INHALABLE FORMULATIONS FOR KINASE INHIBITION
The invention relates to inhalable formulations configured to inhibit target combinations of kinases for the treatment of cardiovascular and pulmonary diseases such as pulmonary arterial hypertension (PAH).
This application claims the benefit of, and priority to, U.S. Provisional Application Nos. 62/849,054, filed May 16, 2019; 62/849,056, filed May 16, 2019; 62/849,058, filed May 16, 2019; 62/849,059, filed May 16, 2019; 62/877,575, filed Jul. 23, 2019; 62/942,408, filed Dec. 2, 2019; 62/984,037, filed Mar. 2, 2020; and 62/958,481, filed Jan. 8, 2020; the content of each of which is hereby incorporated by reference herein in its entirety.
FIELD OF THE INVENTIONThe invention generally relates to inhalable formulations of kinase inhibitors to treat disease.
BACKGROUNDPulmonary arterial hypertension (PAH) is a condition involving elevated blood pressure in the arteries of the lungs with unknown causes and is differentiated from systemic hypertension. PAH is a progressive disease where resistance to blood flow increases in the lungs causing damage to the lungs, the pulmonary vasculature and the heart that can eventually lead to death. While symptoms are treatable with vasodilators and other medications, there is no known disease modifying therapy or cure and advanced cases can eventually require lung transplants.
It has been hypothesized that PDGFR plays a significant role in the pathobiology of PAH and that the PDGFR-inhibiting effect of imatinib was therefore thought to contribute to its efficacy in treating PAH. However, other tyrosine kinase inhibitors active against PDGFR have not shown the same efficacy. In fact, in certain instances PDGFR inhibitors such as dasatinib and nintedanib were found to induce or worsen PAH.
Therefore, up to this point, the exact mechanism of action for treating PAH has remained unknown. Similarly, it is not known what combination of kinases might be linked to PAH proliferation and might prove useful treatment targets. Accordingly, an effective treatment for PAH remains elusive.
SUMMARYThe invention is based on an in-depth molecular characterization of numerous different compounds. Results of those characterizations have led to discovery of a molecular profile associated with pulmonary arterial hypertension (PAH), which profile may be associated with other condition of the pulmonary cardiovascular system. Particularly, the invention recognizes that treating conditions such as PAH requires inhibiting activity of more than just PDGFR, and in fact requires inhibiting activity of a particular set of kinases.
Accordingly, compositions and methods of the invention target specific kinase combinations for inhibition to treat pulmonary and cardiovascular diseases such as PAH. By characterizing the binding affinity of imatinib and other PAH-effective compounds against a variety of kinases, new treatment targets and methods have been identified. Methods of the invention use the targeted inhibition of kinases such as platelet-derived growth factor receptors (PDGFRs) and discoidin domain receptor tyrosine kinase 1 (DDR1), preferably with a single compound, to treat PAH and other pulmonary and cardiovascular diseases.
Methods of the invention may include treating PAH or other pulmonary or cardiovascular diseases through the inhibition of one or more of PDGFR-β, PDGFR-α, DDR1, colony stimulating factor 1 receptor (CSF1R), tyrosine-protein kinase KIT (KIT), discoidin domain receptor tyrosine kinase 2 (DDR2), lymphocyte-specific protein tyrosine kinase (LCK), Abelson murine leukemia viral oncogene homolog 1 (ABL1), Abelson murine leukemia viral oncogene homolog 2 (ABL2), and phosphatidylinositol 5-phosphate 4-kinase type-2 gamma (PI42C). In certain embodiments, pulmonary or cardiovascular diseases such as PAH may be treated through inhibition of one or more of vascular endothelial growth factor receptor 2 (VEGFR-2), HCK proto-oncogene, Src family tyrosine kinase (HCK), fms related receptor tyrosine kinase 4 (FLT4), ret proto-oncogene (RET), SRC proto-oncogene, non-receptor tyrosine kinase (SRC), PDGFR-α, PDGFR-β, DDR1, KIT, CSF1R, fyn related Src family tyrosine kinase (FRK), DDR2, LCK, LYN proto-oncogene, Src family tyrosine kinase (LYN), FYN proto-oncogene, Src family tyrosine kinase (FYN), and FGR proto-oncogene, Src family tyrosine kinase (FGR).
By inhibiting combinations of the above kinases, through the administration of one or more compounds, methods of the invention provide effective treatments for various pulmonary and cardiovascular diseases such as PAH, pulmonary veno-occlusive disease (PVOD), idiopathic pulmonary fibrosis (IPF), and pulmonary capillary hemangiomatosis (PCH) as well as lung transplant rejection and pulmonary hypertension secondary to other diseases like heart failure with preserved ejection fraction (HFpEF) or schistosomiasis.
