and homogenized tissue samples, together with a set of standard and quality control samples, were extracted on Ostroplate (Waters) using an acetonitrile solution of 1% formic acid with an internal standard by means of a protein precipitation technique. 2 μL of filtered samples were injected using an autosampler and chromatographic separation was performed using the following conditions: Phenomenex, Synergi TM, 2.5 μm, 50×3 mm, Polar-RP column, an isocratic mobile phase combination of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (40/60), flow rate 0.8 mL/min. A Sciex Qtrap 6500 mass spectrometer was used as a detector, positive electrospray was performed in MRM ionization mode, and the ion source temperature was 650° C., and a calibration curve established with standards of corresponding analyte concentrations, retention times and mass properties was used for quantification of compound A.

Data analysis

Statistical analysis was performed using GraphPad Prism 9.1.0. Data are reported as mean ± SD. Generally, a two-tailed unpaired Student's t test was used when comparing two groups, and p values < 0.05 were considered significant, and significance levels were indicated as *p < 0.05; **p < 0.01; and ***p < 0.001. For in vivo experiments, n = number of animals. Samples were randomly assigned to experimental groups, and data were not excluded in the analysis.

result

The effect of delivering compound A (300ppm or 900ppm) in the diet as a monotherapy or as a combination therapy with an anti-PD1 antibody (RMP1-14) at a dose of 10mg/kg on 4T1 tumor growth was evaluated. At any given measurement time point or at the time of death at the end point (treatment day 14), the tumor volume (Figures 8 and 9) and tumor weight (Figure 10) of the anti-PD1 (10mg/kg) group were no different from the isotype (control). However, relative to the isotype (control), treatment with different compound monotherapy doses or with different anti-PD1 combinations significantly reduced tumor volume or weight, as shown in Figures 8-10. The survival of animals in the monotherapy and combination groups was also improved (Figure 11). In the absence or presence of anti-PD1, the exposure of compound A in plasma and tissues was comparable in the group receiving the drug in the diet.

Example 3

Materials and methods

In vivo tumor transplantation and treatment

E0771 murine breast cancer cells were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM medium containing 10% fetal bovine serum, 4mM glutamine, 20mM HEPES and 1% penicillin/streptomycin. Cells were cultured in tissue culture flasks at 37°C in a humidified incubator in 5% CO ₂ and 95% air. E0771 tumor cells for transplantation were harvested and resuspended in cold PBS at a concentration of 2.5×10 ⁶ cells/mL. 0.5×10 6 cells in 0.2 mL of cold PBS were injected subcutaneously in the right side of nine-week-old female C57Bl/6 mice (C57Bl/6J, Jackson Laboratory). After 10 days, the mice had tumors of palpable size and had a specific size (approximately 100 mm ^{3 )} for the start of treatment. After measuring the tumors in two dimensions with a vernier caliper to monitor the size, the mice were randomized into groups. Tumor size was calculated using the formula: Tumor volume ( ^mm3 ) = ( ^w2 x L)/2, where w = width and l = length in mm. Tumors were measured twice a week during the study. Mice were individually euthanized when they reached a tumor volume endpoint of 1500 ^mm3 , or if showing any type of discomfort, whichever came first.

Mice were treated with Compound B alone, anti-PD-1 antibody alone, or a combination of Compound B and anti-PD-1 antibody. Compound B was synthesized at Intra-Cellular Therapies Inc. Compound B diets were used to treat mice. 100ppm, 300ppm, or 900ppm Compound B diets (100mg, 300mg, or 900mg of Compound B/kg supplemented to Picolab rodent diet 5053) were produced by Envigo (Madison WI). Vehicle or Compound B was administered to mice 5 days a week.

