Patent Grant
11583541
The present invention relates to glucocorticoid prodrugs and methods of using the prodrugs for treating patients having an inflammatory disease, including but not limited to lupus.
Lupus is a challenging autoimmune rheumatic disease in clinic for which current therapies are unsatisfactory with respect to both remission induction and unwanted toxicities. It is characterized by B and T cell hyperactivation, overproduction of autoantibodies, and the deposition of immune complexes in various tissues/organs. The symptoms of lupus are highly heterogeneous including skin rash, arthritis, pericarditis, neuropsychiatric disorders and nephritis. It is estimated that 1.5 million of Americans are affected by lupus and the number of patients is continuously increasing.
Lupus nephritis (LN), one of the most devastating complications of lupus, and the leading cause of morbidity and mortality in lupus patients, affects between 30-60% of lupus patients in terms of immunosuppression and direct mortality. In the US, approximately 35% adult lupus patients have clinical evidence of nephritis at the time of diagnosis, and an additional 15-25% will develop nephritis within 10 years of diagnosis. LN is initiated by immune complex deposition within the glomeruli and tubules of the kidney and subsequent activation of the immune effector cells (such as macrophages and neutrophils) that leads to damage to renal tissues. If not properly managed, lupus nephritis can rapidly progress to impaired renal function and eventually causing renal failure.
While clinicians have utilized many classes of drugs to manage lupus, only a few have been approved by US FDA specifically for the disease. They include aspirin, belimumab (or Benlysta®, a human monoclonal antibody that inhibits B-cell activating factor), antimalarials (e.g. chloroquine) and glucocorticoids (GC, e.g. prednisone, dexamethasone). Among these treatment options, GC is one of the most potent and widely used drugs for lupus. In American College of Rheumatology (ACR)'s new guidelines for clinical management of lupus nephritis, the recommended treatment regimen consists of a pulse GC treatment followed by low/high-dose daily GC plus an immunosuppressive medication. Compared to the previous guidelines, new immunosuppressants (e.g. mycophenolate mofetil) have been added as alternatives to cyclophosphamide. No alternatives, however, have been recommended for GC. Comparing to the wide applications of GC in most lupus symptoms, the clinical benefits of belimumab in treatment of lupus nephritis has not been well established. NSAIDs, on the other hand, are contraindicated for lupus nephritis due to their renal toxicities.
Due to their potent anti-inflammatory efficacy and the lack of an alternative therapy, GC continues to be the mainstay of clinical management of lupus. Some lupus pathologies, such as arthritis and skin rash can be treated effectively with short-term GC. More severe lupus complications, such as progressive nephritis necessitates long-term GC therapy, which is frequently associated with serious side effects involving the endocrine, cardiovascular, hematopoietic and musculoskeletal systems. These adverse events contribute significantly to morbidity among lupus patients.
The actions of GC are thought to be mediated through two distinct pathways: transactivation and transrepression. It has been postulated that transrepression primarily mediates the anti-inflammatory effects of GC whereas transactivation is responsible for the GC-associated side effects. Compounds that can preferentially activate the transrepression relative to the transactivation pathway have been developed. Nevertheless, these compounds do not exhibit strict pathway selectivity and still elicit GC-related side effects.
To address the various foregoing problems, the present application discloses glucocorticoid (GC) prodrugs and methods of using them for treatment of lupus and LN. These include a polyethylene glycol (PEG)-based macromolecular prodrug (PEG-DiDex) of dexamethasone (Dex), which self-assembles into micelles.
In one aspect, the present invention provides a compound of formula (I) or (XII):
or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein:
n is an integer from 3 to 500;
m is an integer from 1 to 5;
w is an integer from 1 to 5;
GC is a moiety of a glucocorticoid drug molecule
A is absent, C1-C6 alkylene, or C6-C10 arylene;
B is absent, NR4, O, or C(O);
D is absent, NR4, O, C(O), or CR5R5;
E is absent, C1-C6 alkylene, or a linker comprising a branched structure capable of connecting to two or more R2 groups, said linker optionally comprising one, two, or more heteroatoms independently selected from the group consisting of O, S, and N;
G is absent, NR4, or O;
P is absent or C(O);
Q is absent, C6-C10 arylene, or C1-C6 alkylene;
T is absent, C1-C6 alkylene, C6-C10 arylene, or C(O);
X is absent, O, S, or NR4;
Y is absent, C(O), C6-C10 arylene, or C1-C6 alkylene;
Z is absent, NR4, O, C1-C6 alkylene, or a linker comprising a branched structure capable of connecting to one or more dexamethasone moieties, said linker optionally comprising one or more heteroatoms independently selected from the group consisting of O, S, and N;
R1 is H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl, each group except H optionally substituted by one to five substituents independently selected from the group consisting of C1-C4 alkyl, C1-C4 haloalkyl, halogen, oxo (═O), —NRaRb, —NO2, —CN, —OR3, and —SR3; or alternatively, R1 is —CH2-A-B-D-E-(R2)m;
R3 at each occurrence is independently H, C1-C4 alkyl, or C1-C4 haloalkyl;
R4 at each occurrence is independently H or C1-C4 alkyl;
R5 at each occurrence is independently H, C1-C4 alkyl, or C1-C4 haloalkyl;
Ra and Rb are each independently H or C1-C4 alkyl; and
wherein when any of groups A, B, D, E, G, P, Q, T, X, Y, and Z is absent, its two available adjacent groups are single-bonded to each other directly.
In another aspect, the present invention provides a pharmaceutical composition comprising a compound according to any one embodiment described herein, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier, adjuvant, diluent, or vehicle.
In another aspect, the present invention provides a method of treating an autoimmune disease and/or inflammatory disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a compound according to any embodiment disclosed here, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
In another aspect, the present invention provides a method of treating a disease or disorder associated with lupus, comprising administering to a subject in need of treatment a therapeutically effective amount of the pharmaceutical composition of any embodiment disclosed herein.
In another aspect, the present invention provides use of a compound according to any embodiment or any embodiment combinations disclosed here, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, in the manufacture of a medicament for treatment of a disease or disorder, especially those associated with lupus.
The PEG-based macromolecular prodrug of dexamethasone (Dex) overcomes various challenges, for example, their accompanying severe toxicities, through GC prodrug nanomedicine development, in order to more fully realize the therapeutic potential of GC in clinical management of lupus nephritis. Without intending to be bound by theory, the present invention is in part based on a hypothesis that the Extravasation of the nanomedicine through Leaky Vasculature at inflammation and subsequent Inflammatory cell-mediated Sequestration (ELVIS) would dramatically alter the pharmacokinetics/biodistribution (PK/BD) profile of the parent drug, favoring specific accumulation in the inflamed tissues/organs, and enhanced molecular weight associated with the non-immunogenicity of PEG chains would potentially improve drug retention in kidney and prolong the blood circulation time. Specifically, the amphiphilic molecule self-assembles into micelles. When tested in lupus prone NZB/W F1 mice with severe nephritis, the monthly treatment demonstrated superior therapeutic efficacy in improving kidney function than dose equivalent daily Dex treatment, with no GC-associated side effect observed.
Other aspects, benefits, and advantages of the present invention will be better appreciated in view of the following drawings, detailed description, and claims.
The glucocorticoid prodrugs conjugated to polyethylene glycol have been found to possess superior therapeutic efficacy and greatly reduced toxicity as compared with the parent drug.
Specifically, optical imaging, immunohistochemistry and flow cytometry studies reveal that the near-infrared dye labeled PEG-DiDex micelle primarily distributes to the inflamed kidneys after systemic administration, with intraglomerular mesangial cells and proximal tubule epithelial cells chiefly responsible for the intracellular sequestration of the prodrug inside the kidneys. For efficacy and safety evaluation, the prodrug micelle was given monthly to NZB/W F1 female mice (28 weeks old) via tail vein injection. Dose equivalent daily dexamethasone phosphate sodium i.v. administration and saline were used as controls. When compared to Dex treatment, PEG-DiDex markedly improved the survival of NZB/W F1 mice and is significant more effective in normalizing albuminuria; no significant systemic toxicity of GCs (i.e. WBC reduction, total IgG reduction, adrenal gland atrophy and osteopenia) was observed in the prodrug treated group. PEG-DiDex treated animals also exhibit lower serum levels of proinflammatory cytokines (e.g. MCP-1, IFN-β, IFN-γ, etc.) and clear histological indication of nephritis resolution after 2 months of treatment. But it has no impact on the serum anti-dsDNA antibody level. Collectively, these evidences suggest that the novel prodrug micelle design of PEG-DiDex can dramatically alter the biodistribution profile of Dex by passively targeting the drug to the inflamed kidneys. The nephrotropic distribution pattern of PEG-DiDex potentiates and prolongs the local anti-inflammatory effect of Dex within kidney pathology. Its outstanding safety profile may be attributed to the substantially reduced systemic exposure to Dex.