Any route of administration may be applicable and certain formulations and routes of administration are exemplified here. For example, in various embodiments, compounds are administered in inhalable form as dry powder or through a nebulizer. Inhalable formulations can offer greater lung exposure than equivalent doses administered through conventional oral routes or by IV. Accordingly, where a relatively high oral dose would be required to achieve a target lung exposure, the same exposure can be achieved with much lower concentrations of drug delivered by inhalation. By avoiding high systemic loads associated with other conventional administration routes, methods of the invention circumvent some of the adverse effects associated with those high systemic concentrations. For example, as discussed above, systemically administered imatinib, while proving promising in the treatment of PAH, suffered from unacceptable rates of adverse effects including subdural hematoma. In order to minimize the potential for similar risks based on the similar kinase-inhibition profiles, certain methods of the invention provide inhalable compounds.
Methods of the invention recognize that the inhibition of various combinations of specific kinases as discussed above are useful in treating certain cardiovascular and pulmonary ailments. Kinase-binding characterization has been performed for compounds A and B, as detailed below, and those compounds have been found to inhibit those various kinase combinations. Accordingly, in certain embodiments, inhalable formulations of compounds A and/or B may be administered to treat cardiovascular and pulmonary diseases according to the invention.
In certain embodiments, formulations of the invention may be provided with a higher ratio of API (active pharmaceutical ingredient) than found in conventional formulations. In certain embodiments, formulations comprising 50% or more kinase-inhibiting API are provided. Large volumes may be difficult or dangerous for patients to inhale. Therefore, minimizing the amount of non-API components in the formulation can improve patient comfort, safety, and compliance by reducing the overall amount of compound that is inhaled while still providing a therapeutically effective API concentration in target tissue.
Furthermore, aerodynamic properties important to inhalable drug uptake can more easily be managed when less of the formulation is required for carriers or other additives. By providing functional inhalable formulations with high concentrations of kinase inhibitors or salts thereof, compositions and methods of the invention can provide the load-reducing benefits discussed above while still delivering therapeutic results and avoiding the severe adverse events associated with other drug delivery routes.
In various embodiments, the kinase inhibitor(s) or salts thereof used in the high-API compositions and methods of the invention can consist of entirely or almost entirely a single crystal form (e.g., greater than 80%, 85%, 90%, 95%, 99% or 100% of a single crystal form), thereby allowing for controlled and predictable dosing and patient response. In certain embodiments, greater than 95% of the kinase inhibiting compound (e.g., compound A or B) or a salt thereof in the inhalable formulation may be present in a single crystal form.
In certain embodiments inhalable kinase-inhibiting compounds may be micronized through wet or dry milling (e.g., jet milling) to achieve the desired particle size for dry powder formulations for inhalation. Compounds or appropriate salts thereof may be micronized to particle sizes of about 0.5 μm to about 5 μm mass median aerodynamic diameter (MMAD) for desired deep lung penetration.
Inhaled products may be limited in terms of the mass of powder that can be administered and certain salts will contribute significantly to the molecular weight of inhaled formulations. Accordingly, in certain embodiments, the free base of the kinase-inhibiting compound may be preferred over any salts thereof for efficient delivery of the active moiety to lung tissue. If required, various excipients or carriers can be added to the kinase inhibitor(s) or salts thereof before or after micronization depending on application. For example, carriers, excipients, conditioners, and force control agents such as lactose (which when used as a carrier may be conditioned with various solvents to increase separation of imatinib during inhalation), magnesium stearate, leucine, isoleucine, dileucine, trileucine, lecithin, distearylphosphatidylcholine (DSPC) or other lipid-based carriers, or various hydrophilic polymers where they exhibit appropriate physico-chemical properties may be included. The skilled artisan will appreciate that excipients or carriers are optional and that many embodiments of the invention do not require excipients or carriers. In compounds including carriers or excipients, API:carrier ratios may be greater than 50:50, 75:25, or 90:10. Additional ratios are contemplated as discussed below.
In certain embodiments, methods of the invention may include administering kinase-inhibiting inhalable formulations that exclude all or most amorphous forms of the compounds. Because crystal form can be important to drug pharmacokinetics and dosing, as well as physicochemical stability and avoiding amorphous content can therefore be important to providing predictable and efficient therapy.
Treatment methods of the invention may include the administration of kinase-inhibiting compounds to treat a variety of pulmonary and cardiovascular diseases. Doses may vary depending on the characteristics of the compound used (e.g., its kinase-binding profile) and the disease being treated. In various embodiments, dose ranges can include between about 10 mg to about 100 mg per dose for inhalation on a twice to four times per day schedule. About 0.1 mg to about 80 mg of the active imatinib compound may then be deposited within the lungs after inhalation. The use of relatively high concentrations of API (e.g., 50% or greater) allows for the above doses to be achieved with less overall volume of inhalable compared to conventional formulations having 1%-3% API.
Methods of the invention may include administration of spray-dried kinase-inhibiting compounds or salts thereof for inhalation. While carriers such as lactose may be used after micronization to aid in delivery via inhalation, those carriers may generally comprise larger diameter particles and complication in the separation of the active imatinib compound may result in lower amounts of the inhaled compound reaching the lungs. Furthermore, the amount of active compound reaching the lungs may be less predictable using such carriers and methods, making dosing more complicated. Accordingly, spray-dried methods may be used wherein the active kinase-inhibiting compound(s) or salts thereof along with various excipients or other additives may be micronized to a desired particle size and suspended or solubilized for spray-drying and inhalation.
