WO2019170912A1 - Bioresorbable controlled-release compositions with sting modulating molecules - Google Patents

Abstract

The present invention relates to novel pharmaceutical compositions comprising a non- densified and porous or highly densified and non-porous, and at least partly hydrated hydrating ceramic, such as calcium sulfate, for controlled-release of one or more STING modulating molecules preferably STING agonists, for therapeutically, prophylactically and/or diagnostically active purposes.

Description

Bioresorbable controlled-release compositions with STING modulating molecules

Field of the invention

The present invention relates to novel pharmaceutical compositions comprising a non- densified and porous or highly densified and non-porous, and at least partly hydrated hydrating ceramic, such as calcium sulfate, for controlled-release of an agent that can modulate the STING pathway, preferably activate, for therapeutically, prophylactically, and/or diagnostically active purposes.

The STING modulator may be a small molecule, a polypeptide, or a nucleic acid derivative, such as a cyclic dinucleotide (CDN).

The pharmaceutical compositions of the present invention are useful for targeted and controlled local prolonged release of STING modulators.

Background of the invention

Stimulator of interferon genes (STING), is a transmembrane protein that is expressed in various endothelial, epithelial and haematopoietic cells. STING, an endoplasmic reticulum-resident protein, upon binding of cGAMP or other cyclic dinucleotide (CDN) structures, phosphorylates the adaptor protein TBK1 , which, in turn, phosphorylates IRF-3 and initiates the transcription of a number of interferon stimulated genes, including type I interferons and other co-regulated host defense pathways and cytokines. STING can be activated by bacterial and viral DNA as well as self-DNA. The ability of STING activation to promote an immune response to tumors can lead to a T cell response which is required for antitumor effect of checkpoint inhibitor-based immunotherapies. As such STING modulation is a promising new treatment modality for different cancers.

However, treatment with STING modulators, in particular STING activators or agonists, can induce severe adverse events, e.g. cytokine release, if given systemically. In addition, STING agonists might be required to be given at multiple time points over the treatment course in order to achieve desired effects. This would require multiple local, intralesional injections which might be costly, time consuming and painful to the receiving patient. In addition, the drug profile achieved with multiple injections could be less than optimal. Therefore, new release formulations of STING modulating molecules, agents or ligands are warranted.

Summary of the Invention

The present invention relates to drug delivery systems composed of bioresorbable inorganic and hydratable carriers combined with STING modulating molecules, especially cyclic dinucleotides that are STING modulating molecules, for prolonged delivery of these large molecules in a human body. The prolonged delivery of STING modulating molecules is of interest in the administration of active substances in immunotherapy of diseases.

The bioresorbable ceramics have many favorable properties for pharmaceutical formulations in controlled release applications as compared to polymers, such as biocompatibility and biodegradability. In general, the bioresorbable ceramics are non- toxic and are based on molecules which normally is present in the living tissues of mammals. Calcium sulphate is particularly attractive since it is a resorbable and biocompatible material, i.e. it disappears over time.

One aspect of the present invention relates to encapsulation of STING modulating molecules, such as cyclic dinucleotides (CDN) in depots consisting of a matrix of partly hydrated or fully hydrated, porous or dense calcium sulfate. Such encapsulated STING modulating molecules may thereafter be slowly released from the depot in a biological environment of living cells, such as a human or animal body, and maintaining the therapeutically, prophylactically, and/or diagnostically properties and effects of the STING modulating molecules on said cells. The STING modulating molecules, such as cyclic dinucleotides, will thus act as an active pharmaceutical ingredient (API) and the matrix of partly hydrated or fully hydrated, porous or dense calcium sulfate will act as a pharmaceutical excipient.

Embodiment 1. A controlled-release pharmaceutical composition comprising one or more hydratable and bioresorbable ceramics or hydrating inorganic compound and one or more therapeutically, prophylactically, and/or diagnostically active STING modulating molecules. Embodiment 2. A controlled-release pharmaceutical composition, wherein the hydratable and bioresorbable ceramics is selected from the group consisting of non- hydrated, partly hydrated and/or fully hydrated calcium sulfate.

Embodiment 3. A controlled-release pharmaceutical composition according to the present invention, wherein the hydratable and bioresorbable ceramic has been fully or partly compressed to a high density.

Embodiment 4. A controlled-release pharmaceutical composition according to the present invention, wherein the hydratable and bioresorbable ceramic has been fully or partly compressed in an isostatic press.

Embodiment 5. A controlled-release pharmaceutical composition according to the present invention, comprising a mixture of non-compressed and compressed ceramic material, such as calcium sulphate.

Embodiment 6. A controlled-release pharmaceutical composition according to the present invention, wherein the STING modulating agent is selected from the group consisting of one or more, nucleic acids and/or small molecules.

Embodiment 7. A controlled-release pharmaceutical composition according to the present invention, wherein the STING modulating molecule is a cyclic dinucleotide.

Embodiment 8. A controlled-release pharmaceutical composition according to the present invention, wherein the STING modulating molecule wherein the cyclic dinucleotide is chosen from ADU-S100 (MIW815), MK-1454, cyclic diguanylate monophosphate (c-di-GMP), cyclic [G(2',5')pA(3',5')p] (cGAMP) and ML RR-S2 CDA.