In one aspect, the present invention provides a compound of formula (I):
or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein:
n is an integer from 3 to 500;
m is an integer from 1 to 5;
w is an integer from 1 to 5;
A is absent, C1-C6 alkylene, or C6-C10 arylene;
B is absent, NR4, O, or C(O);
D is absent, NR4, O, C(O), or CR5R5;
E is absent, C1-C6 alkylene, or a linker comprising a branched structure capable of connecting to two or more R2 groups, said linker optionally comprising one, two, or more heteroatoms independently selected from the group consisting of O, S, and N;
G is absent, NR4, or O;
P is absent or C(O);
Q is absent, C6-C10 arylene, or C1-C6 alkylene;
T is absent, C1-C6 alkylene, C6-C10 arylene, or C(O);
X is absent, O, S, or NR4;
Y is absent, C(O), C6-C10 arylene, or C1-C6 alkylene;
Z is absent, NR4, O, C1-C6 alkylene, or a linker comprising a branched structure capable of connecting to one or more dexamethasone moieties, said linker optionally comprising one or more heteroatoms independently selected from the group consisting of O, S, and N;
R1 is H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl, each group except H optionally substituted by one to five substituents independently selected from the group consisting of C1-C4 alkyl, C1-C4 haloalkyl, halogen, oxo (═O), —NRaRb, —NO2, —CN, —OR3, and —SR3; or alternatively, R1 is —CH2-A-B-D-E-(R2)m;
R3 at each occurrence is independently H, C1-C4 alkyl, or C1-C4 haloalkyl;
R4 at each occurrence is independently H or C1-C4 alkyl;
R5 at each occurrence is independently H, C1-C4 alkyl, or C1-C4 haloalkyl;
Ra and Rb are each independently H or C1-C4 alkyl; and
wherein when any of groups A, B, D, E, G, P, Q, T, X, Y, and Z is absent, its two available adjacent groups are single-bonded to each other directly.
In some embodiments of this aspect, E is a linker comprising a branched structure capable of covalently bonding to two or more R2 groups, said linker optionally comprising one or more heteroatoms independently selected from the group consisting of O, S, and N.
In some embodiments of this aspect, Z is a linker comprising a branched structure capable of covalently bonding to two or more dexamethasone moieties, said linker optionally comprising one or more heteroatoms independently selected from the group consisting of O, S, and N.
In some embodiments of this aspect, E and Z are each independently selected from the group consisting of absent, C1-C4 alkylene, an amino acid-based linker, a citric acid-based linker, a glycerol-based linker, a tris(2-aminoethyl)amine-based linker, a pentaerythritol-based linker, and a pentetic acid-based linker, respectively having a formula as follows:
amino acid-based, citric acid-based, glycerol-based, Tris(2-aminoethyl)amine-based,
wherein i and j are each independently 0 or an integer select from 1 to 5.
In some embodiments of this aspect,
w is 1;
Z is absent or C1-C4 alkylene;
E is selected from the group consisting of:
In some embodiments of this aspect, m is 1; w is 1; and A, B, D, E, and Z are all absent, characterized by formula (II):
In some embodiments of this aspect, R1 is CH3; Y is C(O); X is NH; and T, Q, P, and G are all absent, characterized by formula:
In some embodiments of this aspect, A is C(O); B and D are absent; E is the amino acid based linker; m is 2; w is 1; Z and Y are absent, and X is NH, characterized by formula (III):
or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein:
i=0 or an integer from 1 to 5;
j=0 or an integer from 1 to 5;
G=absent, NR4, or O;
P=absent or C(O);
Q=absent, C6-C10 arylene, or C1-C6 alkylene;
T=absent, C1-C6 alkylene, C6-C10 arylene, or C(O);
X=absent, O, S, or NR4;
Y=absent or C1-C6 alkylene;
R1=H or C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C10 aryl, or 5-10 membered heteroaryl, 5-10 membered heterocyclyl, each group except H optionally substituted by one to three substituents independently selected from the group consisting of C1-C4 alkyl, C1-C4 haloalkyl, halogen, oxo (═O), —NRaRb, —NO2, —CN, —OR3, and —SR3;
R3 at each occurrence is independently H, C1-C4 alkyl, or C1-C4 haloalkyl;
R4 at each occurrence is independently H or C1-C4 alkyl;
Ra and Rb are each independently H or C1-C4 alkyl; and
wherein when any of groups G, P, Q, T, X, and Y is absent, its two available adjacent groups are single-bonded to each other directly.
In some embodiments of this aspect, i is 0; j is 2; E is a glutamic acid based linker having a formula:
In some embodiments of this aspect, i is 0, j is 2, X is NH; and Q is methylene, P is C(O), G is NH, the compound characterized by formula:
In some embodiments of this aspect, A is methylene, B is NH, m is 1, w is 2, E is a citric acid based linker, Z is NH, the compound characterized by formula (IV):
or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein:
i=0 or an integer from 1 to 5;
j=0 or an integer from 1 to 5;
G=absent, NR4, or O;
P=absent or C(O);
Q=absent, C6-C10 arylene, or C1-C6 alkylene;
T=absent, C1-C6 alkylene, C6-C10 arylene, or C(O);
X=absent, O, S, or NR4;
Y=absent or C1-C6 alkylene;
R1═H or C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C10 aryl, or 5-10 membered heteroaryl, 5-10 membered heterocyclyl, each group except H optionally substituted by one to three substituents independently selected from the group consisting of C1-C4 alkyl, C1-C4 haloalkyl, halogen, oxo (═O), —NRaRb, —NO2, —CN, —OR3, and —SR3;
R3 at each occurrence is independently H, C1-C4 alkyl, or C1-C4 haloalkyl;
R4 at each occurrence is independently H or C1-C4 alkyl;
Ra and Rb are each independently H or C1-C4 alkyl; and
wherein when any of groups G, P, Q, T, X, and Y is absent, its two available adjacent groups are single-bonded to each other directly.
In some embodiments of this aspect, G, P, Q, T, X, and Y are all absent, the compound characterized by formula:
In some embodiments of this aspect, m is 3, w is 1, A is methylene, B is NH, D is C(O) and E is a pentaerythritol-based linker, the compound is characterized by formula (V):
In some embodiments of this aspect, Y is C(O); X is NH; and G, P, Q, and T are all absent, the compound characterized by formula:
In some embodiments of this aspect, the compound has a structure of any one of formulas (VI)-(XI):
wherein R is selected from the group consisting of:
wherein k at each occurrence is independent an integer selected from 1 to 10. In some embodiments, k is an integer selected from 1 to 8; in some preferred embodiments, k is an integer selected from 1 to 6; and in some more preferred embodiments, k is an integer selected from 1 to 4; and in some more preferred embodiments, k is an integer selected from 1 to 2.
In some embodiments of this aspect, E is a glutamic acid based linker having a formula:
In another aspect, the present invention provides a compound of formula (XII):
or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein:
n is an integer from 3 to 500;
m is an integer from 1 to 5;
w is an integer from 1 to 5;
GC is a moiety of a glucocorticoid drug molecule;
A is absent, C1-C6 alkylene, or C6-C10 arylene;
B is absent, NR4, O, or C(O);
D is absent, NR4, O, C(O), or CR5R5;
E is absent, C1-C6 alkylene, or a linker comprising a branched structure capable of connecting to two or more R2 groups, said linker optionally comprising one, two, or more heteroatoms independently selected from the group consisting of O, S, and N;
G is absent, NR4, or O;
P is absent or C(O);
Q is absent, C6-C10 arylene, or C1-C6 alkylene;
T is absent, C1-C6 alkylene, C6-C10 arylene, or C(O);
X is absent, O, S, or NR4;
Y is absent, C(O), C6-C10 arylene, or C1-C6 alkylene;
Z is absent, NR4, o, C1-C6 alkylene, or a linker comprising a branched structure capable of connecting to one or more dexamethasone moieties, said linker optionally comprising one or more heteroatoms independently selected from the group consisting of O, S, and N;
R1 is H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl, each group except H optionally substituted by one to five substituents independently selected from the group consisting of C1-C4 alkyl, C1-C4 haloalkyl, halogen, oxo (═O), —NRaRb, —NO2, —CN, —OR3, and —SR3; or alternatively, R1 is —CH2-A-B-D-E-(R2)m;
R3 at each occurrence is independently H, C1-C4 alkyl, or C1-C4 haloalkyl;
R4 at each occurrence is independently H or C1-C4 alkyl;
R5 at each occurrence is independently H, C1-C4 alkyl, or C1-C4 haloalkyl;
Ra and Rb are each independently H or C1-C4 alkyl; and
wherein when any of groups A, B, D, E, G, P, Q, T, X, Y, and Z is absent, its two available adjacent groups are single-bonded to each other directly.
