NAD(H) NANOPARTICLES AND METHODS OF REDUCING ISCHEMIAREPERFUSION INJURY IN KIDNEY TRANSPLANTS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S. Provisional Appl. No. 63/623,143, filed January 19, 2024, the contents of each of which are incorporated herein by reference in their entirety for any and all purposes.
GOVERNMENT RIGHTS
[0002] This invention was made with government support under AI155816 and AI165977 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD
[0003] The present technology relates generally to the field of bioavailable nanoparticles comprising NAD(H) (i.e., NAD+ and/or NADH) and use thereof in methods of reducing ischemia-reperfusion injury in kidney transplants. The nanoparticles optionally include a human cell membrane such as a human platelet cell membrane.
BACKGROUND
[0004] Ischemia-reperfusion (IR) injury is a major complication for all solid organ transplants, necessitating new therapeutic strategies to suppress IR injury and thereby improve kidney function and expand the availability of donor organs. In kidneys, IR injury is associated with the disruption of mitochondrial homeostasis, including depletion of NAD+ leading to diminished ATP levels which cells need to facilitate prolonged cell survival. However, NAD(H) are poorly bioavailable because of their inability to cross cell membranes in therapeutically significant amounts, making their direct use impractical as a therapeutic regime for IR. SUMMARY
[0005] The present technology provides bioavailable nanoparticles (NPs) that include the oxidized and/or reduced forms of nicotinamide dinucleotide, z.e., NAD+ and NADH respectively, or NAD(H) for short. It has been discovered that the present NPs and methods advantageously reduce IR injury in kidney transplants.
[0006] In one aspect, the present technology provides methods of reducing ischemiareperfusion (IR) injury in a kidney to be transplanted comprising administering an effective amount of a bioavailable nanoparticle comprising NAD+ and/or NADH to the kidney prior to surgical transplantation of the kidney into a subject. In some embodiments of the methods, administering an effective amount of the bioavailable nanoparticle comprises injecting an aqueous solution comprising the effective amount of the bioavailable nanoparticle into a renal blood vessel and/or storing the kidney in the aqueous solution prior to transplantation of the kidney in the subject.
[0007] In another aspect, the present technology provides methods of reducing ischemiareperfusion (IR) injury to a transplanted kidney in a subject (such as a human subject) comprising administering an effective amount of a bioavailable nanoparticle comprising NAD+ and/or NADH to the subject after transplantation of the kidney into the subject. The effective amount of bioavailable nanoparticles may be administered by injection, e.g., intravenously, to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a schematic illustration of an illustrative embodiment of a bioavailable nanoparticle structure.
[0009] FIGS. 2A-2C show the mouse kidney IRI study experimental design of the Examples. FIG. 2A is a schedule of direct kidney injection surgery. FIG. 2B shows schedule of IP injection surgery. FIG. 2C shows schedule of IV injection surgery.
[0010] FIGS. 3A-3D shows data for illustrative embodiments of bioavailable nanoparticles of the present technology. FIG. 3A shows the hydrodynamic size of NAD+-NP measured by DLS. FIG. 3B shows the morphology of NAD+-NP characterized by TEM. FIG. 3C shows the hydrodynamic size of NAD+-NP dispersed in aqueous solution at 4 °C monitored by DLS. FIG. 4D shows the hydrodynamic size of lyophilized NAD+-NP lyophilized containing 10 % sucrose as cryoprotectant and stored at -20 °C.
[0011] FIG. 4 shows a summary graph comparing blood creatinine values for all treatment groups of Example 4. Statistical analysis performed via Tukey’s comparisons test, *P < 0.05, **P < 0.01, *** P < 0.001, **** < 0.0001.
[0012] FIGS. 5A-5F shows representative kidney H&E micrographs of sham (FIGS. 5A and 5B), empty nanoparticle (FIGS. 5C and 5D), and NAD+ loaded nanoparticle (FIGS. 5E and 5F) treated mice.
[0013] FIG. 6 is a summary graph of the illustrative embodiment of Example 4 comparing tubule lesion scores for all treatment groups. Statistical analysis performed via Tukey’s comparisons test, *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001. f NAD+ Direct injection combined data for groups treated with 2.5mg/kg and 5mg/kg NAD+.
DETAILED DESCRIPTION
[0014] The following terms are used throughout as defined below. All other terms and phrases used herein have their ordinary meanings as one of skill in the art would understand.
[0015] As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
[0016] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.
[0017] As used herein, the terms “effective amount” or “therapeutically effective amount,” or “pharmaceutically effective amount” refer to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the full or partial amelioration of disease, disorder, condition or symptoms of a subject (e.g., a human) in need thereof. For example, the effective amount may be an amount that reduces or prevents IR injury to kidneys before and after transplantation, and/or promotes transplanted kidney health. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity, and type of disease. A person of ordinary skill in the art will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional compounds. Multiple doses may be administered. Additionally or alternatively, multiple therapeutic compositions or compounds may administered. In the methods described herein, the compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder described herein.
[0018] As used herein, the term “subject” refers to any animal that can experience IR injury to a transplanted kidney, such as a mammal. In any embodiments, the mammal may be selected from primates, dogs, cats, rodents, horses, cattle, or pigs. In any embodiments, the subject (i.e., primate subject) is a human.
[0019] “Treating,” “treat,” “treated,” or “treatment” as used herein covers the treatment of a disease or disorder described herein (e.g., restenosis), in a subject, such as a human, and includes: (i) inhibiting or preventing a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, preventing, relieving, ameliorating, or slowing progression of one or more symptoms of the disease or disorder. Symptoms may be assessed by methods known in the art or described herein, for example, biopsy, histology, and blood tests to determine relevant enzyme levels, metabolites or circulating antigen or antibody (or other biomarkers), quality of life questionnaires, patient-reported symptom scores, and imaging tests.