Embodiment 9. A controlled-release pharmaceutical composition according to the present invention for use in the treatment of cancer.

Embodiment 10. A controlled-release pharmaceutical composition according to the present invention, for injection into a solid cancer tumor - i.e. intra-tumoral injection.

Embodiment 1 1. A method of treating a human being afflicted with a cancer tumor, comprising administering to a tumor site one or more hydratable and bioresorbable ceramics or hydrating inorganic compounds and one or more therapeutically, prophylactically and/or diagnostically active STING modulating molecules.

Embodiment 12. A method according to the present invention, wherein the hydratable and bioresorbable ceramics is selected from the group consisting of non-hydrated, partly hydrated and/or fully hydrated calcium sulfate.

Embodiment 13. A method according to the present invention, wherein the hydratable and bioresorbable ceramic has been compressed to a high density.

Embodiment 14. A method according to the present invention, wherein the hydratable and bioresorbable ceramic has been compressed in an isostatic press.

Embodiment 15. A method according to the present invention, comprising a mixture of non-compressed and compressed ceramic material.

Embodiment 16. A method according to the present invention, wherein the STING modulating molecule is a cyclic dinucleotide.

Embodiment 17. A method according to the present invention, wherein the cyclic dinucleotide is a chosen from ADU-S100 (MIW815), MK-1454, cyclic diguanylate monophosphate (c-di-GMP), cyclic [G(2',5')pA(3',5')p] (cGAMP) and ML RR-S2 CDA.

Brief description of the drawings

Figure 1 shows the tumor growth of syngeneic tumors following intratumoral adminsitration of a controlled release formulation of a STING agonist according to Example 3.

Figure 2 shows the survival of animals treated in Example 3.

Figure 3 shows the tumor volume in relation to days elapsed for the respective groups in Example 4, where figure 3a shows group 1 , figure 3b shows group 2, figure 3c shows group 3, figure 3d shows group 4 and figure 3e shows group 5, respectively. Figure 4 shows the terminal tumor weight, as recorded following excision on the final day of the study in Example 4. Detailed description of the Invention

A range of drug delivery systems for local, controlled and/or targeted delivery therapy has been developed in the past. Many are based on bioresorbable (or biodegradable) polymers, bioresorbable ceramics and/or hydrogel(s) as carriers for the therapeutically active substance. Commonly used biodegradable polymers are polylactic acids and polylactic-co-glycolide-acids. Various calcium-salt based ceramics, e.g. calcium phosphate or calcium sulphate systems, or hydroxylapatite, have been described in the form of beads, granules, scaffolds, or moldable pastes, to carry and release drugs. These ceramics are often referred to as hydratable or hydrating ceramics due to their ability to react chemically with water to form solid hydrates. See e.g. Royer US

6,391 ,336, US 6,630,486, and US 2003/0170307.

W02005039537 discloses a pharmaceutical composition comprising a bioresorbable hydratable ceramic, sorbed aqueous medium, and an active substance.

Pharmaceutical compositions based on calcium sulfate are described in WO

2007/104549, Bioresorbable controlled release composition.

However, treatment with STING modulators, in particular, STING activators or agonists, can induce severe adverse events, e.g. cytokine release, if given

systemically. Therefore, STING agonists should be given locally, intratumorally when treating cancers, to avoid systemic side-effects. In addition, STING agonists might be required to be given at multiple time points over the treatment in order to achieve desired effects. This would require multiple local, intralesional injections which might be costly, time consuming and painful to the receiving patient. In addition, the drug profile achieved with multiple injections could be less than optimal. To address and circumvent these issues, a STING modulating agent, for example an agonist, could be formulated in a drug delivery system that allows for a local administration leading to a controlled release of the active agent over an extended time.

Immunotherapy is the treatment of a disease by inducing, enhancing, or suppressing an immune response. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppressive immunotherapies.

Immunomodulatory regimens often have fewer side effects than existing drugs, including less potential for creating resistance when treating microbial disease. In order to increase the cellular uptake of cyclic dinucleotide STING agonists, different encapsulating techniques have been investigated. These formulations or

complexations can e.g. involve formation of nanoparticles, micelles, or lipophilic cation complexes. For examples see, Nakamura et al Journal of Controlled Release 216 (2015) 149-157, Junkins et al Journal of Controlled Release 270 (2018) 1-13, Wilson et al Nanomedicine: Nanotechnology, Biology, and Medicine 14 (2018) 237-246,

Koshy et al Adv. Biosys. 2017, 1 , 1600013 and WO 2017/18671 1 A1.

These above-mentioned methods attempt to solve the issue of delivering the negatively charged cyclic dinucleotide STING agonists into cells. However, less attention has been given to avoiding the issue of repeated intratumoral injections by developing an injectable controlled release formulation of STING agonists. An injectable peptide hydrogel formulation of a cyclic dinucleotide has been developed (Leah et al

Biomaterials 163 (2018) 67e75), but this technology has a limited slow release capability with release equilibrium being reach at 14 hours. Furthermore, the peptide matrix used in the hydrogel might have inherent pharmacological properties.