In this aspect, the term “moiety of a glucocorticoid drug molecule” refers to the drug portion of the prodrug linked with PEG through various linkers through a C═N double bond as disclosed herein. In particular, these moieties of glucocorticoid drug molecules include, but are not limited to the following:
or the like.
In some embodiments of this aspect, the compound a structure selected from formulae (XIII) to (XVIII):
wherein R comprises said moiety of a glucocorticoid drug molecule.
In some embodiments, the glucocorticoid drug molecule is selected from the group consisting of:
The size of PEG suitable for compounds of the present invention can vary in a range so that the prodrug can serve the purpose disclosed herein. Typical size can be in the range of 100 to 20,000 Da. molecular weight, or n can be in the range of about 3 to about 500. In some embodiments, n is in the range of 10 to 300. In some embodiments of this aspect, n is 20 to 200. In some embodiments of this aspect, n is 10 to 100.
In some preferred embodiments of this aspect, n is 40-45; and in some particular embodiments, n is 42.
As a person of ordinary skill in the art would understand, in any aspects or embodiments of the compounds disclosed herein, any two adjacent atoms or bonds must comply with the basic bond principles. While basic bond principles are compliant with, any potential combination of the limitations defined in any two or more embodiments disclosed herein are encompassed by the present invention.
In another aspect, the present invention provides a pharmaceutical composition comprising a compound according to any one embodiment described herein, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier, adjuvant, diluent, or vehicle.
In some embodiments of this aspect, the pharmaceutical composition is in oral, nasal, ophthalmic drop, topical, buccal, sublingual, rectal, vaginal, intravenous, or other parenteral form.
In some embodiments of this aspect, the pharmaceutical composition is in vaporization-ready, nebulization-ready, nanoparticle formulation, or liposomal formulation.
In some embodiments of this aspect, the composition of further comprises a second therapeutic agent, including but not limited, a NSAID (e.g. Aspirin, Naproxen, Celebrex), a Glucocorticoid (e.g. Dexamethasone, Prednisone, Betamethasone), a DMARD (e.g. Methotrexate, Leflunomide, Sulfasalazine, Hydroxychoroquine), or the like.
In another aspect, the present invention provides a method of treating an autoimmune disease and/or inflammatory disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a compound according to any embodiment disclosed here, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
In some embodiments of this aspect, the disease or disorder is systemic lupus erythematosus, lupus nephritis, minimal change disease, focal segmental glomerulosclerosis, IgA nephropathy, transplant rejection, rheumatoid arthritis, osteoarthritis, psoriasis, ankylosing spondylitis, vasculitis, multiple sclerosis, systemic sclerosis, gout, uveitis, asthma, cystic fibrosis, chronic obstructive pulmonary disease, atopic dermatitis (eczema), sepsis, inflammatory bowel disease, trauma brain injury, spinal cord injury, ischemia reperfusion injury, heterotopic ossification, or granuloma, etc.
In some embodiments of this aspect, the method of treating disease or disorder further includes administering to the subject a second therapeutic agent, such as a NSAID (e.g. Aspirin, Naproxen, Celebrex), a Glucocorticoid (e.g. Dexamethasone, Prednisone, Betamethasone), a DMARD (e.g. Methotrexate, Leflunomide, Sulfasalazine, Hydroxychoroquine), a biologic (e.g. Belimumab, Etanercept, Anakinra, Infliximab, Rituximab, etc.), or the like.
In another aspect, the present invention provides a method of treating a disease or disorder associated with lupus, comprising administering to a subject in need of treatment a therapeutically effective amount of the pharmaceutical composition of any embodiment disclosed herein.
In some embodiments of this aspect, the subject treated is a mammalian animal, such as human, dog, cat, horse, and so on. In a preferred embodiment, the subject is a human.
In another aspect, the present invention provides use of a compound according to any embodiment or any embodiment combinations disclosed here, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, in the manufacture of a medicament for treatment of a disease or disorder, especially those associated with lupus.
The disease or disorder that can be treated using the present invention includes, but is not limited to, systemic lupus erythematosus, cutaneous lupus erythematosus, photosensitivity, cutaneous vascular disease, nonscarring alopecia, oral ulcer, nail and capillary changes, populonodular mucinosis, bullous lupus erythematosus, sweet syndrome, pyoderma gangrenosum, palisaded neutrophilic granulomatous dermatitis, aseptic meningitis, cerebrovascular disease, demyelinating syndrome, headache, movement disorder, myelopathy, seizure disorder, acute confusional state, anxiety disorder, cognitive dysfunction, mood disorder, psychosis, Guilain-Barré syndrome, autonomic neuropathy, mononeuropathy, myasthenia gravis, cranial neuropathy, plexopathy, polyneuropathy, lupus nephritis, pericarditis, coronary vasculitis, coronary atherosclerosis, vasculitis, pleurisy, pleural effusion, acute lupus pneumonitis, diffuse alveolar hemorrhage, chronic interstitial lung disease shrinking lung syndrome, pulmonary hypertension, thromboembolism, cricoarytenoid arthritis, small airways disease, chronic active and lupoid hepatitis, Sjögren's syndrome, esophagitis, watermelon stomach, eosinophilic gastroenteritis, abdominal pain, intestinal thrombosis, inflammatory bowel disease, protein-losing enteropathy, fat malabsorption, Celiac Sprune, Chronic intestinal pseudo-obstruction, amyloidosis, peritoneal inflammation, pancreatitis, splenomegaly, autoimmune hemolytic anemia, immune thrombocytopenic purpura, leukopenia, arthralgia, arthritis, tendon rupture, myositis, osteonecrosis, osteoporosis, etc.
Without being limited, in some embodiments, the molecular weight of PEG used in the present invention is in the range from about 200 to about 10,000 Da. The glucocorticoid (GC) drug molecule can be conjugated to a chain terminus, or both chain termini of PEG. In addition to linear PEG, branched PEG, brush like PEG or even dendritic PEG can also be used as the prodrug carrier for conjugation with molecules such as Dex. The number of drug molecule such as dexamethasone (Dex) can range from 1 to 10 in each molecule of PEG prodrug conjugate. To increase the micelle stability, the hydrophobic terminus may be crosslinked with weak chemical bonds.
In addition to dexamethasone, other glucocorticoids (e.g. prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, beclometasone), anti-inflammatory agents, and low molecular weight immune suppressant can be used to conjugate to PEG according to the principle and methods disclosed herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as are commonly understood by one of ordinary skill in the art to which this invention pertains.
Unless otherwise specified, a reference to a particular compound includes all of its isomeric forms. For example, when a compound has a C═N—X moiety (X═NHR, OR, or R, wherein R is any suitable group, e.g., H, alkyl, aryl, or acyl, etc.), such as hydrazone (X═NHR), imine (X═R), or oxime (X═OR) derivatives, the X group can exist in syn- (E-) or anti- (Z-) configuration relative to the other portion of the molecule, in particular the groups on both sides of the C═N bond, as would be understood by a person or ordinary skill in the art. When a compound has multiple such C═N—X moieties, multiple isomers would be possible. In the present disclosure, all such isomers, in pure or mixture forms or any combinations thereof, are encompassed by the name or structure of such compounds, as would be understood by those of ordinary skill in the pertinent art, even though they are not explicitly called by names or shown in the structures presented. Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate (e.g., hydrate), protected forms, and other stereoisomers thereof, for example, as discussed herein.
In the compounds disclosed herein, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. For example, substitution with heavier isotopes, such as replacing hydrogen (H) with deuterium (i.e., 2H or D) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. In addition, certain isotopically-labeled compounds (e.g., with 3H and 14C) are useful in compound and/or substrate tissue distribution assays. Isotopically labeled compounds can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples below, by substituting an appropriate isotopically labeled reagent for a non-isotopically labeled reagent. The present invention is meant to include all suitable isotopic variations of the compounds disclosed.
It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, “Pharmaceutically Acceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19.
When the term “about” is applied to a number or parameter, the number or parameter can vary by ±10%, inclusive. As would be understood by a person skilled in the art, when a parameter is not critical, a number is often given only for illustration purpose, instead of being limiting.
The term “a,” “an,” or “the,” as used herein, represents both singular and plural forms. In general, when either a singular or a plural form of a noun is used, it denotes both singular and plural forms of the noun.
The term “treating,” “treatment,” or “therapy,” or the like, as used herein in the context of treating a disease or condition, pertains generally to treatment and therapy of an animal subject, preferably a human, in which some desired therapeutic effect is achieved. For example, therapy can include the inhibition of the progress of the condition, reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, absolute or partial prevention of a delayed complication, and cure of the condition. Treatment also includes prophylactic measure as well as adjunct treatments to a standard treatment regimen established in the art.
The term “therapeutically effective amount,” as used herein, pertains to that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.
