Patent Application
20220031665
The present invention relates to the Nano-formulation of compounds that modulate the physiological action of the proprotein convertase subtilisin kexin type 9 (PCSK9), including its interaction with the low-density lipoprotein receptor (LDLR). In certain embodiments, the invention relates to compositions and associated methods for hepatic-targeted delivery of PCSK9 inhibitors or antagonists such as SBC-115,418 or its analogues (see, e.g., WO 2017/222953) to the liver of a subject. In certain embodiments, compositions are provided which include hydrophobic nanoparticles, a liver-targeting moiety attached or linked (e.g., covalently) to the exterior of each nanoparticle (see, e.g., U.S. Pat. No. 9,682,085), and at least one PCSK9 inhibitor or antagonist (e.g., SBC-115,418) encapsulated within each nanoparticle. The nanoparticles may include chitosan hybrid nanoparticles, amine modified poly-(lactic-co-glycolic acid) (PLGA) nanoparticles, solid lipid nanoparticles (SLNs), and/or combinations thereof. Examples of liver-targeting moieties include Glycyrrhetinic acid (GA), Lactobionic acid (LA), Alginic acid, and/or combinations thereof. The small molecule modulators of PCSK9 function can be used therapeutically to lower LDL-cholesterol levels in blood and can be used in the prevention and/or treatment of cholesterol, lipid, and lipoprotein metabolism disorders, including hypercholesterolemia, familial hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis, and more generally, cardiovascular disease (CVD), diabetes, and for obese subjects with high cardiovascular risk.
Cardiovascular diseases (CVDs) are the leading cause of death, with atherosclerosis being the leading cause of cardiovascular diseases. Atherosclerosis is a disease of the arteries and is responsible for coronary heart disease associated with many deaths in industrialized countries. Several risk factors for coronary heart disease have now been identified including, without limitation: dyslipidemia, hypertension, diabetes, smoking, poor diet, inactivity and stress. Dyslipidemia is elevation of plasma cholesterol (hypercholesterolemia) and/or triglycerides (TGs) or a low high-density lipoprotein (HDL) level that contributes to the development of atherosclerosis, which is a metabolic disorder proven to contribute to cardiovascular disease. In the blood, cholesterol is transported in lipoprotein particles, where the low-density lipoprotein (LDL) cholesterol (LDL-C) is considered “bad” cholesterol, while HDL-cholesterol (HDL-C) is known as “good” cholesterol. Lipid and lipoprotein abnormalities are extremely common in the general population and are regarded as a highly modifiable risk factor for cardiovascular disease, due to the influence of cholesterol on atherosclerosis. There is a significant unmet need with respect to CVD with 60-70% of cardiovascular events, heart attacks and strokes occurring despite the treatment with statins (the current standard of care in atherosclerosis). Moreover, new guidelines suggest that even lower LDL levels should be achieved in order to protect high-risk patients from premature CVD (1).
The establishment of a link between PCSK9 and cholesterol metabolism was rapidly followed by the discovery that selected mutations in the PCSK9 gene caused autosomal dominant hypercholesterolemia (2), suggesting that the mutations confer a gain-of-function (3) by increasing the normal activity of PCSK9. This was supported by the experiment in which wild type and mutant PCSK9 (S127R and F216L) were expressed at high levels in the livers of mice and it was found that hepatic LDLR protein levels fell dramatically in mice receiving either the wild type or mutant PCSK9 (4, 5). No associated reductions in LDLR mRNA levels were observed, indicating that overexpression of PCSK9, whether mutant or wild type reduces LDLRs through a post-transcriptional mechanism.
Given that gain-of-function mutations in PCSK9 cause hypercholesterolemia, it was reasonable to ask if loss-of-function mutations would have the opposite effect and result in hypocholesterolemia. Three loss-of-function mutations in PCSK9 (Y142X, L253F, and C679X) were identified in African-Americans (6). These mutations reduce LDL-C levels by 28% and were shown to decrease the frequency of coronary heart disease (defined as myocardial infarction, coronary death or coronary revascularization) by 88%. Rashid et al. (7) studied the mechanism of loss-of-function mutations in mice where PCSK9 was inactivated. They reported that these knockout mice showed increased hepatic LDLR protein (but not mRNA), increased clearance of circulating lipoproteins and reduced plasma cholesterol levels. Structure-function relationship analysis of the naturally occurring mutations in PCSK9 has also provided insights into the mechanism of action of PCSK9. Interestingly, mutations in PCSK9 that were found to be associated with the greatest reductions in LDL-C plasma levels are those that prevent the secretion of mature PCSK9 by disrupting its synthesis (Y142X), autocatalytic processing (L253F), or folding (C679X) (8). The Y142X mutation produces no detectable protein because it occurs early in the transcript and is predicted to initiate nonsense-mediated mRNA decay. Mutations in the catalytic domain (L253F) interfere with the autocatalytic cleavage of the protein. In cells expressing the PCSK9-253F, the amount of mature protein was reduced compared to that in cells expressing PCSK9-WT, suggesting that the mutation inhibits autocatalytic cleavage. The L253F mutation is near the catalytic triad (PCSK9 is a serine protease), therefore it might disrupt the active site (8). Inasmuch as autocatalytic cleavage of PCSK9 is required for export of the protein out of the endoplasmic reticulum (ER), the L253F mutation delays transport of PCSK9 from the ER to the cell surface. The nonsense mutation (C679X) in PCSK9, which truncates the protein by 14 amino acids, did not interfere with protein processing, but the mature protein accumulates in the cells and none is secreted, suggesting that the protein is cleaved normally but is misfolded and is retained in the ER (8, 9).