In certain embodiments, the micronized kinase-inhibiting compound(s) may be suspended in a feedstock for the purposes of spray-drying to avoid the creation of amorphous or polymorphic imatinib content that may occur if dissolved in a solution (e.g. in an appropriate organic solvent or within an acidified aqueous solution) upon spray-drying. By creating a stable suspension of micronized compound for spray-drying, once dried, the inhalable formulation can retain the desired crystal structure, particle size, and low levels of amorphous content obtained before the micronization process.
Stable suspensions for spray-drying may be obtained through manipulation of factors affecting the solubility of the active compound such as pH, ionic strength, and dispersing agents or surfactants. Excipients that may be used before micronization in the spray-drying methods described above include, for example, leucine, dileucine, trileucine, bulking agents such as trehalose or mannitol, lecithin, DSPC or other lipid-based carriers, citrate, or acetate.
Aspects of the invention include methods of treating pulmonary arterial hypertension (PAH) that may comprise providing to a subject a therapeutically effective amount of an inhalable formulation of a compound in order to inhibit activity of a plurality of kinases comprising one or more platelet-derived growth factor receptors (PDGFRs) and discoidin domain receptor tyrosine kinase 1 (DDR1). The one or more PDGFRs can include PDGFR-β. In certain embodiments, the one or more PDGFRs may include PDGFR-α.
The plurality of kinases can include colony stimulating factor 1 receptor (CSF1R). In some embodiments, the plurality of kinases may include tyrosine-protein kinase KIT (KIT). The plurality of kinases can include discoidin domain receptor tyrosine kinase 2 (DDR2). Methods of the invention may include inhibiting lymphocyte-specific protein tyrosine kinase (LCK).
In certain embodiments, the plurality of inhibited kinases can comprise at least Abelson murine leukemia viral oncogene homolog 1 (ABL1), Abelson murine leukemia viral oncogene homolog 2 (ABL2), colony stimulating factor 1 receptor (CSF1R), discoidin domain receptor tyrosine kinase 2 (DDR2), tyrosine-protein kinase KIT (KIT), lymphocyte-specific protein tyrosine kinase (LCK), and phosphatidylinositol 5-phosphate 4-kinase type-2 gamma (PI42C). Each of the plurality of kinases may be inhibited with a Kd of 500 nM or lower.
Certain aspects of the invention include methods of treating pulmonary arterial hypertension (PAH) that can include providing to a subject a therapeutically effective amount of an inhalable formulation of a compound in order to inhibit activity of two or more of vascular endothelial growth factor receptor 2 (VEGFR-2), HCK proto-oncogene, Src family tyrosine kinase (HCK), fms related receptor tyrosine kinase 4 (FLT4), ret proto-oncogene (RET), SRC proto-oncogene, non-receptor tyrosine kinase (SRC), platelet-derived growth factor receptor α (PDGFR-α), platelet-derived growth factor receptor β (PDGFR-β), discoidin domain receptor tyrosine kinase 1 (DDR1), tyrosine-protein kinase KIT (KIT), colony stimulating factor 1 receptor (CSF1R), fyn related Src family tyrosine kinase (FRK), discoidin domain receptor tyrosine kinase 2 (DDR2), lymphocyte-specific protein tyrosine kinase (LCK), LYN proto-oncogene, Src family tyrosine kinase (LYN), FYN proto-oncogene, Src family tyrosine kinase (FYN), and FGR proto-oncogene, Src family tyrosine kinase (FGR).
DETAILED DESCRIPTIONThe invention relates to the treatment of various pulmonary and cardiovascular diseases through the targeted inhibition of specific combinations of kinases. Diseases including pulmonary arterial hypertension (PAH), pulmonary veno-occlusive disease (PVOD), idiopathic pulmonary fibrosis (IPF), and pulmonary capillary hemangiomatosis (PCH) may be treated as well as lung transplant rejection and pulmonary hypertension secondary to other diseases like heart failure with preserved ejection fraction (HFpEF) or schistosomiasis using inhalable formulations of imatinib and salts thereof.
Through comprehensive profiling of the kinase-binding properties of various compounds thought to effectively treat cardiovascular and pulmonary diseases like PAH, specific combinations of kinases have been identified as targets for inhibition. By treating patients with inhalable compounds that inhibit those target kinases, methods of the invention can provide effective treatment of many such diseases.
In certain embodiments, methods of the invention can include targeted inhibition of combinations of Abelson murine leukemia viral oncogene homolog 1 (ABL1), colony stimulating factor 1 receptor (CSF1R), discoidin domain receptor tyrosine kinase 1 (DDR1), discoidin domain receptor tyrosine kinase 2 (DDR2), tyrosine-protein kinase KIT (KIT), lymphocyte-specific protein tyrosine kinase (LCK), platelet-derived growth factor receptor-α (PDGFR-α), and platelet-derived growth factor receptor-β (PDGFR-β). The above kinases were found to be inhibited by both compound A and compound B.