It may be convenient or desirable to prepare, purify, and/or handle the active compound in the form of a prodrug. The term “prodrug” as used herein, pertains to a compound which, when metabolized, yields the desired active compound or in itself is the active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties. For example, some prodrugs are ethers or esters of the active compound; during metabolism the ether group is cleaved to yield the active drug. Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. Thus, in the methods of treatment of the present invention disclosed herein, the term “administering” shall encompass the treatment of the various conditions described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient. Metabolites of these compounds include active species produced upon introduction of compounds of this invention into the mammalian subject.
Any of the compounds of the present invention may be contemplated for administration to the human subject in the form of a drug, prodrug or even active metabolite.
In particular, although the compounds disclosed herein are prodrugs themselves relative to the parent drug compound, e.g., glucocorticoids (e.g. dexamethasone, prednisone, etc.), these compounds themselves may exist in their prodrug forms. For example, any of the hydroxyl or amino groups on the molecule may be protected by an acyl (e.g., acetyl) or other group that can be readily hydrolyzed under the physiological conditions to become the parent polyethylene glycol-glucocorticoid (PEG-GC) drug compound. Such additional level of prodrug may even be preferred under certain circumstances to further control the release mode and rate of the parent drug compound in a subject.
As noted herein, the salts of the compounds of this invention refer to non-toxic “pharmaceutically acceptable salts.” Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. When the compounds of the present invention contain a basic group, salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts include any such salt known in the art. Where compounds of the present invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
To treat a human patient, an effective amount of one or more compounds of the present invention, or a pharmaceutically-acceptable salt thereof, is administered to the human subject in need so as to promote exposure to or contact of the tissue at risk or the targeted region of the body or nerves, synapses, or neuromuscular junctions, or organ systems including but not limited to the autonomic and central nervous systems. Effective dosage forms, modes of administration and dosage amounts may be determined empirically, and making such determinations is within the skill of the art.
As discussed herein, the PEG-GC drug compounds disclosed herein can be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders (e.g., reconstitutable lyophilized powder), micronized compositions, granules, elixirs, tinctures, suspensions, ointments, vapors, liposomal particles, nanoparticles, syrups and emulsions. Likewise, they may also be administered in intravenous (bolus or infusion), intraperitoneal, topical (e.g., dermal, epidermal, transdermal, ophthalmically such as ocular eyedrop), intranasally, subcutaneous, inhalation, intramuscular or transdermal (e.g., patch, microneedles) form, all using forms well known to those of ordinary skill in the pharmaceutical arts. Again, the ordinarily skilled physician, veterinarian or clinician or a clinical pharmacist may readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
As noted herein, the compounds of the present invention may be used in combination with other drugs or therapies having similar or complementary effects to those of the compounds disclosed herein. The individual components of such combinations can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms to patients or regions of such patients in need of such therapy. The instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term “administering” is to be interpreted accordingly.
As used herein, the term “composition,” “pharmaceutical composition,” or the like, is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
The amount of the active ingredient(s) which will be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration and all of the other factors described above. The amount of the active ingredient(s) which will be combined with a carrier material to produce a single dosage form will generally be that amount of the active ingredient(s) which is the lowest dose effective to produce a therapeutic effect.
Pharmaceutical formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders (e.g., reconstitutable lyophilized powder), granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup or tincture, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and the like, each containing a predetermined amount of the active ingredient(s). The active ingredient(s) may also be administered as a bolus, electuary or paste.
The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, or in specialized capsules for vapor or nebulized administration and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water or oil for injection, immediately prior to use. Extemporaneous injection solutions and suspensions maybe prepared from sterile powders, granules and tablets of the type described above.
PEG-DiDex is an amphiphilic macromolecular prodrug with a Dex dimer constitutes the hydrophobic section. A mPEG 1900 was employed as the hydrophilic section. The utility of this short PEG as the carrier will not only reduce WBC internalization but also significantly reduce the serum half-life of the prodrug, which may greatly limit the prodrug's distribution to the mononuclear phagocyte system (MPS). Due to the use of PEG as the drug carrier, PEG-DiDex activation is slow (
Treatment of NZB/W F1 mice with severe nephritis with PEG-DiDex monthly effectively attenuated albuminuria and maintained 100% animal survival for the entire experiment duration. On the other hand, dose equivalent daily Dex treatment only presented with moderate efficacy and 80% survival (
Additional therapeutic benefit of PEG-DiDex treatment was also found in its capacity to more effectively attenuate systemic proinflammatory cytokine/chemokine levels than Dex treatment (
This effective regulation of systemic proinflammatory cytokine/chemokine may also explain the better-persevered bone quality in mice treated with PEG-DiDex (
PEG-DiDex also compares favorably with the N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-based Dex prodrug (the “P-Dex”) disclosed previously (see, e.g., WO 2005/097073A1, CN 101518654A, and CN 103059220A) on adverse effects. First, PEG-DiDex is a prodrug amphiphile that can self-assemble into micelles with the drug conjugated to chain terminus. The micelles disintegrate into monomers upon dilution in the circulation; whereas in P-Dex, the drug is conjugated to a polymer side chain. P-Dex does not form micelles and cannot disintegrate. Second, because of the use of relatively short PEG (˜2000 Da), PEG-DiDex is predicted to have a much shorter serum half-life than P-Dex, which constitutes its kidney targeting capacity and extremely low systemic toxicity as supported by the toxicity data. Third, the imaging results have shown that PEG-DiDex targets exclusively nephritis, whereas P-Dex has high accumulation in liver and spleen in addition to kidney. Moreover, when dPEG (a commercially available PEG with a single molecular weight) is used, the PEG-DiDex should behave like a small-molecule or biologic drug with a single molecular weight, without the polydispersity that polymer drug conjugates typically have. In addition, when Dex is conjugated to a multifunctional PEG carrier via a hydrazone bond, the prodrug activation rate is slower than P-Dex. Therefore, by design the PEG-Dex hereby disclosed greatly reduces serum free Dex concentration by limiting the prodrug's sequestration by WBC and its deposition in the liver and spleen; whereas the decreased molecular weight and serum half-life of P-Dex would lead to lower liver and spleen distribution.
Optical imaging-based in vivo biodistribution data (
Immunohistological and flow cytometry analyses revealed that Alexa Fluor 488-labeled PEG-DiDex was mainly sequestered by CD11-b+ (monocytes), F4/80+ (macrophages), CD146+ (endothelial cells) and CD326+ (epithelial cells) in the kidneys of NZB/W F1 mice. After 8 weeks of treatment, the kidney of PEG-DiDex treated NZB/W F1 mice showed a significantly lower monocyte/macrophage population and local inflammatory cytokine level than dose equivalent Dex treated animals, suggesting the much effective and sustained local anti-inflammatory effect of PEG-DiDex. The prodrug's internalization, subcellular activation and inflammation resolution were further validated with in vitro cell culture systems.
In summary, the present invention provides a novel micelle-forming PEG-based dexamethasone prodrug (PEG-DiDex) with superior and sustained efficacy against lupus nephritis, but without typical glucocorticoid side effects. While it is hardly perceivable that the Dex released from PEG-DiDex would attenuate inflammation via a different molecular mechanism, this newly developed prodrug indeed altered the Dex's pharmacology on the physiology level by restricting the Dex's distribution to the inflamed kidney and providing a sustained local concentration of Dex via the gradual activation of PEG-DiDex within the endosomal/lysosomal compartments. Given its outstanding therapeutic efficacy and safety profile, PEG-DiDex can provide better clinical management of lupus nephritis. Moreover, the novel glucocorticoid prodrugs may also find wider applications in other renal pathologies, such as minimal change disease, IgA nephropathy, focal segmental glomerulosclerosis, and kidney transplant, since glucocorticoids are commonly used in managing these conditions as well.
The following non-limiting Examples further illustrate certain aspects of the present invention.
Synthesis and Characterization of PEG-DiDex
Polyethylene glycol monomethyl ether 1900 (mPEG, 1.9 kDa) and N-Fmoc-L-glutamic acid were purchased from Alfa Aesar (MA, USA). Dexamethasone (Dex) was obtained from Tianjin Pharmaceuticals Group Co., Ltd. (Tianjin, China). Dexamethasone phosphate was purchased from Hawkins, Inc. (Minneapolis, Minn., USA). Dexamethasone phosphate disodium was purchased from BUFA (The Netherlands). Peperidine was purchased from Sigma-Aldrich (St. Louis, Mo., USA). Dess-Martin periodinane was obtained from Oakwood Products, Inc. (Estill, S.C., USA). IRDye 800CW NHS ester was purchased from LI-COR Biosciences (Lincoln, Nebr., USA). Alexa Fluor 488 NHS ester was obtained from Life Technologies (Carlsbad, Calif., USA). Sephadex LH-20 resins were purchased from GE HealthCare (Piscataway, N.J., USA). All solvents and other reagents if not specified were purchased from Fisher Scientific or ACROS and used without further purification.