The mechanism by which PCSK9 causes the degradation of the LDLR has not been fully elucidated. However, it is clear that the protease activity of PCSK9 is not required for LDLR degradation (10, 11). Li et al. (10) have co-expressed the pro-domain and the catalytic domain and showed that the secreted PCSK9 was catalytically inactive, yet it is functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDLR levels. McNutt et al. (11) also reported similar studies. Furthermore, Zhang et al. (12) has mapped PCSK9 binding to the EGF-A repeat of the LDLR and showed that such binding decreases the receptor recycling and increases its degradation. They also reported that binding to EGF-A domain was calcium-dependent and increased dramatically with reduction in pH from 7 to 5.2. Kwon et al. (13) determined the crystal structure of PCSK9 in complex with the LDLR-EGF-AB (EGF-A and EGF-B). The structure shows a well-defined EGF-A domain, but the EGF-B domain is disordered and absent from their electron density map. The EGF-A domain binds to the PCSK9 catalytic domain at a site distant from the catalytic site and makes no contact with either the C-terminal domain or the pro-domain (14).
Several strategies have been proposed for targeting PCSK9 (15). Strategy 1: mRNA knockdown approaches including the use of antisense oligonucleotides or RNAi. Antisense oligonucleotides administered to mice reduced PCSK9 expression by >90% and lowered plasma cholesterol levels by 53% (16). A single intravenous injection of an RNAi delivered in lipidoid nanoparticles to cynomolgous monkeys reduced plasma PCSK9 levels by 70% and plasma LDL-C levels by 56% (17). Strategy 2: the development of small-molecule inhibitors of PCSK9 processing. Despite evidence that the catalytic activity of PCSK9 is not required for LDLR degradation (11), an intracellular inhibitor of PCSK9 catalytic activity should be effective, since autocatalytic processing of PCSK9 is required for secretion of the protein from the ER. Following its synthesis, PCSK9 undergoes an autocatalytic cleavage reaction that clips off the pro-domain, but the pro-domain remains attached to the catalytic domain (18, 19). The autocatalytic processing step is required for the secretion of PCSK9 (20), likely because the pro-domain serves as a chaperone and facilitates folding. The continued attachment of the pro-domain partially blocks the substrate-binding pocket of PCSK9 (18, 19). Strategy 3: is to prevent binding of PCSK9 to the LDLR on the cell surface with a small molecule, a peptide, or an antibody directed against PCSK9. McNutt et al. (21) demonstrated that antagonism of secreted PCSK9 increases LDLR expression in HepG2 cells. They show that an FH-associated LDLR allele (H306Y) that results in a gain-of-function mutation is due to an increase in the affinity of PCSK9 to the LDLR, which would lead to enhanced LDLR destruction, and decreased plasma LDL-C clearance. Furthermore, they were able to show that blocking the secreted PCSK9 with LDLR (H306Y) sub-fragment resulted in an increase in the level of LDLR in cultured HepG2 cells. Therefore, PCSK9 acts as a secreted factor to cause LDLR degradation, and a small molecule inhibitor that interferes with the binding of PCSK9 to the LDLR will diminish LDLR destruction and increase plasma LDL-C clearance.
Currently (22-24), there are FDA approved injectable PCSK9 monoclonal antibody antagonists on the market. These are Regeneron/Sanofi's PRALUENT (alirocumab) and Amgen's REPATHA (evolocumab), both of which are fully human anti-PCSK9 monoclonal antibodies. These monoclonal antibody approaches follow Strategy 3 using injectable antibodies instead of oral small molecules.
This invention relates to therapeutic applications of Nanoformulated small molecules, optionally hepatic-targeted, that selectively interact with and down modulate PCSK9 function. In a first embodiment, the compounds used in the practice of this invention have the general Formula I:
including pharmaceutically acceptable salts and stereoisomers of the compounds, wherein R1 is independently selected from the group consisting of H and CH3; R2 and R3 are independently selected from the group consisting of H, halogen, (C1-C3)-alkyl and (C1-C3)-alkoxy; and R4 is independently selected from the group consisting of CO2R5, CONR5R6, aryl and heteroaryl, wherein R5 and R6 are independently selected from the group consisting of H and (C1-C3)-alkyl. In a particular embodiment, R4 is an aryl or heteroaryl. In a particular embodiment, R4 is selected from the group consisting of 2-oxazole, 2-oxazoline, 2-benzoxazole and 2-benzimidazole.
In a particular embodiment, the present invention provides for compounds of Formula II:
including pharmaceutically acceptable salts and stereoisomers of the compounds, wherein R1 is independently selected from the group consisting of H and CH3; R2 is H or methoxy; R3 is H or halogen; and R7 is independently selected from the group consisting of H and (C1-C2)-alkyl or taken together form an optionally substituted 6-membered carbo-cycle, including aryl. In a particular embodiment, when R1 is H then R2 is H. In a particular embodiment, when R1 is methyl then R2 is methoxy. In a particular embodiment, R3 is fluorine (e.g., 2-F or 3-F7). In a particular embodiment, the oxazole is replaced with imidazole (i.e., the oxygen is replaced with a nitrogen).