[0020] “Ameliorate,” “ameliorating,” and the like, as used herein, refer to inhibiting, relieving, eliminating, or slowing progression of one or more symptoms. [0021] “Metal organic framework” or “MOF” as used herein refers to the three-dimensional, porous, crystalline structure formed by metal ions and small organic ligands that coordinate to the metal ions. Thus, a “metal organic framework component” refers collectively to the individual component parts of the MOF, i.e., a metal ion and a coordinating ligand. For example, a zinc ion and 2-methylimidazole would be, respectively, the metal ion and coordinating ligand of the metal organic framework component for the MOF, zeolitic imidazolate framework-8, i.e., ZIF-8.
[0022] “Molecular weight” as used herein with respect to polymers refers to numberaverage molecular weights (Mn) and can be determined by techniques well known in the art including gel permeation chromatography (GPC). GPC analysis can be performed, for example, on a D6000M column calibrated with poly(methyl methacrylate) (PMMA) using triple detectors including a refractive index (RI) detector, a viscometer detector, and a light scattering detector, and 7V,7V’-dimethylformamide (DMF) as the eluent. “Molecular weight” in reference to small molecules and not polymers is actual molecular weight, not numberaverage molecular weight.
[0023] The terms “preventing” and “prophylaxis” as used herein refer to administering a pharmaceutical compound or medicament or a composition including the pharmaceutical compound or medicament to a subject before a disease, disorder, or condition fully manifests itself, to forestall the appearance and/or reduce the severity of one or more symptoms of the disease, disorder or condition. The person of ordinary skill in the art recognizes that the term “prevent” is not an absolute term. In the medical art it is understood to refer to the prophylactic administration of a drug to diminish the likelihood or seriousness of a disease, disorder or condition, or a symptom thereof, and this is the sense that such terms are used in this disclosure.
[0024] The phrase “targeting ligand” refers to a ligand that binds to “a targeted receptor” that distinguishes the cell being targeted from other cells. The ligands may be capable of binding due to expression or preferential expression of a receptor for the ligand, accessible for ligand binding, on the target cells. While use of a bioavailable nanoparticle including a human cell membrane may not require a separate targeting ligand, other types of bioavailable nanoparticles may benefit from the attachment of targeting ligands thereto. Examples of such ligands include various oligo and polysaccharides, e.g., mannan, heparain, levan, chondroitin sulfate, glycogen, alginate, and galactan (targeting infected tissues); galactose (targeting liver); those targeting macrophages such as mannose (targeting mannose receptor, CD206); ferritin (targeting ferritin receptor); and hyaluronic acid (targeting CD44 over-expressed macrophages); those targeting immune system cells such as MPLA (targeting TLR4), and imiquimod (targeting TLR7); those targeting endothelial cells such as anti-EGFR nanobody (targeting EGFR receptor); avP3 integrin ligand LXW7 (targeting avP3 integrin); avP3 integrin ligand GRGD peptide (targeting avP3 integrin); thiophosphate modified aptamer or E-selectin binding peptide (DITWDQLWDLMK or KYDGDITWDQLWDLMK) (targeting E-Selectin).
[0025] The phrase “a targeted receptor” refers to a receptor expressed by a cell that is capable of binding a cell targeting ligand. The receptor may be expressed on the surface of the cell. The receptor may be a transmembrane receptor. Examples of such targeted receptors include galactose receptors expressed by hepatocytes, receptors for C-type lectins (e.g., mannose receptor) receptors expressed by macrophages, EGFR expressed by endothelial cells, and a variety of lectin receptors expressed by bacteria.
[0026] A “cell penetrating peptide” (CPP), also referred to as a “protein transduction domain” (PTD), a “membrane translocating sequence,” and a “Trojan peptide”, refers to a short peptide e.g., from 4 to about 40 amino acids) that has the ability to translocate across a cellular membrane to gain access to the interior of a cell and to carry into the cells a variety of covalently and noncovalently conjugated cargoes, including proteins, oligonucleotides, and liposomes. They are typically highly cationic and rich in arginine and lysine amino acids. Examples of such peptides include TAT cell penetrating peptide (GRKKRRQRRRPQ); MAP (KLALKLALKALKAALKLA); Penetratin or Antenapedia PTD (RQIKWFQNRRMKWKK); Penetratin- Arg: (RQIRIWFQNRRMRWRR); antitrypsin (358- 374): (CSIPPEVKFNKPFVYLI); Temporin L: (FVQWFSKFLGRIL-NH2); Maurocalcine: GDC(acm) (LPHLKLC); pVEC (Cadherin-5): (LLIILRRRIRKQAHAHSK); Calcitonin: (LGTYTQDFNKFHTFPQTAIGVGA P); Neurturin:
(GAAEAAARVYDLGLRRLRQRRRLRRERVRA); Penetratin: (RQIKIWFQNR
RMKWKKGG); TAT-HA2 Fusion Peptide: (RRRQRRKKRGGDIMGEWGNEIFGAIAGFLG); TAT (47-57) Y(GRKKRRQRRR); SynBl (RGGRLSYSRRRFSTSTGR); SynB3 (RRLSYSRRRF); PTD-4 (PIRRRKKLRRL); PTD-5 (RRQRRTSKLMKR); FHV Coat-(35-49) (RRRRNRTRRNRRRVR); BMV Gag-(7- 25) (KMTRAQRRAAARRNRWTAR); HTLV-II Rex-(4-16) (TRRQRTRRARRNR); HIV- 1 Tat (48-60) or D-Tat (GRKKRRQRRRPPQ); R9-Tat (GRRRRRRRRRPPQ); Transportan (GWTLNSAGYLLGKINLKALAALAKKIL chimera); SBP or Human Pl (MGLGLHLLVLAAALQGAWSQPKKKRKV); FBP (GALFLGWLGAAGS TMGAWSQPKKKRKV); MPG (ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya (wherein cya is cysteamine)); MPG(ANLS) (ac- GALFLGFLGAAGSTMGAW SQPKSKRKV-cya); Pep-1 or Pep- 1 -Cysteamine (ac-KETWWETWWTEWSQPKKKRKV- cya); Pep-2 (ac-KETWFET WFTEWSQPKKKRKV-cya); Periodic sequences, Polyarginines (RxN (4<N<17) chimera); Polylysines (KxN (4<N<17) chimera); (RAca)6R; (RAbu)6R; (RG)6R; (RM)6R; (RT)6R; (RS)6R; RIO; (RA)6R; and R7.