Instruments
1H and 13C NMR spectra were recorded on a 500 MHz NMR spectrometer (Varian, Palo Alto, Calif., USA). Electrospray ionization mass spectrometry was performed on a LCQ Classic Mass Spectrometer (Finnigan MAT, San Jose, Calif., USA). HPLC analyses were done on an Agilent 1100 HPLC system (Agilent Technologies, Inc., Santa Clara, Calif., USA) with a reverse phase C18 column (Agilent, ZORBAX 300SB-C18, 4.6×250 mm, 5 μm). In vivo near-infrared fluorescence (NIRF)-based optical imaging was accomplished on a LI-COR Pear™ Impulse Small Animal Imaging System (Lincoln, Nebr., USA). Bone qualities were analyzed using a high-resolution micro-CT system (Skyscan 1172, Skyscan, Aartselaar, Belgium). The average hydrodynamic diameter, polydispersity index (PDI) and zeta potential of micelles were determined by dynamic light scattering (DLS) experiments using a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK). The micelles morphology was observed using a Tecnai G2 Spirit transmission electron microscope (TEM) (FEI, Hillsboro, Oreg., USA) at an acceleration voltage of 80 kV. Digital images were acquired using a KeenView high-resolution camera and analyzed using Soft Imaging Solutions AnalySIS ITEM digital software. The quantification of fluorescence signal intensities of IRDye 800 CW, Alexa Fluor 488 and pyrene were measured using Spectramax M2 spectrofluorometer (Molecular Devices, Sunnyvale, Calif.). The flow cytometry analyses were performed using a FACSCalibur flow cytometer (BD Biosciences). A Waters e2695 system equipped with a Waters 2489 absorption detector and a Waters Qtof Micro electrospray ionization mass spectrometer was used to perform high perfor-mance liquid chromatography/mass spectrometry analyses.
The Synthesis of Amphiphilic Macromolecular Dexamethasone Prodrug (PEG-DiDex)
PEG-DiDex, a polyethylene glycol (PEG)-based amphiphilic dexamethasone prodrug, was successfully synthesized according to the route illustrated in Scheme 1. The identity of the polymeric prodrug and the absence of free Dex were confirmed using LC-MS/MS. The multi-step synthesis is straightforward with high yield at each step. Due to the utility of hydrazone as the prodrug activation trigger that links Dex to glutamic acid and the overall dimer design, at least 4 syn/anti hydrazone configure isomers can be formed. These isomers of the Dex dimer (compound 6) were chromatographically separated using LC-MS/MS with chromatography conditions stated in the Instruments section. Mass spectra (positive ion ESI) for these isomers showed the molecular ion [M+H]+ at 1066.7, which confirms their monoisotopic mass of 1065.7. The theoretical Dex content in PEG-DiDex is calculated as 26.7 wt %. After complete hydrolysis of the prodrug, the HPLC analysis showed that 26.4 wt % of the prodrug was released in the form of intact dexamethasone, suggesting the PEG-DiDex prodrug micelle synthesized has a ˜99% purity.
As shown in Scheme 1, the prodrug was synthesized by conjugating a Dex dimer to polyethylene glycol (PEG) 2000 chain terminus via a glutamate/glycine/hydrazone linker system.
Dexamethasone (7.84 g, 20 mmol) and imidazole (2.72 g, 40 mmol) were dissolved in anhydrous DMF (40 mL) and the solution was cooled to 0° C. tert-butylmethylsilyl chloride (TBSCl, 3.3 g, 22 mmol) was added. The solution was stirred at 0° C. for 3 hours and then allowed to room temperature for 2 hours. Ethyl acetate (100 mL) was added and washed with brine (80 mL×4). The organic phase was dried over MgSO4 and then the solvent was removed. The residue was purified with flash chromatography (ethyl acetate/hexanes=1/2) to give 9.98 g of compound 1 (98.5% yield).
1H NMR (500 MHz, CDCl3): δ (ppm)=7.26 (d, J=10.0, 1H), 6.32 (d, J=10.0, 6.10 (s, 1H), 4.63 (d, J=18.0, 1H), 4.38 (d, J=18.0, 1H), 4.37 (m, 1H), 3.23 (s, 1H), 3.05 (m, 1H), 2.62 (td, J=13.5, 6.0, 1H), 2.51 (s, 1H), 2.38 (m, 3H), 2.22 (m, 1H), 1.82 (m, 1H), 1.75 (m, J=7.0, 1H), 1.56 (m, 1H), 1.55 (s, 3H), 1.45 (d, J=13.5, 1H), 1.24 (m, 1H), 1.06 (s, 3H), 0.92 (s, 9H), 0.91 (d, J=7.0, 3H), 0.103 (s, 3H), 0.098 (s, 3H).
13C NMR (125 MHz, CDCl3): δ (ppm)=209.474, 186.768, 166.725, 152.226, 129.617, 124.904, 91.147, 72.159, 71.853, 69.462, 48.503, 43.635, 37.164, 36.146, 34.240, 34.084, 32.289, 31.045, 27.296, 25.814, 22.931, 22.886, 18.442, 17.168, 14.810, −5.328, −5.4662.
The starting material 1 (2.53 g, 5 mmol) and NH2NH2 monohydrate (750 mg, 15 mmol) were dissolved in methanol (25 mL). Acetic acid (60 mg, 1 mmol) was added and the solution was stirred at room temperature for 5 hours. Ethyl acetate (100 mL) was added and washed with brine (80 ml×4). The organic phase was dried over MgSO4 and then the solvent was removed. The residue was purified with flash chromatography (ethyl acetate/hexanes=1/1) to give 1.14 g of compound 2. A total of 1.24 g starting material was recovered. The final yield was calculated as 85.8%. Due to the formation of hydrazone bond, the product is a mixture of two syn/anti hydrazone configure isomers, which can't be separated by flash chromatography. NMR analysis suggests the molar ratio of the two isomers is 1.75:1.
1H NMR (500 MHz, d6-DMSO): δ (ppm)=6.66 (dd, J=10.2 Hz, 1.5 Hz,), 6.34 (s, J=10.2 Hz, 6.28 (dd, J=10.2 Hz, 1.9 Hz), 6.14 (d, J=10.2 Hz), 5.99 (s), 5.24 (br, s), 4.61 (d, J=18.0 Hz), 4.59 (d, J=18.0 Hz), 4.38 (d, J=18.0 Hz), 4.36 (m), 3.05 (m, 1H), 3.03 (s), 2.97 (t, J=4.8 Hz), 2.62 (td, J=13.5, 5.8 Hz), 2.51 (td, J=13.5, 5.8 Hz), 2.40-2.20 (m), 1.70-1.60 (m), 1.55-1.50 (m), 1.476 (s), 1.472 (s), 1.40 (s), 1.37 (s), 1.24-1.20 (m), 1.04 (s), 0.92 (s), 0.90 (d, J=7.3 Hz), 0.103 (s). MS (ESI): m/z=521.5 (M+H+), calculated: 520.3.
Compound 2 (2.86 g, 5.5 mmol), dimethylaminopyridine (DMAP, 201 mg, 1.65 mmol) were dissolved in anhydrous DMF (15 mL) and the solution was cooled to 0° C. Then Fmoc-glycine (2.12 g, 7.15 mmol), dicyclohexylcarbodiimide (DCC, 1.70 g, 8.25 mmol) were added to the solution. The solution was stirred at 0° C. for 3 hours. Ethyl acetate (100 mL) was added and washed with brine (80 mL×4). The organic phase was dried over MgSO4 and then the solvent was removed. The residue was purified with flash chromatography (ethyl acetate/hexanes=1:1) to give 3.72 g of compound 3 (84.5% yield).
Compound 3 (3.0 g, 3.75 mmol) was dissolved in dichloromethane (DCM, 10 mL). The solution was cooled to 0° C. with ice-water bath. Piperidine (1 mL) was added. The solution was stirred at 0° C. for 1 hour. Ethyl acetate (100 mL) was added and washed with brine (80 mL×3). The organic phase was dried over MgSO4 and then the solvent was removed. Toluene (50 mL) was added and then evaporated to remove the residue piperidine. The residue was then purified with flash chromatography (ethyl acetate and then ethyl acetate/methanol=2.5/1) to give 1.96 g of compound 4 (90.6% yield).