In certain embodiments, methods of the invention can include targeted inhibition of combinations of PDGFR-β, PDGFR-α, DDR1, CSF1R, KIT, DDR2, LCK, ABL1, Abelson murine leukemia viral oncogene homolog 2 (ABL2), and phosphatidylinositol 5-phosphate 4-kinase type-2 gamma (PI42C). The above kinases were found to be inhibited by compound A.
In certain embodiments, methods of the invention can include targeted inhibition of combinations of vascular endothelial growth factor receptor 2 (VEGFR-2), HCK proto-oncogene, Src family tyrosine kinase (HCK), fms related receptor tyrosine kinase 4 (FLT4), ret proto-oncogene (RET), SRC proto-oncogene, non-receptor tyrosine kinase (SRC), PDGFR-α, PDGFR-β, DDR1, KIT, CSF1R, fyn related Src family tyrosine kinase (FRK), DDR2, LCK, LYN proto-oncogene, Src family tyrosine kinase (LYN), FYN proto-oncogene, Src family tyrosine kinase (FYN), and FGR proto-oncogene, Src family tyrosine kinase (FGR). The above kinases were found to be inhibited by compound B
In certain embodiments, methods of the invention may include targeted inhibition of combinations of PDGFR-β, PDGFR-α, DDR1, CSF1R, KIT, DDR2, LCK, ABL1, ABL2, and PI42C while not significantly inhibiting (e.g. less than 500 nM Kd) one or more of VEGFR-2, HCK, FLT4, RET, SRC, FRK, LYN, FYN, and FGR.
In certain embodiments, methods of the invention may include targeted inhibition of combinations of VEGFR-2, HCK, FLT4, RET, SRC, PDGFR-α, PDGFR-β, DDR1, KIT, CSF1R, FRK, DDR2, LCK, LYN, FYN, and FGR while not significantly inhibiting (e.g. less than 500 nM Kd) one or more of ABL2, and PI42C.
In various embodiments, methods of the invention may include targeted inhibition of any combination of the above kinases via administration of a compound having an equilibrium dissociation constant (Kd) of less than about 1000 nM, 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 15 nM, or less than about 5 nM with respect to each of the targeted kinases.
Compound A, as characterized below, is free base imatinib having the following structure:
The kinase-binding profile including the equilibrium dissociation constant (Kd) was determined for compound A as detailed in Example 1 below. The results of that characterization are shown in Table 1.
Compound B, as characterized below, has the following structure:
Compound B and some of its potential applications in treating pulmonary and cardiovascular disorders are discussed in U.S. Pat. Nos. 9,815,815; 9,925,184; 10,231,966; and 10,246,438; the content of each of which is incorporated herein by reference.
The kinase-binding profile including the equilibrium dissociation constant (Kd) was determined for compound B as detailed in Example 3 below. The results of that characterization are shown in Table 2.
As shown in the tables above, both compounds significantly bind and therefore inhibit a number of the same kinases while each further inhibits additional kinases not significantly affected by the other compound. By analyzing those combined profiles, in certain embodiments, methods of the invention are able to identify target kinases for inhibition that are common to both compounds and that, therefore may be more likely to provide a therapeutic effect in treating cardiovascular or pulmonary diseases.
Furthermore, the differences in kinase-inhibition between the two compounds may account for differences in compound efficacy in treating certain pulmonary and cardiovascular diseases and account for various adverse effects. Accordingly, in certain embodiments, methods of the invention may target only kinases inhibited by one or the other of the compounds to improve treatment outcomes and reduce the risk of adverse effects.
In certain embodiments methods and compositions described herein may provide greater concentrations of a kinase-inhibiting compound in target lung tissue than obtained with equivalent doses administered orally or through IV. Furthermore, those doses, comprising a high percentage of the overall formulation, can be delivered in lower volume formulations than conventional formulations of between 1% and 3% API. Reducing the volume a patient must inhale can increase patient comfort and compliance, thereby improving results. Additionally, a higher percentage of API content can improve the API distribution and blend uniformity. Accordingly, methods and compositions of the invention allow for treatment of conditions of the pulmonary cardiovascular system (e.g., PAH) with lower doses and less inhalable volume than would be required in systemic administration, thereby lowering the risk of adverse events including subdural hematoma (See, Frost et al.). Thus, the invention provides viable treatment methods for life threatening diseases that were heretofore too risky for practical application.
In certain embodiments, compounds of the invention may include formulations of a kinase inhibitor such as imatinib or salts thereof targeting combinations of kinases as discussed above. In certain embodiments, the free base of the kinase-inhibiting compound may be used in a formulation (either in dry powder or suspension) for inhalation to treat a condition of the pulmonary cardiovascular system such as PAH. Certain salt forms are also contemplated. In various embodiments, kinase inhibitor salts that were found to exhibit suitable thermal stability and few or single polymorphic forms include glycollate, isethionate, malonate, tartrate, and malate. Other salt forms contemplated herein are xinafoate, furoate, trifenatate, HCl, sulfate, phosphate, lactate, maleate, fumarate, succinate, adipate, mesylate, and citrate.