[0027] A “dye” refers to small organic molecules having a molecular weight (actual, not number average) of 2,000 Da or less or a protein which is able to emit light. Non-limiting examples of dyes include fluorophores, chemiluminescent or phosphorescent entities. For example, dyes useful in the present technology include but are not limited to cyanine dyes (e.g., Cy2, Cy3, Cy5, Cy5.5, Cy7, and sulfonated versions thereof), fluorescein isothiocyanate (FITC), ALEXA FLUOR® dyes e.g., ALEXA FLUOR® 488, 546, or 633), DYLIGHT® dyes e.g., DYLIGHT® 350, 405, 488, 550, 594, 633, 650, 680, 755, or 800) or fluorescent proteins such as GFP (Green Fluorescent Protein).
[0028] A “metal chelating ligand” as used herein refers to ligands that chelate metal isotopes for use in imaging. Non-limiting examples of metal chelating ligands include triazacyclononane-phosphinic acid (i.e., TRAP), 1,4, 7,10-tetraazacyclododecane- 1,4, 7,10- tetraacetic acid (i.e., DOTA), 1,4,7-triazacyclononane-triacetic acid (i.e., NOTA), diethylenetriaminepentaacetic acid i.e., DTP A), or chelating peptides). Thus, the metal chelating ligand may be one for use in PET or MRI.
[0029] In one aspect, the present technology provides methods of reducing ischemiareperfusion (IR) injury in a kidney to be transplanted comprising administering an effective amount of a bioavailable nanoparticle comprising NAD+ and/or NADH to the kidney prior to surgical transplantation of the kidney into a subject. “Bioavailable nanoparticles comprising NAD+ and/or NADH” of the present technology are synthetic nanoparticles (and do not include naturally occurring particles such as cells or organelles) that are capable of delivering NAD+ and/or NADH across a cell membrane, e.g., an endothelial cell membrane, at a level at least two times that of NAD(H) alone in solution. In some embodiments, administering an effective amount of the bioavailable nanoparticle comprises comprises injecting an aqueous solution comprising the effective amount of the bioavailable nanoparticle into a renal blood vessel and/or storing the kidney in the aqueous solution prior to transplantation of the kidney in the subject. The injection may take place prior to or concurrently with storage in the aqueous solution, and the kidney may be flushed one or more times (e.g., once, twice, or more) with the aqueous solution containing the effective amount of NAD+ and/or NADH.
[0030] In the present methods of reducing IR injury in a kidney, the aqueous solution may include saline and/or one or more additives selected from the group consisting of potassium lactobionate, potassium hydrogen phosphate, magnesium sulfate, raffinose, denosine, glutathione, allopurinol, and hydroxyethyl starch. The aqueous solution may be used at room temperature or may be cooled, e.g., in an ice bath or using a chiller. Thus, in some embodiments of the present methods, the aqueous solution may have a temperature from 0°C to about 25°C, including 0°C or any of about 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or a range between and including any two of the foregoing values. For example, the aqueous solution may be used at a temperature from 0°C to about 5 °C.
[0031] In some embodiments of the present methods, the effective amount of NAD+ and/or NADH may be about 1 pM to about 10 mM. For example, the effective amount of NAD+ and/or NADH may be about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 125 pM, 150 pM, 200 pM, 300 pM, 400 pM, 500 pM, 750 pM, 1 mM, 2 mM, 5 mM, 10 mM or a range between and including any two of the foregoing values. For example, the effective amount of NAD+ and/or NADH may be about 2 pM to about 100 pM or from about 5 pM to about 20 pM.
[0032] In another aspect, the present technology provides methods of reducing ischemiareperfusion (IR) injury to a transplanted kidney in a subject comprising administering an effective amount of a bioavailable nanoparticle comprising NAD+ and/or NADH to the subject after transplantation of the kidney into the subject.
[0033] In the methods of IR injury to a transplanted kidney in a subject, the effective amount of NAD+ and/or NADH may vary with the application but typically falls within the range of about 0.1 mg/kg (of the subject being treated) to about 200 mg/kg. Thus, in some embodiments the effective amount may be any of about 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 mg/kg or a range between and including any two of the foregoing values. For example, the effective amount of NAD+ and/or NADH may be about 1 mg/kg to about 100 mg/kg or about 5 mg/kg to about 20 mg/kg.
[0034] A variety of bioavailable nanoparticles may be used to deliver the NAD(H) in methods of the present technology. For example, the nanoparticles may include an inorganic core and NAD+ and/or NADH, and a coating including a lipid bilayer. The inorganic core may be calcium phosphate or a metal organic framework (MOF). The MOF may include a transition metal ion coordinated to a coordinating ligand, wherein the transition metal ion is selected from the group consisting of zinc, iron, zirconium, copper, and cobalt ions, and the coordinating ligand is selected from an imidazolate ligand or a carboxylate ligand. While not wishing to be bound by theory, the NAD(H)-loaded NPs are belived to be taken up by the cells via endocytosis and directly replenish cellular NAD+. The CaP or MOF cores are believed to dissolve in the acidic environment of the endosome, leading to endosome swelling and bursting (due to an increase in osmotic pressure) to release the entrapped payload into cytosol.