Anti-PD-1RMP1-14 (lot 800121F12A) and isotype (lot 749620N1) antibodies were purchased from BioXCell. The stock solution was diluted in PBS to produce a dosing solution of 0.1 or 1 mg/mL, which was delivered at a dosing volume of 0.2 mL (10 mL/kg for 20 mg mice) of 1 or 10 mg/kg, respectively. Mice were intraperitoneally administered 0.2 mL (10 mL/kg for 20 mg mice) of isotype or anti-PD-1RMP1-14 twice a week.

Animals were weighed twice weekly until completion of the study. Animals were frequently observed for any signs of toxicity, and notable clinical observations were recorded. Animal body weights were monitored, and any animal that lost more than 15% of body weight on any one measurement was euthanized.

LC/MS/MS quantification of compound B

Tumor, liver and fecal samples were diluted in phosphate buffer and homogenized using an ultrasonic processor. Plasma and homogenized tissue samples, together with a set of standards and quality control samples, were extracted on OstroPlate (Waters) using 1% formic acid in acetonitrile with internal standard by means of protein precipitation technique. 2 μL of filtered samples were injected using an autosampler and chromatographic separation was performed using the following conditions: Phenomenex, Synergi TM, 2.5 μm, 50×3 mm, Polar-RP column, 0.1% formic acid in water and 0.1% formic acid in acetonitrile (40/60) isocratic mobile phase combination, flow rate 0.8 mL/min. A Sciex Qtrap 6500 mass spectrometer was used as a detector, positive electrospray was performed in MRM ionization mode, and the ion source temperature was 650°C. Compound B was quantified using a calibration curve established with standards of corresponding analyte concentrations, retention times and mass properties.

Data analysis

Statistical analysis was performed using GraphPad Prism 9.1.0 for PC. Data are reported as mean ± SD. Generally, a two-tailed unpaired Student's t test was used when comparing two groups, and p values < 0.05 were considered significant, and the significance level was expressed as *p < 0.05; **p < 0.01; and ***p < 0.001. For in vivo experiments, n = number of animals. Samples were randomly assigned to experimental groups, and data were not excluded in the analysis.

result

The effect of delivering Compound B (100 ppm, 300 ppm, or 900 ppm) in the diet as a monotherapy or in combination therapy with a 1 mg/kg dose of an anti-PD1 antibody (RMP1-14) on E0771 tumor growth was evaluated.

At any given measurement time point or at the end point of sacrifice (treatment day 17), the tumor volume (Figures 12 and 13) and tumor weight (Figure 14) of mice treated with anti-PD1 (10 mg/kg) alone were not different from those of the isotype (control). However, treatment with different dose levels of Compound B as a monotherapy or in combination with anti-PD1 significantly reduced tumor volume or weight relative to isotype (control) and/or anti-PD1 alone, as shown in Figures 12-14. The exposure of Compound B in plasma and tissues was comparable in the groups receiving the drug in the diet in the absence or presence of anti-PD1.

Example 4

Materials and methods

In vivo tumor transplantation and treatment

4T1 murine breast cancer cells were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI medium containing 10% fetal bovine serum, 4mM glutamine and 1% penicillin/streptomycin. Cells were cultured in a tissue culture bottle at 37°C in a humidified incubator in 5% CO ₂ and 95% air. 4T1 tumor cells for transplantation were harvested and resuspended in cold PBS at a concentration of 50×10 ³ cells/mL. 10×10 ³ cells in 0.2mL cold PBS were injected subcutaneously on the right side of nine-week-old female Balb/c mice (Balb/cJ, Jackson Laboratory). After 10 days, the mice had tumors of palpable size and had a specific size (about 100mm ³ ) for the start of treatment. After measuring the tumor in two dimensions with a vernier caliper to monitor the size, the mice were randomly divided into groups. Tumor size was calculated using the formula: Tumor volume ( ^mm3 ) = ( ^w2 xl)/2, where w = width and l = length in mm. Tumors were measured twice a week during the study. Mice were individually euthanized when they reached a tumor volume endpoint of 1500 ^mm3 , or if showing any type of discomfort, whichever came first.

