Patent Application
20230108186
The present disclosure relates generally to nitric oxide-releasing compounds, their synthesis, and their use in treating disorders mediated by nitric oxide. Pharmaceutical compositions comprising these compounds and their methods of use are also disclosed.
Nitric oxide (NO) is known to play important functional roles in a variety of physiological systems. Within the vasculature, NO induces vasodilation, inhibits platelet aggregation, prevents neutrophil/platelet adhesion to endothelial cells, inhibits smooth muscle cell proliferation and migration, regulates programmed cell death (apoptosis) and maintains endothelial cell barrier function. NO generated by neurons acts as a neurotransmitter, whereas NO generated by macrophages in response to invading microbes acts as an antimicrobial agent. Because neurons, blood vessels and cells of the immune system are integral parts of the reproductive organs, and in view of the important functional role that NO plays in those systems, NO is an important regulator of the biology and physiology of the reproductive system (Rosselli et al., Role of nitric oxide in the biology, physiology and pathophysiology of reproduction, Hum Reprod Update. 1998 January-February; 4(1):3-24).
NO-releasing compounds (i.e., NO donors) have been proposed as therapeutics, often in the form of polymers with side-chains that include nitric oxide-releasing moieties, such as nitrosothiols and diazeniumdiolates. Nitric oxide-releasing polymers have hetetofore been underused as therapeutics, based at least in part on limited NO payloads, NO release rates that are more rapid than desired, and the lack of targeted NO delivery.
It would be advantageous to have new NO-donor compounds, pharmaceutical compositions comprising the NO donor compounds, and methods of treating patients suffering from disorders mediated by nitric oxide using these compounds and compositions. The present invention provides such compounds, compositions, and methods.
Nitric oxide, an endogenously produced diatomic free radical, is associated with numerous biological processes. Exogenous NO delivery can be an effective strategy for treating or preventing a variety of disease states, including baldness, ischemia/reperfusion injury, thrombosis/restenosis, a fibrotic disease, a cancer, a cardiovascular disease, a disease of platelet aggregation and platelet adhesion, sickle cell disease, a disease caused by or characterized by low nitric oxide levels, a metabolic disease, or a response to a medical device, pathological conditions resulting from abnormal cell proliferation, autoimmune diseases, inflammation, vascular diseases, restenosis, pain, fever, gastrointestinal disorders, respiratory disorders, and sexual dysfunctions. Methods for treating these disorders are also disclosed herein.
Nitric oxide-releasing compounds (also referred to as nitric oxide donors or NO donors), compositions containing such compounds, and methods of treating microbial infections using the compounds and compositions, are disclosed.
In one embodiment, the compounds disclosed herein have the following formula:
wherein:
X is selected from the group consisting of H, D, R, and RC(O)—,
R is C1-12 alkyl, aryl, heteroaryl, alkylaryl, or arylalkyl, optionally substituted with one or more substituents as defined herein,
and M+ is a pharmaceutically-acceptable cation.
In some embodiments, M+ is a cation with a valence other than one, for example, +2 or +3, in which case the ratio of the compound of Formula I to the cation is such that the total positive charge equals the total negative charge. So, for a compound with a total charge of negative three, and a cation with a total charge of positive two, there would be two compounds and three cations.
Representative positively charged cations include sodium, potassium, lithium, calcium, magnesium, and quaternary ammonium salts.
Methods for making these compounds are also disclosed. In one embodiment, compounds with an R(CO)— moiety that does not include acidic α C—H (i.e., alpha to the carbonyl), such as aryl, heteraryl, and branched alkyl groups, like t-butyl groups, can be prepared by reacting all acidic α C—H on the methyl group of a compound with the formula R(CO)CH3 with nitric oxide in basic methanol to give trisdiazeniumdiolates. A representative reaction is shown below:
By way of example, the reaction product of acetophenone with nitric oxide in KOH/methanol is:
In another embodiment, compounds where X is H or D can be prepared by reacting acetone with nitric oxide in basic methanol or deuterated methanol to give tris-diazeniumdiolates.
In another embodiment, the NO-releasing compound has the structure of Formula II:
where M+ is as defined above with respect to Formula I. In some embodiments, the cation is sodium, lithium, potassium, or a quaternary ammonium salt.
The compounds of Formula II can be prepared, for example, by reacting acetone, acetonitrile, or ethanol with NO, optionally at high pressures (i.e., pressures above atmospheric pressure, ideally above about 2 ATM of pressure, and preferably above about 10 ATM of pressure) in the presence of a base, such as a methoxide/methanol solution, to form one or more diazeniumdiolate-containing species. In several embodiments, high purity compounds (greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 98%) can be produced according to the methods disclosed herein.
In still another embodiment, the NO-releasing compound has the structure of Formula III:
In some embodiments, the compounds of any of Formulas I, II or III have a purity in excess of 96%, in excess of 97%, in excess of 98%, in excess of 99%, or in excess of 99.5%. The present disclosure also relates to compounds having this purity level.
In one embodiment, the nitric oxide-releasing compound has a NO-release half-life, at normal physiological temperature and pH, of between 0.1 and 24 hours. In another embodiment, the NO-release half-life is at least 15 minutes. In some embodiments, the compound has a total releasable NO storage in a range of 2-10 μmol of NO per mg of NO donor compound. In several embodiments, the compound has a total duration of NO release in the range of 1-60 hours. In several embodiments, the total NO release after 4 hours is in the range between 0.1-1.0 μmol of NO per mg of compound.
The compounds can be formulated in a variety of pharmaceutical compositions, for delivery intravenously, via inhalation, via nebulization, via intranasal administration, via oral administration, via injection, via rectal or vaginal administration, and via topical administration.
In one embodiment, the pharmaceutical compositions comprise one or more nitric oxide-releasing compounds described herein and an aqueous solution. In one aspect of this embodiment, the nitric oxide releasing compound has an aqueous solubility of at least about 25 mg/ml in the aqueous solution, at a physiologically compatible pH.
The pharmaceutical compositions can further include one or more additional active agents, depending on the type of disorder to be treated. For example, where the disorder is cancer, one or more additional anticancer agents can be co-administered. Where the disorder is an inflammatory disorder, one or more additional anti-inflammatory compounds can also be present. Where the disorder is an autoimmune disorder, antibody products and JAK 1/2 inhibitor compounds, such as Jakafi or Baricitinib, can be co-administered. Where the disorder is a CNS disorder associated with low dopamine levels, dopamine agonists or partial agonists can be co-administered.
In some embodiments, the compositions can further include one or more of a chelating agent, a mucoadhesive agent, or a low molecular weight polyethylene glycol.
In certain embodiments, the small molecule nitric oxide donors are provided in dilute solutions (e.g., for nebulization, vaporization or inhalation), and in other embodiments, are provided in the form of gels or viscous liquids, for example, for topical administration.
Methods for treating the disorders listed above are also disclosed. In some embodiments, the methods involve delivering nitric oxide to a subject in need of treatment, by delivering the compounds of any of Formulas I, II or III, and having the compound degrade upon exposure to physiological pH and temperature to release nitric oxide.
The compositions and related methods set forth in further detail below describe certain actions taken by a practitioner; however, it should be understood that they can also include the instructions of those actions by another party. Thus, actions such as “administering a NO-donating compound” include “instructing the administration of a NO-donating compound.”
The embodiments discussed above will be better understood with reference to the following detailed description.
Nitric oxide (NO) is implicated in a wide variety of disorders, including baldness, ischemia/reperfusion injury, thrombosis/restenosis, a fibrotic disease, a cancer, a cardiovascular disease, a disease of platelet aggregation and platelet adhesion, sickle cell disease, a disease caused by or characterized by low nitric oxide levels, a metabolic disease, or a response to a medical device, pathological conditions resulting from abnormal cell proliferation, autoimmune diseases, inflammation, vascular diseases, restenosis, pain, fever, gastrointestinal disorders, respiratory disorders, and sexual dysfunctions, including impotence and hypogonadism, cardiac disorders, and pulmonary disorders.
The role of NO as a therapeutic has heretofore been underused, based at least in part on limited NO payloads of therapeutic compositions, NO release rates that are more rapid than desired, and a lack of targeted NO delivery.
NO-releasing compounds, compositions comprising such compounds, methods of producing such compounds and compositions, and methods of treating or preventing disorders associated with nitric oxide, are disclosed. In some embodiments, the compounds are present in pharmaceutical compositions with desired physical properties, such as viscosity and gelation.
The compounds are small molecules, i.e., have a molecular weight below around 500 g/mol, and, in some embodiments, around 200 g/mol, not including the associated cation. One of the advantages of using small molecules over polymers is that the compounds can be prepared with relatively lower impurity levels than polymeric compounds. Further, relative to polymeric compounds, the NO load can be higher, because the percent composition ratio between NO to the scaffold can be maximized, as described herein.
Small molecule precursor compounds, which can be converted to the NO-releasing compounds described herein, can be selected with a relatively low number of reactive groups, for example, a hydrogens adjacent a carbonyl group, reducing the possibility that many different species will result from a nitrosation reaction. As a result, nitrosylation of the NO-precursor can proceed with little or no partial reaction products, which provides the potential for relatively pure products.
Knowing the structure of the small molecule allows for more predictable release kinetics than that obtainable with polymers.
Before the subject disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments of the disclosure described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.
In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
The present invention will be better understood with reference to the following definitions.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. The terminology used in the description of the subject matter herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the subject matter.
The term “effective amount,” as used herein, refers broadly to that amount of a recited compound effective to treat, prevent, reduce the severity or progression of a disorder associated with nitric oxide. microbial load in a subject afflicted with a microbial infection. This includes improving the subject's condition (e.g., in one or more symptoms), delaying or reducting the progression of the disorder, preventing or delaying the onset of the disorder, and/or changing clinical parameters, disease or illness, etc., as would be well known in the art.
For example, an effective amount can refer to the amount of a composition, compound, or agent that improves a condition in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
In some embodiments, an improvement in a condition can be a reduction in . . . in a subject. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired response for a particular subject. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.
“Treat” or “treating” or “treatment” refers broadly to any type of action that imparts a desired physiological effect, including treating or preventing a microbial infection, reducing the microbial load, improving the condition of the subject (e.g., in one or more symptoms), delaying or reducing the progression of the infection, and/or changing one or more clinical parameters.
The terms “nitric oxide donor” or “NO donor” refer broadly to species and/or molecules that donate, release and/or directly or indirectly transfer a nitric oxide species, and/or stimulate the endogenous production of nitric oxide in vivo and/or elevate endogenous levels of nitric oxide in vivo, such that the biological activity of the nitric oxide species is expressed at the intended site of action.
The terms “nitric oxide releasing” or “nitric oxide donating” refer to species that donate, release and/or directly or indirectly transfer any one (or two or more) of the three redox forms of nitrogen monoxide (NO+, NO−, NO (e.g., •NO)) and/or methods of donating, releasing and/or directly or indirectly transferring any one (or two or more) of the three redox forms of nitrogen monoxide (NO+, NO−, NO). In some embodiments, the nitric oxide releasing is accomplished such that the biological activity of the nitrogen monoxide species is expressed at the intended site of action.
The “patient” or “subject” treated as disclosed herein is, in some embodiments, a human patient, although it is to be understood that the principles of the presently disclosed subject matter indicate that the presently disclosed subject matter is effective with respect to all vertebrate species, including mammals, which are intended to be included in the terms “subject” and “patient.” Suitable subjects are generally mammalian subjects. The subject matter described herein finds use in research as well as veterinary and medical applications. The term “mammal” as used herein includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dog, rabbits, rodents (e.g., rats or mice), monkeys, etc. Human subjects include neonates, infants, juveniles, adults and geriatric subjects. The subject “in need of” the methods disclosed herein can be a subject that is experiencing a disease state and/or is anticipated to experience a disease state, and the methods and compositions of the invention are used for therapeutic and/or prophylactic treatment.
For the general chemical formulas provided herein, if no substituent is indicated, a person of ordinary skill in the art will appreciate that the substituent is hydrogen. A bond that is not connected to an atom, but is shown, indicates that the position of such substituent is variable. A jagged line, wavy line, two wavy lines drawn through a bond or at the end of a bond indicates that some additional structure is bonded to that position. For a great number of the additional monomers disclosed herein, but not explicitly shown in structures, it is understood by those in the art of polymers, that these monomers can be added to change the physical properties of the resultant polymeric materials even where the elemental analysis would not indicate such a distinction could be expected. Such physical properties include solubility, charge, stability, cross-linking, secondary and tertiary structure, and the like. Moreover, if no stereochemistry is indicated for compounds having one or more chiral centers, all enantiomers and diasteromers are included. Similarly, for a recitation of aliphatic or alkyl groups, all structural isomers thereof also are included. Unless otherwise stated, groups shown as A1 through An and referred to herein as an alkyl group, in the general formulas provided herein are independently selected from alkyl or aliphatic groups, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl. The alkyl may be optionally substituted (e.g., substituted or not substituted, as disclosed elsewhere herein). The alkyl may be a substituted alkyl group, such as alkyl halide (e.g. —CX3 where X is a halide, and combinations thereof, either in the chain or bonded thereto), alcohols (e.g. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) or other similarly substituted moieties such as amino-, amino acid-, aryl-, alkyl aryl-, alkyl ester-, ether-, keto-, nitro-, sulfhydryl-, sulfonyl-, sulfoxide modified-alkyl groups.
The term “amino” and “amine” refer to nitrogen-containing groups such as NR3, NH3, NHR2, and NH2R, wherein R can be as described elsewhere herein. Thus, “amino” as used herein can refer to a primary amine, a secondary amine, or a tertiary amine. In some embodiments, one R of an amino group can be a diazeniumdiolate (e.g., NONO).
Whenever a group is described as being “optionally substituted” that group may be unsubstituted or substituted with one or more of the indicated substituents. Likewise, when a group is described as being “unsubstituted or substituted” (or “substituted or unsubstituted”) if substituted, the substituent(s) may be selected from one or more of the indicated substituents. If no substituents are indicated, it is meant that the indicated “optionally substituted” or “substituted” group may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), cycloalkyl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxy, alkoxy, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, nitro, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, an amino, a mono-substituted amine group, a di-substituted amine group, a mono-substituted amine(alkyl), a di-substituted amine(alkyl), a diamino-group, a polyamino, a diether-group, and a polyether-group.
As used herein, “Ca to Cb” in which “a” and “b” are integers refer to the number of carbon atoms in a group. The indicated group can contain from “a” to “b”, inclusive, carbon atoms. Thus, for example, a “C1 to C4 alkyl” or “C1-C4 alkyl” group refers broadly to all alkyl groups having from 1 to 4 carbons, that is, CH3—, CH3CH2—, CH3CH2CH2—, (CH3)2CH—, CH3CH2CH2CH2—, CH3CH2CH(CH3)— and (CH3)3C—. If no “a” and “b” are designated, the broadest range described in these definitions is to be assumed.
As used herein, the term “alkyl” refers broadly to a fully saturated aliphatic hydrocarbon group. The alkyl moiety may be branched or straight chain. Examples of branched alkyl groups include, but are not limited to, iso-propyl, sec-butyl, t-butyl and the like. Examples of straight chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and the like. The alkyl group may have 1 to 30 carbon atoms (whenever it appears herein, a numerical range such as “1 to 30” refers broadly to each integer in the given range; e.g., “1 to 30 carbon atoms” means that the alkyl group may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated). The “alkyl” group may also be a medium size alkyl having 1 to 12 carbon atoms. The “alkyl” group could also be a lower alkyl having 1 to 6 carbon atoms. An alkyl group may be substituted or unsubstituted. By way of example only, “C1-C5 alkyl” indicates that there are one to five carbon atoms in the alkyl chain, e.g., the alkyl chain is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl (branched and straight-chained), etc. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl and hexyl.