When the compounds of the present invention are administered as pharmaceuticals, to humans and mammals, they can be given alone or as a pharmaceutical composition containing, for example, 0.1 to 99.5% of active ingredient (e.g., imatinib or a salt thereof) in combination with a pharmaceutically acceptable carrier. In preferred embodiments, to reduce inhaled volumes for patients and improve patient outcomes, formulations can comprise at least 50% of a kinase-inhibiting compound or a salt thereof.
In certain embodiments, formulations of the invention may include one or more excipients. Excipients may include, for example, lactose in various forms (e.g., roller dried or spray dried). Larger lactose particles can be used as a carrier for inhalation of micronized formulations. The carrier particles, with their larger size, can be used to increase aerodynamic forces on the combined kinase inhibitor/carrier in order to aid in delivery through inhalation. Solvents may be used to condition the lactose surface such that the active component can be effectively separated from the lactose as it leaves the inhaler device and within the oral cavity when being used as a carrier. Magnesium stearate can be used as a force-control agent or conditioning agent in various embodiments. In some embodiments, leucine can be used as a force-control agent including different forms of leucine (e.g. isoleucine) along with dileucine and even trileucine.
Lecithin phospholipids such as DSPC may be used as an excipient for dry powder inhalation. In certain embodiments, excipients may include various hydrophilic polymers. See, for example, Karolewicz, B., 2016, A review of polymers as multifunctional excipients in drug dosage form technology, Saudi Pharm J., 24(5):525-536, incorporated herein by reference.
In the high-API-ratio formulations contemplated herein, carriers or excipients may make up the remainder of the formulation in amounts of 50% or less of the overall composition. In certain embodiments, inhalable formulations may have API:carrier ratios of 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, or 95:5. Certain inhalable formulations may be pure API with no additional components. In various embodiments, formulations may include a kinase inhibitor or salts thereof as the API in amounts greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%. As used herein, API ratios refer to % w/w.
In various embodiments, micronized kinase inhibitor and salts thereof retain crystallinity, even after micronization and spray drying (as discussed in detail below). For example, kinase inhibiting formulations of the invention can include less than 50%, less than 25%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% amorphous API content by mass. In preferred embodiments, formulations of the invention include no observable amorphous content of the kinase-inhibiting compound. Of particular note is, by suspending micronized kinase inhibitor particles in a solution as opposed to solubilizing, the desired crystalline form and low amorphous content obtained during micronization is carried through to the spray-dried inhalable powder because the kinase inhibitor crystals may not be dissolved in the solution to a significant degree.
Another unexpected result obtained with methods and formulations of the invention is that kinase inhibitor formulations of the invention may be significantly less hygroscopic than conventional salt compounds such as imatinib mesylate. Accordingly, the formulations of the invention are better suited for dry powder inhalation and can comprise less than 5% water content, less than 4%, less than 3%, less than 2%, or, in preferred embodiments, less than 1% water content.
As discussed above, in order to accurately and consistently model pharmacokinetics of formulations for proper dosing, low polymorphism is desired. To that end, inhalable formulations of the invention include kinase inhibitors or salts thereof present in a single crystal form. In various embodiments, the kinase inhibitor or a salt thereof may be present at greater than 75%, 80%, 85%, 90%, 95%, or, in preferred embodiments, greater than 99% in a single crystal form by mass. The single crystal form may be, for example, imatinib in type A or type B in various embodiments. Crystalline purity can be estimated using any known method including, for example, x-ray powder diffraction (XRPD).
In various embodiments, kinase inhibitors or salts thereof are provided in dry powder formulations for inhalation. Dry powder can be administered via, for example, dry powder inhalers such as described in Berkenfeld, et al., 2015, Devices for Dry Powder Drug Delivery to the Lung, AAPS PharmaSciTech, 16(3):479-490, incorporated herein by reference. Dry powder compounds may be divided into single doses for single, twice daily, three times daily, or four times daily inhalation to treat disorders such as PAH or other conditions of the pulmonary cardiovascular system. The single doses may be divided into individual capsules or other formats compatible with the dry powder inhaler to be used.
In other embodiments, suspensions having the characteristics described herein (e.g., low polymorphism and amorphous content) can be delivered via inhalation using, for example, a nebulizer. Suspensions may offer advantages over solutions as discussed below. For nebulized suspensions, micronization and particle diameter may be of particular importance for efficient delivery and the active kinase-inhibiting compound may be preferably micronized to a mass median diameter of 2 μm or less. The suspension solution for nebulizer inhalation can be aqueous and doses may be divided into individual containers or compartments for sterile storage prior to use.
Micronized kinase inhibitor particle size can range from about 0.5 μm to about 5 μm depending on application (e.g., dry powder or suspension for inhalation). In preferred embodiments the size range is about 1 μm to about 3 μm in dry powder formulations to achieve deep lung penetration.