[0035] The amount of NAD+ or NADH loaded into the nanoparticle may vary, e.g., from 1 wt% to 50 wt% NAD+, NADH, or both. Thus, in any embodiments, the nanoparticles of the present methods may include 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45wt%, or 50 wt% NAD+ and/or NADH, or a range between and including any two of the foregoing values, e.g., 1 wt% to 25 wt% or 2 wt% to 20 wt%, or 3 wt% to 15 wt% NAD+ and/or NADH among others. [0036] In any embodiments of the present methods where the NPs include a lipid bilayer, any suitable lipids may be used. For example, the lipids of the lipid bilay er(s) may be selected from the group consisting of L-a-phosphatidylcholine (PC), l,2-dioleoyl-sn-glycero-3- phosphate (DOPA), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn- glycero-3 -phosphocholine (DOPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), l,2-distearoyl-sn-glycero-3- phosphorylethanolamine (DSPE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)] (DSPE-PEG), cholesterol, and combinations of two or more thereof. In any embodiments of the NPs, the lipids of the lipid bilayer may include a combination of PC and/or DOPA. In any embodiments, the lipids of the lipid bilayer may include a combination of PC and/or DOPA, and cholesterol. The lipids of the lipid bilayer may include DSPE or DSPE-PEG.
[0037] In any embodiments of the present methods, a portion of lipids in the lipid bilayer of the NPs may be conjugated to poly(ethylene glycol) (PEG). Up to 100 mol% of the lipids in the lipid bilayer may be conjugated to PEG, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mol%, or a range between and including any two of the foregoing values. In any embodiments, the lipids of the lipid bilayer may include DSPE-PEG. The PEG (when conjugated to a lipid) includes a free terminus selected from the group consisting of OH, O-Ci-4 alkyl ether, NH2, NHR, COOH, COOR, wherein R is an alkyl or alkenyl group, a dye, a targeting ligand, and a metal chelating ligand. Thus, the PEG terminus may be optionaly conjugated to a dye, a targeting ligand, or a metal chelating ligand directly or through any suitable linker (e.g., with a molecular weight below about 500 Da) known in the art. The PEG may have a number average molecular weight ranging from 300 to 10000 Da. In any embodiments the PEG may have a number average molecular weight of about 300, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 Da or a range between and including any two of the foregoing values, e.g., 3000-7000 Da. In any embodiments, the lipid of the lipid bilayer(s) includes a cell membrane extracted from a red blood cell, macrophage, neutrophil, or platelet, and combinations of two or more thereof.
[0038] In any embodiments, the nanoparticles of the present methods may include lipid bilayer(s) which coat the surface of the nanoparticle in whole or in part. The nanoparticles may include, e.g., 10 wt% to 75 wt% lipid bilayer(s). Thus, in any embodiments, the present nanoparticles may include 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt% lipid bilayer(s), or a range between and including any two of the foregoing values, e.g., 10 wt% to 40 wt%, 10 wt% to 30 wt%, 15 wt% to 40 wt%, 20 wt% to 75 wt%, 30 wt% to 70 wt%, or 50 wt% to 70 wt% lipid bilayer(s).
[0039] In any embodiments of the present methods where the bioavailable NPs include a lipid bilayer, the lipid bilayer may include or be encapsulated by (in part or in whole) a human cell membrane selected from the group consisting of a human platelet membrane, a human red blood cell membrane, a human white blood cell membrane and a human macrophage cell membrane. For example, the lipid bilayer may include or be encapsulated by (in part or in whole) a human platelet membrane. In any embodiments, the weight ratio of of the human cell membrane coating to the remainder of the bioavailable nanoparticle (e.g., inorganic core, lipid bilayer, and NAD(H)) may be 2: 1 to 1 :2. In some embodiments, the weight ratio of of the human cell membrane coating to the remainder of the bioavailable nanoparticle (e.g., inorganic core, lipid bilayer, and NAD(H)) may be about 1 : 1. In some embodiments, the human cell membrane coating may be a human platelet membrane coating, and the weight ratio of of the human platelet membrane coating to the remainder of the bioavailable nanoparticle (e.g., inorganic core, lipid bilayer, and NAD(H)) may be, e.g., about 2: 1 to about 1 :2 or, e.g., about 1 : 1. In any embodiments
[0040] In any embodiments, cell penetrating peptides, targeting ligands, and/or metal chelating agents may optionally be conjugated directly or indirectly (through any suitable linker of less than 500 Da known in the art) to lipids in the lipid bilayer.
[0041] In any embodiments, the present NPs include 10 wt% to 90 wt% inorganic core, e.g., 10 wt% - 90 wt% calcium phosphate or 10 wt% - 90 wt% MOF. For example, the present nanoparticles may include 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt% inorganic core or a range between and including any two of the foregoing values, e.g., 20 wt% to 80 wt%, or 40 wt% to 60 wt%. [0042] In any embodiments, the inorganic core is calcium phosphate. Calcium phosphate may be a single compound but is more typically a mixture of two or more such compounds having, e.g., a molar ratio of Ca to P from 0.5 to 2 (e.g., 0.5, 1.2, 1.33, 1.5, 1.67, 2 or a range between and including same). In some embodiments the calcium phosphate comprises one or more of hydroxy apatite (Caio(P04)e(OH)2), Ca(H2PO4)2*H2O, CaQHhPCU CaHPO4*2H2O, Ca(HPO4) and the like.