As used herein, the term “alkylene” refers broadly to a bivalent fully saturated straight chain aliphatic hydrocarbon group. Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene and octylene. An alkylene group may be represented by followed by the number of carbon atoms, followed by a “*”. For example,
to represent ethylene. The alkylene group may have 1 to 30 carbon atoms (whenever it appears herein, a numerical range such as “1 to 30” refers broadly to each integer in the given range; e.g., “1 to 30 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 30 carbon atoms, although the present definition also covers the occurrence of the term “alkylene” where no numerical range is designated). The alkylene group may also be a medium size alkyl having 1 to 12 carbon atoms. The alkylene group could also be a lower alkyl having 1 to 6 carbon atoms. An alkylene group may be substituted or unsubstituted. For example, a lower alkylene group can be substituted by replacing one or more hydrogens of the lower alkylene group and/or by substituting both hydrogens on the same carbon with a C3-6 monocyclic cycloalkyl group
The term “alkenyl” used herein refers broadly to a monovalent straight or branched chain radical of from two to twenty carbon atoms containing a carbon double bond(s) including, but not limited to, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and the like. An alkenyl group may be unsubstituted or substituted.
The term “alkynyl” used herein refers broadly to a monovalent straight or branched chain radical of from two to twenty carbon atoms containing a carbon triple bond(s) including, but not limited to, 1-propynyl, 1-butynyl, 2-butynyl and the like. An alkynyl group may be unsubstituted or substituted.
As used herein, “cycloalkyl” refers broadly to a completely saturated (no double or triple bonds) mono- or multi-cyclic (such as bicyclic) hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a fused, bridged or spiro fashion. As used herein, the term “fused” refers broadly to two rings which have two atoms and one bond in common. As used herein, the term “bridged cycloalkyl” refers broadly to compounds wherein the cycloalkyl contains a linkage of one or more atoms connecting non-adjacent atoms. As used herein, the term “spiro” refers broadly to two rings which have one atom in common and the two rings are not linked by a bridge. Cycloalkyl groups can contain 3 to 30 atoms in the ring(s), 3 to 20 atoms in the ring(s), 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s) or 3 to 6 atoms in the ring(s). A cycloalkyl group may be unsubstituted or substituted. Examples of mono-cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Examples of fused cycloalkyl groups are decahydronaphthalenyl, dodecahydro-1H-phenalenyl and tetradecahydroanthracenyl; examples of bridged cycloalkyl groups are bicyclo[1.1.1]pentyl, adamantanyl and norbornanyl; and examples of spiro cycloalkyl groups include spiro [3.3]heptane and spiro [4.5]decane.
As used herein, “cycloalkenyl” refers broadly to a mono- or multi-cyclic (such as bicyclic) hydrocarbon ring system that contains one or more double bonds in at least one ring; although, if there is more than one, the double bonds cannot form a fully delocalized pi-electron system throughout all the rings (otherwise the group would be “aryl,” as defined herein). Cycloalkenyl groups can contain 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s) or 3 to 6 atoms in the ring(s). When composed of two or more rings, the rings may be connected together in a fused, bridged, or spiro fashion. A cycloalkenyl group may be unsubstituted or substituted.
As used herein, “aryl” refers broadly to a carbocyclic (all carbon) monocyclic or multicyclic (such as bicyclic) aromatic ring system (including fused ring systems where two carbocyclic rings share a chemical bond) that has a fully delocalized pi-electron system throughout all the rings. The number of carbon atoms in an aryl group can vary. For example, the aryl group can be a C6-C14 aryl group, a C6-C10 aryl group or a C6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene. An aryl group may be substituted or unsubstituted. As used herein, “heteroaryl” refers to a monocyclic or multicyclic (such as bicyclic) aromatic ring system (a ring system with fully delocalized pi-electron system) that contain(s) one or more heteroatoms (for example, 1, 2 or 3 heteroatoms), that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur. The number of atoms in the ring(s) of a heteroaryl group can vary. For example, the heteroaryl group can contain 4 to 14 atoms in the ring(s), 5 to 10 atoms in the ring(s) or 5 to 6 atoms in the ring(s), such as nine carbon atoms and one heteroatom; eight carbon atoms and two heteroatoms; seven carbon atoms and three heteroatoms; eight carbon atoms and one heteroatom; seven carbon atoms and two heteroatoms; six carbon atoms and three heteroatoms; five carbon atoms and four heteroatoms; five carbon atoms and one heteroatom; four carbon atoms and two heteroatoms; three carbon atoms and three heteroatoms; four carbon atoms and one heteroatom; three carbon atoms and two heteroatoms; or two carbon atoms and three heteroatoms. Furthermore, the term “heteroaryl” includes fused ring systems where two rings, such as at least one aryl ring and at least one heteroaryl ring or at least two heteroaryl rings, share at least one chemical bond. Examples of heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline and triazine. A heteroaryl group may be substituted or unsubstituted.
As used herein, “heterocyclyl” or “heteroalicyclyl” refers broadly to three-, four-, five-, six-, seven-, eight-, nine-, ten-, up to 18-membered monocyclic, bicyclic and tricyclic ring system wherein carbon atoms together with from 1 to 5 heteroatoms constitute said ring system. A heterocycle may optionally contain one or more unsaturated bonds situated in such a way, however, that a fully delocalized pi-electron system does not occur throughout all the rings. The heteroatom(s) is an element other than carbon including, but not limited to, oxygen, sulfur and nitrogen. A heterocycle may further contain one or more carbonyl or thiocarbonyl functionalities, so as to make the definition include oxo-systems and thio-systems such as lactams, lactones, cyclic imides, cyclic thioimides and cyclic carbamates. When composed of two or more rings, the rings may be joined together in a fused, bridged or spiro fashion. As used herein, the term “fused” refers to two rings which have two atoms and one bond in common. As used herein, the term “bridged heterocyclyl” or “bridged heteroalicyclyl” refers to compounds wherein the heterocyclyl or heteroalicyclyl contains a linkage of one or more atoms connecting non-adjacent atoms. As used herein, the term “spiro” refers to two rings which have one atom in common and the two rings are not linked by a bridge. Heterocyclyl and heteroalicyclyl groups can contain 3 to 30 atoms in the ring(s), 3 to 20 atoms in the ring(s), 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s) or 3 to 6 atoms in the ring(s). For example, five carbon atoms and one heteroatom; four carbon atoms and two heteroatoms; three carbon atoms and three heteroatoms; four carbon atoms and one heteroatom; three carbon atoms and two heteroatoms; two carbon atoms and three heteroatoms; one carbon atom and four heteroatoms; three carbon atoms and one heteroatom; or two carbon atoms and one heteroatom. Additionally, any nitrogens in a heteroalicyclic may be quaternized. Heterocyclyl or heteroalicyclic groups may be unsubstituted or substituted. Examples of such “heterocyclyl” or “heteroalicyclyl” groups include but are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiole, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidine N-Oxide, piperidine, piperazine, pyrrolidine, azepane, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone and their benzo-fused analogs (e.g., benzimidazolidinone, tetrahydroquinoline and/or 3,4-methylenedioxyphenyl). Examples of spiro heterocyclyl groups include 2-azaspiro[3.3]heptane, 2-oxaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2-oxaspiro [3.4]octane and 2-azaspiro[3.4]octane.
As used herein, “aralkyl” and “aryl(alkyl)” refer broadly to an aryl group connected, as a substituent, via a lower alkylene group. The lower alkylene and aryl group of an aralkyl may be substituted or unsubstituted. Examples include but are not limited to benzyl, 2-phenylalkyl, 3-phenylalkyl and naphthylalkyl.
As used herein, “cycloalkyl(alkyl)” refer broadly to an cycloalkyl group connected, as a substituent, via a lower alkylene group. The lower alkylene and cycloalkyl group of a cycloalkyl(alkyl) may be substituted or unsubstituted.
As used herein, “heteroaralkyl” and “heteroaryl(alkyl)” refer broadly to a heteroaryl group connected, as a substituent, via a lower alkylene group. The lower alkylene and heteroaryl group of heteroaralkyl may be substituted or unsubstituted. Examples include but are not limited to 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl and imidazolylalkyl and their benzo-fused analogs.
A “heteroalicyclyl(alkyl)” and “heterocyclyl(alkyl)” refer broadly to a heterocyclic or a heteroalicyclic group connected, as a substituent, via a lower alkylene group. The lower alkylene and heterocyclyl of a (heteroalicyclyl)alkyl may be substituted or unsubstituted. Examples include but are not limited tetrahydro-2H-pyran-4-yl(methyl), piperidin-4-yl(ethyl), piperidin yl(propyl), tetrahydro-2H-thiopyran-4-yl(methyl) and 1,3-thiazinan-4-yl(methyl).
As used herein, the term “hydroxy” refers broadly to a —OH group.
As used herein, “alkoxy” refers broadly to the Formula —OR wherein R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl) is defined herein. A non-limiting list of alkoxys are methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, phenoxy and benzoxy. An alkoxy may be substituted or unsubstituted.
As used herein, “acyl” refers broadly to a hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl) and heterocyclyl(alkyl) connected, as substituents, via a carbonyl group. Examples include formyl, acetyl, propanoyl, benzoyl and acryl. An acyl may be substituted or unsubstituted.
As used herein, a “cyano” group refers broadly to a “—CN” group.
The term “halogen atom” or “halogen” as used herein, means any one of the radio-stable atoms of column 7 of the Periodic Table of the Elements, such as, fluorine, chlorine, bromine and iodine.
A “thiocarbonyl” group refers broadly to a “—C(═S)R” group in which R can be the same as defined with respect to O-carboxy. A thiocarbonyl may be substituted or unsubstituted. An “O-carbamyl” group refers to a “—OC(═O)N(RARB)” group in which RA and RB can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An O-carbamyl may be substituted or unsubstituted.
An “N-carbamyl” group refers broadly to an “ROC(═O)N(RA)—” group in which R and RA can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An N-carbamyl may be substituted or unsubstituted.
An “O-thiocarbamyl” group refers broadly to a “—OC(═S)—N(RARB)” group in which RA and RB can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An O-thiocarbamyl may be substituted or unsubstituted.
An “N-thiocarbamyl” group refers broadly to an “ROC(═S)N(RA)—” group in which R and RA can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An N-thiocarbamyl may be substituted or unsubstituted.
A “C-amido” group refers broadly to a “—C(═O)N(RARB)” group in which RA and RB can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). A C-amido may be substituted or unsubstituted.
An “N-amido” group refers broadly to a “RC(═O)N(RA)—” group in which R and RA can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An N-amido may be substituted or unsubstituted.
An “S-sulfonamido” group refers broadly to a “—SO2N(RARB)” group in which RA and RB can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An S-sulfonamido may be substituted or unsubstituted.
An “N-sulfonamido” group refers broadly to a “RSO2N(RA)—” group in which R and RA can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). An N-sulfonamido may be substituted or unsubstituted.
An “O-carboxy” group refers broadly to a “RC(═O)O—” group in which R can be hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein. An O-carboxy may be substituted or unsubstituted.
The terms “ester” and “C-carboxy” refer broadly to a “—C(═O)OR” group in which R can be the same as defined with respect to O-carboxy. An ester and C-carboxy may be substituted or unsubstituted.
A “nitro” group refers broadly to an “—NO2” group.
A “sulfenyl” group refers broadly to an “—SR” group in which R can be hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl). A sulfenyl may be substituted or unsubstituted.
A “sulfinyl” group refers broadly to an “—S(═O)—R” group in which R can be the same as defined with respect to sulfenyl. A sulfinyl may be substituted or unsubstituted.
A “sulfonyl” group refers broadly to an “SO2R” group in which R can be the same as defined with respect to sulfenyl. A sulfonyl may be substituted or unsubstituted.
As used herein, “haloalkyl” refers broadly to an alkyl group in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono-haloalkyl, di-haloalkyl, tri-haloalkyl and polyhaloalkyl). Such groups include but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-chloro-2-fluoromethyl, 2-fluoroisobutyl and pentafluoroethyl. A haloalkyl may be substituted or unsubstituted.
As used herein, “haloalkoxy” refers broadly to an alkoxy group in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono-haloalkoxy, di-haloalkoxy and tri-haloalkoxy). Such groups include but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 1-chloro-2-fluoromethoxy and 2-fluoroisobutoxy. A haloalkoxy may be substituted or unsubstituted.
The terms “amino” and “unsubstituted amino” as used herein refer broadly to a —NH2 group.
A “mono-substituted amine” group refers broadly to a “—NHRA” group in which RA can be an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein. The RA may be substituted or unsubstituted. A mono-substituted amine group can include, for example, a mono-alkylamine group, a mono-C1-C6 alkylamine group, a mono-arylamine group, a mono-C6-C10 arylamine group and the like. Examples of mono-substituted amine groups include, but are not limited to, —NH(methyl), —NH(phenyl) and the like.
A “di-substituted amine” group refers broadly to a “—NRARB” group in which RA and RB can be independently an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein. RA and RB can independently be substituted or unsubstituted. A di-substituted amine group can include, for example, a di-alkylamine group, a di-C1-C6 alkylamine group, a di-arylamine group, a di-C6-C10 arylamine group and the like. Examples of di-substituted amine groups include, but are not limited to, —N(methyl)2, —N(phenyl)(methyl), —N(ethyl)(methyl) and the likes used herein, “mono-substituted amine(alkyl)” group refers broadly to a mono-substituted amine as provided herein connected, as a substituent, via a lower alkylene group. A mono-substituted amine(alkyl) may be substituted or unsubstituted. A mono-substituted amine(alkyl) group can include, for example, a mono-alkylamine(alkyl) group, a mono-C1-C6 alkylamine(C1-C6 alkyl) group, a mono-arylamine(alkyl group), a mono-C6-C10 arylamine(C1-C6 alkyl) group and the like. Examples of mono-substituted amine(alkyl) groups include, but are not limited to, —CH2NH(methyl), —CH2NH(phenyl), —CH2CH2NH(methyl), —CH2CH2NH(phenyl) and the like.
As used herein, “di-substituted amine(alkyl)” group refers broadly to a di-substituted amine as provided herein connected, as a substituent, via a lower alkylene group. A di-substituted amine(alkyl) may be substituted or unsubstituted. A di-substituted amine(alkyl) group can include, for example, a dialkylamine(alkyl) group, a di-C1-C6 alkylamine(C1-C6 alkyl) group, a di-arylamine(alkyl) group, a di-C6-C10 arylamine(C1-C6 alkyl) group and the like. Examples of di-substituted amine(alkyl)groups include, but are not limited to, —CH2N(methyl)2, —CH2N(phenyl)(methyl), —CH2N(ethyl)(methyl), —CH2CH2N(methyl)2, —CH2CH2N(phenyl)(methyl), —NCH2CH2(ethyl)(methyl) and the like.
As used herein, the term “diamino-” denotes a “—N(RA)RB—N(RC)(RD)” group in which RA, RC, and RD can be independently a hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein, and wherein RB connects the two “N” groups and can be (independently of RA, RC, and RD) a substituted or unsubstituted alkylene group. RA, RB, RC, and RD can independently further be substituted or unsubstituted.
As used herein, the term “polyamino” denotes a “—(N(RA)RB—)n—N(RC)(RD)”. For illustration, the term polyamino can comprise —N(RA)alkyl-N(RA)alkyl-N(RA)alkyl-N(RA)alkyl-H. In some embodiments, the alkyl of the polyamino is as disclosed elsewhere herein. While this example has only 4 repeat units, the term “polyamino” may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units. RA, RC, and RD can be independently a hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein, and wherein RB connects the two “N” groups and can be (independently of RA, RC, and RD) a substituted or unsubstituted alkylene group. RA, RC, and RD can independently further be substituted or unsubstituted. As noted here, the polyamino comprises amine groups with intervening alkyl groups (where alkyl is as defined elsewhere herein).
As used herein, the term “diether-” denotes an “—ORBO—RA” group in which RA can be a hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein, and wherein RB connects the two “O” groups and can be a substituted or unsubstituted alkylene group. RA can independently further be substituted or unsubstituted.