Dosages for treating PAH and other conditions of the pulmonary cardiovascular system may be in the range of between about 10 mg to about 100 mg per dose for inhalation on once, twice or three times per day schedule. About 0.1 mg to about 80 mg of the kinase inhibitor(s) or salts thereof may then be deposited within the lung after inhalation. In certain embodiments about 10 mg to 30 mg of kinase-inhibiting compound may be given in a capsule for a single dry-powder inhalation dose with about 5 mg to about 10 mg of the compound to be expected to reach the lungs. In inhalable suspension embodiments, kinase inhibitor may be present at about 0.1 to about 1 mg/kg in a dose and may be administered one to four times a day to obtain the desired therapeutic results.
In certain embodiments, the kinase-inhibiting methods of the invention may be used to treat pulmonary hypertension as a result of schistosomiasis. See, for example, Li, et al., 2019, The ABL kinase inhibitor imatinib causes phenotypic changes and lethality in adult Schistosoma japonicum, Parasitol Res., 118(3):881-890; Graham, et al., 2010, Schistosomiasis-associated pulmonary hypertension: pulmonary vascular disease: the global perspective, Chest, 137(6 Suppl):20S-29S, the content of each of which is incorporated herein by reference.
Methods and compositions of the invention may be used to treat lung transplant recipients to prevent organ rejection. See, Keil, et al., 2019, Synergism of imatinib, vatalanib and everolimus in the prevention of chronic lung allograft rejection after lung transplantation (LTx) in rats, Histol Histopathol, 1:18088, incorporated herein by reference.
In certain embodiments, pharmaceutical compositions described herein can be used to treat pulmonary veno-occlusive disease (PVOD). See Sato, et al., 2019, Beneficial Effects of Imatinib in a Patient with Suspected Pulmonary Veno-Occlusive Disease, Tohoku J Exp Med. 2019 February; 247(2):69-73, incorporated herein by reference.
For treatment of any conditions of the pulmonary cardiovascular system for which kinase-inhibiting methods of the invention may produce a therapeutic effect, compounds and methods of the invention may be used to provide greater concentration at the target lung tissue through inhalation along with consistent, predictable pharmacokinetics afforded by low polymorphism and amorphous content. The efficient localization of therapeutic compound at the target tissue allows for lower systemic exposure and avoidance of the adverse events associated with prolonged oral administration of certain kinase inhibitors such as imatinib mesylate.
Methods of the invention can include preparation of kinase-inhibiting formulations. As noted above, kinase inhibitors or salts thereof may be administered via inhalation in suspension or dry powder form. Dry powder formulations may be obtained via any known method including, in preferred embodiments, jet milling. Jet milling can be used to grind active compounds and, potentially, various additives (e.g., excipients) using a jet (or jets) of compressed air or gas to force collisions between the particles as they transit at near sonic velocity around the perimeter of a toroidal chamber. The size reduction is the result of the high-velocity collisions between particles of the process material. Outputs of the jet mill may allow particles to exit the apparatus once a desired size has been reached. As noted herein, desired particle size for dry powder inhalation and other formulations may be in the range of about 0.5 μm to about 5 μm.
In certain embodiments, bulk compounds may be micronized to the desired size for inhalation via wet milling wherein the kinase inhibitor particles are suspended in a slurry and reduced through shearing or impact with a grinding media.
An unexpected finding of the invention is that, once micronized, free base kinase-inhibiting compounds retain crystallinity and are considerably less hygroscopic than certain salt forms (e.g., imatinib mesylate). Furthermore, micronized imatinib obtained using methods of the invention has been found to exhibit no apparent polymorphs other than the designated Type A and very low levels of amorphous content. Accordingly, this can result in improved stability of the drug substance and any drug product upon storage. Single crystal forms of imatinib or other kinase inhibitors such as described may allow for more predictable in vivo behavior and appropriate dosing can be determined.
Once micronized, in dry powder form, kinase inhibitor formulations of the invention, with their low polymorphic and amorphous content, can be prepared for inhalation. In certain embodiments, the dry powder kinase inhibitor can be combined with larger carrier particles such as lactose as discussed above.
In some embodiments a suspension can be formed of the kinase inhibitor compound(s). The suspension may result from dry micronization followed by suspension of the resulting dry powder or can be obtained as the outcome of a wet milling procedure. Suspensions of micronized crystal forms may be used in nebulized inhalation treatment or may be spray dried for dry powder treatments.
Spray drying methods may follow the following procedure. First, bulk kinase inhibitor compound may be micronized as described above to obtain particles in a desired size range. Then the micronized compound can be suspended in a solution such that it does not dissolve and instead retains the desired crystalline features (e.g., low polymorphism and amorphous content). The suspended particles can then be spray dried using any known method. Spray drying techniques are well characterized and described, for example, in Ziaee, et al., 2019, Spray drying of pharmaceuticals and biopharmaceuticals: Critical parameters and experimental process optimization approaches, Eur. J. Pharm. Sci., 127:300-318, and Weers et al., 2019, AAPS PharmSciTech. 2019 Feb. 7; 20(3):103. doi: 10.1208/s12249-018-1280-0, and 2018/0303753, each of which is incorporated herein by reference. Spray drying micronized kinase inhibitor compounds or salts thereof provides for uniform and predictable crystallinity and particle size and can avoid the need for large carrier molecules that may adversely affect the amount of inhaled drug that reaches the target lung tissue.