[0043] In any embodiments, the inorganic core of the present nanoparticles may be a MOF. As noted above the transition metal ion of the MOF may be selected from the group consisting of zinc, iron, zirconium, copper, and cobalt ions. In any embodiments the transition metal ion of the MOF may be zinc ions or iron ions. The coordinating ligand may be an imidazolate ligand or a carboxylate ligand as noted above. Imidazolate ligands are coordinating ligands that contain an imidazole group such as, e.g., imidazole itself, 2-methyl -imidazole, benzimidazole, or 5 -methylbenzimidazole. Carboxylate ligands include, e.g., terephthalic acid, 2-methyl-pterphthalic acid, 2-hydroxy-terphthalic acid, and 2-amino-terphthalic acid. The imidazolate and carboxylate ions are typically but are not necessarily in their anionic forms. Those of skill in the art will recognize which ligands are suitable for use with a particular type of metal to form a metal organic framework component. By way of example only, zinc may be used with imidazolate ligands and iron may be used with carboxylate ligands, especially dicarboxylate ligands. In any embodiments, the coordinating ligand also may be selected from the group consisting of imidazole, 2-methyl-imidazole, benzimidazole, 5-methylbenzimidazole, terephthalic acid, 2-methyl-pterphthalic acid, 2-hydroxy-terphthalic acid, and 2-amino-terphthalic acid, benzene-l,3,5-tricarboxylic acid, l,3,5-tris(4- carboxyphenyl)benzene, 2,6-naphthalenedicarboxylic acid, 4,4',4''- -triazine-2,4,6-triyl- tribenzoic acid, and 2,5-dihydroxyterephthalic acid. In any embodiments, the coordinating ligand is 2-methyl-imidazole. In any embodiments, the the MOF includes zinc ions and imidazolate ligands. In any embodiments, the imidazolate ligand may be selected from imidazole, 2-methyl-imidazole, benzimidazole, or 5-methylbenzimidazole. In any embodiments of the present NPs, the imidiazolate ligand may be 2-methyl-imidazole.
[0044] The present nanoparticles may include a metal organic framework component as described above. Suitable metal ions that may be employed in the metal organic framework component include zinc, iron, zirconium, copper, and cobalt ions. In any embodiments the metal ion may be zinc ion or it may be iron ion. The coordinating ligand may be an imidazolate ligand or a carboxylate ligand as noted above. Imidazolate ligands are coordinating ligands that contain an imidazole group such as, e.g., imidazole itself, 2-methyl- imidazole, benzimidazole, or 5-methylbenzimidazole. Carboxylate ligands include, e.g., terephthalic acid, 2-methyl-pterphthalic acid, 2-hydroxy-terphthalic acid, and 2-amino- terphthalic acid. The imidazolate and carboxylate ions are typically but are not necessarily in their anionic forms. Those of skill in the art will recognize which ligands are suitable for use with a particular type of metal to form a metal organic framework component. By way of example only, zinc may be used with imidazolate ligands and iron may be used with carboxylate ligands, especially dicarboxylate ligands.
[0045] The present nanoparticles may have a hydrodynamic diameter ranging from at least 50 nm to less than 1000 nm. For example, the NPs may have a hydrodynamic diameter of 50, 60, 70, 80, 90, 100, 110, 130, 150, 170, 200, 250, 300, 400, 500, 600, 700, 800, 900, or less than 1000 nm or a range between and including any two of the foregoing values. Such ranges include but are not limited to NPs with a hydrodynamic diameter of 70 to 700 nm or 100 to 400 nm. Alternatively, the present technology provides NPs with a median or average hydrodynamic diameter, also selected from any of the foregoing values or ranges.
[0046] In any embodiments, the bioavailable nanoparticle may be a nanoparticle comprising a disulfide-containing lipopeptide as disclosed in USSN 63/443,675 (filed 2/6/2023 and titled “Disulfide-containing lipopeptides, nanoparticles and methods of use”), incorporated by reference herein and for all purposes. Thus, the bioavailable nanoparticle may include a compound of Formula I:
I a stereoisomer thereof, and/or a pharmaceutically acceptable salt thereof, wherein
X1 and X2 are independently absent or selected from unsubstituted Ci-6 alkylene or C2-6 alkenylene;
X3 is selected from unsubstituted C1-6 alkylene or C2-6 alkenylene;
Y1 and Y2, are each independently absent, C(O)O, or C(O)NH;
Y3 is absent, C(O), C(O)O, or C(O)NH;
Y4 is C(O)O, C(O)NH, NHC(O)O, or NHC(O)NH;
R1 and R2 are independently selected from unsubstituted Cs-24 alkyl or Cs-24 alkenyl groups;
R3 and R4 are independently absent or selected from an amino acid residue, a peptide or isopeptide comprising 2-10 amino acid residues, or a C1-12 alkyl group, each of which is optionally substituted with 1, 2, or 3 ionizable functional groups such that at least one ionizable functional group is present on at least one of R3 and R4, provided that Y3 is absent when R3 is an amino acid, peptide, or isopeptide, and Y4 is absent when R4 is an amino acid residue, peptide, or isopeptide; and further provided that if one of R3 and R4 is absent, the other is present; and n and p are each independently 1, 2, 3, 4, or 5.
[0047] In one embodiment, the present technology provides a bioavailable nanoparticle that includes a compound of Formula I that is a compound of Formula IA, a stereoisomer thereof, and/or a pharmaceutically acceptable salt thereof, wherein
X1 and X2 are independently absent or selected from unsubstituted Ci-6 alkylene or C2-6 alkenylene;
X3 is selected from unsubstituted C1-6 alkylene or C2-6 alkenylene;
Y1 and Y2 are each independently selected from C(O)O or C(O)NH;
R1 and R2 are independently selected from unsubstituted Cs-24 alkyl or Cs-24 alkenyl groups;
R3 and R4 are independently selected from C1-10 alkyl groups substituted with 1, 2, or 3 ionizable functional groups; and n and p are each independently 1, 2, 3, 4, or 5.
[0048] In any embodiments, the bioavailable nanoparticle includes a compound of Formula I (or IA) that is a compound of Formula II,
[0049] In any embodiments, bioavailable nanoparticle includes a compound of Formula I (or IA) that is a compound of Formula III,
[0050] In any embodiments, the present compounds may include 1, 2, or 3 ionizable functional groups. In any embodiments, each ionizable functional group may be independently selected from NH2, NHR, NR2, guanidine, imidazole, or amidine, wherein each R is independently an unsubstituted Ci-6 alkyl, phenyl, or benzyl group. In any embodiments, at least one ionizable functional group is guanidine.