As used herein, the term “polyether” denotes a repeating —(ORB—)nORA group. For illustration, the term polyether can comprise —Oalkyl-Oalkyl-Oalkyl-Oalkyl-ORA. In some embodiments, the alkyl of the polyether is as disclosed elsewhere herein. While this example has only 4 repeat units, the term “polyether” may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units. RA can be a hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein. RB can be a substituted or unsubstituted alkylene group. RA can independently further be substituted or unsubstituted. As noted here, the polyether comprises ether groups with intervening alkyl groups (where alkyl is as defined elsewhere herein and can be optionally substituted).
Where the number of substituents is not specified (e.g. haloalkyl), there may be one or more substituents present. For example, “haloalkyl” may include one or more of the same or different halogens. As another example, “C1-C3 alkoxyphenyl” may include one or more of the same or different alkoxy groups containing one, two or three atoms. As used herein, a radical indicates species with a single, unpaired electron such that the species containing the radical can be covalently bonded to another species. Hence, in this context, a radical is not necessarily a free radical. Rather, a radical indicates a specific portion of a larger molecule. The term “radical” can be used interchangeably with the term “group.”
When a range of integers is given, the range includes any number falling within the range and the numbers defining ends of the range. For example, when the terms “integer from 1 to 20” is used, the integers included in the range are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., up to and including 20.
When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range.
The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “about 10 one millipascal-second” includes “10 one millipascal-second.”
Also as used herein, “and/or” refers broadly to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. The term “consists essentially of” (and grammatical variants), shall be given its ordinary meaning and shall also mean that the composition or method referred to can contain additional components as long as the additional components do not materially alter the composition or method. The term “consists of” (and grammatical variants), shall be given its ordinary meaning and shall also mean that the composition or method referred to is closed to additional components. The term “comprising” (and grammatical variants), shall be given its ordinary meaning and shall also mean that the composition or method referred to is open to contain additional components.
The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
As disclosed elsewhere herein, some embodiments disclosed herein pertain to small molecules capable of delivering NO to achieve microbicidal activity. In some embodiments, the cations present in the small molecules have antimicrobial or other desired physiological properties. In some embodiments, the compounds are water-soluble.
In one embodiment, provided herein is a NO releasing compound which exhibits potent antimicrobial characteristics, comprising the structure of Formula I:
wherein:
X is selected from the group consisting of H, D, R, and RC(O)—,
R is C1-12 alkyl, aryl, heteroaryl, alkylaryl, or arylalkyl, optionally substituted with one or more substituents,
wherein the substituents are independently selected from the group consisting of —OH, —NH2, —OCH3, —C(O)OH, —CH2OH, —CH2OCH3, —CH2OCH2CH2OH, —OCH2C(O)OH, —C H2OCH2C(O)OH, —CH2C(O)OH, —NHC(O)—CH3, —C(O)O((CH2)aO)b—H, —C(O)O((CH2)aO)b—(CH2)cH, —C(O)O(C1-5alkyl), —C(O)—NH—((CH2)dNH)e—H, —C(O)—NH4CH2)dNH)e—(CH2)fH, —O—((CH2) aO)b—H, —O—((CH2)aO)b—(CH2)cH, —O—(C1-5 alkyl), —NH—((CH2)dNH)e—H, and —NH—((CH2)dNH)e—(CH2)fH,
each instance of a, b, c, d, e, f, g, h, i, j, k, and 1 is independently selected from an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,
and M+ is a pharmaceutically-acceptable cation.
In some embodiments, M+ is a cation with a valence other than one, for example, +2 or +3, in which case the ratio of the compound of Formula I to the cation is such that the total positive charge equals the total negative charge. So, for a compound with a total charge of negative three, and a cation with a total charge of positive two, there would be two compounds and three cations.
Representative positively charged cations include sodium, potassium, lithium, calcium, magnesium, and quaternary ammonium salts.
In another embodiment, the compound has the following structure:
wherein M+ refers to a pharmaceutically-acceptable cation. The cation can be any pharmaceutically acceptable, non-toxic cation known to those skilled in the art, including but not limited to sodium, potassium, lithium, calcium, magnesium, ammonium, or substituted ammonium. It will be appreciated by those skilled in the art that when the cation (M) has a valency greater than one, the ratio of negative charge in the methyl trisdiazenium diolate moiety to the positive charge in the cation will balance out. For example, if the cation (M) has a charge of +2, then there is a ratio of 2 methyl trisdiazenium diolate moieties to three M+2 ions, and if the cation (M) has a charge of +3, then there is a 1/1 ratio of cation to methyl trisdiazenium diolate.
One representative compound is shown below:
Formula II is also described as a methane trisdiazeniumdiolate (MTDD), and Formula III as methane trisdiazeniumdiolate sodium salt.
Although various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, metal nitrosyls, organic nitrates) are known to provide for controlled exogenous NO delivery, the diazeniumdiolate moieties in the compounds disclosed herein are attractive because of their good stability and facile storage, and because they spontaneously undergo proton-triggered dissociation under physiological conditions to regenerate nitric oxide, including NO radicals.
The C-diazeniumdiolates described herein are pH-triggered NO-release donors. Reacting with protons under physiological conditions (e.g., 37° C., pH 7.4), 1 mole of Formula III generates two moles of NO radicals and 2 to 3 moles of nitroxyl compounds.
Several embodiments disclosed herein have one or more of the following advantages: efficient and unique synthesis routes and resultant chemical composition of small molecules.
In several embodiments, the NO-releasing compounds are stable at a variety of temperatures 20° C. (e.g., 40° C., 45° C., 55° C., 60° C., 80° C., etc.) and are stable for prolonged storage periods (e.g., 10 hours, 20 hours, 22 hours, 25 hours, 30 hours, etc., days such as 1 day, 3 days, 5 days, 6 days, 7 days, 15 days, 30 days, 45 days, etc., weeks such as 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, etc., months such as 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc., or even years (1 year or greater)).
In some embodiments, the compounds have NO storage capacities (in μmol NO/mg of the compounds) of greater than or equal to about: 0.25, 0.4, 0.5, 1.0, 1.5, 2.0, 3.0, or ranges including and/or spanning the aforementioned values. In some embodiments, within 2 h of being added to a PBS buffer solution, the compounds release greater than or equal to about: 25%, 50%, 75%, 85%, 90%, 95%, 100%, or ranges including and/or spanning the aforementioned values, their total wt % of bound NO. In several embodiments, NO release in use for reducing or eliminating a biofilm occurs in similar amounts, e.g., about 20-25%, about 30-50%, about 60-75%, at least 80%, at least 85%, at least 90%, at least 95%, ranges including and/or spanning the aforementioned values, of the total wt % of bound NO.
In some embodiments, the NO release may occur over a period of about 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 hours, 60 hours, or ranges including and/or spanning the aforementioned values. In several embodiments, the NO release half-life is equal to or at least about: 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or ranges including and/or spanning the aforementioned values. In some embodiments, the NO release occurs in less than or equal to about: 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours or ranges including and/or spanning the aforementioned values.
In some embodiments, the compounds have a degradation rate per hour in an amylase enzyme exposure assay of less than or equal to about: 0.2%, 0.5%, 1.0%, 1.5%, 2.5%, 5.0%, 10%, or ranges including and/or spanning the aforementioned values.
Several embodiments disclosed herein provide the synthesis and characterization of the diazeniumdiolate NO donor-modified compounds described herein. The synthesis of compounds capable of controlled NO storage and release is important for taking advantage of NO's role in physiology and for developing NO-based therapeutics.
Several embodiments disclosed herein have one or more of the following advantages: efficient and unique synthesis routes and resultant chemical composition of constructs. Certain compounds described herein have been previously disclosed, but the present disclosure describes their synthesis and utility in pharmaceutical compositions and methods of treating or preventing microbial infections, or reducing microbial loads.
There are a number of ways to make the compounds of Formula I. In one embodiment, compounds with an R(CO)— moiety that does not include acidic α C—H (i.e., alpha to the carbonyl), such as aryl, heteraryl, and branched alkyl groups, like t-butyl groups, can be prepared by reacting all acidic α C—H on the methyl group of a compound with the formula R(CO)CH3 with nitric oxide in basic methanol to give trisdiazeniumdiolates. A representative reaction is shown below:
By way of example, the reaction product of acetophenone with nitric oxide in KOH/methanol is:
In another embodiment, compounds where X is H or D can be prepared by reacting acetone with nitric oxide in basic methanol or deuterated methanol to give tris-diazeniumdiolates.
Formula II can be prepared via a number of different approaches, including the reaction of ethanol, acetonitrile, or acetone with nitric oxide gas, in the presence of a basic methanol solution. Ideally, the nitric oxide gas is present at a pressure greater than atmospheric pressure, more ideally, greater than two atmospheres of pressure, and, preferably, greater than ten atmospheres of pressure. Higher pressures help ensure complete reaction. Where there is an incomplete reaction, one of the by-products of the reaction is methane bis-diazeniumdiolate:
Methane bis-diazeniumdiolate does not release NO or NHO under physiological conditions.
The reaction of acetone with nitric oxide, at relatively high pressures, in the presence of basic methanol solutions such as sodium or potassium hydroxide in methanol, tends to provide the purest compound. The 13C NMR for methane tris-diazenium diolate prepared using this approach are shown in
The proposed reaction mechanism behind this reaction, using sodium hydroxide in methanol to provide the compound where M+ is Na+, is provided below:
The following proposed mechanism relates to the degradation of this compound to form nitric oxide:
1H and 13C NMR spectra for the degradation products are shown in
The degradation pathway is of particular interest, because it provides clarity in how NO is being produced from a carbon bound diazeniumdiolate. Typically, carbon bound diazeniumdiolates do not generate NO. Instead, they produce HNO, which dimerizes to form nitrous oxide, N2O. In this case, the initial decay of Formula III follows the expected HNO pathway, which in turn leads to the formation of an intermediate alcohol. Upon rearrangement of the alcohol intermediate, 2 moles of NO gas can be released. This matches the experimental results, which show that one mole of Formula III releases approximately 2 moles of NO. The NMR of the degraded Formula III by-product (shown in
Pharmaceutical compositions comprising one or more compounds of Formulas I, II or III, along with a suitable pharmaceutically acceptable carrier or excipient, are also disclosed.
According to several embodiments, the compounds described herein can be present in aqueous solutions comprising concentrations equal to or at least about 100 μg/mL, and can be higher, e.g. about 1 mg/ml, about 5 mg/ml, about 10 mg/ml, about 20/ml, or about 40 mg/ml or higher. The amount of the second compound in the aqueous composition can be at least about 10% by weight, based on the weight of the first compound, and may be higher, e.g., at least about 20% by weight, at least about 30% by weight, or at least about 50% by weight, same basis. The compounds in an aqueous composition are selected such that compounds are mutually miscible.
In some embodiments, the compositions disclosed herein provide NO-releasing compounds discussed herein having NO storage capacities (in μmol NO/mg powder) of greater than or equal to about: 2.0, 4.0, 6.0, 8.0, or 10.0 or ranges including and/or spanning the aforementioned values. In some embodiments, within 2 h of being added to a PBS buffer solution as described in the Examples, the NO-releasing compounds, release greater than or equal to about: 25%, 50%, 75%, 85%, 90%, 95%, 100%, or ranges including and/or spanning the aforementioned values, their total wt % of bound NO.
In some embodiments, the compositions are in the form of a liquid, a dry powder, a gel, or an aerosol. The compositions may be provided in the form of a formulation loaded into a delivery device, such as an inhaler.
In some embodiments, the composition includes a concentration of less than or equal to about: 1 mg/ml, 10 mg/ml, 20 mg/ml, 50 mg/ml, 100 mg/ml, 250 mg/ml of the compounds described herein, or ranges including and/or spanning the aforementioned values.
Formulations for Pulmonary Administration
In some embodiments, the compounds are administered to the pulmonary tract (i.e., via pulmonary administration). In one specific embodiment, pulmonary administration comprises inhalation of the compounds, typically in the form of particles or droplets, such as by nasal, oral inhalation, or both. The formulations can be developed to be aerosolized via a metered dose inhaler, a dry powder inhaler, a liquid spray or a nebulizer devises. Nebulization can be accomplished by compressed air, ultrasonic energy, or vibrating mesh to form a plurality of liquid droplets or solid particles comprising the NO-releasing compounds.
In one aspect of this embodiment, particles may be formulated as an aerosol (i.e.: liquid droplets of a stable dispersion or suspension of particles which include one or more of the compounds described herein in a gaseous medium). Particles delivered by aerosol may be deposited in the airways by gravitational sedimentation, inertial impaction, and/or diffusion.
Whether administered by inhalation or nebulization, the particles or droplets can be administered in two or more separate administrations (doses).
In one embodiment, the compositions are administered via inhalation to treat bacterial infections related to cystic fibrosis. Cystic fibrosis-related bacterial infections include, but are not limited to stenotrophomonis, mybacterium avium intracellulaire and M. abcessus, Burkhoderia cepacia and Pseudomonas aeruginosa (P. aeruginosa) infections.
Biodegradable particles can be used for the controlled-release and delivery of the compounds described herein. Aerosols for the delivery of therapeutic agents to the respiratory tract have been developed. Adjei, A. and Garren, J. Pharm Res. 7, 565-569 (1990); and Zanen, P. and Lamm, J.-W. J. Int. J. Pharm. 114, 111-115 (1995).
Porous Particles
The respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung. Gonda, I. “Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract,” in Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313, 1990. The deep lung, or alveoli, are the primary target of inhaled therapeutic aerosols for systemic drug delivery.
Accordingly, it can be important to deliver antiviral particles to the deep lung (i.e., the alveolar regions of the lung). Relatively large particles tend to get trapped in the oropharyngeal cavity, which can lead to excessive loss of the inhaled drug. Relatively smaller particles can be delivered to the deep lung, but can be phagocytosed. One way to deliver relatively large particles (sized to avoid phagocytosis), which are light enough to avoid excessive entrapment in the oropharyngeal cavity, is to use porous particles.
In one embodiment, the particles for delivering the compounds described herein to the alveolar regions of the lung are porous, “aerodynamically-light” particles, as described in U.S. Pat. No. 6,977,087. Aerodynamically light particles can be made of a biodegradable material, and typically have a tap density less than 0.4 g/cm3 and a mass mean diameter between 5 μm and 30 μm. The particles may be formed of biodegradable materials such as biodegradable polymers. For example, the particles may be formed of a functionalized polyester graft copolymer consisting of a linear alpha-hydroxy-acid polyester backbone having at least one amino acid group incorporated herein and at least one poly(amino acid) side chain extending from an amino acid group in the polyester backbone. In one embodiment, aerodynamically light particles having a large mean diameter, for example greater than 5 can be used for enhanced delivery of one or more of the compounds described herein to the alveolar region of the lung.
Aqueous Solutions
In several embodiments, the compounds disclosed herein are administered as aqueous solutions, for delivery, for example, topically, intranasally, intraveneously, by injection, and by nebulization. In several embodiments, the solutions comprise one or more salts and are isotonic.
Oral Drug Delivery Vehicles
In several embodiments, compositions can take the form of, for example, tablets, pills, or capsules, prepared using conventional techniques, with pharmaceutically acceptable excipients. Representative excipients include binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycollate), wetting agents (e.g., sodium lauryl sulphate), suspending agents, solubilizers, and mixtures thereof.
The tablets can be coated by methods known in the art. For example, a therapeutic agent can be formulated in combination with hydrochlorothiazide, and as a pH-stabilized core having an enteric or delayed release coating which protects the therapeutic agent until it reaches the target organ.
Liquid preparations for oral or topical administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or another suitable vehicle before use. Such liquid preparations can be prepared by conventional techniques with pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations also can contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to provide controlled release of the active compound. For buccal administration the compositions can take the form of tablets or lozenges formulated in a conventional manner.