1H NMR (500 MHz, d6-DMSO): δ (ppm)=7.01 (d, J=10.2 Hz), 6.83 (d, J=10.2 Hz), 6.77 (s), 6.66 (d, J=10.2 Hz), 6.57 (d, J=10.2 Hz), 6.56 (s), 6.43 (d, J=10.2 Hz), 6.38 (d, J=9.8 Hz), 6.27 (d, J=10.2 Hz), 6.18 (d, J=9.8 Hz), 6.00 (s), 5.92 (s), 5.15 (s), 5.13 (s), 4.94 (s), 4.76 (d, J=9.0 Hz), 4.27 (d, J=9.0 Hz), 4.11 (br), 2.88 (br), 2.70-2.50 (m), 2.49 (s), 2.40-2.20 (m), 2.15-2.05 (m), 1.73-1.63 (m), 1.63-1.53 (m), 1.41 (s), 1.40 (s), 1.37 (s), 1.35-1.25 (m), 1.10-1.00 (m), 0.87 (s), 0.84 (s), 0.76 (d, J=6.8 Hz), 0.03 (s), 0.02 (s). MS (ESI): m/z=578.3 (M+H+), calculated: 577.3.
Compound 4 (444 mg, 0.768 mmol) was dissolved in anhydrous DMF (3 mL), Fmoc-glutamic acid (135 mg, 0.366 mmol), DCC (226 mg, 1.098 mmol) and hydroxybenzotriazole (HOBt, 148 mg, 1.098 mmol) were added. The solution was stirred at room temperature for 4 hours. Ethyl acetate (100 mL) was added and washed with brine (80 mL×3). The organic phase was dried over MgSO4 and then the solvent was removed. The residue was then purified with flash chromatography (ethyl acetate/methanol=10/1) to give 471 mg of compound 5 (86.5% yield). Due to the presence of multiple syn/anti-hydrazone groups in the Dex dimer, the assignment of peaks in the 1H NMR spectrum of compound 5 was complex, so LC-MS/MS was used to confirm the identity of compound 5.
Compound 5 (450 mg, 0.3 mmol) was dissolved in DCM (4.5 mL). The solution was cooled to 0° C. with ice-water bath. Piperidine (1.5 mL) was added. The solution was stirred at 0° C. for 1 hour. Ethyl acetate (100 mL) was added and washed with brine (80 mL×3). The organic phase was dried over MgSO4 and then the solvent was removed. The residue was then purified with flash chromatography (ethyl acetate and followed by ethyl acetate/methanol=3/1) to give 330 mg of compound 6 (86.4% yield). MS (ESI): m/z=1266.7 (M+H+), calculated: 1265.7.
mPEG-COOH (100 mg, 0.052 mmol), HOBt (70.2 mg, 0.52 mmol) and DCC (107 mg, 0.52 mmol) were dissolved in DMF (3 mL) and the solution was stirred at room temperature for 1 hour. Compound 6 (428 mg, 0.34 mmol) was added. The solution was stirred at room temperature for 24 hours and then applied to LH-20 column to separate the polymeric fraction. After evaporation of the solvent, the residue was dissolved in tetra-n-butylammonium fluoride (TBAF, 1 M, 2 mL). The solution was stirred for 2 hours. The resulting solution was again applied onto LH-20 column to give 118.7 mg of compound 7 (PEG-DiDex, 77.8% yield).
MS (ESI): two clusters of peaks were observed in the mass spectrum: one appears at m/z≈1550, representing the diion peaks of PEG-DiDex; and the other at m/z≈1100, representing triion peaks of PEG-DiDex. The appearance of multiple peaks at each m/z value can be attributed to the polydispersity of mPEG used. For example, the peak at 1546.7 represents diionic peak of PEG-DiDex with 44 repeating ethylene glycol unit in mPEG; the peak at 1068.5 represents triionic peak PEG-DiDex with 46 repeating ethylene glycol units in mPEG.
The monomethyl citrate (0.206 g, 1 mmol) is dissolved in anhydrous DMF (15 mL) and the solution is cooled to 0° C. DCC (0.494 g, 2.4 mmol), compound 2 (1.144 g, 2.2 mmol) and DMAP (48.8 mg, 0.4 mmol) are added. The solution is stirred at 0° C. for 3 hours. Ethyl acetate (100 mL) is added and washed with brine (80 mL×4). The organic phase is dried over MgSO4 and then the solvent is removed. The residue is purified with column chromatography (ethyl acetate/hexanes=1/1) to give compound 8.
Compound 3 (0.9 g, 0.75 mmol) and mPEG-NH2 (0.285 g, 0.15 mmol) are dissolved in anhydrous DMF (5 mL) and the solution is heated to 80° C. for 6 hours under the protection of Argon. Then the solution is allowed to room temperature and TBAF (3 mL, 1 M in THF) is added. The solution was stirred for 1 hour at room temperature. The solution is then concentrated and then purified by LH-20 chromatography to give Compound 9.
The Pentaerythritol derivative (0.41 g, 1 mmol) is dissolved in anhydrous DMF (5 mL). DCC (0.82 g, 4 mmol), HOBt (0.405 g, 3 mmol) and Et3N (0.30 g, 3 mmol) are added and the solution is stirred for 30 min, and mPEG-NH2 (0.38 g, 0.2 mmol) is added. The solution is stirred at room temperature for 20 hours. It is then purified by LH-20 to give compound 10.
Compound 10 (0.23 g, 0.1 mmol) and sodium hydroxide (12 mg, 0.3 mmol) are dissolved in a mixture solution of methanol and water (1:1, 5 mL). The solution is stirred at room temperature for 5 hours. Then HCl (3 mL, 1M) is added. The solvent is then removed and the residue is purified by LH-20 to give the deprotected intermediate. It is then dissolved in anhydrous dichloromethane (5 mL). DCC (0.30 g, 1.5 mmol), HOBt (0.20 g, 1.5 mmol) and Et3N (0.15 g, 1.5 mmol) are added and the solution is stirred for 30 min, then compound 2 (0.62 g, 1.2 mmol) is added. The solution is stirred at room temperature for 20 hours. The solution is then loaded on to a silica gel column and ethyl acetate/hexanes=1:1 is used as eluent to recover the unreacted compound 2. Then methanol is used as eluent to get the crude product. The solvent is then removed and the residue is dissolved in THF (5 mL) and TBAF (1.5 mL, 1 M in THF) is added. The solution is stirred at room temperature for 1 hour. The solution is then concentrated and purified by LH-20 to give the final product compound 11.
mPEG-CH2COOH (0.19 g, 0.1 mmol) was dissolved in anhydrous DMF (5 mL). DCC (0.21 g, 1 mmol), HOBt (0.135 g, 1 mmol) and Et3N (0.1 g, 1 mmol) were added and the solution was stirred for 30 min, and then the compound 2 (0.26 g, 0.5 mmol) was added. The solution was stirred at room temperature for 20 hours. TBAF (0.5 mL, 1 M in THF) was added and the solution was stirred for 1 hour, followed by LH-20 purification to give compound 12.
HOOCCH2-PEG-CH2COOH (0.14 g, 0.07 mmol) was dissolved in anhydrous DMF (5 mL). DCC (0.142 g, 0.7 mmol), HOBt (0.093 g, 0.7 mmol) were added and the solution was stirred for 30 min, and then the compound 2 (0.398 g, 0.7 mmol) was added. The solution was stirred at room temperature for 15 hours. TBAF (1 mL, 1 M in THF) was added and the solution was stirred for 1 h and then purified by LH-20 to give compound 13.
Fmoc-L-glutamic acid (93 mg, 0.25 mmol) is dissolved in anhydrous DMF (5 mL). DCC (0.62 g, 3 mmol), HOBt (0.41 g, 3 mmol) are added and the solution is stirred for 30 min, and then compound 6 (0.79 g, 0.625 mmol) is added. The solution is stirred at room temperature for 15 hours. Ethyl acetate (100 mL) is added and washed with brine (80 mL×4). The organic phase is dried over MgSO4 and then the solvent was removed. The residue is purified with flash chromatography (ethyl acetate/methanol=5/1) to give the product which is dissolved in dichloromethane (3 mL), piperidine (1 mL) is then added and the solution is stirred for 1 hour and then the solution is purified by column chromatography (ethyl acetate/methanol=2.5/1) to give compound 14.
The mPEG-CH2COOH (50 mg, 0.025 mmol) is dissolved in anhydrous DMF (3 mL). DCC (0.10 g, 0.5 mmol), HOBt (68 mg, 0.5 mmol) are added and the solution is stirred for 30 min, and then the compound 14 (0.43 g, 0.15 mmol) is added. The solution is stirred at room temperature for 15 hours. TBAF (0.5 mL, 1 M in THF) is added and the solution is stirred for 1 hour and then the solution is purified by LH-20 to give compound 15.
Characterization and Testing of PEG-Dex Dimer Prodrug (PEG-DiDex)
Formation of PEG-DiDex Micelles
With an amphiphilic structural design, PEG-DiDex can form micelles through self-assembly. PEG-DiDex (26.5 mg) was dissolved in distilled water (1 mL) and equilibrated at 37° C. for 4 hours to allow micelles formation. The micelle solution was then diluted to 3 mg/mL (1.02×10−3 M) by adding double distilled water and then used for the following characterization.