Therefore, new controlled release formulations of STING modulating molecules, agents or ligands are warranted in order to achieve a controlled release of a STING ligand over an extended period of time following a single intratumoral or local injection. In addition, such a controlled release formulation should preferably be innocuous and bioresorbable.

The bioresorbable ceramics have many favorable properties for pharmaceutical formulations in controlled release applications as compared to polymers, such as biocompatibility and biodegradability. In general, the bioresorbable ceramics are non- toxic and are based on molecules which normally is present in the living tissues of mammals. Calcium sulphate is particularly attractive since it is a resorbable and biocompatible material, i.e. it disappears over time.

One aspect of the present invention relates to encapsulation of STING modulating molecules, such as cyclic dinucleotides (CDN) in depots consisting of a matrix of partly hydrated or fully hydrated, porous or dense calcium sulfate. Such encapsulated STING modulating molecules may thereafter be slowly released from the depot in a biological environment of living cells, such as a human or animal body, and maintaining the therapeutically, prophylactically, and/or diagnostically properties and effects of the STING modulating molecules on said cells. The STING modulating molecules, such as cyclic dinucleotides, will thus act as an active pharmaceutical ingredient (API) and the matrix of partly hydrated or fully hydrated, porous or dense calcium sulfate will act as a pharmaceutical excipient.

In a further aspect, the present invention relates to the ability to manufacture a homogeneous dry powder which contains STING modulating molecules, such as cyclic dinucleotides (CDN) that can be long-term stored at room temperature. This powder can then, at the time for administration, be reconstituted with an aqueous diluent prior administration to form depots in vivo. A preferred example is described in Example 5.

The STING modulating molecules, used in the present invention, may for example be embedded into the calcium sulfate matrix by mixing the calcium sulfate powder and the STING modulating molecules and thereafter make the calcium sulfate react with water, i.e. hydrate, to a solid form. This solid form is used as a drug depot. The drug depot dissolves slowly in a water-based environment such as a human or animal body or in a cell medium and is thus suitable for immunotherapy and the like.

The STING modulating molecules / active pharmaceutical ingredient (API) may be loaded onto the calcium sulphate matrix, i.e. calcium sulphate hemihydrate, in different ways. The calcium sulphate hemihydrate can for example be mixed with the STING modulating molecules creating a STING modulating molecule-loaded binary mixture. This may be performed by dry mixing. This may also be performed by dissolving the STING modulating molecules in a solvent to which the calcium sulphate is inert (such as an alcohol, preferably 2-propanol). This leads to a slurry of calcium sulphate powder in a solute of STING modulating molecules and solvent. By evaporating the solvent while agitating the slurry a dry powder of STING modulating molecules and calcium sulphate is formed.

The loading of the STING modulating molecules can further be done simultaneously in the recrystallization process to calcium sulphate dihydrate using an aqueous solution/dispersion of the STING modulating molecules as the calcium sulphate hemihydrate is converted into other forms of calcium sulphate, such as recrystallized into calcium sulphate dihydrate. The STING modulating molecules -loaded calcium sulphate dihydrates in such a formulation may have different physical properties and release rates / release profiles depending on how the precursor calcium sulphate hemihydrate was prepared, e.g. particle size distribution.

The curing time of such a calcium sulphate mixture may be controlled by the presence of a retardant. It is preferred that the resulting calcium dehydrate paste may be ejected using a regular syringe equipped with a regular or small cannula. Examples of such retardant is carboxymethylcellulose, notably the sodium salt thereof (Na-CMC). The amount of such Na-CMC is usually about 0.1 to 5 wt%, such as 0.25%. Such a calcium sulphate mixture may comprise a mixture of non-compressed and compressed calcium sulphate. Such mixture comprising a mixture of non-compressed and compressed calcium sulphate may have different physical properties and release rates / release profiles depending on the ratio between non-compressed and compressed calcium sulfate. Such ratio may vary from about 100:1 to about 1 : 100.

Such binary mixture can further be conventionally compressed in STING modulating molecules -loaded dense granules (or bodies). The STING modulating molecules - loaded dense granules (or bodies) will have different physical properties and release rates / release profiles depending on how it is prepared, for example which pressure that was used in the densifying step. Suitable pressures are 1000 - 5000 bar for about 1-3 hours.

Preferably, such STING modulating molecules -loaded dense granules (or bodies) are prepared by wetting calcium sulphate hemihydrate, with enough water to convert it to calcium sulphate dihydrate and simultaneously apply a pressure of about 4000 bar for about 1 hour.

A controlled-release pharmaceutical composition according to the present invention may comprise a mixture of non-compressed and compressed ceramic material, such as calcium sulphate.

The STING modulating molecules can be either agonists or antagonists, of which agonists are preferred.