Nanoparticulate Compositions
The compounds described herein can also be administered in the form of nanoparticulate compositions. In one embodiment, the controlled release nanoparticulate formulations comprise a nanoparticulate active agent to be administered and a rate-controlling polymer which functions to prolong the release of the agent following administration. In this embodiment, the compositions can release the active agent, following administration, for a time period ranging from about 2 to about 24 hours or up to 30 days or longer. Representative controlled release formulations including a nanoparticulate form of the active agent are described, for example, in U.S. Pat. No. 8,293,277.
Nanoparticulate compositions comprise particles of the active agents described herein, having a non-crosslinked surface stabilizer adsorbed onto, or associated with, their surface. The average particle size of the nanoparticulates is typically less than about 800 nm, more typically less than about 600 nm, still more typically less than about 400 nm, less than about 300 nm, less than about 250 nm, less than about 100 nm, or less than about 50 nm. In one aspect of this embodiment, at least 50% of the particles of active agent have an average particle size of less than about 800, 600, 400, 300, 250, 100, or 50 nm, respectively, when measured by light scattering techniques.
A variety of surface stabilizers are typically used with nanoparticulate compositions to prevent the particles from clumping or aggregating. Representative surface stabilizers are selected from the group consisting of gelatin, lecithin, dextran, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulo se, hydroxyethylcellulo se, hydroxypropylcellulo se, hydroxypropylmethyl-cellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, tyloxapol, poloxamers, poloxamines, poloxamine 908, dialkylesters of sodium sulfosuccinic acid, sodium lauryl sulfate, an alkyl aryl polyether sulfonate, a mixture of sucrose stearate and sucrose distearate, p-isononylphenoxypoly-(glycidol), SA9OHCO, decanoyl-N-methylglucamide, n-decyl-D-glucopyranoside, n-decyl-D-maltopyranoside, n-dodecyl-D-glucopyranoside, n-dodecyl-D-maltoside, heptanoyl-N-methylglucamide, n-heptyl-D-glucopyranoside, n-heptyl-D-thioglucoside, n-hexyl-D-glucopyranoside, nonanoyl-N-methylglucamide, n-nonyl-D-glucopyranoside, octanoyl-N-methylglucamide, n-octyl-D-glucopyranoside, and octyl-D-thioglucopyranoside. Lysozymes can also be used as surface stabilizers for nanoparticulate compositions.
Representative rate controlling polymers into which the nanoparticles can be formulated include chitosan, polyethylene oxide (PEO), polyvinyl acetate phthalate, gum arabic, agar, guar gum, cereal gums, dextran, casein, gelatin, pectin, carrageenan, waxes, shellac, hydrogenated vegetable oils, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcelluose (HPMC), sodium carboxymethylcellulose (CMC), poly(ethylene) oxide, alkyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydrophilic cellulose derivatives, polyethylene glycol, polyvinylpyrrolidone, cellulose acetate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose acetate trimellitate, polyvinyl acetate phthalate, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose acetate succinate, polyvinyl acetaldiethylamino acetate, poly(alkylmethacrylate), poly(vinyl acetate), polymers derived from acrylic or methacrylic acid and their respective esters, and copolymers derived from acrylic or methacrylic acid and their respective esters.
Methods of making nanoparticulate compositions are described, for example, in U.S. Pat. Nos. 5,518,187 and 5,862,999, both for “Method of Grinding Pharmaceutical Substances;” U.S. Pat. No. 5,718,388, for “Continuous Method of Grinding Pharmaceutical Substances;” and U.S. Pat. No. 5,510,118 for “Process of Preparing Therapeutic Compositions Containing Nanoparticles.”
Controlled Release Formulations
In a preferred embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including but not limited to implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. For example, enterically coated compounds can be used to protect cleavage by stomach acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Suitable materials can also be obtained commercially.
Liposomal suspensions (including but not limited to liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 (incorporated by reference). For example, liposome formulations can be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
Mucoadhesive Agents
Mucoadhesion is presently defined as the adhesion between two materials, at least one of which is a mucosal surface. Compounds, such as NO donors, are often delivered locally because their half-life is often below the time required for systemic distribution. The mucoadhesive agents described herein enable formulations suitable for mucoadhesive drug delivery systems (buccal, nasal, ocular, gastro, vaginal, and rectal). Mucoadhesive-containing topical and local systems have been shown to exhibit enhanced bioavailability. For example, it typically provides enhanced absorption (compared to a non-mucoadhesive formulation) and taking advantage of mucous tissues having high surface area and high blood flow.
In some embodiments, the mucoadhesive agent is a mucoadhesive polymer. In some embodiments, the mucoadhesive agent has numerous hydrophilic groups, such as hydroxyl, carboxyl, amide, and sulfate. These groups enable attachment to mucus or the cell membrane through physical and chemical interactions such as hydrogen bonding, hydrophobic, electrostatic, or conformational interactions. Hydrophilicity augments through drawing water for greater hydration and physically swell if in a gelatinous state. Aspects considered in selecting an appropriate mucoadhesive agent include the following:
In one embodiment, the mucoadhesive agent adheres to the mucosal surface through nonspecific, noncovalent interactions which are primarily electrostatic in nature. In another embodiment, the mucoadhesive agent adheres to the mucosal surface through hydrophilic functional groups that hydrogen bond with similar groups on biological substrates. In another embodiment, the mucoadhesive agent adheres to the mucosal surface through specific receptor sites on the cell or mucus surface. For example, lectins and thiolated polymers adhere to mucosal surfaces through specific receptor sites on the cell or mucus surfaces. As used herein, lectins are proteins or glycoprotein complexes of nonimmune origin that are able to bind sugars selectively in a noncovalent manner. It is proposed that lectins attach to carbohydrates on the mucus or epithelial cell surface. Thiolated polymers, or thiomers, have pendant thiols providing hydrophilicity, for example to polyacrylates or cellulosic polymeric backbones. The thiol group may form stable covalent bonds with mucus glycoproteins resulting in increased residency and improved bioavailability.
Many such mucoadhesive agents are known in the art. Useful mucoadhesive polymers include but are not limited to carbopols, N-isopropylacrylamide, polyvinyl alcohol/polyvinyl pyrrolidone, dextran, hydroxyethylmethacrylate/methacrylic acid, polyvinyl alcohol, polyacrylamide, polyethylene glycol/poly lactic acid, carboxymethyl chitosan and collagen. The mucoadhesive agent may include a polycarbophil and other acrylate/methacrylate polymers, anionic polymers based on methacrylic acid esters, which form pH selectably dissolvable hydrogels that dissolve (enabling physiological conditions to interact with and further initiate the release of NO) within physiochemically specified pH ranges, generally between about pH 5.5 to about pH 7.5. Such formulations dissolving in the pH range from about 5.5 to about 6.0 is useful for targeting the duodenum. Dissolution at higher pH generally targets lower sections of the intestine. For example, a pH of dissolution of between about 6.5 to about 7.0 may be useful for targeting the colon.
In some embodiments, the mucoadhesive agent comprises a water-soluble polymer. In particular, while a water-soluble polymer may or may not form a hydrogel to some extent when hydrated, it is capable of forming a flowable aqueous solution. Mucoadhesive agents of this type include but are not limited to polyols and polycarbohydrates, hydroxylated celluloses (hydroxypropylmethyl cellulose and hydroxymethyl cellulose).
In certain embodiments, the mucoadhesive agent enhances resonance time of the nitric oxide donor at the targeted site, for example, the respiratory tract.
In other certain embodiments, the mucoadhesive agent may also possess adhesion specificity to a biofilm comprising a pathogenic species. For example, some alginate oligomers are known to interact with pseudomonas Aeruginosa biofilms.
Chelating Agents
In illustrative embodiments, compositions disclosed herein may include one or more chelating agents. According to one aspect, a chelating agent is included to scavenge trace metals, so as to quench their potentially deleterious effects on the NO donor compound. Exemplary chelating agents are known in the art and examples include Ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA), yet other compounds described by Baldari et al., “Current Biomedical Use of Copper Chelation Therapy,” Int J Mol Sci. 2020; 21(3):1069.
Implantation
In several embodiments, the disclosed compositions also can be formulated as a preparation for implantation. Thus, for example, the compositions can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt). The compositions also can be formulated in rectal compositions (e.g., suppositories or retention enemas containing conventional suppository bases, such as cocoa butter or other glycerides), creams or lotions, or transdermal patches.
Composition Properties
In several embodiments, the combination of all the various components of the pharmaceutical composition, including the molecular weight, concentrations, or other chemical features of the compounds, and other components in the compositions, contribute to the tunability of the properties of the compositions disclosed herein. In several embodiments, by changing one or more of these features, one or more properties of the compositions can be tuned according to the preferred properties described herein. In several embodiments, the NO release rate, antimicrobial effect, water solubility, degradation rate, viscosity, gel firmness (where the formulation forms a gel), viscoelasticity, modulus, etc. are tunable.
In those embodiments where polymers are present in the formulations along with the small molecule NO-releasing compounds described herein, the properties of compositions can be tuned by adjusting the molecular weight of certain polymers used in the formulation. In several embodiments, the weight-average molecular weight (Mw) in kDa of polymers disclosed herein are greater than or equal to about: 2.5, 5.0, 7.0, 10, 15, 30, 50, 100, 200, 500, 750, 1,000, 2,000, 10,000, or ranges including and/or spanning the aforementioned values. In several embodiments, the number-average molecular weight (Mn) in kDa of polymers disclosed herein are greater than or equal to about: 2.5, 5.0, 7.0, 10, 15, 30, 50, 90, 100, 200, 500, 700, 1,000, 2,000, 10,000, or ranges including and/or spanning the aforementioned values. In several embodiments, the polymers disclosed herein may have n repeat units. In several embodiments, n equal to or at least about: 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, or ranges including and/or spanning the aforementioned values. In several embodiments, size exclusion chromatography (SEC) can be used to measure the molecular weight of the scaffold structures disclosed herein. In several embodiments, multi-angle light scattering (SEC-MALS) detectors can be used. In several embodiments, the scaffold structures can be characterized using their polydispersity index. The polydispersity index (PDI) is a measure of the distribution of molecular mass in a given polymer sample. PDI can be calculated by dividing the weight average molecular weight and the number average molecular weight. In several embodiments, the scaffold structures have a PDI of greater than or equal to about: 1.05, 1.1, 1.2, 1.3, 1.5, 1.7, 1.8, 1.9, 2.0, or ranges including and/or spanning the aforementioned values.
Representative polymers include those disclosed in each of U.S. Patent Application No. 62/441,742, U.S. Patent Application No. 62/483,505 International Application No. PCT/IB2018/050051, U.S. Patent Application No. 62/447,564, International Application No. PCT/IB2018/052144, U.S. patent application Ser. No. 14/421,525, U.S. Patent Application No. 62/639,119, and U.S. Patent Application No. 62/737,603 are used. Each of these applications and publications is incorporated by reference in its entirety for all purposes.
In several embodiments, the compositions (including all the components) may be water soluble and/or mutually miscible. In several embodiments, the compositions are soluble in water (at about 20° C.) at a concentration of greater than or equal to about: 1 mg/ml, 10 mg/ml, 20 mg/ml, 50 mg/ml, 100 mg/ml, 200 mg/ml, 300 mg/ml, 400 mg/ml, 500 mg/ml, or ranges including and/or spanning the aforementioned values.
According to several embodiments, the NO donor can be formulated within a pharmaceutical formulation at a concentration equal to or at least about: 100 μg/mL, and can be higher, e.g. about 1 mg/ml, about 5 mg/ml, about 10 mg/ml, about 20 mg/ml, 25 mg/ml, 50 mg/ml, 75 mg/ml, 100 mg/ml or about 200 mg/ml or higher. In illustrative embodiments, a polymeric species can be formulated within a pharmaceutical formulation at a concentration equal to or at least about: 100 μg/mL, and can be higher, e.g. about 1 mg/ml, about 5 mg/ml, about 10 mg/ml, about 20 mg/ml, 25 mg/ml, 50 mg/ml, 75 mg/ml, 100 mg/ml or about 200 mg/ml or higher. The amount of the polymer in the aqueous composition can be at least about 10% by weight, based on the weight of the NO donor, and may be higher, e.g., at least about 20% by weight, at least about 30% by weight, or at least about 50% by weight, same basis. Any combinations of NO donors and polymers in an aqueous composition are selected to be mutually miscible. As noted above, the NO donor and the polymer are considered mutually miscible if at least about 90% of the polymeric components remain mutually soluble 24 hours after mixing and maintaining at room temperature in water at a concentration of each polymer of 1 mg/ml, upon visible examination. Surprisingly, such mutual miscibility of the water-soluble polymers with the NO donors can be achieved, despite an expectation of phase separation at the 1 mg/ml concentrations and molecular weights described herein. The aqueous compositions described herein can be prepared by intermixing the individual formulation components with water, e.g., at room temperature with stirring.
In several embodiments, the composition disclosed herein have properties characteristic of a viscous fluid and/or of a gel. In several embodiments, a composition has a gelling point at room temperature (in water or PBS) at a concentration (in w/w %) of less than or equal to about: 0.5%, 1%, 2.5%, 5%, 10%, or ranges including and/or spanning the aforementioned values. In several embodiments, the composition may have a gelling point in water. In several embodiments, the composition gels in water (at about 20° C.) at a concentration of greater than or equal to about: 0.5 mg/ml, 1 mg/ml, 10 mg/ml, 20 mg/ml, 50 mg/ml, 100 mg/ml, 250 mg/ml, or ranges including and/or spanning the aforementioned values. In several embodiments, at a concentration of 5% w/w solution, the polymers have a viscosity (in cPa·s at 20° C.) of equal to or at least about: 10, 50, 100, 1,000, 2,000, 5,000, 10,000, or ranges including and/or spanning the aforementioned values. In several embodiments, the polymers have an intrinsic viscosity of equal to or greater than about: 0.5 m3/kg, 1.0 m3/kg, 2.0 m3/kg, 4.0 m3/kg, 8.0 m3/kg, or ranges including and/or spanning the aforementioned values.
In several embodiments, at a concentration of 5% w/w solution, the compositions have a firmness of equal to or at least about: 1.0 mN, 2.5 mN, 5 mN, 10 mN, 15 mN, 20 mN, 30 mN, 50 mN, or ranges including and/or spanning the aforementioned values. In several embodiments, at a concentration of 5% w/w solution, the formulations have a work of adhesion (in mN*mm) of equal to or at least about: 1.0, 2.5, 5, 10, 15, 20, 30, 50, 100, or ranges including and/or spanning the aforementioned values. In several embodiments, at a concentration of 5% w/w solution, the compositions have a storage modulus (G′) in Pa of equal to or at least about: 250, 500, 1,000, 2,000, 4,000, 5,000, 10,000, or ranges including and/or spanning the aforementioned values. In several embodiments, at a concentration of 5% w/w solution, the compositions have an elastic modulus (G″) in Pa of equal to or at least about: 25, 50, 100, 200, 400, 500, 1,000, 2,000, 5,000, 10,000, or ranges including and/or spanning the aforementioned values. In several embodiments, the aqueous composition is characterized by a barrier activity, as measured by a decrease in the diffusion rate of an anionic dye of more than 2 logs at a total scaffold concentration of 40 mg/ml or less.
In several embodiments, the formulation is a gel and the gel are stable at a variety of temperatures 20° C. (e.g., 40° C., 45° C., 55° C., 60° C., 80° C., etc.) and are stable for prolonged storage periods (e.g., 10 hours, 20 hours, 22 hours, 25 hours, 30 hours, etc., days such as 1 day, 3 days, 5 days, 6 days, 7 days, 15 days, 30 days, 45 days, etc., weeks such as 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, etc., months such as 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc., or even years (1 year or greater)).