The present invention further provides a composition for hepatic-targeted delivery of a PCSK9 antagonist to a liver of a subject. In a particular embodiment, the composition comprises nanoparticles (e.g., hydrophobic nanoparticles), at least one liver targeting moiety attached to the exterior of each nanoparticle (e.g., covalently bonded), and at least one PCSK9 antagonist (e.g., a compound of Formula I or II) encapsulated within each nanoparticle. In a particular embodiment, the hydrophobic nanoparticles are selected from the group consisting of chitosan hybrid nanoparticles, amine-modified poly-(lactic-co-glycolic acid) (PLGA) nanoparticles, solid lipid nanoparticles, and/or combinations thereof. In a particular embodiment, the liver targeting moiety is selected from the group consisting of Glycyrrhetinic acid (GA; enoxolone), Lactobionic acid (LA), Alginic acid, and/or combinations thereof.
The present invention further provides methods for targeted delivery of a PCSK9 antagonist (e.g., SBC-115,418 and analogs thereof, compounds of Formula I or II) to the liver of a subject. In a particular embodiment, the method comprises administering a composition to the subject, wherein the composition comprises nanoparticles (e.g., hydrophobic nanoparticles), at least one liver targeting moiety attached to the exterior to each nanoparticle (e.g., covalently bonded), and at least one PCSK9 antagonist (e.g., a compound of Formula I or II) encapsulated within each nanoparticle. The methods can be to treat, inhibit, and/or prevent hypercholesterolemia (e.g., familial hypercholesterolemia), dyslipidemia (e.g., atherogenic dyslipidemia), atherosclerosis, and/or cardiovascular disease (CVD) in a subject in need thereof.
In a particular embodiment, the hydrophobic nanoparticles are as described in U.S. Pat. No. 9,956,291 (incorporated herein by reference). In a particular embodiment, the hydrophobic nanoparticles are positively charged. In a particular embodiment, the hydrophobic nanoparticles have a diameter less than 1 μm, particularly 1 nm to about 500 nm, particularly 50 nm to about 300 nm. In a particular embodiment, the hydrophobic nanoparticles are chitosan hybrid nanoparticles, amine-modified poly-(lactic-co-glycolic acid) (PLGA) nanoparticles, solid lipid nanoparticles (SLNs), polyvinyl pyrrolidone (PVP) nanoparticles, hydroxypropyl methylcellulose acetate succinate (HPMC-AS) nanoparticles, or combinations thereof. In a particular embodiment, the hydrophobic nanoparticles are selected from the group consisting of chitosan hybrid nanoparticles, amine-modified poly-(lactic-co-glycolic acid) (PLGA) nanoparticles, solid lipid nanoparticles, and/or combinations thereof. In a particular embodiment, the nanoparticles comprise chitosan and poly-(lactic-co-glycolic acid) (PLGA). In a particular embodiment, the nanoparticles comprise polyvinyl pyrrolidone (PVP) and hydroxypropyl methylcellulose acetate succinate (HPMC-AS). In a particular embodiment, the nanoparticles comprise polyvinyl pyrrolidone (PVP) and chitosan. In a particular embodiment, the nanoparticles comprise DSPE-PEG and/or PLGA.
In a particular embodiment, the liver targeting moiety is selected from the group consisting of Glycyrrhetinic acid (GA), Lactobionic acid (LA), Alginic acid, and/or combinations thereof. In a particular embodiment, the liver targeting moiety is coated onto the nanoparticle. In a particular embodiment, the liver targeting moiety is conjugated with the nanoparticle. In a particular embodiment, the liver targeting moiety is attached to the nanoparticle by ionic conjugation (e.g., COO− with NH3+). In a particular embodiment, the liver targeting moiety is covalently attached to the nanoparticle.
In a particular embodiment, the PCSK9 antagonist is of Formula I or II. In a particular embodiment, the PCSK9 antagonist is selected from the group consisting of SBC-115,418, SBC-115,433, SBC-115,448, SBC-115,462 and SBC-115,477. In a particular embodiment, the PCSK9 antagonist is a compound of Formula I-V described in WO 2017/222953 (incorporated herein by reference).
In a particular embodiment, the compositions and/or methods further comprise at least one LDL-lowering substance and/or anti-dyslipidemia agent. The anti-dyslipidemia agent may be contained within (encapsulated) and/or outside the nanoparticle. In a particular embodiment, the method comprises administering the anti-dyslipidemia agent separately from the above composition. In a particular embodiment, the anti-dyslipidemia agent is selected from the group consisting of statin, ezetimibe, bempedoic acid, a thyroid hormone receptor beta agonist (TR-β agonist), and/or combinations thereof.
Entrapment efficiency (loading)=([Drug]f)/([Drug]t)×100,
where [Drug]f is the concentration of SBC-115,418 in the nanoparticles and [Drug]t is the theoretical concentration of drug (meaning total amount of SBC-115,418 added initially). In both the nanoparticles (Formulation A & Formulation E), the entrapment was found to be more than 90%. The loading (w/w) was determined by weighing the total amount of Nano-formulations on a balance and determining the corresponding amount of SBC-115,418 with UV/VIS spectroscopy. The overall loading of SBC-115,418 in nanoparticles was found be around 4.0% w/w for Formulation A and around 6.5% w/w for Formulation E. Shown is the determination of entrapment/loading efficiency of SBC-115,418 encapsulated in nanoparticles. Top: UV-VIS spectra used to construct the standard curve of SBC-115,418 (Inset: Concentrations of SBC-115,418 from 0.3, 0.625, 1.25, 2.5, 5 and 10 μg/mL). Middle & Bottom: Determination of entrapment efficiency by comparing OD from UV-Vis spectra of total amount SBC-115,418 (free+encapsulated) and encapsulated SBC-115,418 in Formulation A and Formulation E, respectively.