Micelle Characterization
The average hydrodynamic diameter, polydispersity index (PDI) and ζ-potential of the micelles were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK). The intensity of scattered light was measured at a 1730 scattering angle. To understand the micelles morphology, transmission electronic microscopy (TEM) was used to visualize micelles deposited on formvar/silicone monoxide coated 200 mesh copper grids surface.
Pyrene-based fluorescence polarization method was used to determine the critical micelle concentration (CMC) for PEG-DiDex. For sample preparation, known amounts of stock solution of pyrene in acetone were added to empty wells of 96-well plate. Aqueous solutions of PEG-DiDex at different concentrations were added to the wells and evaporate for 2 hours at room temperature. The pyrene concentration in the final solution was 0.6 μM, which is slightly below its water solubility in water at room temperature. Before measurement, the samples from each well were transferred to a quartz 96-well plate, and the fluorescence intensity was measured with excitation wavelength of 334 nm and emission wavelength at 373 nm (I1) and 384 nm (I3) using a fluorescence microplate spectrofluorometer (Molecular Devices, Sunnyvale, Calif.). Ratio of fluorescence intensity I1/I3 was plotted against prodrug concentration to obtain the CMC value.
To understand the impact of pH values and the presence of serum proteins on the release of Dex from PEG-DiDex, the prodrug (3 mg/mL) was dissolved in acetate buffers (pH=5.0, 6.5 and 7.4) and mouse serum (0.2 wt % sodium azide as bacteriostat). Pluoronic F127 was added as in order to create sink condition. The micelle solutions were placed in a shaking incubator (60 r/min) at 37° C. At selected time points, the releasing solution (0.5 mL) was withdrawn, neutralized with NaOH (0.1 M) and analyzed with HPLC to determine the free Dex concentration. The analysis of each sample was performed in triplicate. The accumulative release of Dex from PEG-DiDex micelles was calculated according the following equation, where Ci refers to the concentration of Dex at time i.
To quantify the Dex content in PEG-DiDex, the prodrug (1 mg) was dissolved in HCl (0.5 mL, 0.1 N) overnight. The sample (50 μL) was withdrawn and neutralized by addition of NaOH (50 μL, 0.1N), then diluted in acetonitrile (CAN, 0.9 mL). The sample (in triplet) was analyzed using an Agilent 1100 HPLC system equipped with a reverse phase C18 column (Agilent, ZORBAX 300SB-C18, 4.6×250 mm, 5 μm). Mobile phase: acetonitrile/water=30/70; detection wavelength, 240 nm; flow rate, 1 mL/min; Injection volume, 10 μL. The Dex content in PEG-DiDex was then calculated based on the HPLC analysis result.
Testing Results
Dynamic light scattering (DLS) measurement indicates that the PEG-DiDex can indeed form micelle with an average micelle diameter of ˜11 nm, a polydispersity index (PDI) of 0.345 and a ζ-potential of −5±4.61 mV. As shown in the TEM images (
As the Dex activation trigger, the hydrazone bond in the PEG-DiDex design can only be cleaved via acidic environment. This was confirmed by the in vitro drug release experiment. As shown in
Treating Lupus Nephritis with PEG-DiDex
Beginning at 20 weeks of age, NZB/W F1 female mice (Jackson Laboratories, Bar Harbor, Me.) were randomized into three test groups (saline control, Dex and PEG-DiDex). Their urine protein level was monitored weekly using Albustix Reagent Strips (Siemens Healthineers). Only mice with established nephritis, as evidenced by sustained albuminuria (≥100 mg/dL) over 2 weeks, were enrolled in the study. PEG-DiDex treatment (106 mg/kg, containing 28 mg/kg of dexamethasone, n=10) and saline (n=12) were administered as a monthly i.v. injection. The Dex treatment (dexamethasone 21-phosphate disodium, 1.32 mg/kg, containing 1.00 mg/kg of dexamethasone, n=11) was given as daily i.v. injection. All treatments continued for 8 weeks. The body weight and proteinuria level of the animals was monitored on a weekly basis. Peripheral blood was collected from saphenous vein every 4 weeks for serum analyses. Mice that developed severe proteinuria (≥2000 mg/dl) or showed signs of distress (e.g. reduced mobility, weight loss >20%, edema, unkempt appearance) were sacrificed immediately. The surviving mice were monitored for an additional two weeks after the last treatment. The mice were then euthanized by CO2 asphyxiation, with all major tissues and organs isolated, weighted and processed at necropsy. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of University of Nebraska Medical Center (UNMC).
Analysis of Bone Quality
Femoral bone quality was analyzed using a Skyscan 1172 micro-CT system. The micro-CT scanning parameters were set as the following: voltage 48 kV, current 187 μA, exposure time 620 msec, resolution 6.07 μm, and aluminum filter 0.5 mm. Three-dimensional reconstructions were performed with NRecon and DataViewer software (SkyScan). Trabecular bone was selected for analysis based on a polygonal region of interest within the center of the femur, starting at 20 slices (0.25 mm) proximal from the growth plate and extending proximally 80 slices (0.99 mm) further. Trabecular bone volume/tissue volume (BV/TV), the mean bone mineral density (BMD), trabecular number and thickness were quantified with CTAn software (SkyScan).
Near-Infrared Imaging Study
After the proteinuria was established, NZB/W F1 mice (n=6) were given IRDye 800 CW-labeled PEG-DiDex (IRDye 800 CW dose at 148 nmol/kg, Dex equivalent dose of 28 mg/kg) via tail vein injection. The same dose of IRDye 800 CW-labeled PEG-DiDex was also given intravenously to NZW mice (n=6, healthy control). At selected time points (1 and 4 days post administration), the mice were euthanized and perfused with saline. All major organs (i.e. heart, lung, liver, spleen, kidney and adrenal gland) were isolated and imaged using a LI-COR Pearl™ Impulse Small Animal Imaging System to evaluate the distribution and retention of PEG-DiDex.
Flow Cytometry Analysis
After the proteinuria was established, NZB/W F1 mice (n=6) and NZW mice (healthy control) were given Alexa Fluor 488-labeled PEG-DiDex (Alexa Fluor 488 dose at 300 nmol/kg, Dex equivalent dose of 28 mg/kg) via tail vein injection. At selected time points (1 and 4 days post administration), the animals were euthanized and perfused. White blood cells were isolated from peripheral blood. Bone marrow, kidney, spleen and liver were harvested, macerated, and passed through a 70-μm strainer to prepare single-cell suspensions. Cells were marked by the following antibodies: PE-labeled anti-mouse CD3e(17A2), CD11b, F4/80, NK1.1, CD146, prominin (Miltenyi Biotec) and CD19 (eBioscience Inc.); APC-labeled anti-mouse CD11c, Ly-6G, CD326, CD117; anti-mouse GL7-eFluor660. The cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences).
Statistics
Most of the statistical analyses were performed using SPSS software (version 19.0). The data that does not assume to follow a normal distribution was compared using the Kruskal-Wallis test, a nonparametric alternative to one-way analysis of variance. To evaluate specific differences between experimental groups, Tukey's post hoc test and Mann-Whitney U test were used in the comparisons of normal-distributed and non-normal distributed data respectively. Two-tailed P values≤0.05 were considered significant.
For inflammatory cytokine/chemokine analysis, the data obtained were log 2 transformed to make them more normally distributed. The mixed effects model with AR(1) correlation among repeated measures over time of the same animal were used to fit the log transformed cytokine expression values for each cytokine separately. Three different comparisons were conducted to evaluate (1) difference between the treatment groups at each observation time; (2) difference between different times within each treatment group; and (3) difference in the change of the cytokines expression from baseline among different treatment groups. The Benjamini-Hochberg method was used to control the false discovery rate for multiple comparisons. The results on the differences at log-scaled data, the standard error, the raw p value without adjustment for false discovery rate, and the adjusted p value for BH false discovery rate control were reported.
PEG-DiDex Effectively Ameliorated Proteinuria and Improved the Survival of NZB/W F1 Mice with Severe Nephritis
To evaluate the therapeutic potential of PEG-DiDex, it was given was administered monthly to NZB/W F1 female mice (˜28 wks) with fully developed nephritis, as evidenced by sustained albuminuria. The treatment was continued for 8 weeks. Dose equivalent daily Dex treatment and monthly saline administration were used as controls. As shown in
For additional evidence of PEG-DiDex's superior therapeutic efficacy, the kidneys from the tested animals were further sectioned and stained with PSA. They were then examined by a pathologist (KWF), who was blind to the group design. The periodic-acid schiff (PAS) stained kidney sections were also graded by a histopathologic score system with a 4-point scale. As shown in
PEG-DiDex Treatment Attenuates Tissue-Damaging Proinflammatory Cytokines/Chemokines.