In several embodiments, the viscosity of the composition increases with increasing temperature, or decreases with decreasing temperature. For example, if the composition is above the gelling temperature, then the composition has a relatively high viscosity, such as in the form of a gel, and if cooled to below the gelling temperature, then the composition decreases in viscosity, such as in the form of a liquid. In several embodiments, as such, the polymers as disclosed herein may be reversible polymers (e.g., thermoreversible polymers), where the transition from liquid to gel may be reversed upon exposure to appropriate conditions. For instance, as described above, compositions of the present disclosure include thermoreversible polymers, where the viscosity of the composition may be changed depending on the temperature of the composition. In several embodiments, the tunability of the viscosity enables a tailored composition profile upon delivery (e.g., more liquid at a delivery temperature and more viscous at, for example, body temperature).
In several embodiments, the compositions are characterized by a degree of swelling when exposed to water. In some embodiments, the swelling degree % of the composition disclosed herein is equal to or at least about: 100, 250, 500, 1,000, 2,000, 5,000, or ranges including and/or spanning the aforementioned values. In other words, the composition may swell or otherwise expand by 2×, 4×, 5×, 10×, 20×, 50×, 100×, or more.
In certain embodiments, the compositions disclosed herein have a gelling temperature similar to the normal body temperature of a subject, such as similar to human body temperature, or 37° C. By gelling temperature is meant the point on intersection between the plot for the elastic modulus and the plot for the viscous modulus. In some cases, if the composition is below the gelling temperature, then the composition has a relatively low viscosity, such as in the form of a liquid. In some instances, if the composition is above the gelling temperature, then the composition increases in viscosity (e.g., polymerizes), such that the composition is in the form of a gel. Compositions that transition from a liquid to a gel may facilitate administration of the composition to the subject, for example by facilitating injection of a low viscosity (e.g., liquid) composition at a temperature below the gelling temperature. After injection of the composition to the target treatment site, the temperature of the composition may increase due to absorption of heat from the surrounding body tissue, such that the composition increases in viscosity (e.g., transitions from a liquid to a gel, or polymerizes), thus providing structural and/or geometric support to the body tissue at the target treatment site. In some instances, gelling of the composition at the target treatment site may also facilitate retention of the composition at the treatment site by reducing the diffusion and/or migration of the composition away from the treatment site. In certain embodiments, the composition has a gelling temperature of 30° C. to 40° C., such as from 32° C. to 40° C., including from 35° C. to 40° C. In certain instances, the composition has a gelling temperature of 37° C.
Combination Therapy
The compounds described herein can be combined with active agents conventionally used to treat the disorders being treated using the compounds described herein. For example, in addition to administering one or more of the compounds described herein, a patient can also be administered a conventional agent.
Combination Therapy for Particular Use in Treating Covid-19 Infections
The compounds described herein can be combined with additional compounds useful for treating the disease states also treated by the release ofNO. In particular, the compounds discussed below can be used in combination therapy to treat Covid-19 infections, or other respiratory infections with similar pathology.
Various compounds that can be combined with the compounds described herein are discussed below.
Several embodiments pertain to a method of treating a disease state. In several embodiments, an effective amount of the compounds or compositions is administered to a subject in need thereof, wherein said disease state is selected from the group consisting of baldness, ischemia/reperfusion injury, thrombosis/restenosis, a fibrotic disease, a cancer, a cardiovascular disease, a microbial, fungal, or viral infection, a disease of platelet aggregation and platelet adhesion, a disease caused by or characterized by low nitric oxide levels, a metabolic disease, pathological conditions resulting from abnormal cell proliferation, autoimmune diseases, inflammation, vascular diseases, scar tissue, wound contraction, restenosis, pain, fever, gastrointestinal disorders, respiratory disorders, sexual dysfunctions, and sexually transmitted diseases. Reducing implant-related infections.
The term “effective amount,” as used herein, refers to that amount of a recited compound that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the condition, prevention or delay of the onset of the disorder, and/or change in clinical parameters, disease or illness, etc., as would be well known in the art. For example, an effective amount can refer to the amount of a composition, compound, or agent that improves a condition in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired response for a particular subject and/or application. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.
“Treat” or “treating” or “treatment” refers to any type of action that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the condition, and/or change in clinical parameters, disease or illness, curing the illness, etc.
The “patient” or “subject” treated as disclosed herein is, in some embodiments, a human patient, although it is to be understood that the principles of the presently disclosed subject matter indicate that the presently disclosed subject matter is effective with respect to all vertebrate species, including mammals, which are intended to be included in the terms “subject” and “patient.” Suitable subjects are generally mammalian subjects. The subject matter described herein finds use in research as well as veterinary and medical applications. The term “mammal” as used herein includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dog, rabbits, rodents (e.g., rats or mice), monkeys, etc. Human subjects include neonates, infants, juveniles, adults and geriatric subjects. The subject “in need of” the methods disclosed herein can be a subject that is experiencing a disease state and/or is anticipated to experience a disease state, and the methods and compositions of the invention are used for therapeutic and/or prophylactic treatment.
Some embodiments provide a method for treating a tissue defect comprising positioning any of the compounds or compositions described herein, i.e., compound of Formula I-III, at, over, or into the tissue defect. In several embodiments, the tissue defect is a wound. Several embodiments provide a method for treating a wound, for performing tissue repair, and/or for providing tissue and organ supplementation. In several embodiments, the first step of treating a tissue defect, wound, and/or supplementing and replacing tissue involves identifying a patient in need of an antimicrobial compound or composition to aid in the remedying and healing of a tissue defect, healing of a wound, or in need of a tissue supplement.
A non-limiting list of patients in need of an antimicrobial compound or composition includes patients suffering tissue defects. In several embodiments, the patients in need of an antimicrobial compound or composition suffer from wounds including those from burns, skin ulcers, lacerations, bullet holes, animal bites, and other wounds prone to infection. Antimicrobial compounds or compositions can also be used in the treatment of diabetic foot ulcers, venous leg ulcers, pressure ulcers, amputation sites, in other skin trauma, or in the treatment of other wounds or ailments. Patients in need of an antimicrobial scaffold also include patients in need of repair and supplementation of tendons, ligaments, fascia, and dura mater. Degradable antimicrobial compounds or compositions can be used in supplement tissue in procedures including, but not limited to, rotator cuff repair, Achilles tendon repair, leg or arm tendon or ligament repair (e.g., torn ACL), vaginal prolapse repair, bladder slings for urinary incontinence, breast reconstruction following surgery, hernia repair, staple or suture line reinforcement, bariatric surgery repair, pelvic floor reconstruction, dural repair, gum repair, bone grafting, and reconstruction. Further, a patient in need of an antimicrobial compound or composition also includes one in need of tissue or organ replacement.
In several embodiments, the antimicrobial compounds or polymers described herein can be used as fillers and/or to supplement and/or replace tissue by acting as an artificial extracellular matrix. In such an application, an antimicrobial compound or composition can be used to support cell and tissue growth. Briefly, cells can be taken from a patient or a viable host and seeded on an antimicrobial scaffold either in vivo or ex vivo. Then as the patient's natural tissues invade the material, it is tailored to degrade and leave only naturally occurring tissues and cells free of bacterial infection.
In some embodiments, the compounds and/or compositions discussed herein (i.e., compound of any of Formulas I-III) may be administered by direct injection or application to, for example, an injured tissue. Suitable routes also include injection or application to a site adjacent to the injured tissue. Administration may include parenteral administration (e.g., intravenous, intramuscular, or intraperitoneal injection), subcutaneous administration, administration into vascular spaces, and/or administration into joints (e.g., intra-articular injection). Additional routes of administration include intranasal, topical, vaginal, rectal, intrathecal, intraarterial, and intraocular routes. In several embodiments, the compounds and compositions disclosed herein can be applied as a gel to a site of treatment. In several embodiments, the compounds and compositions can be applied as a liquid.
NO is the endothelium-derived relaxing factor responsible for vasodilation and blood pressure regulation, and that NO plays key physiological roles in the cardiovascular (1) and nervous (2 and 3) systems, and as a signaling molecule capable of modulating cytokine production (4) in the immune response (5).
The specific role of NO in the various disease states referred to above is discussed in more detail below.
Cardiovascular Disorders
Organic nitrates, such as isosorbide mononitrate and glyceryl trinitrate, are widely used to treat angina, heart failure, and pulmonary hypertension, but are disadvantaged by the risk of hypotension, headaches and evolving tolerance (Miller and Megson, Br. J. Pharmacol., 2007, 151, 305-321). The metal nitrosyl sodium nitroprusside is also a potent vasodilator but requires co-administration with other drugs to prevent cyanide poisoning.
The compounds described herein are low molecular weight NO-donor compounds, with a relatively long half-life. As such, they can deliver precise NO doses to cardiac tissues, providing an improved therapeutic output and reduced side effects relative to the organic nitrate drugs.
Cancer Treatments
Nitric oxide is also intricately involved in cancer biology where both NO concentration and lifetime governs whether it acts as a tumor progressor or suppressor (Mocellein, et al., Med. Res. Rev., 2007, 27, 317-352).
Large concentrations of NO (i.e., micromolar) produce reactive nitrogen species, which along with reactive oxygen species result in oxidative and nitrosative stress, leading to DNA base deamination, nitrosylation of enzymes, impaired cellular function, enhanced inflammatory reactions, inhibited mitochondrial respiration and cell apoptosis (Wink, et al., Carcinogenesis, 1998, 19, 711-721). Lower NO concentrations (i.e., picomolar) present anti-apoptotic effects and promote angiogenesis thereby increasing nutrient delivery and facilitating tumor growth.
The main advantage of NO over other antitumor therapies is a decreased toxicity toward healthy cells, particularly at concentrations toxic to tumor cells.
The therapeutic consequence of any NO-based drug depends strongly on the concentration and duration of NO delivered. For example, micromolar NO concentrations are required to inhibit the growth of tumor cells, while picomolar NO concentrations have an angiogenic effect leading to cell proliferation (Mocellein, et al., Med. Res. Rev., 2007, 27, 317-352).
Accordingly, to be effective, NO-donor compounds must be capable of delivering high concentrations of NO specifically to the tumor site without the possibility of residual low concentrations that promote tumor growth.
As a result, effective NO-based therapies must store and deliver only relevant NO doses for specific durations. In addition, it is preferable that NO delivery is selective, due to NO's short half-life (seconds) (Ignarro, Nitric Oxide: Biology and Pathobiology, Academic Press, San Diego, Calif., 2000).
Control over these parameters (i.e., delivery site, NO concentration, and rate ofNO release) remains key for developing useful therapeutics. Pro-NO cancer therapies aim to increase NO concentrations at the tumor site to initiate apoptosis and/or necrosis of cancer cells (Hirst and T. Robson, Curr. Pharm. Des., 2010, 16, 411-420). Many LMW NO donors have shown anti-tumor efficacy, including diethylenetriamine NONOate, GTN, sodium nitroprusside, furoxan-based derivatives, and NO-releasing aspirin.
While extensive in vitro and in vivo testing of LMW NO donors for cancer therapy has been reported (Mocellein, et al., Med. Res. Rev., 2007, 27, 317-352; Hirst and Robson, Curr. Pharm. Des., 2010, 16, 411-420; Hickok and Thomas, Curr. Pharm. Des., 2010, 16, 381-391; Rigas, Biochem. Soc. Trans., 2007, 35, 1364-1368), few drugs have proceeded to clinical trials. The sensitive concentration dependence of NO's tumoricidal properties are likely the cause for the insecurity about pro-NO cancer therapies.
Indeed, both the concentration and delivery of NO must be precisely controlled for NO-based chemotherapies to be successful. When low molecular weight (LMW) donor compounds have been tested in the past, most of the NO payload was lost while the drugs were still in circulation.
The compounds described herein have a relatively high NO concentration, due to their having three diazeniumdiolate moieties per molecule, and due to the relatively small size of the molecules (i.e., 200-500 g/mol), so overcome the limitations of prior low molecular weight (LMW) NO-donor compounds. While not wishing to be bound to a particular theory, it is believed that the NO-donor compounds disclosed herein have an improved effect on cancer cells, relative to other LMW NO-donor compounds that have been tried, because the NO-release is relatively slow. This can allow for a delay of NO release until the compounds have reached their intended target (i.e., tumor cells). Accordingly, it is advantageous to use the NO-donors disclosed herein, both for thei relatively long NO half-lives, and their relatively high NO storage capacity.
To specifically target tumor cells, the compounds can be encapsulated, for example, in small unilamellar vesicles (SULVs), with an average diameter of around 30 nm to around 120 nm. These vesicles are small enough to travel through arteries and veins, but are trapped in capillary beds surrounding tumors. The compounds described herein can be effectively delivered to tumors in these vesicles before they release all of their NO payload.
Other targeting approaches can also be used, for example, particles that encapsulate the compounds described herein, and which include targeting ligands, such as antibodies, for receptors which are overexpressed by certain tumors.
The compounds described herein can be used to treat or prevent certain types of cancers, including, but not limited to, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer or cancer of the anal region, stomach cancer, colon cancer, breast cancer, gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system (e.g., cancer of the thyroid, parathyroid or adrenal glands), sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphonas, cancer of the bladder, cancer of the kidney or ureter (e.g., renal cell carcinoma, carcinoma of the renal pelvis), and neoplasms of the central nervous system (e.g., primary CNS lymphoma, spinal axis tumors, brain stem gliomas and pituitary adenomas).
The use of NO alone for cancer treatment can be complicated by its dual role as a tumor promoter and repressor, so in some embodiments, the NO-based antitumor therapy disclosed herein involve co-administration with other therapies.
Anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II (cyclooxygenase II) inhibitors, can be used in conjunction with a compound of formula 1 and pharmaceutical compositions described herein. Examples of useful COX-II inhibitors include CELEBREX (alecoxib), valdecoxib, and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are described in WO 96/33172 (published Oct. 24, 1996), WO 96/27583 (published Mar. 7, 1996), European Patent Application No. 97304971.1 (filed Jul. 8, 1997), European Patent Application No. 99308617.2 (filed Oct. 29, 1999), WO 98/07697 (published Feb. 26, 1998), WO 98/03516 (published Jan. 29, 1998), WO 98/34918 (published Aug. 13, 1998), WO 98/34915 (published Aug. 13, 1998), WO 98/33768 (published Aug. 6, 1998), WO 98/30566 (published Jul. 16, 1998), European Patent Publication 606,046 (published Jul. 13, 1994), European Patent Publication 931,788 (published Jul. 28, 1999), WO 90/05719 (published May 331, 1990), WO 99/52910 (published Oct. 21, 1999), WO 99/52889 (published Oct. 21, 1999), WO 99/29667 (published Jun. 17, 1999), PCT International Application No. PCT/IB98/01113 (filed Jul. 21, 1998), European Patent Application No. 99302232.1 (filed Mar. 25, 1999), Great Britain patent application number 9912961.1 (filed Jun. 3, 1999), U.S. Provisional Application No. 60/148,464 (filed Aug. 12, 1999), U.S. Pat. No. 5,863,949 (issued Jan. 26, 1999), U.S. Pat. No. 5,861,510 (issued Jan. 19, 1999), and European Patent Publication 780,386 (published Jun. 25, 1997), all of which are incorporated herein in their entireties by reference. Preferred MMP inhibitors are those that do not demonstrate arthralgia. More preferred are those that selectively inhibit MMP-2 and/or MMP-9 relative to the other matrix-metalloproteinases (i.e. MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).
The compounds described herein can also be used with signal transduction inhibitors, such as agents that can inhibit EGFR (epidermal growth factor receptor) responses, such as EGFR antibodies, EGF antibodies, and molecules that are EGFR inhibitors; VEGF (vascular endothelial growth factor) inhibitors, such as VEGF receptors and molecules that can inhibit VEGF; and erbB2 receptor inhibitors, such as organic molecules or antibodies that bind to the erbB2 receptor, for example, HERCEPTIN™ (Genentech, Inc. of South San Francisco, Calif., USA).