The size distribution of the nanoparticles in aqueous dispersions was determined using a Malvern zeta sizer (Malvern Instrumentation Co, Westborough, Mass.). 50 mg of the lyophilized nanoparticles were re-suspended in 2 mL of water. This nanoparticles solution was placed into a 3 mL, 4-sided, clear plastic cuvette, and measured directly. Shown are the HPLC chromatograms of SBC-115,418 used to construct the standard curve at different concentrations. Calibrator series of SBC-115,418 were prepared in DMSO by a 2-fold series dilution including 3.9, 7.8, 15.6, 31.5, 62.5, 125, and 250 μg/mL (left). 80% Acetonitrile does not fully dissolve 100 μg/mL of SBC-115,418.
The present invention provides potent LDL-lowering agents and methods of use thereof including Nano-hepatic targeting approaches. To improve the efficacy of small molecule PCSK9/LDLR antagonists, the present invention uses a nanotechnology platform to target PCSK9 antagonists such as SBC-115,418 and its analogs, with or without statin or other LDL-C lowering agents, for differential targeted delivery to the liver in order to maximize efficacy and minimize its systemic distribution.
Nano-hepatic targeted PCSK9 antagonists (e.g., SBC-115,418) of the instant invention can be combined with a statin or other lipid lowering substance. In a particular embodiment, the drugs (e.g., PCSK9 antagonist and lipid lowering (e.g., LDL-lowering) substance) are Nano-encapsulated into hydrophobic nanoparticles. Examples of hydrophobic nanoparticles include, without limitation, amine-modified poly-(lactic-co-glycolic acid) (PLGA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or EPA/DHA, or solid lipid nanoparticles (SLNs) using HDL with or without cross-linkage or grafting to chitosan. Chitosan, particularly low to ultralow molecular weight chitosan, may be conjugated to fatty acids (including, but not limited to, EPA, DHA, and/or combinations in different amounts) or other acids (e.g., amino acids, hyaluronic acid, and/or linoleic acids), leading to the generation of a hydrophobic polymer that still retains mucoadhesive properties and positive charges for long residence time on cell membranes. Encapsulation of SBC-115,418 into hydrophobic PLGA with or without chitosan and polyethylene glycol (PEG), chitosan-EPA, chitosan-DHA or chitosan-EPA/DHA nanoparticles, as well as SLNs conjugated to hepatic targeting moieties such as Glycyrrhetinic acid, Lactobionic acid, and Alginic acid are exemplified hereinbelow.
As used herein, the term “subject” includes both humans and animals. As used herein, the term “PCSK9” refers to any form of the protein PCSK9, including PCSK9 mutants and variants, which retain at least part of PCSK9 activity or function. Unless otherwise indicated, such as by specific reference to human PCSK9, PCSK9 refers to all mammalian species of native sequence PCSK9, e.g., human, porcine, bovine, equine, canine and feline. One exemplary human PCSK9 sequence is found as Uniprot Accession Number Q8NBP7. An exemplary amino acid sequence is:
As used herein, a “modulator of PCSK9 function” refers to a small molecule that is able to inhibit PCSK9 biological activity or function, and/or downstream pathway(s) mediated by PCSK9 signaling, including PCSK9-mediated down-regulation of the LDLR, and PCSK9-mediated inhibition of the decrease in LDL blood clearance. A modulator of PCSK9 function encompasses compounds that block, antagonize, suppress or reduce (to any degree including significantly) PCSK9 biological activity, including downstream pathways mediated by PCSK9 signaling, such as LDLR interaction and/or elicitation of a cellular response to PCSK9. For purpose of the present invention, it will be explicitly understood that the term “modulator of PCSK9 function” encompasses all the previously identified terms, titles, and functional states and characteristics whereby the PCSK9 itself, a PCSK9 biological activity (including but not limited to its ability to mediate any aspect of interaction with the LDLR, down regulation of LDLR, and inhibit the decrease in blood LDL clearance), or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any measurable degree. In some embodiments, a modulator of PCSK9 function binds PCSK9 and prevents its interaction with the LDLR or its secretion. In other embodiments, a modulator of PCSK9 function binds to the active site of PCSK9 to stabilize its zymogen and prevent auto-processing. In further embodiments, a modulator of PCSK9 function decreases or blocks PCSK9 mediated down-regulation of the LDLR; inhibits the PCSK9-mediated decrease in LDL blood clearance; increases LDL clearance in media by cultured hepatocytes; increases blood LDL clearance by the liver in vivo; improves patients' sensitivity to other LDL lowering drugs, including statins; is synergistic to other LDL lowering drugs, including statins; and blocks PCSK9 interaction with other yet to be identified factors. Examples of modulators of PCSK9 function are provided herein. In a particular embodiment, the “modulator of PCSK9 function” is a PCSK9 antagonist.
The compounds of the instant invention can be administered as salts, which are also within the scope of this invention. Pharmaceutically acceptable (i.e., non-toxic, physiologically compatible) salts are preferred. If the compounds of the method of the present invention have, for example, at least one basic center, they can form acid addition salts. These can be formed, for example, with strong inorganic acids, such as mineral acids, for example sulfuric acid, phosphoric acid or a hydrochloric acid, with strong organic carboxylic acids, such as alkane carboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for example, by halogen, for example acetic acid, such as saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or terephthalic acid, such as hydroxyl carboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid, such as amino acids, for example aspartic or glutamic acid or lysine or arginine, or benzoic acid, or with organic sulfonic acids, such as (C1-C4) alkyl or arylsulfonic acids which are unsubstituted or substituted, for example by halogen, for example methyl- or para-toluene-sulfonic acid. Corresponding acid addition salts can also be formed having plural basic centers, if desired. The compounds used in the method of the present invention having at least one acid group (for example COOH) can also form salts with suitable bases. Representative examples of such salts include metal salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium or magnesium salts, or salts with ammonia or an organic amine, such as morphine, thiomorpholine, piperidine, pyrrolidine, a mono, di- or tri-lower alkylamine, for example ethyl, tert-butyl, diethyl, diisopropyl, triethyl, tributyl or dimethyl-propylamine, or a mono, di- or trihydroxy lower alkylamine, for example mono, di- or triethanolamine. Corresponding internal salts may also be formed.