[0051] Thus, the bioavailabl nanoparticle includes any of the disulfide-containing lipopeptides disclosed herein, including but not limited to compounds of Formulas I, II, and III. In any embodiments, the bioavailable nanoparticle may further include a PEG-lipid, e.g., PEG-DSPE and/or PEG-DMG. In any embodiments, the nanoparticles may further include a structural lipid, e.g., cholesterol and/or P-sitosterol. In any embodiments, the nanoparticles may further include a phospholipid, e.g., DSPC and/or DOPE.
[0052] In any embodiments, the bioavailable nanoparticle may be a nanoparticle comprising a poly(amidoamine) oligomer, as disclosed in USSN 63/546,129 (filed 10/27/2023 and titled “Lipid nanoparticis formed by lipidoids for efficient delivery of nucleotide drugs and biologies”), incorporated by reference herein and for all purposes. Thus, the bioavailable nanoparticle may include in one aspect, the present technology provides a compound of Formula IV, wherein
PAO is a linear or branched polyamine or poly(amidoamine) oligomer, each having 2 to 32 amine groups and 2 to 100 carbon atoms, wherein each ester side-chain is connected to the PAO by one of the amine groups;
R at each occurrence is independently selected from a Ce-20 alkyl group, a Ce-20 alkenyl group, or TG; TG is a monosaccharide selected from the group consisting of glucose, O-protected glucose, galactose, O-protected galactose, fructose, O-protected fructose, O-protected N-acetylgalactosamine, and N-acetylgalactosamine; and n is 2 to 32.
[0053] The PAO of structure IV may be a linear or branched polyamine or poly(amidoamine) oligomer having 2 to 32 amine groups, i.e., 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 32 amine groups or a range between any two of the foregoing values. For example, the PAO may have 4 to 30, 5 to 28, 6 to 26, or 8 to 26 amine groups. The PAO may have 2 to 120 carbon atoms, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or a range between any two of the foregoing values. For example, the PAO may have 4 to 120 carbon atoms, 6 to 120 carbon atoms, 8 to 110 carbon atoms, 4 to 100 carbon atoms, 6 to 100 carbon atoms, or 10 to 100 carbon atoms. In any embodiments the PAO may be linear. In any embodiments, the PAO may be branched. In any embodiments, the PAO may have a carbon chain of 2-4 carbons connecting a pair of amine groups. The carbon chain of 2-4 carbons may be a linear alkylene group. In any embodiments, each pair of amine groups may be connected by a carbon chain of 2-4 carbons. In any embodiments, the PAO may have 6 to 24 or 8 to 26 amine groups. In any embodiments, the PAO may have 10 to 100 carbon atoms. In any embodiments, the PAO may have 10 to 16 amine groups and 18 to 60 carbon atoms. In any embodiments, the PAO may be branched.
[0054] In the compound of Formula IV, n may also be 2 to 32, i.e., any of 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, 30, 31, or 32 or a range between and including any two of the foregoing values. For example, n may be 4 to 8 or
[0055] In any embodiments, the PAO may be a linear polyamine oligomer. In any embodiments, the PAO may be a branched polyamine oligomer, e.g., a polyethyleneimine (PEI) oligomer. In any embodiments, the PAO (including but not limited to the PEI oligomer) may have a number average molecular weight of about 200 Da to about 1800 Da, e.g., any of about 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 or a range between and including any two of the foregoing values. Thus, in any embodiments, the PAO may have a number average molecular weight of about 400 Da to about 800 Da.
[0056] In any embodiments, the PAO may comprise a poly(amidoamine) oligomer (PAMAM), e.g., a PAMAM dendrimer with an ethylenediamine core. In any embodiments, the PAMAM dendrimer may be a generation 0 or 1. In any embodiments, the PAMAM dendrimer may be capped with a C2-4 alkylenediamine, e.g., ethylenediamine. Thus, in any embodiments, the PAO may have the structure of Formula V:
[0057] It will be understood by those skilled in the art that the squiggly lines indicate the attachment points to the ester side chains of Formula IV.
[0058] As a small polymer, the PAO may or may not be polydisperse. Typically, the PAO may have a poly dispersity of about 1 to about 2. For example, the poly dispersity may be any of about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or may be within a range between and including any two of the foregoing values, such as about 1 to about 1.5.
[0059] In any embodiments of the compound of Formula IV, at least one occurrence of R is a C6-20 alkylene group. In any embodiments at least one occurrence of R is a Cio-18 alkylene group. In any embodiments, at least one occurrence of R is an C6-20 alkenyl group. In some such embodiments, the C6-20 alkenyl group may have 1, 2 or 3 carbon-carbon double bonds. In any embodiments, at least one occurrence of R is a Cio-18 alkenyl group having 1 or 2 carbon-carbon double bonds. In any embodiments, at least one occurrence of R is TG. In any embodiments, TG may be glucose, galactose, fructose, or N-acetylgalactosamine. In any embodiments, TG is glucose. In any embodiments, TG may be O-protected galactose, fructose, O-protected fructose, or O-protected N-acetylgalactosamine. In some such embodiments, the O-protected moieties are protected as ketals, e.g., acetonides.
[0060] The compound of Formula IV may include various amounts of different R groups. For example, about 10% to about 90% or about 25% to about 75% of R groups may be Ce-20 alkyl groups (e.g., Cio-is alkyl groups or any of those disclosed herein), and the remaining R groups may be TG. In any embodiments, about 25% to about 50% of R groups may be a Ce- 20 alkyl group (e.g., Cio-is alkyl groups or any of those disclosed herein) and the remaining R groups may be TG. In any embodiments, about 50% of R groups may be Ce-20 alkyl groups, e.g., Cio-18 alkyl groups or any of those disclosed herein.