Mice were treated with Compound B alone, anti-PD-1 antibody alone, or a combination of Compound B and anti-PD-1 antibody. Compound B was synthesized at Intra-Cellular Therapies Inc. Compound B diets were used to treat mice. 100ppm, 300ppm, or 900ppm Compound B diets (100mg, 300mg, or 900mg of Compound B/kg supplemented to Picolab rodent diet 5053) were produced by Envigo (Madison WI). Vehicle or Compound B was administered to mice 5 days a week.

Anti-PD-1RMP1-14 (lot 800121F12A) and isotype (lot 749620N1) antibodies were purchased from BioXCell. The stock solution was diluted in PBS to produce a dosing solution of 0.1 or 1 mg/mL, which was delivered at a dosing volume of 0.2 mL (10 mL/kg for 20 mg mice) of 1 or 10 mg/kg, respectively. Mice were intraperitoneally administered 0.2 mL (10 mL/kg for 20 mg mice) of isotype or anti-PD-1RMP1-14 twice a week.

Animals were weighed twice weekly until completion of the study. Animals were frequently observed for any signs of toxicity, and notable clinical observations were recorded. Animal body weights were monitored, and any animal that lost more than 15% of body weight on any one measurement was euthanized.

Flow cytometry and cell sorting

Flow cytometry and cell sorting were performed essentially as in Example 1.

RNAseq

RNAseq was performed essentially according to Example 1.

LC/MS/MS quantification of compound B

Tumor, liver and fecal samples were diluted in phosphate buffer and homogenized using an ultrasonic processor. Plasma and homogenized tissue samples, together with a set of standards and quality control samples, were extracted on OstroPlate (Waters) using 1% formic acid in acetonitrile with internal standard by means of protein precipitation technique. 2 μL of filtered samples were injected using an autosampler and chromatographic separation was performed using the following conditions: Phenomenex, Synergi TM, 2.5 μm, 50×3 mm, Polar-RP column, 0.1% formic acid in water and 0.1% formic acid in acetonitrile (40/60) isocratic mobile phase combination, flow rate 0.8 mL/min. A Sciex Qtrap 6500 mass spectrometer was used as a detector, positive electrospray was performed in MRM ionization mode, and the ion source temperature was 650°C. Compound B was quantified using a calibration curve established with standards of corresponding analyte concentrations, retention times and mass properties.

Data analysis

Statistical analysis was performed using GraphPad Prism 9.1.0. Data are reported as mean ± SD. Generally, a two-tailed unpaired Student's t test was used when comparing two groups, and p values < 0.05 were considered significant, and significance levels were indicated as *p < 0.05; **p < 0.01; and ***p < 0.001. For in vivo experiments, n = number of animals. Samples were randomly assigned to experimental groups, and data were not excluded in the analysis.

result

The effect of delivering compound B (100ppm, 300ppm or 900ppm) in the diet as a monotherapy or as a combination therapy with an anti-PD1 antibody (RMP1-14) at a dose of 10mg/kg on 4T1 tumor growth was evaluated. During the experiment after day 5 or at the time of death at the end point (treatment day 14), the tumor volume (Figures 15 and 16) and tumor weight (Figure 17) of mice treated with anti-PD1 (10mg/kg) alone were not different from the isotype (control). However, compared with the isotype (control) and/or anti-PD1 alone, treatment with different doses of compound B as a monotherapy or in combination with anti-PD1 significantly reduced tumor volume or weight, as shown in Figures 15-17. In the absence or presence of anti-PD1, the exposure of compound B in plasma and tissues was comparable in the group receiving the drug in the diet.