Exemplary salts of the compounds described herein, which contain a basic group, include monohydrochloride, hydrogensulfate, methanesulfonate, phosphate or nitrate.
Exemplary salts of the compounds described herein, which contain an acid group, include sodium, potassium and magnesium salts and pharmaceutically acceptable organic amines.
All stereoisomers of the compounds, which may be used in the methods described herein, either in a mixture or in pure or substantially pure form, are considered to be within the scope of this invention. The compounds of the present invention can have asymmetric centers at any of the carbon atoms including any one of the R substituents. Consequently, compounds used in the method of the invention can exist in enantiomeric or diastereomeric forms or in mixtures thereof. The processes for preparation of such compounds can utilize racemates, enantiomers or diastereomers as starting materials. When diastereomeric or enantiomeric products are prepared, they can be separated by conventional methods, for example chromatographic, chiral HPLC or fractional crystallization.
As used herein, the term “pharmacophore” refers to the ensemble of steric and electronic features that are necessary to ensure the optimal supramolecular interactions with a specific biological target structure and to trigger, activate, block, inhibit or modulate the biological target's biological activity, as the case may be. See, IUPAC, Pure and Applied Chemistry (1998) 70: 1129-1143.
As used herein, the term “pharmacophore model” refers to a representation of points in a defined coordinate system wherein a point corresponds to a position or other characteristic of an atom or chemical moiety in a bound conformation of a ligand and/or an interacting polypeptide, protein, or ordered water molecule. An ordered water molecule is an observable water in a model derived from structural determination of a polypeptide or protein. A pharmacophore model can include, for example, atoms of a bound conformation of a ligand, or portion thereof. A pharmacophore model can include both the bound conformations of a ligand, or portion thereof, and one or more atoms that interact with the ligand and are from a bound polypeptide or protein. Thus, in addition to geometric characteristics of a bound conformation of a ligand, a pharmacophore model can indicate other characteristics including, for example, charge or hydrophobicity of an atom or chemical moiety. A pharmacophore model can incorporate internal interactions within the bound conformation of a ligand or interactions between a bound conformation of a ligand and a polypeptide, protein, or other receptor including, for example, van der Waals interactions, hydrogen bonds, ionic bonds, and hydrophobic interactions. A pharmacophore model can be derived from two or more bound conformations of a ligand.
As used herein, the term “ligand” refers to any compound, composition or molecule that interacts with the ligand-binding domain of a receptor and modulates its activity. A “ligand” may also include compounds that modulate the receptor without binding directly to it.
In carrying out the method of the invention, the above-described compounds may be administered as such, or in a form from which the active agent can be derived, such as a prodrug. A prodrug is a derivative of a compound described herein, the pharmacologic action of which results from the conversion by chemical or metabolic processes in vivo to the active compound. The term “prodrug esters” as employed herein includes esters and carbonates formed by reacting one or more hydroxyls of compounds used in the method of the invention with alkyl, alkoxy, or aryl substituted acylating agents employing procedures known to those skilled in the art to generate acetates, pivalates, methylcarbonates, benzoates and the like. Any compound that can be converted in vivo to provide the bioactive agent (e.g., a compound of Formula I or II) is a prodrug within the scope and spirit of the invention. Various forms of prodrugs are well known in the art. A comprehensive description of prodrugs and prodrug derivatives are described in: (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., Ch 31 (Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard (Elsevier, 1985); (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds., Ch. 5, pp. 113-191 (Harwood Academic Publishers, 1991).
The therapeutic agent used in practicing the method of the invention is generally administered in an amount sufficient to induce the desired therapeutic effect in the recipient thereof. Thus, the term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent which is sufficient to treat or prevent a condition treatable by administration of one or more of the compounds of Formula I or II or a prodrug thereof. In a particular embodiment, the therapeutically effective amount refers to the amount appropriate to treat a PCSK9-associated condition, i.e. to bring a detectable therapeutic, preventative, or ameliorative effect. The effect may include, for example, treatment or prevention of the conditions described herein.
The compound(s) described herein may be administered at a dose in range from about 0.01 mg to about 200 mg/kg of body weight per day. A dose of from 0.1 to 100 mg/kg per day, and particularly from 1 to 30 mg/kg per day in one or more applications per day or week should be effective to produce the desired result. By way of example, a suitable dose for oral administration would be in the range of 1-30 mg/kg of body weight per day, whereas a typical dose for intravenous administration would be in the range of 1-10 mg/kg of body weight per day. Of course, as those skilled in the art will appreciate, the dosage actually administered will depend upon the condition being treated, the age, health and weight of the recipient, the type of concurrent treatment, if any, and the frequency of treatment. Moreover, the effective dosage amount may be determined by one skilled in the art based on routine empirical activity testing to measure the bioactivity of the compound(s) in a bioassay, and thus establish the appropriate dosage to be administered.