STING agonists include naturally occurring and synthetic cyclic dinucleotides. The term cyclic dinucleotide represents a class of cyclic molecules where two nucleotides are connected by two phosphodiester linkages, two phosphorothioate diester linkages or one phosphodiester and one phosphorothioate diester linkage. The linkages can be (3’- 5’)-(3’-5’), (3’-5’)-(2’-5’), (2’-5’)-(3’-5’) or (2’-5’)-(2’-5’) and all are within the scope of the present invention.

Phosphorothioate linkages are inherently chiral and cyclic dinucleotides can therefore exist in in chiral or diastereomeric forms.

Such diastereomeric form of a phosphorothioate is preferably an optically pure diastereoisomer or optically pure form. As used herein, the term“optically pure” means an isomeric excess over other isomers greater than about 80%, preferably greater than about 90%, preferably greater than about 95%, more preferably greater than about 97%, even more preferably greater than about 99%, more preferably greater than about 99.5% or more, and most preferably up to 100%, wherein the remainder may be one or more of the other isomers.

Cyclic dinucleotides include, but are not limited to, c-di-adenosine monophosphate (AMP), c-di-guanosine monophosphate (GMP), c-di-inosine monophosphate (IMP), c- AMP- GMP, c- AMP-IMP, and c-GMP-IMP, and analogs thereof including, but not limited to, phosphorothioate analogues.

In one embodiment of the present invention the cyclic nucleotide is of the general structure:

wherein Bi and B₂ are purine bases independently selected from adenosine, guanine, or hypoxanthine;

Qi and Q₂ are independently selected from H, OH or F; and

Yi and Y₂ are independently selected from O or S, or a pharmaceutically acceptable salt thereof.

Further, but not limiting examples of cyclic dinucleotides that act as STING modulating molecules include:

In addition STING modulating agents or molecules maybe small molecule analogs or derivatives of flavone acetic acid (FAA), 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and 10-(carboxymethyl)-9(10H)acridone.

The STING modulating molecules can also be dispiro diketopiperazine (DSDP) derivatives.

The STING modulating molecules can be immunomodulatory compounds that can be used in combination therapy methods.

A further aspect is to administer the pharmaceutical compositions of the present invention, i.e. containing a therapeutically, prophylactically, and/or diagnostically active STING modulating molecule, to a human or animal subject in need of such treatment. Example of such treatments include, but is not limited to, hyperproliferative diseases such as cancer and including advanced cancer. Typically, such a cancer is selected from (a) solid cancers including, but not limited to, sarcomas, breast cancer, prostate cancer, head and neck cancer, brain tumors, colorectal cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, melanoma, gastric cancers, renal cell carcinoma, endometrial cancer, sarcomas and hepatocellular carcinomas; (b) hematological cancers including, but not limited to, chronic myelogenous leukemia, acute myelogenous leukemia, cutaneous T-cell lymphoma, Hodgkin’s disease, anaplastic large-cell lymphoma and Burkitt’s lymphoma.

Pharmaceutical compositions of the present invention may be injected into a tumor site, preferably intratumorally or adjacent to the tumor, (such as at a distance of 0.1 -10 mm), to a solid tumor. The tumor may be a primary tumor or a metastasis.

In a preferred version of the invention the formulation is injected intratumorally where it solidifies to a solid local depot (comparable to a drug eluting implant) which over time dissolves and releases the active substance locally. The local solidification of the formulation facilitates a controlled local administration of the active substance.

The locally solidifying formulation forming a local drug depot may also be injected adjacent to a tumor or to a location characterized by the presence of cancer cells and cancerous tissues.

The injected formulation may be administered in different ways to the tumor or cancerous tissue, depending on the size, shape and distribution of the tumor or cancer tissue. The injection may be focused to one solid depot to optimally focus the release to the interior of one tumor; or several depots of similar or different shapes may be injected to cover a larger area or treat several tumors; or the depot may be injected as strings or range of smaller depots to adapt to a tumor of undefined or irregular shape. Any such injected formulation may consist of a mixture of a non-compressed and compressed ceramic material, such as calcium sulphate.

The present invention thus provides a method of treating a subject, such as a human being, afflicted with a tumor, comprising administering to a tumor site a hydratable and bioresorbable ceramic, preferably calcium sulphate, comprising a STING modulating molecule. Such method of treating a subject may result in (a) reducing the volume of the tumor; (b) reducing the growth of the tumor; (c) reducing metastasis of the tumor; (d) increasing the survival of the subject; (e) increasing the progression free survival of the subject; and/or (f) increasing a T cell response to an antigen within the tumor.

One aspect of the present invention provides a method to reducing the volume of a tumor at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 percent, by administering to a tumor site a hydratable and bioresorbable ceramic, preferably calcium sulphate, comprising a STING modulating molecule. Such reduction of volume usually occurs within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 21 , 28, 35, 42, 49, 180, 365 or 1 -365 days or within about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 months.

Pharmaceutical compositions of the present invention are advantageous since they have a controlled-release profile and thus requires less frequent and/or less number of injections compared to compositions of the prior art. Each administration is costly, time consuming and painful to the receiving patient and the compositions of the present invention are thus likely to improve a patient’s compliance.