Next, the drug-related changes in immune cells (macrophages, T cells, and NK cells) in the tumor microenvironment from the isotype (control), compound B (900ppm) alone, anti-PD1 antibody (10mg/kg) alone, and compound B (900ppm) + anti-PD1 antibody (10mg/kg) combination groups were analyzed by flow cytometry (Figures 18 and 19). The results showed that the combination treatment of compound B + anti-PD1 antibody did not change the total number of macrophages in the tumor microenvironment (Figure 18A), but the combination treatment had an effect on the M1/M2 ratio (Figure 18B). M1 macrophages are pro-inflammatory, while M2 macrophages are anti-inflammatory. Compound B + anti-PD1 antibody combination treatment significantly increased the M1/M2 macrophage ratio compared to the isotype group (Figure 18B). As measured by flow cytometry, the combination treatment had no significant effect on T cell distribution (Figure 19).

RNAseq analysis was performed to explore drug-related gene expression changes in compound B (900ppm) + anti-PD1 (10mg/kg) tumors compared to the isotype (control) group. The comparative volcano plot is shown in Figure 20. The volcano plot shows that 708 genes were downregulated and 281 genes were upregulated in compound B (900ppm) + anti-PD1 (10mg/kg) tumors (fold change <-1.5 or >1.5; P<0.05). In order to characterize the role of these genes differentially expressed in compound B (900ppm) + anti-PD1 (10mg/kg) tumors, pathway analysis was performed. Figure 21A shows pathways enriched in genes differentially expressed in compound B + anti-PD1 (10 mg/kg) tumors (including genes regulating inflammatory processes, such as chemokine signaling pathways, including type II interferon signaling or cytokines and inflammatory responses) and pathways enriched in genes downregulated in compound B + anti-PD1 (10 mg/kg) tumors (including those involved in cell proliferation, survival and migration pathways). Figure 21B shows transcriptional regulators associated with upregulated or downregulated genes in compound B (900 ppm) + anti-PD1 (10 mg/kg) tumors. RNaseq analysis showed that the combination therapy significantly downregulated genes (TFAP2A, SP1, TEAD2 and FOS) involved in cell growth, survival and migration pathways, while upregulated genes (PRDM1, IRF8, NFKβ1, HIF1A, STAT1 and NR3C1) involved in inflammatory pathways. These results suggest that the combination of compound B and anti-PD1 antibodies promotes anti-tumor immunity, leading to tumor growth inhibition.

Claims (20)