The compounds used in the method of the invention will typically be administered from 1-2 times a day up to 1-2 times a week, to deliver the above-mentioned daily dosage. However, the exact regimen for administration of the compounds described herein will necessarily be dependent on the needs of the individual subject being treated, the type of treatment administered, and the judgment of the attending medical specialist.
In one aspect, the invention provides a method for treating or preventing hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, CVD or coronary heart disease, in an individual comprising administering to the individual an effective amount of a modulator of PCSK9 function that antagonizes circulating PCSK9.
In a further aspect, the invention provides an effective amount of a modulator of PCSK9 function that antagonizes intracellular, extracellular or circulating PCSK9 for use in treating or preventing hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, CVD or coronary heart disease, in an individual. The invention further provides the use of an effective amount of a modulator of PCSK9 function that antagonizes intracellular, extracellular or circulating PCSK9 in the manufacture of a medicament for treating or preventing hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, CVD or coronary heart disease, in an individual.
The methods of the invention use a modulator of PCSK9 function, which refers to any molecule that blocks, suppresses or reduces (including significantly reduces) PCSK9 biological activity, including downstream pathways mediated by PCSK9 signaling, such as elicitation of a cellular response to PCSK9.
A modulator of PCSK9 function should exhibit any one or more of the following characteristics: (a) bind to PCSK9; (b) decrease or block PCSK9 interaction with the LDLR; (c) decrease or block secretion of PCSK9; (d) decrease or block PCSK9 mediated down-regulation of the LDLR; (e) inhibit the PCSK9-mediated decrease in LDL blood clearance, (f) increase LDL clearance in media by cultured hepatocytes, (g) increase blood LDL clearance by the liver in vivo, (h) improve patients' sensitivity to other LDL lowering drugs, including statins, (i) is synergistic to other LDL lowering drugs, including statins; and (j) block PCSK9 interaction with other yet to be identified factors.
In general, the compound(s) used in the method of the invention can be administered to achieve modulation of PCSK9 function by using any acceptable route known in the art, either alone or in combination with one or more other therapeutic agents. Thus, the active agent(s) can be administered orally, buccally, parenterally, such as by intravenous or intra-arterial infusion, intramuscular, intraperitoneal, intrathecal or subcutaneous injection, by liposome-mediated delivery or nanoparticle encapsulation, rectally, vaginally, by inhalation or insufflation, transdermally or by optic delivery.
The orally administered dosage unit may be in the form of tablets, caplets, pills, semisolids, soft or hard gelatin capsules, aqueous or oily solutions, emulsions, suspensions or syrups. Suitable dosage forms for parenteral administration include injectable solutions or suspensions, suppositories, powder formulations, such as nanocrystals, microcrystals or aerosol spray. The active agent may also be incorporated into a conventional transdermal delivery system.
As used herein, the expression “physiologically compatible carrier medium” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, fillers and the like as suited for the particular dosage form desired. Remington: The Science and Practice of Pharmacy, 20th edition (A. R. Genaro et al., Part 5, Pharmaceutical Manufacturing, pp. 669-1015 (Lippincott Williams & Wilkins, Baltimore, Md./Philadelphia, Pa.) (2000)) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional pharmaceutical carrier medium is incompatible with the PCSK9 modulators used in the present invention, such as by producing an undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of a formulation comprising such compounds, its use is contemplated to be within the scope of this invention.
For the production of solid dosage forms, including hard and soft capsules, the therapeutic agent may be mixed with pharmaceutically inert, inorganic or organic excipients, such as lactose, sucrose, glucose, gelatin, malt, mannitol, silica gel, starch or derivatives thereof, talc, stearic acid or its salts, dried skim milk, vegetable, petroleum, animal or synthetic oils, wax, fat, polyols, and the like. For the production of liquid solutions, emulsions or suspensions or syrups one may use excipients such as water, alcohols, aqueous saline, aqueous dextrose, polyols, glycerin, lipids, phospholipids, cyclodextrins, vegetable, petroleum, animal or synthetic oils. For suppositories, one may use excipients, such as vegetable, petroleum, animal or synthetic oils, wax, fat and polyols. For aerosol formulations, one may use compressed gases suitable for this purpose, such as oxygen, nitrogen and carbon dioxide. The pharmaceutical composition or formulation may also contain one or more additives including, without limitation, preservatives, stabilizers, e.g., UV stabilizers, emulsifiers, sweeteners, salts to adjust the osmotic pressure, buffers, coating materials and antioxidants.
The present invention further includes controlled-release, sustained-release, or extended-release therapeutic dosage forms for administration of the active agent, which involves incorporation of the active agent into a suitable delivery system. This dosage form controls release of the active agent(s) in such a manner that an effective concentration of the active agent(s) in the bloodstream may be maintained over an extended period of time, with the concentration in the blood remaining relatively constant, to improve therapeutic results and/or minimize side effects. Additionally, a controlled-release system would provide minimum peak to trough fluctuations in blood plasma levels of the active agent.
Compounds for use in practicing this invention include PCSK9 antagonists such as those of Formula I and particularly Formula II, above. In a particular embodiment, the PCSK9 antagonist is selected from the group consisting of SBC-115,418, SBC-115,433, SBC-115,448, SBC-115,462 and SBC-115,477 (see, e.g.,
The methods of the present invention will normally include medical follow-up to determine the therapeutic or prophylactic effect brought about in the subject undergoing treatment with the compound(s) and/or composition(s) described herein.