[0061] Thus, the present technology provides lipid nanoparticles (LNP) that include a compound of Formula IV as disclosed herein and a PEG-lipid. In any embodiments, the LNP includes about 75 wt% to about 95 wt% of the compound of Formula IV and about 5 wt% to about 25 wt% of the PEG-lipid. In any embodiments, the LNP includes about 85 wt% to about 90 wt% of the compound and about 10 wt% to about 15 wt% of the PEG-lipid.
[0062] LNPs of the present technology may include a variety of PEG-lipids. For example, the LNP may include DMG-PEG, DSPE-PEG and/or ceramide-PEG. In any embodiments, the PEG-lipid may include DMG-mPEG. The PEG-lipid may further include CPP-DMG- mPEG and/or glucose-DMG-mPEG.
[0063] A pharmaceutical composition comprising bioavailable nanoparticle as described herein and a pharmaceutically acceptable carrier and/or excipient. In any embodiments of the present methods, the bioavailable nanoparticles may be administered to the subject, e.g., a human subject, as a pharmaceutical composition.
[0064] The compositions described herein can be formulated for various routes of administration, for example, by injection, including by not limited to intravenous injection, or by parenteral, intravitreal, intrathecal, intracerebroventricular, rectal, nasal, vaginal administration, or via a temporary and/or implanted reservoir (e.g., a stent comprising a reservoir of bioavailable nanoparticles. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, and intramuscular injections. The following dosage forms are given by way of example and should not be construed as limiting the instant present technology.
[0065] Injectable dosage forms generally include solutions or aqueous suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent so long as such agents do not interfere with formation of the nanoparticles described herein. Inj ectable forms may be prepared with acceptable solvents or vehicles including, but not limited to sterilized water, phosphate buffer solution, Ringer's solution, 5% dextrose, or an isotonic aqueous saline solution.
[0066] Besides those representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the instant present technology. Such excipients and carriers are described, for example, in “Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference. Exemplary carriers and excipients may include but are not limited to USP sterile water, saline, buffers (e.g., phosphate, bicarbonate, etc.), tonicity agents (e.g., glycerol),
[0067] Specific dosages may be adjusted depending on conditions of disease, the age, body weight, general health conditions, sex, and diet of the subject, dose intervals, administration routes, excretion rate, and combinations of drug conjugates. Any of the above dosage forms containing effective amounts are well within the bounds of routine experimentation and therefore, well within the scope of the instant present technology. By way of example only, such dosages may be used to administer effective amounts of the present bioavailable nanoparticles to the patient and may include 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 mg/kg or a range between and including any two of the forgoing values such as about 0.1 to about 200 mg/kg, or about 1 mg/kg to aboutlOO mg/kg, or about 2.5 mg/kg to about 40 mg/kg or even 20 mg/kg. Such amounts may be administered parenterally as described herein and may take place over a period of time including but not limited to 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 12, hours, 15 hours, 20 hours, 24 hours or a range between and including any of the foregoing values. The frequency of administration may vary, for example, once per day, per 2 days, per 3 days, per week, per 10 days, per 2 weeks, or a range between and including any of the foregoing frequencies. More frequent administration is also possible. Alternatively, the compositions may be administered once per day on 2, 3, 4, 5, 6 or 7 consecutive days. A complete regimen may thus be completed in only a few days or over the course of 1, 2, 3, 4 or more weeks.
[0068] The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the nanoparticles compositions of the present technology. To the extent that the compositions include ionizable components, salts such as pharmaceutically acceptable salts of such components may also be used. The examples herein are also presented in order to more fully illustrate the preferred aspects of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations or aspects of the present technology described above. The variations or aspects described above may also further each include or incorporate the variations of any or all other variations or aspects of the present technology.
EXAMPLES
Materials and General Procedures
[0069] Materials. Calcium chloride (CaC12), P-nicotinamide adenine dinucleotide (NAD+), and IGEPAL CO-520 (polyoxyethylene nonylphenyl ether, a non-ionic surfactant) were sourced from Sigma-Aldrich (St. Louis, MO, USA). Disodium hydrogen phosphate (Na2HPO4) was purchased from Dot Scientific Inc. (Burton, MI, USA). L-a- phosphatidylcholine (Soy PC) and dioleoylphosphatydic acid (DOPA) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). N-(Methylpolyoxyethylene oxy carbonyl)- 1,2- distearoyl-sn-glycero-3-phosphoethanol-amine (DSPE-mPEG2k) was purchased from NOF America (New York, NY, USA). Chloroform and cyclohexane were purchased from Thermo Fisher Scientific (Fitchburg, WI, USA). [0070] Characterization. Dynamic light scattering (DLS) was used to measure the hydrodynamic diameter and zeta potential of NAD+-NP and related nanoparticles using a ZetaSizer Nano ZS90 spectrometer (Malvern Instruments, USA). Transmission electron microscopy (TEM, FEI Tecnai G2 F30 TWIN 300 KV, E.A. Fischione Instruments, Inc., USA) was used to determine the morphology of these nanoparticles. A schematic illustration of the nanoparticle structure is shown in FIG. 1. To evaluate the loading efficiency and loading content of the NAD(H)-loaded NPs, the NPs were dissolved in 0.01 M HC1 to release NAD(H). The pH of the solutions was adjusted to neutral, and the NAD(H) concentration was determined using HPLC or an Amplite™ Colorimetric Total NAD and NADH Assay Kit (AAT Bioquest, Sunnyvale, CA, USA). For the HPLC method, mobile phase A (20 mM Na^PCU) and mobile phase B (acetonitrile/acetic acid 0.01%) were delivered in a gradient elution mode at 1 mL/min. Phase B increased linearly from 0% to 60% from 0 to 10 min. The NAD(H) signals were detected at 260 nm. The loading efficiency and loading content in percentages were calculated according to the following equations: Loading efficiency (%) = (weight of encapsulated NAD(H) / total weight of NAD(H) added) x 100%. Loading content (%) = weight of encapsulated drug / total weight of nanoparticles x 100%.