Pharmaceutical compositions of the present invention are further advantageous since they induce fewer adverse events compare to an immediate-release formulation.

Adverse events are most likely to occur at or near the maximum (or peak) serum concentration (C_max) of a drug.

Pharmaceutical compositions of the present invention are yet further advantageous since they combine a pharmacologically active STING modulating with a bioresorbable carrier that has been proven safe and tolerated in humans. The compositions of the present invention comprise a hydratable and bioresorbable ceramic, preferably calcium sulphate.

One aspect of the present invention is a controlled-release composition comprising a non-densified and porous hydratable and bioresorbable ceramic.

One aspect of the present invention is a controlled-release composition comprising highly densified and non-porous hydratable and bioresorbable ceramic, such as calcium sulphate.

One aspect of the present invention is a controlled-release composition comprising a mixture of a non-densified and porous with a highly densified and non-porous hydratable and bioresorbable ceramic, such as calcium sulphate.

One aspect of the present invention is to provide a controlled-release composition (or depot) that releases the STING modulating molecules over a total period of at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 weeks, whereby the release rate of STING modulating molecules is approximately constant during the total time period either in vivo or when subjected to an in vitro dissolution test according to established using USP or European Pharmacopeia methods, or similar laboratory in vitro release methods, such as USP apparatus 7 (37 degrees physiological saline, 5 dips per minute) or similar; or e.g. USP apparatus 4 (37 degrees physiological saline, large cell, 19 ml medium, minimal flow) or similar. Such a controlled-release formulation provides a pharmacological acceptable and useful balance between safety and efficacy.

One aspect of the present invention is to provide a controlled-release composition (or depot) that releases the STING modulating molecules over a total period of at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or 13 weeks, whereby a release of 20-80%, such as 30-60% or 40-50% of the total amount of STING modulating molecules in the composition is released within the first half of the time period, or during the first third of the time period either in vivo or when subjected to an in vitro dissolution test according to established using USP or European Pharmacopeia methods, or similar laboratory in vitro release methods, such as USP apparatus 7 (37 degrees physiological saline, 5 dips per minute) or similar; or e.g. USP apparatus 4 (37 degrees physiological saline, large cell, 19 ml medium, minimal flow) or similar. Such a controlled-release formulation provides a pharmacological acceptable and useful balance between safety and efficacy.

One aspect of the present invention is to provide a controlled-release composition with a release profile over an extended period of time defined by a faster initial release during the first half of the total treatment time, or during the first third of the total treatment time, followed by a slower release for the remaining duration of the treatment, when tested according to established USP or European Pharmacopeia drug release methods, such as USP apparatus 7 (37 degrees physiological saline, 5 dips per minute) or similar; or e.g. USP apparatus 4 (37 degrees physiological saline, large cell, 19 ml medium, minimal flow) or similar. Such a controlled-release formulation provides a pharmacological acceptable and useful balance between safety and efficacy.

One aspect of the present invention is to provide a controlled-release composition with a release profile over an extended period of time defined by a slower initial release that may or may not include an initial time period with no release at all during the first half of the total treatment time, such as during the first third of the total treatment time, followed by a faster release for the remaining duration of the treatment, when tested according to established USP or European Pharmacopeia drug release methods, such as USP apparatus 7 (37 degrees physiological saline, 5 dips per minute) or similar; or e.g. USP apparatus 4 (37 degrees physiological saline, large cell, 19 ml medium, minimal flow) or similar. Such a controlled-release formulation provides a

pharmacological acceptable and useful balance between safety and efficacy.

One aspect of the present invention is to provide a controlled-release composition (or depot) that releases the STING modulating molecules over a total period of at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or 13 weeks, whereby two or more different release rates are present either in vivo or when subjected to an in vitro dissolution test according to established using USP or European Pharmacopeia methods, or similar laboratory in vitro release methods, such as USP apparatus 7 (37 degrees

physiological saline, 5 dips per minute) or similar; or e.g. USP apparatus 4 (37 degrees physiological saline, large cell, 19 ml medium, minimal flow) or similar. Such a controlled-release formulation provides a pharmacological acceptable and useful balance between safety and efficacy. The present invention thus delivers a pharmaceutical formulation with a slow release capability and a release equilibrium well beyond 14 hours.

The precise dose to be employed in the formulation will depend on the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient’s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

However, suitable doses of the pharmaceutically active agent range from about 0.01 to 100 pg active compound per kilogram body weight. Fixed doses of the

pharmaceutically active agent vary from 0.7 to 7000 pg.

Examples of STING modulating molecules contemplated for use in the invention are given in Table 1.

Table 1.

In the experiments of Examples 1-4 we have used ML RR-S2 CDA as our STING agonist. ML-RR-S2 CDA is a potent STING-activating cyclic di-adenosine (CDA or c- diAMP) nucleotide ligand that contains non-canonical 2',5'-phosphodiester bond and doubly substituted thiophosphate (S2) for optimal phosphodiesterase-resistance and thereby enhanced cellular potency. ML-RR-S2 CDA effectively induces IRF3, NF-kB, and STAT6 transcription activities in a STING-dependent manner in both murine and human cell cultures (5-100 pM) as well as displays profound anti-tumor efficacy in vivo (100% growth inhibition of established B16.F10, 4T-1 or CT26 tumor with 3X 50 pg/mouse/96 hr intratumoral injections) with lasting immune-mediated tumor rejection.