The following definitions are provided to facilitate an understanding of the present invention:
The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term “alkyl” is a branched or unbranched saturated hydrocarbon chain moiety. “Lower alkyl” denotes branched or unbranched hydrocarbon chains, having 1 to about 8 carbons, such as, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, 2-methylpentyl pentyl, hexyl, isohexyl, heptyl, 4,4-dimethyl pentyl, octyl, 2,2,4-trimethylpentyl and the like. “Substituted alkyl” includes an alkyl group which may be substituted with one or more substituent groups which are attached commonly to such chains, such as, hydroxy, halogen, mercapto or thio, cyano, alkylthio, carboxy, carbalkoxy, amino, nitro, alkoxy, or optionally substituted, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl, and the like to form alkyl groups such as trifluoro methyl, 3-hydroxyhexyl, 2-carboxypropyl, 2-fluoroethyl, carboxymethyl, cyanobutyl, phenethyl, benzyl and the like.
The term “halogen” or “halo” as used herein alone or as part of another group refers to chlorine, bromine, fluorine, and iodine.
The term “alkoxy” refers to alkyl-O—, in which alkyl is as defined above.
Unless otherwise indicated, the term “cycloalkyl” as employed herein alone or as part of another group includes saturated or partially unsaturated (containing 1 or more double bonds) cyclic hydrocarbon groups (‘carbocycle’) containing 1 to 3 rings, including monocyclic alkyl, bicyclic alkyl and tricyclic alkyl, containing a total of 3 to 20 carbons forming the rings, particularly 3 to 10 carbons, forming the ring and which may be fused to 1 or 2 aromatic rings as described for aryl, which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl and cyclohexenyl.
“Substituted cycloalkyl” includes a cycloalkyl group which may be substituted with 1 or more substituents such as halogen, alkyl, substituted alkyl, alkoxy, hydroxy, aryl, substituted aryl, aryloxy, cycloalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, thiol and/or alkylthio and/or any of the substituents included in the definition of “substituted alkyl.”
Unless otherwise indicated, the term “aryl” or “Ar” as employed herein alone or as part of another group refers to monocyclic and polycyclic aromatic groups containing 6 to 10 carbons in the ring portion (such as phenyl or naphthyl including 1-naphthyl and 2-naphthyl) and may optionally include one to three additional rings fused to a carbocyclic ring, such as a cycloalkyl ring or fused to an aryl or heterocyclic ring or substituted forms thereof.
“Substituted aryl” includes an aryl group which may be substituted with one or more substituent groups, such as halo, alkyl, haloalkyl (e.g., trifluoromethyl), alkoxy, haloalkoxy (e.g., difluoromethoxy), alkenyl, alkynyl, cycloalkyl-alkyl, heterocyclo-alkyl, aryl, heteroaryl, arylalkyl, aryloxy, aryloxyalkyl, arylalkoxy, alkoxycarbonyl, alkylcarbonyl, arylcarbonyl, arylalkenyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, aminocarbonylaryl, arylthio, arylsulfinyl, arylazo, heteroarylalkyl, heteroarylalkenyl, heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro, cyano, amino, substituted amino wherein the amino includes 1 or 2 substituents (which are optionally substituted alkyl, aryl or any of the other substituents mentioned in the definitions), thiol, alkylthio, heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, alkylcarbonylamino, arylcarbonylamino, arylsulfinylalkyl, arylsulfonylamino or arylsulfonaminocarbonyl and/or any of the alkyl substituents referred to above.
Unless otherwise indicated, the term “heteroaryl” or “Het” as used herein alone or as part of another group refers to a 5- or 7-membered aromatic ring which includes 1, 2, 3 or 4 hetero atoms such as nitrogen, oxygen or sulfur and such rings fused to an aryl, cycloalkyl, heteroaryl or heterocycloalkyl ring and includes possible N-oxides. Examples of heteroaryl groups include pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isooxazolyl, thiazolyl, isothiazolyl, thiadiazolyl and oxadiazolyl. Examples of fused heteroaryl groups include quinoline, isoquinoline, indole, isoindole, carbazole, acridine, benzimidazole, benzofuran, benzoxazole, isobenzofuran, benzothiophene, phenanthroline, purine, and the like. “Substituted heteroaryl” includes a heteroaryl group, which may be substituted with 1 to 4 substituents, such as the substituents included above in the definition of “substituted alkyl,” “substituted cycloalkyl,” and “substituted aryl.”
The term “heterocyclo”, “heterocycle” or “heterocyclic ring,” as used herein alone or as part of another group, represents an unsubstituted or substituted stable 5- to 7-membered monocyclic ring system which may be saturated or partially unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from N, O, or S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized. “Substituted heterocyclo” (or heterocycle or heterocyclic ring) includes a heterocyclic group which may be substituted with 1 to 4 substituents, such as the substituents included above in the definition of “substituted alkyl,” “substituted cycloalkyl,” and “substituted aryl.” The heterocyclic ring may be attached at any heteroatom or carbon atom, which results in the creation of a stable structure. Examples of such heterocyclic groups include, but are not limited to, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, tetrahydropyranyl, thiamorpholinyl, thiamorpholinyl sulfoxide and thiamorpholinyl sulfone.