In spray-dried embodiments, micronized drug particles may be suspended within a non-aqueous solvent or within an emulsion of a non-aqueous solvent which, in turn is emulsified or dispersed within an aqueous environment (e.g. oil in water) and spray-dried, resulting in crystalline drug particles. The non-aqueous component may or may not be fugitive and thus could be removed completely during spray drying or, it could be retained, depending on the desired properties required. In such embodiments, each atomized droplet (mass median diameter ˜10 μm) contains dispersed drug crystals. During the initial moments of the drying process, the more volatile aqueous phase begins to evaporate. The rapidly receding atomized droplet interface drives enrichment of the slowly diffusing drug and emulsion particles at the interface. This leads to formation of a void space in the center of the drying droplet. As the drying process continues, the less volatile oil phase in the emulsion droplets evaporates, resulting in formation of hollow pores in their place. Overall, the resulting hollow spray-dried composite particles contain drug crystals.
As maintaining a stable solution of crystalline kinase inhibitor compound is important to many features of the formulations and methods of the invention, formulation methods include manipulation of the suspension to prevent dissolution of the kinase inhibitor compound. Aqueous solution factors such as pH, ionic strength and dispersing agents may be used to obtain a stable suspension for nebulized inhalation or spray drying. For example, the pH of the aqueous solution may be adjusted to prevent dissolution.
Additionally, the presence of ions in aqueous solution may tend to ‘salt out’ the active compound. The solubility of the kinase inhibitors and any salts thereof may decrease with salinity. Accordingly, salt in the aqueous solution may be used to reduce solubility of the active compound crystals in certain embodiments.
To promote dispersion and thoroughly deagglomerate the API particles, a dispersing agent or surfactant (e.g., Tween 20 or Tween 80) may be added but should not cause dissolution of the kinase inhibitor in suspension.
In certain embodiments, excipients can be added to the suspension before spray drying. In various embodiments, the excipient may be a water-soluble excipient, such as leucine, dileucine, trileucine, trehalose, mannitol, citrate or acetate. In other embodiment, the excipient may be a water insoluble excipient, such as lecithin, distearylphosphatidylcholine (DSPC) or limonene. Such insoluble excipients may be dissolved in a non-aqueous medium that is miscible or immiscible with water, thereby creating an emulsion. Alternatively, a liposomal dispersion could be created into which the suspended kinase inhibitor could be added and homogenized or where it could be spray dried in separate feedstocks.
The effective dosage of each agent can readily be determined by the skilled person, having regard to typical factors such as the age, weight, sex and clinical history of the patient. In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce the desired therapeutic effect. Such an effective dose will generally depend upon the factors described above.
If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
The pharmaceutical compositions of the invention include a “therapeutically effective amount” or a “prophylactically effective amount” of one or more of the compounds of the present invention, or functional derivatives thereof. An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., a diminishment or prevention of effects associated with PAH. A therapeutically effective amount of a compound of the present invention or functional derivatives thereof may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the therapeutic compound to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to, or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount. A prophylactically or therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the beneficial effects.
Dosage regimens may be adjusted to provide the optimum desired response (e.g. a therapeutic or prophylactic response). For example, a single inhalable bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigency of the therapeutic situation. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the patient.
The term “dosage unit” as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the compound, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
In some embodiments, therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually rats, non-human primates, mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in other subjects. Generally, the therapeutically effective amount is sufficient to reduce PAH symptoms in a subject. In some embodiments, the therapeutically effective amount is sufficient to eliminate PAH symptoms in a subject.
Dosages for a particular patient can be determined by one of ordinary skill in the art using conventional considerations, (e.g. by means of an appropriate, conventional pharmacological protocol). A physician may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. The dose administered to a patient is sufficient to effect a beneficial therapeutic response in the patient over time, or, e.g., to reduce symptoms, or other appropriate activity, depending on the application. The dose is determined by the efficacy of the particular formulation, and the activity, stability, or half-life of the compounds of the invention or functional derivatives thereof, and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose is also determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, formulation, or the like in a particular subject. Therapeutic compositions comprising one or more compounds of the invention or functional derivatives thereof are optionally tested in one or more appropriate in vitro and/or in vivo animal models of disease, such as models of PAH, to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art. In particular, dosages can be initially determined by activity, stability or other suitable measures of treatment vs. non-treatment (e.g., comparison of treated vs. untreated cells or animal models), in a relevant assay. Administration can be accomplished via single or divided doses.
In certain embodiments, in which an aqueous suspension is part of the manufacturing process, the aqueous suspension may contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents dispersing or wetting agents such as a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such a polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, mannitol, or trehalose.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
The term “pharmaceutical composition” means a composition comprising a compound as described herein and at least one component comprising pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, taste-masking agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms.
The term “pharmaceutically acceptable carrier” is used to mean any carrier, diluent, adjuvant, excipient, or vehicle, as described herein. Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Examples of suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and organic esters such as ethyl oleate. Examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate. Examples of disintegrating agents include starch, alginic acids, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols.
The term “pharmaceutically acceptable” means it is, within the scope of sound medical judgment, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
INCORPORATION BY REFERENCEReferences and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.