ML RR-S2 CDA, also known as ADU-S100 and MIW815, is a cyclic dinucleotide with the following structure:

ML RR-S2 CDA or ADU-S100 is an inducer of STING (stimulator of interferon genes). ADU-S100 has enhanced binding affinity to STING and activate all known human STING alleles.

The calcium sulfate formulation loaded with STING modulating molecules can be used alone or in combination with systemic immunotherapies. More specifically be used with checkpoint inhibitors including anti-CTLA4, anti-PD-1 or anti-PD-L1 antibodies (e.g. Ipilimumab, Tremelimumab, Pembrolizumab, Nivolumab (PD1 ), Atezolizumab

(MPDL3280A), MEDI4736, Avelumab, PDR001 ). Such combination therapy or combined administration may be in a fixed combination product or using concomitant and/or sequential administration.

The advantages of immunotherapy using calcium sulfate formulation loaded with STING modulating molecules include decreased systemic side-effects, increased efficacy through direct activation of the innate immune system in the tumor

microenvironment and increased tissue penetration and decreased number of invasive and painful injection increasing patient compliance.

The term“controlled-release” as used herein relates to a mechanism that delivers an active pharmaceutical ingredient (API) with a delay after its administration or for a prolonged period after its administration or with a release profile that is substantially different than an immediate release profile.

The terms“extended-release”,“delayed release” or“prolonged release” as used herein has the same meaning as“controlled release”.

The term“immunotherapeutic agent” as used herein relates to a medicament capable of inducing, enhancing, or suppressing an immune response.

The term“immunotherapy” as used herein relates to the treatment of a disease by inducing, enhancing, or suppressing an immune response. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppressive immunotherapies

The term“STING modulating molecule” as used herein relates to a molecule that can activate or inhibit the stimulator of interferon genes (STING) protein or STING pathway.

The term“small molecule” as used herein relates to a low molecular weight (< 900 daltons) organic compound that may regulate a biological process.

Examples

Example 1

The purpose of the example is a preliminary evaluation of the possibilities to

incorporate a STING modulating molecule in a calcium sulfate matrix, and release the STING modulating molecules in a relevant medium with maintained bio-functionality.

STING agonists are potent mediators of immune activation and anti-tumor effects. However, there is a need to reduce systemic toxicity and maintain local concentration at a sufficiently high level.

The purpose of the experiment was to assess a potential local immunotherapy as a means to reduce toxicity in humans in a future locally injectable drug product.

Method/Procedure, Example 1 :

The STING agonist (ML RR-S2 CDA ammonium salt) is dissolved in 2-propanol in the ratio of 1 :450. This solution is then mixed under magnetic stirring at 35°C during 15 minutes making a clear solution. Micronized calcium sulfate hemihydrate is added to the solution to an amount that generate a final dry powder containing 0.6 wt.% STING agonist. The suspension is evaporated for 20 hours to form a“cake-like” dry powder adhered to the inside of the evaporation glass vessel. The cake is broken and sieved through a 200 pm mesh sieve to achieve a free-flowing powder.

Prior to administration the powder is mixed with an aqueous diluent in the weight ratio of 1 :1 , where the diluent is 0.25 wt.% sodium carboxymethylcellulose (Na-CMC) in purified water. The diluent is mixed thoroughly by docking the male Luer-Lok syringe containing diluent with the female Luer-Lok powder syringe and then pushing the pistons back and forth several times to create a homogeneous suspension. The powder syringe is then changed to a suitable injection needle and after priming, the drug product (reconstituted suspension) is prepared to be used for injection. The depot formulation is injected locally with a suitable volume into the tumor, where the formulation solidifies within approximately 10-20 minutes.

Table 2, Composition, Example 1

Example 2

The purpose of the experiment was to examine the release of a STING agonist into a solution from the depot formulation.

Results from Example 2

The formed tablet was placed in a solution of PBS at 37°C. The concentration of the STING agonist was measured initially and after 7 days.

The relative release was measured to approximately 60% after 7 days.

Example 3

The purpose of Example 3 is to evaluate the antitumor effect of a STING agonist formulated in calcium sulfate prepared as in Example 1. The results are shown in Figure 1 and 2.

To assess the in vivo anti-tumor efficacy of the calcium sulfate formulation of a STING agonist, a syngeneic murine melanoma model with B16F10 melanoma cells implanted subcutaneously in B6D2F1 mice was used.

A preparation of 5x10⁵ B16F10 cells in PBS was injected subcutaneously into the flank of female B6D2F1 mice in a total injection volume of 100 pi. Animals bearing tumors of preferably >100 mm³ were assigned to treatment groups and therapy as conducted as outlined in Table 3.