The term “optionally substituted” is used herein to signify that a chemical moiety referred to, e.g., alkyl, aryl, heteroaryl, may be unsubstituted or substituted with one or more groups including, without limitation, lower alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl, aryl, haloaryl, heterocycle, heterocycloalkyl, heteroaryl, hydroxyl, amino, monoalkylamino, dialkylamino, alkoxy, halogen, haloalkoxy, aryloxy, aryloxyalkyl, alkylaryloxy, arylalkoxy, alkoxyaryl, carboxy, carbalkoxy, carboxamido, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, monoalkylaminosulfinyl, dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl, alkylsulfonylamino, hydroxysulfonyloxy, alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl, monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl, dialkylaminosulfinylalkyl and the like. The chemical moieties of formulas I and II, above, that may be optionally substituted include lower alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl, aryl, heterocycle, and heteroaryl. For example, optionally substituted alkyl would comprise both propyl and 2-chloro-propyl. Additionally, “optionally substituted” is also inclusive of embodiments where the named substituent or substituents have multiple substituents rather than simply a single substituent. For example, optionally substituted aryl would comprise both phenyl and 3-bromo-4-chloro-6-ethyl-phenyl.
Unless expressly indicated otherwise, all references herein to alkyl and aryl groups also include the substituted forms thereof.
The activities of compounds described herein have been experimentally demonstrated. The following examples are provided to describe the invention in further detail. These examples are provided for illustrative purposes only and are not intended to limit the invention in any way.
Testing of SBC-115,418 and analogs was performed in a binding assay to determine their ability to inhibit the PCSK9/LDLR interaction. SBC-115,418 inhibited the PCSK9/LDLR interaction with an IC50 in the sub-micro-molar range. Experimental details are provided hereinabove and in WO 2017/222953. Several new analogs (see
SBC-115,418 and its analogs exhibited no effect on the synthesis, processing and secretion of PCSK9 either in the cells or into the media. Experimental details are provided hereinabove and in WO 2017/222953.
SBC-115,418, SBC-115,433, SBC-115,448 and SBC-115,462 exhibited an increase in the level of LDLR as compared to cells treated with the same volume of DMSO (control) with a significant upregulation of LDLR. In addition, SBC-115,418 and SBC-115,462 exhibited significant increase in the Dil-LDL uptake in HepG2 cells at 10 μM concentrations (
Several SBC-115,418 Nanoformulations were prepared and optimized (
Table 2 illustrates the size, zeta potentials and Entrapment Efficiency (%) of optimized Nanoformulations no.'s 1-3. Table 2 further includes no. 3 where 5% mannitol was added as cryoprotectant to prevent aggregation. Nanoformulation no. 3 was desirable because of its smaller size.
SBC-115,418/Nanoformulation D (SBC-115,418, PVP, Alginic acid, Chitosan oligosaccharide lactate) was used to determine PK and PD. For PK and oral bioavailability of SBC-115,418, the data showed increased concentrations of SBC-115,418 (5 mg/kg oral and i.p.) observed from 30 min to 1 hour in mice plasma. The data shows that SBC-115,418 has 18% oral bioavailability (
HPMC-AS is a widely used excipient that possesses increased solubility, capable of forming a solid dispersion and inhibiting the crystallization of the API from the dispersion matrix as well as a rate-controlling polymer for sustained-release dose form. The dispersions were prepared as follows: SBC-115,418 was solubilized in HPMC-AS using sonication for 10 minutes. Using SBC-115,418/HPMC-AS as a Dispersion formulation versus the Nanocrystal/Hepatic Targeting optimal formulation, Nano SBC-115,418 (SBC-115,418/PVP/HPMC-AC/Alginic Acid) (
Mice were housed as four animals per cage under climate-controlled conditions of temperature (20-24° C.), humidity (60-70%), and alternating 12 hour light/dark cycles. Mice were fed a high-fat diet (TD.06414, Harlan Research Diet, Inc., Indianapolis, Ind.) that provides 60 calories from fat sources to increase total cholesterol. The nutritionally induced mouse is therefore a suitable model for examining the effects of Nano SBC-115,418 for liver targeting in lowering LDL-C levels. Male C57BL/6 mice were fed either a commercial chow diet (Prolab RMH 3000, PMI feeds, St. Louis, Mo.) to serve as a negative control, or a high-fat diet (TD.06414, Harlan Research Diet, Inc., Indianapolis, Ind.). Plasma was collected once weekly to monitor the level of LDL-C and PCSK9 levels. After 4 weeks of feeding on the high-fat diet, mice were randomly assigned to one of the different groups such that the average of each biomarker level are comparable among the different groups. One group was treated with vehicle, and the other groups are treated with Nano SBC-115,418 at different doses (Table 4). Blood samples (75 μl) were collected from the retro-orbital venous plexus via heparinized capillary tubes containing 2 USP units of ammonium heparin per tube (Carolina, Burlington, N.C.). Plasma was separated immediately by centrifugation (5,000×g) for 5 minutes at room temperature and then kept at −80° C. until assayed for lipid profile. Plasma total and free cholesterol, LDL-C and free PCSK9 levels were measured.
The data demonstrates that Nano SBC-115,418 is highly effective at lowering LDL-C and PCSK9 in mice fed high-fat diet (
The specification includes citations to certain publications, which are provided to indicate the state of the art to which this invention pertains. The entire disclosure of each of the cited publications is incorporated by reference herein.
While certain embodiments of the present invention have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. The present invention is, therefore, not limited to the particular embodiments described and/or exemplified but is capable of considerable variation and modification without departure from the scope of the appended claims. Furthermore, the transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what un-recited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, un-recited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinarily associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All compositions and methods of use thereof that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/755,709, filed Nov. 5, 2018. The foregoing application is incorporated by reference herein.
This invention was made with government support under SBIR Grant No. HL137449 awarded by the National Heart, Lung and Blood Institute (NHLBI). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/059777 | 11/5/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62755709 | Nov 2018 | US |