EquivalentsVarious modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.
EXAMPLES Example 1An inhalable form of imatinib (compound A) was screened against 468 quantifiable kinases in the human kinome (the set of protein kinases encoded in the human genome) to characterize binding affinity for each kinase.
Kinase binding was profiled using KINOMEscan® (Eurofins DiscoverX Corporation). KINOMEscan™ is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand. The assay is performed by combining three components: DNA-tagged kinase; immobilized ligand; and a test compound. The ability of the test compound to compete with the immobilized ligand is measured via quantitative PCR of the DNA tag.
Kinase-tagged T7 phage strains were prepared in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32° C. until lysis. The lysates were centrifuged and filtered to remove cell debris. Some kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection.
Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The ligand-bound beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce nonspecific binding. Binding reactions were assembled by combining kinases, ligand-bound affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements were distributed by acoustic transfer (non-contact dispensing) in 100% DMSO.
The compounds were then diluted directly into the assays such that the final concentration of DMSO was 0.9%. All reactions were performed in polypropylene 384-well plates. Each was a final volume of 0.02 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.
Example 2Compound B, as detailed above, was screened against 468 quantifiable kinases in the human kinome (the set of protein kinases encoded in the human genome) to characterize binding affinity for each kinase.
Kinase binding was profiled using KINOMEscan® (Eurofins DiscoverX Corporation). KINOMEscan™ is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand. The assay is performed by combining three components: DNA-tagged kinase; immobilized ligand; and a test compound. The ability of the test compound to compete with the immobilized ligand is measured via quantitative PCR of the DNA tag.
Kinase-tagged T7 phage strains were prepared in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32° C. until lysis. The lysates were centrifuged and filtered to remove cell debris. Some kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection.
Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The ligand-bound beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce nonspecific binding. Binding reactions were assembled by combining kinases, ligand-bound affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements were distributed by acoustic transfer (non-contact dispensing) in 100% DMSO.
The compounds were then diluted directly into the assays such that the final concentration of DMSO was 0.9%. All reactions were performed in polypropylene 384-well plates. Each was a final volume of 0.02 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.
Claims
1. A method of treating pulmonary arterial hypertension (PAH), the method comprising providing to a subject a therapeutically effective amount of an inhalable formulation of a compound in order to inhibit activity of a plurality of kinases comprising one or more platelet-derived growth factor receptors (PDGFRs) and discoidin domain receptor tyrosine kinase 1 (DDR1).
2. The method of claim 1 wherein the one or more PDGFRs comprises PDGFR-β.
3. The method of claim 2 wherein the one or more PDGFRs comprises PDGFR-α.
4. The method of claim 3 wherein the plurality of kinases comprises colony stimulating factor 1 receptor (CSF1R).
5. The method of claim 4 wherein the plurality of kinases comprises tyrosine-protein kinase KIT (KIT).
6. The method of claim 5 wherein the plurality of kinases comprises discoidin domain receptor tyrosine kinase 2 (DDR2).
7. The method of claim 6 wherein the plurality of kinases comprises lymphocyte-specific protein tyrosine kinase (LCK).
8. The method of claim 1 wherein the plurality of kinases comprise Abelson murine leukemia viral oncogene homolog 1 (ABL1), Abelson murine leukemia viral oncogene homolog 2 (ABL2), colony stimulating factor 1 receptor (CSF1R), discoidin domain receptor tyrosine kinase 2 (DDR2), tyrosine-protein kinase KIT (KIT), lymphocyte-specific protein tyrosine kinase (LCK), and phosphatidylinositol 5-phosphate 4-kinase type-2 gamma (PI42C).
9. The method of claim 8 wherein each of the plurality of kinases are inhibited with a Kd of 500 nM or lower.
10. A method of treating pulmonary arterial hypertension (PAH), the method comprising providing to a subject a therapeutically effective amount of an inhalable formulation of a compound in order to inhibit activity of a plurality of kinases comprising vascular endothelial growth factor receptor 2 (VEGFR-2), HCK proto-oncogene, Src family tyrosine kinase (HCK), fms related receptor tyrosine kinase 4 (FLT4), ret proto-oncogene (RET), SRC proto-oncogene, non-receptor tyrosine kinase (SRC), platelet-derived growth factor receptor α (PDGFR-α), platelet-derived growth factor receptor β (PDGFR-β), discoidin domain receptor tyrosine kinase 1 (DDR1), tyrosine-protein kinase KIT (KIT), colony stimulating factor 1 receptor (CSF1R), fyn related Src family tyrosine kinase (FRK), discoidin domain receptor tyrosine kinase 2 (DDR2), lymphocyte-specific protein tyrosine kinase (LCK), LYN proto-oncogene, Src family tyrosine kinase (LYN), FYN proto-oncogene, Src family tyrosine kinase (FYN), and FGR proto-oncogene, Src family tyrosine kinase (FGR).
Type: Application
Filed: May 14, 2020
Publication Date: Nov 19, 2020
Inventor: Ben Dake (Boston, MA)
Application Number: 16/874,190