Table 3

To prepare for injection, a dry powder containing ML-RR-S2 CDA finely distributed in Ca₂S0₄ was mixed with a 0.25% Na-CMC aqueous solution at proportions of 1 ml of solution to 1 g of powder, forming a viscous but injectable paste. 50 pL of Nanozolid was injected intratumorally using a 21 G syringe on the day of randomization.

Tumor volumes and body weights were determined three times a week. For animal welfare regulations (fast growth of syngeneic models), animals that reached a tumor volume of 1500 mm³were terminated.

Figure 1 shows the tumor growth of syngeneic tumors following intratumoral adminsitration of a controlled release formulation of a STING agonist according to Example 3. As can be seen in Figure 1 , calcium sulfate formulated STING agonist significantly reduced the tumor growth when given as a single intratumoral injection. This resulted in an increased survival (Figure 2), Figure 2 showing survival of animals treated in Example 3. Example 4.

The purpose of Example 4 is to evaluate the antitumor effect of different doses of a STING agonist formulated in calcium sulfate prepared as in Example 1. The results are shown in Figure 3 and 4.

To assess the in vivo anti-tumor efficacy of the calcium sulfate formulations of a STING agonist, female C57BL/6J mice were inoculated with 5 x 10⁵ B16-F10 cells, in a volume of 100 pl_, via subcutaneous (s.c.) injection in the rear flank. When the majority of the tumors had reached a volume of 100 mI_, 25 animals were stratified into five

experimental groups of 5 animals/group, with equal distributions of tumor size, as shown in Table 4. The results on tumor growth of the groups, group 1 Nanozolid (vehicle), group 2 STING comparator 3x50 pg, group 3 Nanozolid-STING 50 pg in 50 pL, group 4 Nanozolid-STING 150 pg in 50 mI_, Nanozolid-STING 200 pg in 50 mI_, is shown in figure 3a-3e.

Table 4

To prepare for injection, a dry powder containing ML-RR-S2 CDA finely distributed in Ca₂S0₄ was mixed with a 0.25% Na-CMC aqueous solution at proportions of 1 ml of solution to 1 g of powder, forming a viscous but injectable paste. 50 pl_ of Nanozolid was injected intratumorally (it) using a 21 G syringe on the day of randomization.

Immunohistochemistry on cryopreserved tumor samples was performed using the following primary antibodies:

Table 5

Microscopical analysis

The number of positive cells in tumors was judged as; negative (0), few positive cells (1 +), few to moderate numbers (1 -2+), moderate numbers of positive cells (2+), moderate to high numbers (2-3+) or high numbers of positive cells (3+).

On Day 1 , all animals were injected intratumorally with test item. Animals in Group 2 were additionally injected intratumorally with test item on Days 5 and 8. Mice were weighed and monitored with regard to health status on all treatment days, as well as on Days 4 and 6; tumor volume was measured with a digital calliper. The final measurements of body weight, health status and tumor volume were performed on Day 1 1 , after which mice were euthanized. T umors were excised and weighed, after which they were divided into two equal parts for histopathological and immunohistochemical analyses.

All animals demonstrated a general increase in body weight from Day -8 to Day 10. Treatment with test item had no effect on body weight. Treatment with test item had a significant effect on tumor volume (p<0.0001 ) (Figure 3). Whereas animals in Group 1 (NZ-Vehicle) demonstrated a significant increase in tumor size from the first day on which tumors were measurable (Day -2) to the final day of the study, this was not evident in the remaining groups. Specifically, on the final day of the study, tumor volumes were significantly smaller in all treatment groups, as compared to the NZ- Vehicle group (Group 1 ). The effect of treatment was further corroborated by tumor weight, where tumors in Groups 2, 3 and 5 were significantly smaller than tumors in Group 1 (NZ-Vehicle). Immunohistochemical analysis revealed similar levels of CD4+ and CD8+ and IFN-beta production were evident in Groups 1 and 2. In comparison cellular infiltration was more extensive in Groups 3 - 5 In conclusion, administration NZ-STING, particularly at a dose of 0.05 mg, was associated with a significant decrease in B16-F10 tumor growth and size.

Administration of NZ-STING was associated with infiltration of CD4+ and CD8+ cells, and the immunohistochemistry scoring results can be seen in table 6. Table 6

Figure 3 shows the tumor growth of individual syngeneic tumors following intratumoral adminsitration of a a STING agonist according to Example 4 for the respective groups 1-5. As can be seen in Figure 3, calcium sulfate formulated STING agonist significantly reduced the tumor growth when given as a single intratumoral injection.

Figure 4 shows the terminal tumor weight, as recorded following excision on the final day of the study in Example 4. As can be seen in Figure 4, animals treated with a single intratumoral injection of calcium sulfate formulated STING agonist had tumors of significantly reduced size compared to untreated animals.

Example 5

The purpose of the example was a preliminary evaluation of the possibilities to long- term store a powder consisting of an incorporated STING modulating molecule in a calcium sulfate matrix at room temperature, and to be able to release the STING modulating molecules both in vitro and in vivo with the intended release profile and maintained bio-functionality, also after storage. The concentration of 0.46 wt.% in the powder was chosen in order to be able to inject a dose of 150 pg STING modulating molecule per 50 pi reconstituted homogeneous suspension.