EGFR inhibitors are described in, for example in WO 95/19970 (published Jul. 27, 1995), WO 98/14451 (published Apr. 9, 1998), WO 98/02434 (published Jan. 22, 1998), and U.S. Pat. No. 5,747,498 (issued May 5, 1998), and such substances can be used as described herein. EGFR-inhibiting agents include, but are not limited to, the monoclonal antibodies C225 and anti-EGFR 22Mab (ImClone Systems Incorporated of New York, N.Y., USA), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck KgaA), EMD-5590 (Merck KgaA), MDX-447/H-477 (Medarex Inc. of Annandale, N.J., USA and Merck KgaA), and the compounds ZD-1834, ZD-1838 and ZD-1839 (AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis), PTK 787 (Novartis), CP 701 (Cephalon), leflunomide (Pharmacia/Sugen), CI-1033 (Warner Lambert Parke Davis), CI-1033/PD 183,805 (Warner Lambert Parke Davis), CL-387,785 (Wyeth-Ayerst), BBR-1611 (Boehringer Mannheim GmbH/Roche), Naamidine A (Bristol Myers Squibb), RC-3940-II (Pharmacia), BIBX-1382 (Boehringer Ingelheim), OLX-103 (Merck & Co. of Whitehouse Station, N.J., USA), VRCTC-310 (Ventech Research), EGF fusion toxin (Seragen Inc. of Hopkinton, Mass.), DAB-389 (Seragen/Lilgand), ZM-252808 (Imperical Cancer Research Fund), RG-50864 (INSERM), LFM-A12 (Parker Hughes Cancer Center), WHI-P97 (Parker Hughes Cancer Center), GW-282974 (Glaxo), KT-8391 (Kyowa Hakko) and EGFR Vaccine (York Medical/Centro de Immunologia Molecular (CIM)). These and other EGFR-inhibiting agents can be used in combination with the compounds described herein.
VEGF inhibitors, for example CP-547,632 (Pfizer Inc., N.Y.), AG-13736 (Agouron Pharmceuticals, Inc. a Pfizer Company), SU-5416 and SU-6668 (Sugen Inc. of South San Francisco, Calif., USA), and SH-268 (Schering) can also be combined with the compound of the present invention. VEGF inhibitors are described in, for example in WO 99/24440 (published May 20, 1999), PCT International Application PCT/IB99/00797 (filed May 3, 1999), in WO 95/21613 (published Aug. 17, 1995), WO 99/61422 (published Dec. 2, 1999), U.S. Pat. No. 5,834,504 (issued Nov. 10, 1998), WO 98/50356 (published Nov. 12, 1998), U.S. Pat. No. 5,883,113 (issued Mar. 16, 1999), U.S. Pat. No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat. No. 5,792,783 (issued Aug. 11, 1998), WO 99/10349 (published Mar. 4, 1999), WO 97/32856 (published Sep. 12, 1997), WO 97/22596 (published Jun. 26, 1997), WO 98/54093 (published Dec. 3, 1998), WO 98/02438 (published Jan. 22, 1998), WO 99/16755 (published Apr. 8, 1999), and WO 98/02437 (published Jan. 22, 1998), all of which are incorporated herein in their entireties by reference. Other examples of some specific VEGF inhibitors useful in the present invention are IM862 (Cytran Inc. of Kirkland, Wash., USA); anti-VEGF monoclonal antibody of Genentech, Inc. of South San Francisco, Calif.; and angiozyme, a synthetic rib ozyme from Ribozyme (Boulder, Colo.) and Chiron (Emeryville, Calif.). These and other VEGF inhibitors can be used in the present invention as described herein.
ErbB2 receptor inhibitors, such as CP-358,774 (OSI-774) (Tarceva) (OSI Pharmaceuticals, Inc.), GW-282974 (Glaxo Wellcome plc), and the monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc. of The Woodlands, Tex., USA) and 2B-1 (Chiron), can furthermore be combined with the compound of the invention, for example those indicated in WO 98/02434 (published Jan. 22, 1998), WO 99/35146 (published Jul. 15, 1999), WO 99/35132 (published Jul. 15, 1999), WO 98/02437 (published Jan. 22, 1998), WO 97/13760 (published Apr. 17, 1997), WO 95/19970 (published Jul. 27, 1995), U.S. Pat. No. 5,587,458 (issued Dec. 24, 1996), and U.S. Pat. No. 5,877,305 (issued Mar. 2, 1999), which are all hereby incorporated herein in their entireties by reference. ErbB2 receptor inhibitors useful in the present invention are also described in U.S. Provisional Application No. 60/117,341, filed Jan. 27, 1999, and in U.S. Provisional Application No. 60/117,346, filed Jan. 27, 1999, both of which are incorporated in their entireties herein by reference. The erbB2 receptor inhibitor compounds and substance described in the aforementioned PCT applications, U.S. patents, and U.S. provisional applications, as well as other compounds and substances that inhibit the erbB2 receptor, can be used with the compounds described herein.
The compounds can also be used with other agents useful in treating abnormal cellular proliferation or cancer, including, but not limited to, agents capable of enhancing antitumor immune responses, such as CTLA4 (cytotoxic lymphocite antigen 4) antibodies, and other agents capable of blocking CTLA4; and antiproliferative agents such as other farnesyl protein transferase inhibitors, and the like. Specific CTLA4 antibodies that can be used in the present invention include those described in U.S. Provisional Application 60/113,647 (filed Dec. 23, 1998) which is incorporated by reference in its entirety, however other CTLA4 antibodies can be used.
Other anti-angiogenesis agents, including, but not limited to, other COX-II inhibitors, other MMP inhibitors, other anti-VEGF antibodies or inhibitors of other effectors of vascularization can also be used.
Pathological conditions resulting from abnormal cell proliferation are not always cancer. Abnormal cell proliferation can occur either due to abnormal cell division or by abnormal cell differentiation. Abnormal cell proliferation results in the formation of neoplasms, which are abnormal masses of tissue where the growth and division of the cells are uncoordinated and continue in the same excessive manner even after the cessation of the stimuli that caused it. Like cancers, these neoplasms can also be treated using NO-donor compounds.
Cardiovascular Disease
In the vascular endothelium, NO is generated to maintain proper blood flow and pressure (Loscalzo and J. A. Vita, Nitric oxide and the cardiovascular system, Humana Press, Inc., Totowa, N.J., 2000).
When NO is produced from vascular endothelial cells, it influences the cellular activities of smooth muscle cells, platelets, and immune cells. After generation, NO diffuses into vascular smooth muscle cells and reacts with the iron of soluble guanylate cyclase. This activation of guanylate cyclase results in the production of cyclic guanosine monophosphate (cGMP), leading to relaxation of the smooth muscle cells and an overall dilation of blood vessels.
Deficiencies in NO occur when the endothelium is injured or not functioning properly as is the case for several cardiovascular conditions, including atherosclerosis, heart failure, hypertension, arterial thrombotic disorders, coronary heart disease, and stroke (Ferrari, et al., J. Appl. Biomed., 2009, 7, 163-173). Accordingly, NO-donor compounds can be used to treat these disorders.
LMW organic nitrates and nitrites have been used for centuries as cardiovascular therapeutics, particularly to initiate dilation of vascular smooth muscle (Loscalzo and Vita, Nitric oxide and the cardiovascular system, Humana Press, Inc., Totowa, N.J., 2000).
Glyceryl trinitrate (GTN) is perhaps the most popular and widely used organic nitrate for the regulation of blood pressure, although its success is hindered by the induction of tolerance. The pathophysiology of GTN tolerance is complex and dependent on several factors including GTN bioactivation, desensitization of vascular soluble guanylate cyclase, oxidative stress, and mitochondrial aldehyde dehydrogenase inactivation. Sodium nitroprusside has also been used clinically, but its success is limited by cyanide toxicity.
The compounds described herein provide not only a sustained release of nitric oxide, due to their relatively long NO half-lives, but also have relatively large payloads of nitric oxide precursors. As such, they can offer distinct advantages over nitrates and nitrites for these indications.
Circulatory Dysfunctions
The systemic delivery of exogenous NO to the vasculature for treating circulatory dysfunctions is particularly difficult due to the rapid scavenging of NO by hemoglobin, which restricts the ability to maintain sustained NO levels throughout the vasculature (Loscalzo and Vita, Nitric oxide and the cardiovascular system, Humana Press, Inc., Totowa, N.J., 2000).
Nitric oxide release from the NO donor compounds described herein allows for much longer and continuous delivery of NO at therapeutic levels than the nitrates and nitrites in use today, and thus represents a promising alternative to those NO donors for blood pressure regulation.
Even with the relatively long NO half-lives, patients may benefit from further increased circulation times. One way to achieve this is to encapsulate the compounds, for example, in pegylated liposomes, also known as “stealth” liposomes, as they tend to avoid metabolism by the liver. Stealth liposomes are liposomes that evade detection by the immune system, hence, they are also known as immunoliposomes. Conventional liposomes are not stable for long periods of time, especially when injected into a body, but stealth liposomes use poly-(ethylene glycol) as a steric stabilizer. The stealth liposomes are not fully inert vesicles; they can eventually become detected by the immune system, just more slowly than non-pegylated liposomes. As such, stealth liposomes tend to provide relatively slow release of therapeutic compounds in vivo. The relatively slow release, coupled with the NO-donor compounds' relatively long NO half-life and large NO capacity, can improve the efficacy of the compounds described herein, for example, in providing a more sustained reduction in mean arterial pressure.
As a result of the steady delivery of therapeutically relevant NO concentrations, the compounds can be used to obtain decreased blood pressure over an extended period of time. Prolonged circulation and proper regulation of blood pressure with minimal negative side-effects (i.e., toxicity and tolerance development) are some of the advantages the compounds described herein can provide when used to treat circulatory disorders.
Ischemia/Reperfusion Injury
When proper circulation is restored following a period of restricted blood flow, oxidative stress leads to inflammation and tissue damage. This condition is referred to as ischemia/reperfusion (I/R) injury. Due to its antioxidant and anti-inflammatory properties, endogenous NO is known to be a mediator/protector of I/R injury, and can be used to treat this cardiovascular dysfunction. Compared to the systemic NO release required for blood pressure regulation, targeted NO delivery is necessary for reducing I/R injury, as the cytoprotective activity is only relevant at the compromised tissue.
The compounds can be targeted to the liver, for example, by incorporating them in particles, and attaching appropriate targeting ligands to the particles.
The compounds can be used to reduce I/R injury in a heart. The amount of infarcted (i.e., necrotic) tissue can be significantly reduced upon pretreatment (i.e., before the onset of ischemia) with the NO-releasing compounds. When delivered to the heart, NO diffusion to intracellular organelles leads to cardioprotective effects.
Another area where I/R injuries are seen is in the carotid artery. Ischemic Stroke (IS) occurs as a result of a clot in the artery blocking the flow of blood to the brain leading to dysfunction or death of the brain tissue. Carotid artery disease is caused by a buildup of plaque in carotid arteries that deliver blood to the brain. These disorders can also be treated using the compounds described herein.
Blood clots can cause heart attacks and strokes, and while the compounds of Formulas I-III can minimize ischemic damage, it can also be advantageous to administer an anti-coagulant/blood thinner, such as tissue plasminogen activator, integrilin, coumarin or heparin.
Thrombosis and Restenosis
The introduction and/or removal of intravenous devices often leads to thrombosis (i.e., blood clots within a vessel) or restenosis (i.e., re-narrowing of vessels). In healthy vasculature, endothelial cells generate NO to prevent thrombosis and platelet activation/adhesion. However, interventional procedures such as angioplasty and stenting often cause damage to, or removal of the endothelial cells lining the artery. As a result, normal NO production from the endothelium required for vascular homeostasis is disrupted with thrombosis resulting as activated platelets aggregate and adhere along with proteins (i.e., fibrin) to the device and/or injured site. Following thrombosis at vascular injuries, smooth muscle cells migrate to the site, and their proliferation results in neointimal hyperplasia (i.e., proliferation of the cells of the inner most lining of an artery) ultimately leading to restenosis.
Due to NO's innate role in the cardiovasculature, application of exogenous NO can address problems arising at the site of vascular device implantation and vascular injury. To prevent thrombosis due to platelet adhesion to a surface, implanted devices, such as drug-loaded stends, may be coated with a sustained release polymer that incorporates the NO-releasing compounds described herein. The resulting NO release can effectively decrease platelet adhesion and activation on the interior walls of excorporeal blood circulatory tubes. Thus, lumen surfaces coated with NO-releasing compounds exhibit decreased platelet adhesion.
In response to vascular injury, thrombosis may also prove problematic by initiating excessive platelet activation and aggregation. Stasko et al. reported the ability of NO-releasing dendrimers (G4-SNAP) to inhibit platelet aggregation relative to the LMW NO donor SNAP (Stasko, et al., Biomacromolecules, 2008, 9, 834-841). However, since the LMW NO donor compounds described herein provide enhanced NO half-lives, and have relatively large NO payloads, they can be as advantageous, or more so, as the polymeric NO donors, as they can provide a large, localized concentration of NO.
The recruitment and proliferation of vascular smooth muscle cells (VSMC) and endothelial cells may lead to neointimal hyperplasia following thrombosis in the restenosis cascade. As NO inhibits VSMC proliferation, the prevention of restenosis with exogenous NO is achieved by decreasing platelet activation/aggregation and reducing neointimal hyperplasia. Optimal treatment requires that NO is delivered directly to the vascular injury either by direct application or by some NO release trigger.
Site-specific delivery may be achieved using NO-release vehicles capable of targeted NO release.
As detailed above, NO-based therapies have a long history in cardiovascular diseases. Compounds that release NO have a positive effect on blood flow regulation, ischemia/reperfusion injury, thrombosis and restenosis.
Autoimmune Diseases
Nitric oxide (NO) is an intercellular messenger that performs a number of functions, including neurotransmission, vasodilatation, inhibition of platelet aggregation, and modulation of leukocyte adhesion. NO has recently been shown to act as a potent cytotoxic effector molecule as well as to play an important role in the pathogenesis of organ-specific autoimmunity. NO may also modulate the immune response by interfering with Th1/Th2 balance in autoimmune diseases.
Autoimmune disorders include multiple organ-specific to systemic disorders, including type 1 diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, scleroderma, thyroiditis, and others. There are also implications of autoimmune pathology in common health problems such as arteriosclerosis, inflammatory bowel diseases, psoriasis, schizophrenia, and certain types of infertility.
Protein expression of the inducible nitric oxide synthase (iNOS) has been found in the majority of the above mentioned human autoimmune diseases or in animal models of these diseases, mostly in inflammatory cell infiltrates like activated macrophages, but also in organ-specific epithelial cells or in parenchymal cells. The role of NO in inflammation is very complex. On the one hand, NO mediates important direct cytotoxic effects, and on the other hand it modulates immunological functions, which in the setting of autoimmune diseases can be beneficial (Klaus-Dietrich Kroncke, “The Role of Nitric Oxide in Autoimmune Diseases”, Current Medicinal Chemistry—Anti-Inflammatory & Anti-Allergy Agents (2004) 3: 223).
Accordingly, the NO-donor compounds described herein can be used to treat a variety of autoimmune disorders, including type 1 diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, scleroderma, thyroiditis, and psoriasis.
When used to treat psoriasis, local delivery of the compounds via topical formulations may be preferable to systemic delivery, as the local concentration of NO on the tissue of interest can be enhanced.
When used to treat rheumatoid arthritis, intra-articular delivery of the compounds may be beneficial, as NO release will occur directly in the affected joint.
Inflammation
Recently, researchers have been finding correlations between nitric oxide and inflammation. Nitric oxide, in many studies, acts as an anti-inflammatory, and can help inflamed joints and muscles, and help reduce the severity of symptoms from certain ailments.
Nitric oxide (NO) is a signaling molecule that plays a key role in the pathogenesis of inflammation. It gives an anti-inflammatory effect under normal physiological conditions. However, on the other hand, NO is considered as a pro-inflammatory mediator that induces inflammation due to over production in abnormal situations. Accordingly, NO can be used to treat inflammation, but care must be taken to avoid abnormally large concentrations of NO that might induce inflammation.