As shown in
PEG-DiDex Treatment does not Lead to Typical GC Toxicities
To understand the impact of PEG-DiDex treatment on bones, the femoral mean bone mineral density (BMD) and micro-architecture were evaluated using a high resolution μ-CT (Skyscan 1172). The BMD value and trabecular thickness in the femoral trabecular bone of PEG-DiDex treated mice were significantly higher than that observed in both saline and Dex group (
Chronic exposure to GC therapy is known to be associated with systemic immunosuppression. To understand if PEG-DiDex as a GC prodrug would be similarly immune suppressive, we evaluated the end point total serum IgG level and the peripheral white blood cell (WBC) counts during the time course of the experimental. As shown in
GC exposure, even in short term may suppress hypothalamic-pituitary-adrenal (HPA) axis, leading to clinical atrophy of the adrenal gland. To understand if PEG-DiDex treatment would cause adrenal gland atrophy, we analyzed the end point adrenal gland mass. The mean adrenal gland mass in the Dex group was significantly lower than the PEG-DiDex group (
PEG-DiDex Passively Targeted to Nephritic Kidneys in NZB/W F1 Mice
To understand the potentiated therapeutic efficacy and greatly reduced GC-associated toxicities of PEG-DiDex (a GC prodrug), the in vivo biodistribution of PEG-DiDex was analyzed using near-infrared optical imaging. Both NZB/W F1 mice and NZW mice received intravenous injections of IRDye 800 CW-labeled PEG-DiDex. The animals were sacrificed on day 1 and day 4 post injection and all vital organs (i.e. heart, lung, liver, spleen, kidney and adrenal gland) were harvested for the optical imaging. As shown in
PEG-DiDex Treatment does not Alter Serum Anti-dsDNA Level
GCs are known to exert its therapeutic effect against lupus partially through down regulation of anti-dsDNA antibody level. Therefore, it would be of great interest to see if a Dex prodrug, such as PEG-DiDex, treatment would ameliorate lupus symptoms according to a similar pharmacology. As shown in
Characterization of PEG-DiDex and Drug Loading Efficiency
Effective hydrodynamic diameters (Deff) and polydispersity index (PDI) of PEG-DiDex dimer were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK). PEG-DiDex dimer was dissolved into PBS (pH 7.4) at a concentration of 2 mg/mL. The DLS analysis showed the hydrodynamic size for PEG-DiDex dimer was 273.5 nm with a PDI value of 0.464.
To quantify Dex loading in PEG-DiDex, 1 mg of the prodrug was dissolved in 0.5 ml of buffer (HCL, 0.1N) overnight. 50 μl of the sample was neutralized by 50 μl NaOH (0.1N), and then diluted in 0.9 ml ACN. 1 ml of the sample (triplet) was analyzed with an Agilent 1100 HPLC system with a quaternary pump (with degasser), an autosampler and a diode-array based UV detector. Mobile phase, acetonitrile/water=30/70; Detection, UV 240 nm; Flow rate, 1 ml/min; Injection volume, 10 μl. The mean value and standard deviation were obtained with Microsoft Excel. The HPLC analysis showed that 26.38% of the prodrug was released as the form of dexamethasone. Since the theoretical drug content of PEG-DiDex is 26.75%, 98.6% of theoretical Dex content was covalently conjugated to the prodrug.
PEG-DiDex Dimer Treatment of Lupus Nephritis
Experimental Animals and Drug Treatment
Beginning at 28 weeks of age, (NZB×NZW)F1 female mice (Jackson Laboratories, Bar Harbor, Me.) groups of mice were randomized into saline, Dex and PEG-DiDex groups and monitored weekly for albuminuria using Albustix dipsticks (Siemens Corp., Washington D.C.). Only the mice in each group with established nephritis, evidenced by sustained albuminuria (100 mg/dl) over a monitoring period of 2 weeks, were officially enrolled in the study. PEG-DiDex (106 mg/kg, containing 28 mg/kg of dexamethasone, n=10) and saline groups (n=12) were administered via a single injection per month. The free Dex group was given daily i.v. injections of dexamethasone 21-phosphate disodium (n=11, Dex, 1.32 mg/kg, containing 1.00 mg/kg of dexamethasone, Hawkins, Inc., Minneapolis, Minn.). Treatment continued for 8 weeks. Mice were monitored for an additional two weeks after cessation of treatment. After that, the mice were euthanized and the spleen, kidneys, adrenal glands and left femur were harvest. PEG-DiDex reverses established albuminuria, extends survival rates and reduces incidence of severe nephritis
To determine if PEG-DiDex could ameliorate established nephritis, PEG-DiDex was administered monthly to (NZB×NZW)F1 females beginning at ˜28 weeks of age, after they had developed nephritis, as evidenced by sustained albuminuria. Treatment was continued for 8 weeks. Two control groups, one receiving dose equivalent daily Dex and the other receiving a monthly dose of saline, were also treated for 8 weeks. Mice were monitored for an additional two weeks after cessation of treatment. Over the entire experimental time course, albuminuria not only persisted in 100% of the mice in the saline treated group, but also increased in severity in most of these mice (75%) (
By contrast, albuminuria resolved in 60% of the mice in the PEG-DiDex group (
Prior to the end of the experiment, ˜42% of mice in the saline group and ˜18% of Dex treated mice were euthanized due to severe nephritis (
Evaluation of Treatment-Induced Side Effects
PEG-DiDex Treatment does not Affect Bone Quality
Peripheral dual x-ray absorptiometry was performed with a scanning speed of 20 mm/second and resolution of 0.2×0.2 mm. The micro-CT scanning parameters were as follows: voltage 48 kV, current 187 μA, exposure time 620 msec, resolution 6.07 μm, and aluminum filter 0.5 mm. Three-dimensional reconstructions were performed with NRecon and DataViewer software (SkyScan). Trabecular bone was selected for analysis based on a polygonal region of interest within the center of the femur, starting at 20 slices (0.25 mm) proximal from the growth plate and extending proximally 80 slices (0.99 mm) further. Trabecular bone volume/tissue volume (BV/TV), the mean bone mineral density (BMD), trabecular number, and trabecular thickness were quantified with CTAn software (SkyScan).
Osteoporosis is a major adverse side effect of long-term use of GCs. To investigate the impact of PEG-DiDex on the skeleton, the femoral BMD and micro-architecture were evaluated. Trabecular bone volume/tissue volume (BV/TV) and trabecular thickness did not differ significantly from the means in the saline group (
PEG-DiDex Treatment does not Lead to Peripheral White Blood Cells Reduction
GC therapy is associated with immunosuppression. Therefore, we monitored peripheral white blood cell (WBC) counts and during the experimental time course. In comparison of healthy control mice, peripheral white blood cell (WBC) counts were significantly lower in the other three groups (data not shown). But the PEG-DiDex group exhibited significantly greater WBC counts compared to the Dex group (
PEG-DiDex Treatment does not Induce Adrenal Gland Atrophy
GC therapy causes suppression of hypothalamic-pituitary-adrenal (HPA) axis and atrophy of the adrenal glands. Therefore, at necropsy, we determined the mass of the adrenal glands in each mouse. The mean adrenal mass in the Dex group was significantly lower than the PEG-DiDex group (
In summary, PEG-DiDex treatment would potentiate lupus nephritis resolution in terms of prolonged lifespan and reduced incidence of severe nephritis, as well as a reduced risk of systemic toxicities and side effects.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as claimed in the appending claims.
All publications, patents, and patent applications cited in this application are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference.
This application is a Continuation of U.S. patent application Ser. No. 16/694,722, filed on Nov. 25, 2019, which is a Continuation of U.S. patent application Ser. No. 15/775,647, filed on May 11, 2018, now U.S. Pat. No. 10,485,809, which is the U.S. National Phase of International Patent Application No. PCT/US2016/061728, filed on Nov. 12, 2016, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/254,512, filed on Nov. 12, 2015, all of which are herein incorporated by reference in their entirety.
| Number | Name | Date | Kind |
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| 8796248 | Zhang et al. | Aug 2014 | B2 |
| 10485809 | Wang et al. | Nov 2019 | B2 |
| 10933071 | Wang | Mar 2021 | B2 |
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| 103764172 | Apr 2014 | CN |
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| Liu et al., “Syntheses of Click PEG-Dexamethasone Conjugates for teh Treatment of Rheumatoid Arthritis,” Biomacromolecules (Oct. 11, 2010); 11(10);2621-2628. |
| Number | Date | Country | |
|---|---|---|---|
| 20210252018 A1 | Aug 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62254512 | Nov 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16694722 | Nov 2019 | US |
| Child | 17188309 | US | |
| Parent | 15775647 | US | |
| Child | 16694722 | US |