Method/Procedure, Example 5:

The STING agonist (ML RR-S2 CDA ammonium salt) was dissolved in 2-propanol in the ratio of 1 : 1400. This solution was then mixed under magnetic stirring at 35°C for 40 minutes making a clear solution. Micronized calcium sulfate hemihydrate was added to the solution to an amount that generate a final dry powder containing 0.46 wt.% STING agonist. The suspension was evaporated for 2.5 hours to form a“cake-like” dry powder adhered to the inside of the evaporation glass vessel. The cake was broken and sieved through a 125 pm mesh sieve to achieve a free-flowing powder. The powder was then filled into plastic female Luer-Lok syringes, where the piston is positioned to have a minimal dead space without compressing the powder, and then closed with a plastic Luer-Lok stopper.

Prior to administration the powder was mixed with an aqueous diluent in the weight ratio of 1 :1 , where the diluent was 0.25 wt.% sodium carboxymethylcellulose (Na-CMC) in purified water. The diluent was mixed thoroughly by docking the male Luer-Lok syringe containing diluent with the female Luer-Lok powder syringe and then pushing the pistons back and forth several times to create a homogeneous suspension. The powder syringe was then changed to a suitable injection needle and after priming, the reconstituted suspension was prepared to be used for injection. The depot formulation was injected locally with a suitable volume into the tumor, where the formulation solidified within approximately 10-20 minutes.

Table 7, Composition, Example 5

The in vivo and in vitro characteristics were essentially similar as for Example 1 above. Assay was measured after six months of storage. In conclusion, the analyses of Example 5 showed that the powder can be stored as a dry powder for at least six months at room temperature in plastic containers with maintained functionality.

Claims

1. A controlled-release pharmaceutical composition comprising one or more hydratable and bioresorbable ceramics or hydrating inorganic compounds and one or more therapeutically, prophylactically and/or diagnostically active STING modulating molecules.

2. A pharmaceutical composition according to claim 1 , wherein the hydratable and bioresorbable ceramics is selected from the group consisting of non-hydrated, partly hydrated and/or fully hydrated calcium sulfate.

3. A pharmaceutical composition according to claim 1-2, wherein the hydratable and bioresorbable ceramic solidifies in vivo to a solid depot that locally dissolves and produces a controlled local release of the active STING modulating molecules.

4. A pharmaceutical composition according to any of the preceding claims, wherein the hydratable and bioresorbable ceramic has been fully or partly compressed to a high density.

5. A pharmaceutical composition according to any of the preceding claims, wherein the hydratable and bioresorbable ceramic has been fully or partly compressed in an isostatic press.

6. A pharmaceutical composition according to any of the preceding claims, comprising a mixture of non-compressed and compressed ceramic material.

7. A pharmaceutical composition according to any of the preceding claims, wherein the STING modulating molecule is a cyclic dinucleotide.

8. A pharmaceutical composition according to claim 7, wherein the cyclic dinucleotide is a chosen from ADU-S100 (MIW815), MK-1454, cyclic diguanylate monophosphate (c-di-GMP), cyclic [G(2',5')pA(3',5')p] (cGAMP) and ML RR-S2 CDA.

9. A pharmaceutical composition according to claim 7, wherein the cyclic dinucleotide is a compound of Formula I,

wherein Bi and B₂ are purine bases independently selected from adenosine, guanine or hypoxanthine;

Qi and Q₂ are independently selected from H, OH or F; and

Yi and Y₂ are independently selected from O or S,

or a pharmaceutically acceptable salt thereof.

10. A method of treating a human being afflicted with a cancer tumor, comprising administering to a tumor site one or more hydratable and bioresorbable ceramics or hydrating inorganic compounds and one or more therapeutically, prophylactically and/or diagnostically active STING modulating molecules.

1 1. A method according to claim 10, wherein the hydratable and bioresorbable ceramics is selected from the group consisting of non-hydrated, partly hydrated and/or fully hydrated calcium sulfate.

12. A method according to claim 10 or 1 1 wherein the hydratable and bioresorbable ceramic has been compressed to a high density.

13. A method according to any of claims 10-12, wherein the hydratable and

bioresorbable ceramic has been compressed in an isostatic press.

14. A method according to any of claims 10-13, comprising a mixture of non- compressed and compressed ceramic material.

15. A method according to any of claims 10-14, wherein the STING modulating molecule is a cyclic dinucleotide.

16. A method according to claim 15, wherein the cyclic dinucleotide is a chosen from ADU-S100 (MIW815), MK-1454, cyclic diguanylate monophosphate (c-di-GMP), cyclic [G(2',5')pA(3',5')p] (cGAMP) and ML RR-S2 CDA.

17. A method according to claim 15, wherein the cyclic dinucleotide is a compound of Formula I,

wherein Bi and B₂ are purine bases independently selected from adenosine, guanine or hypoxanthine;

Qi and Q₂ are independently selected from H, OH or F; and

Yi and Y₂ are independently selected from O or S,

or a pharmaceutically acceptable salt thereof.