1. A method for treating breast cancer, the method comprising administering to an individual in need thereof a pharmaceutically acceptable amount of a PDE1 inhibitor alone or in combination with a pharmaceutically acceptable amount of an immune checkpoint inhibitor. 2. The method according to claim 1, wherein the breast cancer is triple negative breast cancer (TNBC). 3. The method according to claim 2, wherein the TNBC is high-risk early-stage TNBC. 4. The method according to claim 2, wherein the treatment is adjuvant therapy following surgical removal of the TNBC. 5. The method according to claim 2, wherein the individual has locally recurrent unresectable or metastatic TNBC, and the individual's tumor expresses PD-L1 (combined positive score (CPS) ≥ 1). 6. The method according to any of the preceding claims, wherein the immune checkpoint inhibitor is selected from one or more CTLA-4, PD-1 and/or PD-L1 inhibitors. 7. The method according to any one of the preceding claims, wherein the immune checkpoint inhibitor is a PD-1 inhibitor. 8. The method according to any one of the preceding claims, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. 9. The method according to any of the preceding claims, wherein the immune checkpoint inhibitor comprises one or more members selected from the group consisting of nivolumab, pembrolizumab, cemiplizumab, ipilimumab, avelumab, durvalumab, atezolizumab, and spartalizumab. 10. The method according to any of the preceding claims, wherein the individual suffers from a systemic inflammatory response, a gastrointestinal inflammation-related disorder, an endocrine inflammation-related disorder, a skin inflammation-related disorder, an ophthalmic inflammation-related disorder, a neurological inflammation-related disorder, a blood inflammation-related disorder, a genitourinary inflammation-related disorder, a respiratory system inflammation-related disorder, a musculoskeletal inflammation-related disorder, a cardiac inflammation-related disorder, or a well-defined systemic inflammation-related disorder. 11. The method according to any one of the preceding claims, wherein administration of a pharmaceutically acceptable amount of a PDEl inhibitor to an individual alone or in combination with a pharmaceutically acceptable amount of an immune checkpoint inhibitor increases the macrophage M1/M2 ratio in the tumor microenvironment. 12. A method for preventing or ameliorating a disease, disorder or adverse reaction following administration of an immune checkpoint inhibitor therapy to an individual with breast cancer, the method comprising reducing the amount of the checkpoint inhibitor administered to the individual and administering to the individual a pharmaceutically acceptable amount of a PDE1 inhibitor in combination with the immune checkpoint inhibitor therapy. 13. The method according to claim 12, wherein the breast cancer is triple negative breast cancer (TNBC). 14. The method according to any one of claims 12-13, wherein the checkpoint inhibitor is selected from one or more CTLA-4, PD-1 and/or PD-L1 inhibitors. 15. The method according to any one of claims 12-14, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. 16. The method according to any one of the preceding claims, wherein the PDE1 inhibitor is a compound selected from the group consisting of: (A) Formula I: in (i) R ₁ is H or C _1-4 alkyl (e.g., methyl); (ii) R ₄ is H or C _1-4 alkyl and R ₂ and R ₃ are independently H or C _1-4 alkyl (e.g., R ₂ and R ₃ are both methyl, or R ₂ is H and R ₃ is isopropyl), aryl, heteroaryl, (optionally hetero)arylalkoxy, or (optionally hetero)arylalkyl; or _R2 is H and _R3 and _R4 together form a di-, tri- or tetramethylene bridge (preferably, wherein _R3 and _R4 together have a cis configuration, e.g., wherein the carbons carrying _R3 and _R4 have the R and S configuration, respectively); (iii) R ₅ is substituted heteroarylalkyl, for example, substituted with haloalkyl; or _R5 is attached to a nitrogen on the pyrazolo moiety of formula I, and R ₅ is a moiety of formula A wherein X, Y, and Z are independently N or C, and R ₈ , R ₉ , R ₁₁ , and R ₁₂ are independently H or halogen (e.g., Cl or F), and R ₁₀ is halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluoromethyl), aryl (e.g., phenyl), heteroaryl optionally substituted with halogen (e.g., pyridyl (e.g., pyridin-2-yl), or thiadiazolyl (e.g., 1,2,3-thiadiazol-4-yl)), diazolyl, triazolyl, tetrazolyl, arylcarbonyl (e.g., benzoyl), alkylsulfonyl (e.g., methylsulfonyl), heteroarylcarbonyl, or alkoxycarbonyl, provided that when X, Y, or Z is nitrogen, R ₈ , R ₉ , or R ₁₀ , respectively, is absent; (iv) _R is H, alkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), arylamino (e.g., phenylamino), heteroarylamino, N,N-dialkylamino, N,N-diarylamino, or N-aryl-N-(arylalkyl)amino (e.g., N-phenyl-N-(1,1'-biphenyl-4-ylmethyl)amino); (v) n = 0 or 1; and (vi) When n=1, A is -C(R ₁₃ R ₁₄ )- wherein R ₁₃ and R ₁₄ are independently H or C _1-4 alkyl, aryl, heteroaryl, (optionally hetero)arylalkoxy or (optionally hetero)arylalkyl; The compounds of formula I are in free form, salt form or prodrug form, including their enantiomers, diastereomers and racemates; (B) Formula II: (i) X is C _1-6 alkylene (e.g., methylene, ethylene or prop-2-yn-1-yl); (ii) Y is a single bond, an alkynylene group (e.g., —C≡C—), an arylene group (e.g., phenylene) or a heteroarylene group (e.g., pyridylene); (iii) Z is H, aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, e.g., pyridin-2-yl), halogen (e.g., F, Br, Cl), halogenated C _1-6 alkyl (e.g., trifluoromethyl), —C(O)—R ¹ , —N(R ² )(R ³ ), or C _3-7 cycloalkyl optionally containing at least one atom selected from N or O (e.g., cyclopentyl, cyclohexyl, tetrahydro-2H-pyran-4-yl, or morpholinyl); (iv) R ¹ is C _1-6 alkyl, halogenated C _1-6 alkyl, —OH or —OC _1-6 alkyl (e.g., —OCH ₃ ); (v) ^R2 and ^R3 are independently H or _C1-6 alkyl; (vi) ^R4 and ^R5 are independently H, _C1-6 alkyl, or aryl (e.g., phenyl) (the aryl is optionally substituted with one or more halogen atoms (e.g., fluorophenyl, e.g., 4-fluorophenyl), hydroxy (e.g., hydroxyphenyl, e.g., 4-hydroxyphenyl or 2-hydroxyphenyl)) or _C1-6 alkoxy; and (vii) wherein X, Y and Z are independently and optionally substituted by one or more halogen atoms (e.g., F, Cl or Br), Ci _-6 alkyl (e.g., methyl), halo-Ci _-6 alkyl (e.g., trifluoromethyl), for example, Z is heteroaryl, for example, pyridyl, substituted by one or more halogen atoms (e.g., 6-fluoropyridin-2-yl, 5-fluoropyridin-2-yl, 6-fluoropyridin-2-yl, 3-fluoropyridin-2-yl, 4-fluoropyridin-2-yl, 4,6-dichloropyridin-2-yl), halo-Ci _-6 alkyl (e.g., 5-trifluoromethylpyridin-2-yl) or Ci- ₆ -alkyl (e.g., 5-methylpyridin-2-yl), or Z is aryl, for example, phenyl, substituted by one or more halogen atoms (e.g., 4-fluorophenyl); The compounds of formula II are in free form, salt form or prodrug form, including their enantiomers, diastereomers and racemates; (C) Formula III: in (i) R ₁ is H or C _1-4 alkyl (e.g., methyl or ethyl); (ii) R ₂ and R ₃ are independently H or C _1-6 alkyl (e.g., methyl or ethyl); (iii) R ₄ is H or C _1-4 alkyl (e.g., methyl or ethyl); (iv) R ₅ is aryl (eg, phenyl), which is optionally substituted with one or more groups independently selected from -C(=O)-C _1-6 alkyl (eg, -C(=O)-CH ₃ ) and C _1-6 -hydroxyalkyl (eg, 1-hydroxyethyl); (v) _R and _R are independently H or aryl (e.g., phenyl) optionally substituted by one or more groups independently selected from C _1-6 alkyl (e.g., methyl or ethyl) and halogen (e.g., F or Cl), such as unsubstituted phenyl, or phenyl substituted by one or more halogen (e.g., F), or phenyl substituted by one or more C _1-6 alkyl and one or more halogen, or phenyl substituted by one C _1-6 alkyl and one halogen, such as 4-fluorophenyl or 3,4-difluorophenyl or 4-fluoro-3-methylphenyl; and (vi) n is 1, 2, 3, or 4, The compounds of formula