The NO-donor compounds described herein have a relatively long NO half-life, so can provide sustained release of NO at therapeutically effective concentrations that can control inflammation. This can help avoid relatively high concentrations, as might be observed using NO-donor compounds with significantly shorter NO half-lives, and the corresponding higher concentrations of NO resulting from faster release from compounds with shorter NO half-lives. Thus, the compounds are uniquely suited for treating inflammatory disorders.
In the central nervous system, representative inflammatory disorders include CNS encephalitis, myelitis, meningitis arachnoiditis, PNS neuritis, eye dacryoadenitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, uveitis, ear otitis externa, otitis media, labyrinthitis, and mastoiditis.
In the cardiovascular system, representative inflammatory disorders include carditis, endocarditis, myocarditis, pericarditis, vasculitis arteritis, phlebitis, and capillaritis.
In the respiratory system, representative inflammatory disorders include chronic obstructive pulmonary disorder (COPD), upper sinusitis, rhinitis, pharyngitis, laryngitis, lower tracheitis, bronchitis, bronchiolitis, pneumonitis, pleuritic, and mediastinitis.
In the mouth, representative inflammatory disorders include stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, sialadenitis/parotitis, cheilitis, pulpitis, and gnathitis.
In the gastrointestinal tract, representative inflammatory disorders include esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, duodenitis, ileitis, caecitis, appendicitis and proctitis.
In the accessory digestive organs, representative inflammatory disorders include hepatitis, such as non-alcoholic steatohepatitis, ascending cholangitis, cholecystitis, pancreatitis and peritonitis.
In the integumentary system, representative inflammatory disorders include dermatitis folliculitis, cellulitis, and hidradenitis.
In the musculo skeletal system, representative inflammatory disorders include arthritis, dermatomyositis, soft tissue myositis, synovitis/tenosynovitis, bursitis, enthesitis, fasciitis, capsulitis, epicondylitis, tendinitis, panniculitis, osteochondritis, osteitis/osteomyelitis, spondylitis, periostitis, and chondritis.
In the urinary system, representative inflammatory disorders include nephritis glomerulonephritis, pyelonephritis, ureteritis, cystitis and urethritis.
In females, representative inflammatory disorders include oophoritis, salpingitis, endometritis, parametritis, cervicitis, vaginitis, vulvitis, and mastitis. During pregnancy, inflammatory disorders a pregnant woman might experience include chorioamnionitis, funisitis, and omphalitis.
In males, representative inflammatory disorders include orchitis, epididymitis, prostatitis, seminal vesiculitis, balanitis, posthitis, and balanoposthitis.
In the endocrine system, representative inflammatory disorders include insulitis, hypophysitis, thyroiditis, parathyroiditis, and adrenalitis. In the lymphatic system, representative inflammatory disorders include lymphangitis and lymphadenitis.
Any of these disorders can be treated using the compounds described herein.
Fibrotic Disorders
Systemic sclerosis (scleroderma: SSc) is a multisystem, connective tissue disease of unknown aetiology characterized by vascular dysfunction, autoimmunity, and enhanced fibroblast activity resulting in fibrosis of the skin, heart, and lungs, and ultimately internal organ failure, and death. One of the most important and early modulators of disease activity is thought to be oxidative stress. Evidence suggests that the free radical nitric oxide (NO), a key mediator of oxidative stress, can profoundly influence the early microvasculopathy, and possibly the ensuing fibrogenic response. Animal models and human studies have also identified dietary antioxidants, such as epigallocatechin-3-gallate (EGCG), to function as a protective system against oxidative stress and fibrosis. Hence, targeting EGCG may prove a possible candidate for therapeutic treatment aimed at reducing both oxidant stress and the fibrotic effects associated with SSc. Audrey Dooley, K. Richard Bruckdorfer, David J. Abraham, “Modulation of Fibrosis in Systemic Sclerosis by Nitric Oxide and Antioxidants”, Cardiology Research and Practice, vol. 2012, Article ID 521958, 9 pages, 2012.
Pain
Certain types of pain, including peripheral nerve pain, can benefit from enhanced nitric oxide levels. Peripheral neuropathy occurs when the part of the nervous system that carries signals from the brain to the muscles, skin, and other tissues begins to weaken and die. One of the conditions that can cause the peripheral nerves to die is neuropathy. Neuropathy often occurs when the tissues around nerves fail to get sufficient circulation, and this is a common condition in diabetic patients. The nerves begin to die when tissues become starved of oxygen and nutrients.
Nitric oxide causes blood vessels to open, increasing the volume of blood that is able to move through them. Along with this increased blood flow, tissues get access to the greater supply of oxygen and vital nutrients. Enhanced levels of nitric oxide can therefore be used to treat neuropathy, so the NO-releasing compounds described herein can treat neuropathy, including peripheral neuropathy.
Nitric Oxide (NO) offers pain relief in a number of ways, and NO is the mediator of the analgesic effect of opioids such as morphine. Accordingly, the NO-donor compounds described herein can be used to provide pain relief.
Fever
Nitric oxide (NO) has a role in thermoregulation and fever. An elevated level of NO in the central nervous system can prevent fever, possibly via positive feedback action of NO on presynaptic glutaminergic neurons (Riedel W. Antipyretic role of nitric oxide during endotoxin-induced fever in rabbits. Int J Tissue React). See also Steiner and Branco, “Nitric oxide in the regulation of body temperature and fever,” Journal of Thermal Biology, Volume 26, Issues 4-5, September 2001, Pages 325-330. Accordingly, the NO-donor compounds described herein can help reduce fevers.
Sexual Dysfunctions
The functional state of the penis, flaccid or erect, is governed by smooth muscle tone. Sympathetic contractile factors maintain flaccidity, and parasympathetic factors induce smooth muscle relaxation and erection. It is generally accepted that nitric oxide (NO) is the principal agent responsible for relaxation of penile smooth muscle (Cartledge et al., “The role of” nitric oxide in penile erection, Expert Opin. Pharmacother., 2001 January; 2(1):95-107. For this reason, NO-releasing compounds like sildenafil are used to treat impotence. More recently, nitroglycerin lotions/creams have been topically applied in order to provide erections.
Researchers at the University College Hospital in London and various other United Kingdom medical centers tested a nitroglycerin gel on a total of 220 male participants. After applying a small amount of the gel, nearly half of the male participants reported getting an erection within five minutes, and 70 percent within 10 minutes. Accordingly, the gel works roughly times faster than Viagra.
Like nitroglycerine, the NO-donor compounds release NO, so can be used orally, like Viagra, or topically, such as in a gel, to induce smooth muscle relaxation and erection.
Hair Growth
Baldness has been treated using helmets that emit light at wavelengths that promote the production or the release of endogenous NO. For example, Revian has an FDA-cleared all-LED hair loss treatment for men and women, clinically proven to grow more hair in less time than other hair loss products.
Topical administration of the compounds described herein to the scalp can also promote hair growth, as the local administration of NO to the tissue surrounding hair follicles can increase blood flow to the hair follicles.
Central Nervous System Disorders
There are a number of CNS disorders, particularly those with an inflammatory component, and/or those associated with poor vascularization in one or more regions of the brain, that can be treated using the compounds described herein. These disorders include cognitive disorders, motor disorders, and behavioral/mood disorders.
In disorders with an inflammatory component, the anti-inflammatory properties of nitric oxide can be leveraged to treat these disorders. In disorders with a vascular component, the release of nitric oxide can help promote increased vascularization.
The treatments described herein can be used to treat a number of different disorders, including neurocognitive disorders, movement disorders, and emotional/behavioral/mood disorders. The treatments can also increase the flow of drugs across the blood brain barrier, so can be used to treat disorders of the brain, such as cancer, or disorders associated with an impaired blood brain barrier.
Disorders associated with an impaired blood brain barrier include Alzheimer's disease (van de Haar H J, et al., “Blood-Brain Barrier Leakage in Patients with Early Alzheimer Disease”. Radiology. 282 (2): 615 (February 2017)), anxiety and depression (Gal Z, Huse R J, Gonda X, Kumar S, Juhasz G, Bagdy G, Petschner P (March 2019). “[Anxiety and depression—the role of blood-brain barrier integrity]”. Neuropsychopharmacologia Hungarica. 21 (1): 19-25), brain abscesses (caused by inflammation and collection of lymphatic cells and infected material originating from a local or remote infection), De Vivo disease (also known as GLUT1 deficiency syndrome, resulting from inadequate transportation of the sugar glucose across the blood—brain barrier, typically caused by genetic defects in glucose transporter type 1 (GLUT1), HIV encephalitis (Ivey N S, MacLean AG, Lackner A A (April 2009). “Acquired immunodeficiency syndrome and the blood-brain barrier”. Journal of Neurovirology. 15 (2): 111-22), Meningitis (associated with inflammation of the membranes that surround the brain and spinal cord, i.e., meninges), multiple sclerosis (Waubant E (2006). “Biomarkers indicative of blood-brain barrier disruption in multiple sclerosis”. Disease Markers. 22 (4): 235-44), and neuromyelitis optica, also known as Devic's disease, which is similar to multiple sclerosis.
Brain cancers, such as astrocytomas, including gliomas, glioblastoma multiforme, and meningiomas, ependymomas, pituitary tumors, such as pituitary adenomas and pituitary carcinomas, craniopharyngiomas, germ cell tumors, such as germinomas, pineal region tumors, including slow growing (pineocytoma) and fast growing (pineoblastoma) tumors, medulloblastomas, and primary CNS lymphomas, can also be treated.
When used to treat brain cancers, conventional anticancer drugs can be coadministered, and their passage across the blood brain barrier can be enhanced, thus maximizing treatment efficiency. Representative anticancer drugs include SFC, Accutane, AEE788 (Novartis), AMG-102, Anti Neoplaston, AQ4N (Banoxantrone), AVANDIA (Rosiglitazone Maleate), Avastin (Bevacizumab) BCNU, BiCNU, Carmustine, Carboplatin, CC-223, CC223, CCI-779, CCNU, CCNU Lomustine, Celecoxib (Systemic), Chloroquine, Cilengitide (EMD 121974), Cisplatin, CPT-11 (CAMPTOSAR, Irinotecan), Cytoxan, Dasatinib (BMS-354825, Sprycel), Etoposide (Eposin, Etopophos, Vepesid), GDC-0449, Gleevec (imatinib mesylate), GLIADEL Wafer, Hydroxychloroquine, Hydroxyurea, IL-13, IMC-3G3, Immune Therapy, Iressa (ZD-1839), Lapatinib (GW572016), Methotrexate, Novocure, OSI-774, PCV, Procarbazine, RAD001 Novartis (mTOR inhibitor), Rapamycin (Rapamune, Sirolimus), RMP-7, RTA 744, Simvastatin, Sirolimus, Sorafenib, SU-101, SU5416 Sugen, Sulfasalazine (Azulfidine), Sutent (Pfizer), Tamoxifen, TARCEVA (erlotinib HCl), Taxol, TEMODAR, TEMODAR Schering-Plough
Thalomid (thalidomide), Toca 511, Topotecan (Systemic), VEGF Trap, VEGF-Trap, Velcade, Vincristine, Vorinostat (SAHA), XL 765, XL-184, XL184, XL765, Zarnestra (tipifarnib), and Zocor (simvastatin). Dexamethasone and furosemide can be used to decrease swelling.
Central nervous system (CNS) vasculitis is inflammation of blood vessel walls in the brain or spine, which make up the central nervous system. CNS vasculitis is often accompanied by other autoimmune diseases such as systemic lupus erythematosus, dermatomyositis, and, rarely, rheumatoid arthritis. It is often caused by a viral or bacterial infection and can be systemic. When the vasculitis is only confined to the brain or the spinal cord, it is referred to as primary angiitis of the CNS (PACNS). The compounds described herein can treat these disorders, due to the ability of NO to treat inflammation, and also to treat the underlying viral or bacterial infection.
There are a number of neurocognitive disorders (also known as cognitive disorders) that can be treated using the compounds described herein. Examples include Alzheimer's disease, amnesia, Binswanger's disease, cerebellar cognitive affective syndrome, clinical dementia rating, clouding of consciousness, cognitive deficit, cognitive slippage, cognitive vulnerability, corticobasal degeneration, corticobasal syndrome, delirium, dementia, disabilities affecting intellectual abilities, frontal assessment Battery, frontotemporal dementia, frontotemporal dementia and parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, HIV-associated neurocognitive disorders, learning problems in childhood cancer, Lewy body dementia association, Lewy body dementia, logopenic progressive aphasia, mild cognitive impairment, paratonia, Pick's disease, post-chemotherapy cognitive impairment, postoperative cognitive dysfunction, primary progressive aphasia, progressive nonfluent aphasia, progressive supranuclear palsy, pseudosenility, REM sleep behavior disorder, semantic dementia, severe cognitive impairment, subcortical dementia, and vascular dementia.
Neurocognitive disorders can have numerous causes, including genetics, brain trauma, stroke, and heart issues. The main causes are neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease because they affect or deteriorate brain functions. Other diseases and conditions that cause NDCs include vascular dementia, frontotemporal degeneration, Lewy body disease, prion disease, normal pressure hydrocephalus, and dementia/neurocognitive issues due to HIV infection. They may also include dementia due to substance abuse or exposure to toxins.
Neurocognitive disorders also include brain trauma, including concussions and traumatic brain injuries, as well as post-traumatic stress and alcoholism. This is referred to as amnesia, and is characterized by damage to major memory encoding parts of the brain such as the hippocampus. Difficulty creating recent term memories is called anterograde amnesia and is caused by damage to the hippocampus part of the brain. Retrograde amnesia is also caused by damage to the hippocampus, but the memories that were encoded or in the process of being encoded in long-term memory are erased.
Movement disorders are clinical syndromes with either an excess of movement or a paucity of voluntary and involuntary movements, unrelated to weakness or spasticity, and are typically divided into two major categories—hyperkinetic and hypokinetic. Hyperkinetic movement disorders refer to dyskinesia, or excessive, often repetitive, involuntary movements that intrude upon the normal flow of motor activity. Hypokinetic movement disorders refer to akinesia (lack of movement), hypokinesia (reduced amplitude of movements), bradykinesia (slow movement), and rigidity. In primary movement disorders, the abnormal movement is the primary manifestation of the disorder. In secondary movement disorders, the abnormal movement is a manifestation of another systemic or neurological disorder.
Representative movement disorders include hypokinetic movement disorders, Parkinson's disease (Primary or Idiopathic Parkinsonism), secondary Parkinsonism, Parkinson plus syndromes, Hallevorden-Spatz Disease, progressive supranuclear ophthalmoplegia, striatonigral deneneration, hyperkinetic movement disorders, dystonia, including drug-induced dystonia, idiopathic familial dystonia, idiopathic non-familial dystonia, ideopathic orofacial dystonia, spasmodic torticollis, blepharospasm, and other other dystonias, extrapyramidal movement disorders, essential tremors, drug induced tremors, myoclonus, opsoclonus, chorea (rapid, involuntary movement), including drug-induced chorea, rheumatic chorea (Sydenham's chorea), and Huntington's Chorea, ballismus (violent involuntary rapid and irregular movements), hemiballismus (affecting only one side of the body), athetosis (contorted torsion or twisting), dyskinesia (abnormal, involuntary movement), tardive dyskinesia, tic disorders (involuntary, compulsive, repetitive, stereotyped), including Tourette's syndrome and drug-induced tics and tics of organic origin, stereotypic movement disorder, paroxysmal nocturnal limb movement, painful legs (or arms), moving toes (or fingers) syndrome, sporadic restless leg syndrome, familial restless leg syndrome, stiff-person syndrome, abnormal head movements, cramp and spasm, and fasciculation.
Emotional and behavioral disorders (EBD; also known as behavioral and emotional disorders (ICD-10)) refer to a disability classification used in educational settings that allows educational institutions to provide special education and related services to students who have displayed poor social and/or academic progress.