III are in free form, salt form or prodrug form, including their enantiomers, diastereomers and racemates; (D) Formula IV in (i) R ₁ is C _1-4 alkyl (eg, methyl or ethyl), or -NH(R ₂ ), wherein R ₂ is phenyl optionally substituted by a halogen atom (eg, fluorine), for example, 4-fluorophenyl; (ii) X, Y and Z are independently N or C; (iii) R ₃ , R ₄ and R ₅ are independently H or C _1-4 alkyl (e.g. methyl); or R ₃ is H and R ₄ and R ₅ together form a trimethylene bridge (preferably, wherein R ₄ and R ₅ together have a cis configuration, e.g., wherein the carbons carrying R ₄ and R ₅ have R and S configurations, respectively); and (iv) R ₆ , R ₇ and R ₈ are independently: H, C _1-4 alkyl (e.g., methyl), pyridin-2-yl substituted by hydroxy, or –S(O) ₂ -NH ₂ ; Provided that when X, Y and/or Z are N, then R ₆ , R ₇ and/or R ₈ are absent; and when X, Y and Z are all C, then at least one of R ₆ , R ₇ or R ₈ is -S(O) ₂ -NH ₂ or pyridin-2-yl substituted with hydroxy, The compounds of formula IV are in free form, salt form or prodrug form, including their enantiomers, diastereomers and racemates; (E) Formula 1a: in (i) _R2 and _R5 are independently H or hydroxy and _R3 and _R4 together form a trimethylene or tetramethylene bridge [preferably, wherein the carbons carrying _R3 and _R4 have the R and S configuration respectively]; or _R2 and _R3 are each methyl and _R4 and _R5 are each H; or _R2 , _R4 and _R5 are H and _R3 is isopropyl [preferably, the carbon carrying _R3 has the R configuration]; (ii) R ₆ is phenylamino (optionally substituted by halogen atoms), benzylamino (optionally substituted by halogen atoms), C _1-4 alkyl, or C _1-4 alkylthio; for example, phenylamino or 4-fluorophenylamino; and (iii) R ₁₀ is C _1-4 alkyl, methylcarbonyl, hydroxyethyl, carboxylic acid, sulfonyl, phenyl (optionally substituted by halogen or hydroxy), pyridyl (optionally substituted by halogen or hydroxy) (e.g. 6-fluoropyridin-2-yl), or thiadiazolyl (e.g. 1,2,3-thiadiazol-4-yl); and X and Y are independently C or N, The compounds of formula Ia are in free form, pharmaceutically acceptable salt form or prodrug form, including their enantiomers, diastereomers and racemates; (F) Formula V in (i) R ₁ is -NH(R ₄ ), wherein R ₄ is phenyl optionally substituted by a halogen atom (eg, fluorine), such as 4-fluorophenyl; (ii) R ₂ is H or C _1-6 alkyl (e.g., methyl, isobutyl or neopentyl); and (iii) R ₃ is -SO ₂ NH ₂ or -COOH, The compound of formula V is in free form, salt form or prodrug form, including their enantiomers, diastereomers and racemates; and/or (G) Type V in (i) R ₁ is -NH(R ₄ ), wherein R ₄ is phenyl optionally substituted by a halogen atom (eg, fluorine), such as 4-fluorophenyl; (ii) R ₂ is H or C _1-6 alkyl (e.g., methyl or ethyl); and (iii) R ₃ is H, halogen (e.g., bromine), C _1-6 alkyl (e.g., methyl), aryl optionally substituted by halogen (e.g., 4-fluorophenyl), heteroaryl optionally substituted by halogen (e.g., 6-fluoropyridin-2-yl or pyridin-2-yl), or acyl (e.g., acetyl), The compounds of formula VI are in free form, salt form or prodrug form, including their enantiomers, diastereomers and racemates. 17. The method according to any one of the preceding claims, wherein the PDE1 inhibitor is selected from any one of the following: The compounds are in free form or in the form of pharmaceutically acceptable salts. 18. The method according to any one of the preceding claims, wherein the PDE1 inhibitor is: The PDEl inhibitor is in free form or in a pharmaceutically acceptable salt form (eg, monophosphate form). 19. The method according to any one of the preceding claims, wherein the PDE1 inhibitor is: The PDEl inhibitor is in free form or in the form of a pharmaceutically acceptable salt. 20. A pharmaceutical combination therapy comprising a pharmaceutically acceptable amount of a PDEl inhibitor and a pharmaceutically acceptable amount of an immune checkpoint inhibitor for use in the method of the preceding claim.