Disruptive behavior disorders include attention-deficit hyperactivity disorder (ADHD), oppositional defiant disorder (ODD), and conduct disorders (CD). Schizophrenia is also included in this definition.
Anxiety disorders are also included. Representative anxiety disorders include generalized anxiety disorder, specific phobia, social anxiety disorder, separation anxiety disorder, agoraphobia, panic disorder, and selective mutism. Anxiety disorders often occur with other mental disorders, particularly major depressive disorder, personality disorder, and substance use disorder.
Depressive disorders include major depressive disorder (MDD, also known as major depression, unipolar depression, or clinical depression), and there are several subtypes or course specifiers, including atypical depression (AD), melancholic depression, psychotic major depression (PMD, or simply psychotic depression), catatonic depression, postpartum depression, premenstrual dysphoric disorder (PMDD), seasonal affective disorder (SAD), dysthymia, double depression, depressive personality disorder (DPD), recurrent brief depression (RBD), minor depressive disorder (minor depression), and depressive disorder not otherwise specified (DD-NOS), which encompasses “any depressive disorder that does not meet the criteria for a specific disorder.”
Bipolar disorders (BD, also called manic depression or manic-depressive disorder), including bipolar I, bipolar II, cyclothymia, and bipolar disorder not otherwise specified (BD-NOS, or “sub-threshold” bipolar).
Certain mood disorders are substance-induced, including alcohol-induced and benzodiazepine-induced.
Medications, such as antidepressants, benzodiazepines, or beta blockers, may be used in combination with the treatments described herein.
Specific pharmaceutical treatment regimens for various disorders discussed herein are outlined below.
Combination Treatment for Parkinson's DiseaseWhile there is no cure for Parkinson's disease, a number of different types of medications provide some relief. The main families of drugs useful for treating motor symptoms are levodopa (typically combined with a dopa decarboxylase inhibitor and sometimes also with a COMT inhibitor), dopamine agonists and MAO-B inhibitors. Representative dopamine agonists include bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine and lisuride. Representative MAO-B inhibitors include safinamide, selegiline and rasagiline.
Those of skill in the art appreciate that the stage of the disease and the age at disease onset determine which group is most useful. Braak staging of Parkinson's disease gives six stages that can be used to identify early stages, later stages, and late stages. The initial stage in which some disability has already developed and requires pharmacological treatment is followed by later stages associated with the development of complications related to levodopa usage, and a third stage when symptoms unrelated to dopamine deficiency or levodopa treatment may predominate.
Typically, only 5-10% of levodopa crosses the blood—brain barrier. Much of the remainder is metabolized to dopamine elsewhere in the body, causing a variety of side effects including nausea, vomiting and orthostatic hypotension.
Carbidopa and benserazide are dopa decarboxylase inhibitors which do not cross the blood-brain barrier and inhibit the conversion of levodopa to dopamine outside the brain, reducing side effects and improving the availability of levodopa for passage into the brain. One of these drugs is usually taken along with levodopa, often combined with levodopa in the same pill.
Because the treatments described herein can increase the passage of drugs through the blood-brain barrier, it is possible to lower the dosage of levodopa while still achieving the same degree of efficacy, and, therefore, reducing side effects, including dyskinesias.
Intestinal infusions of levodopa (Duodopa) can also be used, and can reduce dosage fluctuations relative to oral levodopa.
Catechol-O-methyltransferase (COMT) inhibitors such as opicapone, entacapone and tolcaponecan be used in combination with levodopa and dopamine dearecarboxylase (DDC) inhibitors to inhibit peripheral levodopa metabolism, increasing the amount of levodopa delivered to the brain. Other drugs such as amantadine, anticholinergics, quetiapine, cholinesterase inhibitors, modafinil, pimavanserin, doxepin and rasagline can also be used.
Combination Treatment for Alzheimer's Disease and Other Cognitive Disorders
In addition to the treatments described herein, various drugs have been developed for use with Alzheimer's patients, and these can be used in combination with the treatments. Representative compounds that can be administered include memantine, Solanezumab, aducanumab, compounds which reduce beta-amyloid levels, such as apomorphine and aducanumab, vaccines that train the immune system to recognize, attack, and reverse deposition of amyloid, such as ACC-001 and bapineuzumab, and antisense therapies, neuroprotective agents, such as AL-108, metal-protein interaction attenuation agents, such as PBT2, TNFα receptor-blocking compounds, such as the fusion protein, etanercept, tau aggregation inhibitors, such as methylthioninium chloride and its prodrug LMTX, antihistamines, such as dimebon, and beta-secretase protein inhibitors, such as verubecestat, which reduced amyloid beta concentrations. Medications that reduce oxidative stress can improve memory, and can therefore be co-administered. Treatments that reduce amyloid-β not only improve memory but also reduce oxidative stress.
As discussed elsewhere herein, in animal models, such as the senescence accelerated mouse (SAMP8) model, amyloid precursor protein (APP) is overproduced. The blood—brain barrier is damaged, causing a decreased expulsion of amyloid-β protein from the brain, and causing an increase in oxidative stress in the brain.
The use of the treatments described herein can increase the ability of agents to cross the blood brain barrier, so can enhance the effectiveness of these agents, as well as accelerate expulsion of amyloid-β protein from the brain.
Disease Caused by or Characterized by Low Nitric Oxide Levels
In addition to the other disorders discussed herein, there are other diseases and disorders associated with low nitric oxide levels. Sickle cell anemia is one of them. Low levels of both oxygen and nitric oxide appear to have an unfortunate synergy for patients with sickle cell disease. These two conditions, common in sickle cell disease, dramatically increase red blood cells' adhesion to the lining of blood vessels walls and the debilitating pain crises that can result. The compounds disclosed herein can be used to treat sickle cell disease, as the release of nitric oxide can minimize adhesion of red blood cells to the lining of blood vessel walls, and relieve the pain that otherwise would result.
Methemoglobinemia can also be reduced by the administration of low-dose nitric oxide releasing compounds.
The overall production of nitric oxide (NO) is decreased in chronic kidney disease (CKD), which contributes to cardiovascular events and further progression of kidney damage. Other disorders associated with low NO levels include glomerular hypertension, glomerular ischemia, glomerulosclerosis, tubulointerstitial injury, and proteinuria (Baylis, American Journal of Physiology-Renal Physiology, 2008, Vol. 294, No. 1).
These disorders can be treated using the NO-releasing compounds described herein.
Metabolic Syndrome
Metabolic syndrome (MS) is a cluster of metabolic disorders that collectively increase the risk of cardiovascular disease. Mitochondrial dysfunction is closely associated with obesity, metabolic syndrome and type 2 diabetes mellitus. Impaired nitric oxide synthase (NOS) activity is closely associated with insulin resistance.
Nitric oxide (NO) therefore plays a crucial role in the pathogeneses of MS components and is involved in different mitochondrial signaling pathways that control respiration and apoptosis (Litvinova, et al., “Nitric oxide and mitochondria in metabolic syndrome.” Frontiers in Physiology, 6 (1): 20 (2015)).
Administration of the compounds described herein can increase nitric oxide levels, and therefore treat metabolic syndrome and other metabolic disorders.
Restenosis
The compounds described herein can be used to treat or prevent restenosis, which often occurs after stents are inserted into blood vessels. In one embodiment, the compounds are administered using drug-loaded stents.
Types of Compound Delivery
Depending on the nature of the disorder, the compounds and/or compositions discussed herein can be administered by inhalation, nebulization, intranasal delivery, direct injection or application to, for example, an infected tissue. Administration can also include parenteral administration (e.g., intravenous, intramuscular, or intraperitoneal injection), subcutaneous administration, administration into vascular spaces, and/or administration into joints (e.g., intra-articular injection).
The compounds can also be administered via topical, vaginal, rectal, buccal, intrathecal, and intraarterial administration, or applied as a liquid or gel to a site of treatment.
The compounds can be administered to the mouth, nasal passages, throat, esophagus, larynx, pharynx, trachea, bronchioles, bronchi, upper airways, lower airways, subcutaneously or via an implant (for example, up under the ribs and into the chest cavity), and combinations thereof.
In some embodiments, the compounds are nebulized, inhaled, or delivered intranasally. In one specific embodiment, the methods comprise inhalation of particles including one or more of the compounds described herein aerosolized via nebulization. Nebulizers generally use compressed air or ultrasonic power to create inhalable aerosol droplets of the particles or suspensions thereof. In this embodiment, the nebulizing results in pulmonary delivery to the subject of aerosol droplets of the particles or suspension thereof. In another embodiment, the methods comprise inhalation of particles aerosolized via a pMDI, wherein the particles or suspensions thereof are suspended in a suitable propellant system (including but not limited to hydrofluoroalkanes (HFAs) containing at least one liquefied gas in a pressurized container sealed with a metering valve. Actuation of the valve results in delivery of a metered dose of an aerosol spray of the particles or suspensions thereof.
In one embodiment, the compounds are administered during lung lavage, which can be whole lung lavage or bronchoalveolar lavage (BAL). In BAL, also known as bronchoalveolar washing, a bronchoscope is passed through the mouth or nose into the lungs and fluid is squirted into a small part of the lung and then collected for examination. The compounds can travel through the fluid, and treat the entire fluid-coated portion of the lung.
Bronchoalveolar lavage is commonly used to diagnose infections in people with immune system problems, pneumonia in people on ventilators, some types of lung cancer, and scarring of the lung (interstitial lung disease). It is the most common method used to sample the epithelial lining fluid (ELF) and to determine the protein composition of the pulmonary airways. It is often used in immunological research as a means of sampling cells (for example, T cells) or pathogen levels (for example, influenza virus) in the lung. Whole lung lavage (WLL; or “lung washing”) is a treatment for pulmonary alveolar proteinosis. While the lung is washed, therapy with the compounds can also be administered, and the fluid allows the compounds to contact the entire fluid-coated surface of the lung.
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only.
This example pertains to the synthesis and identification of one embodiment of the compound of Formula III. This embodiment has the following features, advantages, and/or uses.
These molecules have NO-releasing properties. Compounds such as those disclosed were found to be the product of certain high pressure nitric oxide synthetic strategies. In this context, the compounds can form in trace to major components of the reaction, depending on reaction conditions, such as NO pressure, base content, temperature and, reactant content.
The methane trisdiazeniumdiolate, sodium salt, was prepared according to the following procedure in Table 1:
NaOH was dissolved in MeOH
Acetone was added to the stirring solution.
Mixture was transferred to a 400 mL Parr reactor, which was equipped with stir bar.
Parr reactor was sealed.
3× N3 Purge with 100 Psi of N2 while stirring.
2.5 bar of NO charged into Parr vessel and allowed to stir at room temperature for 4 days.
The solution was cloudy with an off-white to slightly yellow precipitate.
The reaction solution was filtered via vacuum filtration using 110 mm GF/F filter paper.
The solids collected during filtration were washed with 150 ml of MeOH.
The solids were dried overnight under vacuum at room temperature.
Recovery: 5.0 g+4.2 g=9.2 g off-white powder
The synthesized compound was isolated and tested to confirm identity. FTIR, HPLC, UV-Vis spectroscopy, 1H NMR and 13C NMR analysis were used to support the conclusions that the synthesis yielded the compound of Formula III.
Referring now to
Analytical results for the compound of Formula III are summarized in Table 2.
Antimicrobial Activity
The NO release of the compound of Formula III at pH 7.4 is shown in
The antibacterial efficacy of the compound of Formula III derived according to the synthesis example against various Pseudomonas bacteria strains are set forth in Tables 3-5. The results from testing varying amounts of the compound mixed with a representative excipient (β-cyclodextrin) is set forth in Table 6.
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
aeruginosa
In some cases, the synthesis of the compound of Formula III also generated an impurity, referred to herein as MD2, or methane bis-diazeniumdiolate, which has the following formula:
Although antimicrobial uses for the compounds are not disclosed in this application, the production of nitric oxide allows for one to effectively kill bacteria. Therefore, in determining the ability of the compound of Formula III and M2D to produce nitric oxide, bactericidal activity is a reasonable measure of nitric oxide release. Accordingly, the activity of mixtures of these compounds was evaluated against P. aeruginosa (PAK), with the goal of determining how the percentage of the compound of Formula III (referenced as MD3 in the table) relative to MD2 in different samples affected the activity of the mixture against P. aeruginosa. The data is shown below:
The results, shown in Table 7, demonstrate that the higher the percentage of the compound of Formula III in the sample relative to the percentage of MD2, the better the activity is against PAK. It should be noted that when samples have degraded under simulated physiological conditions, that MD2 does not not degrade to any significant degree. Nor does it grow in as a degradation product of the compound of Formula III. Therefore, MD2 does not release NO or HNO when subjected to physological temperatures and pHs. In the table, where a percentage of MD3 is shown, the balance, adding up to 100%, is predominantly MD2, with minor amounts of other impurities.
Although antimicrobial efficacy is not the subject matter of this application, it is important to know the optimal pH for releasing NO. Accordingly, in this example, the efficacy of the compound of Formula III against PAK was evaluated at various pHs, 6.4, 7.6, and 8.4, all of which are physiological pHs, though at different places in the human body. For example, tuminal pH in the proximal small bowel ranges from 5.5 to 7.0 and gradually rises to 6.5-7.5 in the distal ileum. There is a decrease in luminal pH from the terminal ileum to the caecum (range 5.5-7.5). The pH in the colon can range from 7.9 to 8.5. A normal blood pH level is 7.40, and this is approximately the pH in the lung. The pH of saliva is ranges from 6.5 to 7.5. The data (not shown) demonstrated that the release of NO from the compound of Formula III is pH-dependent, with higher rates of NO-release at a pH of around 6.5 than around 7.5, and relatively little NO release at a pH of around 8.4.
The compound of Formula III can be prepared using acetone, ethanol or acetonitrile as a starting material as well as other compound with similar functional groups, though when prepared from ethanol or acetonitrile (or any other compound), the impurity profiles for each will be unique. A set of chromatograms are shown of samples of the compound of Formula III prepared from acetone, ethanol, and acetonitrile. In each case a different set of impurities were observed. While the material prepared from ethanol was comparable in purity to the material prepared from acetone (>90% area), the material prepared from acetonitrile was significantly less pure (<40% area).
The process for synthesizing the compound of Formula III starting from acetone was optimized. A series of experiments were run to evaluate the effects of starting acetone concentration (14 vs 30 mg/mL), base equivalents (4 vs 6), NO pressure (2.5 vs 20 bar), and temperature (10 vs 20° C.) on the yield and purity of product produced. Table 8 show the results of the experiments run at an acetone concentration of 14 mg/ml and a temperature of 20° C. for 4 days. Each condition was run in duplicate.
The best overall conditions identified from these experiments are listed here: an acetone concentration of 14 mg/mL, an NO pressure of 20 bar, and a base equivalent of 4. Temperature had no effect between 10 and 20° C. when run at these conditions. At these conditions, an average purity of ˜97% area was achieved with a yield >95%.
It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within one or more of the inventions. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an embodiment can be used in all other embodiments set forth herein.
Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed inventions. Thus, it is intended that the scope of the present inventions herein disclosed should not be limited by the particular disclosed embodiments described above.
Moreover, while the invention is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any methods disclosed herein need not be performed in the order recited.
The methods disclosed herein include certain actions taken by a practitioner; however, they can also include any third-party instruction of those actions, either expressly or by implication. For example, actions such as “administering an NO-donating composition” include “instructing the administration of an NO-donating composition.” In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
The contents of all documents referred to herein are hereby incorporated by reference for all purposes.
The present patent application claims the benefit and priority of U.S. Provisional Patent Application No. 62/971,624, filed on Feb. 7, 2020, titled “NITRIC OXIDE-RELEASING ANTIBACTERIAL COMPOUNDS, FORMULATIONS, AND METHODS PERTAINING THERETO,” the contents of which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/016869 | 2/5/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62971624 | Feb 2020 | US |