[0071] Stability Assay. The freshly made NAD+-NP were stored in 10 mM Tris HC1 buffer (pH 7.4) at 4°C for stability study. The hydrodynamic sizes of the NAD+-NP were measured daily by DLS as above. To prepare lyophilized NAD+-NP, freshly made NAD+- NPs solution was supplemented with 10% (w/v) sucrose as the cryoprotectant and then stored at -20°C post-lyophilization. The lyophilized NAD+-NP were redispersed in DI water and their hydrodynamic sizes were measured weekly by DLS as above.
[0072] Animal Protocol. Male C57BL/6 mice (Charles River Laboratories) weighing 22 to 33 g were used for all experiments. Animals were housed in specific pathogen-free conditions in the animal care facilities at the University of Wisconsin (UW)-Madison Institute for Medical Research in accordance with institutional guidelines. The study protocol (#B0007) was approved by the Institutional Animal Care and Use Committee at the UW School of Medicine and Public Health, and all animals were treated ethically. [0073] Surgical and Treatment Procedures. Mice were randomized into one of fourteen experimental and control groups (Table 1), each consisting of 4 to 10 animals. NAD+-NP, ENP or free NAD+ treatments were administered using three different techniques: direct injection into the kidney via the renal artery and two different systemic applications via intraperitoneal injection (IP), and intravenously (IV) via the vena cava (VC). The schedule by which each technique was administered is shown diagrammatically in FIGS. 2A-2C.
Table 1
[0074] All three treatment methods began with anesthetizing the mouse with 5% isoflurane inhalation, shaving the abdomen with an electric clipper and disinfecting the abdomen with 75% alcohol. The mouse was then positioned in a supine position on a heated surgery pad with its nose in an anesthesia cone and limbs immobilized with tape followed by lowering the isoflurane to 2%.
[0075] Following immobilization on the surgery pad, mice in the kidney injected treatment group received a longitudinal midline skin and muscle incision from the pubis to the xiphoid followed by retractor insertion. The intestines were mobilized to the right side of the abdomen and covered with moistened gauze, leaving the left kidney, renal vessels, and aorta exposed. The tips of two microvascular clamps were placed on the aorta as far proximal and distal to the renal vessels as possible. NAD+-NP, ENP or free NAD+ were raised in 100 pL of saline at concentrations of 2.5, 5.0 or 20 mg/kg and injected into the aorta distal to the renal artery. Successful injection and perfusion of the kidney was judged by the change in color of the kidney from normal red to olive green. Immediately after perfusion, a non-traumatic microvascular clamp was used to occlude the renal hilum as close to the kidney as possible, preventing blood circulation through the kidney, for a 30 min IR injury incubation. A single suture is then used to repair the needle hole in the aorta followed by removal of the micro clamps on the aorta.
[0076] Mice in the systemic IP injected treatment group, once immobilized on the surgery pad, received an injection of 20 mg/kg NAD+-NP, ENP or free NAD+ in 150 pL of saline. After a 30 min incubation, to allow circulation of the NP’s, the left kidney was exposed as described above. A non-traumatic microvascular clamp was then used to occlude the renal hilum as close to the kidney as possible, preventing blood circulation through the kidney, for a 30 min IR injury incubation.
[0077] Mice in the systemic IV injected treatment group, after immobilization on the surgery pad, had their left kidney exposed as described above, and were injected intravenously with 20 mg/kg NAD+-NP, ENP or free NAD+ in 100 pL of saline via the VC. After a 5 min incubation, to allow circulation of the NP’s, a non-traumatic microvascular clamp was used to occlude the renal hilum as close to the kidney as possible, preventing blood circulation through the kidney, for a 30 min IR injury incubation.
[0078] During their 30 min IR injury incubation all animals received a right nephrectomy. At the end of the ischemic period, the renal hilum clamp was removed and reperfusion of the kidney confirmed visually. The abdominal incision was then sutured closed. 24 hours later, the animal was anesthetized, blood collected by cardiac puncture, and a left nephrectomy was performed. Blood creatinine was measured on a VetScan i-STAT 1 Analyzer MN: 300V (Abbott Park, IL, USA) using Abbott CHEM8+ assay chips (Abbott Park, IL, USA) and the kidney was divided, and samples preserved for histology and cytokine analysis.
[0079] Histology. Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin, sections were then mounted onto slides and stained with hematoxylin-eosin. The renal tubular injury of each specimen was semi-quantitatively evaluated by a blinded, board-certified renal pathologist and scored on a scale of 0 - 5 according to the extent and severity of injury. [0080] Statistical Analysis. Statistical analyses were performed via Tukey’s multiple comparisons test using GraphPad Prism v.10.0.03 software. Data are expressed as mean ± SEM.
Example 1 - Preparation of PEGylated lipid-coated NAD+ loaded NP (NAD+-NP)
[0081] The NAD+-NP was prepared using the water-in-oil reverse microemulsion and thin-film hydration methods.1 Two reverse microemulsions, A and B, were prepared, each with a total volume of 50 mL. The organic phase of both microemulsions consisted of cyclohexane and the surfactant IGEPAL CO-520 in a volume ratio of 71 :29. The aqueous phase of microemulsion A contained 1 mL of a 2.5 M CaCh solution and 2 mg of NAD+. Meanwhile, the aqueous phase of microemulsion B included 1 mL of a 25 mM Na2HPO4 solution (pH 9) with 6 mg of DOPA. The two microemulsions were combined and stirred for 30 minutes. Afterwards, 100 mL of ethanol was added to demulsify and remove the oil phase. The nanoparticle was then collected by centrifugation at 10,000g for 15 min. Following sequential washing twice with ethanol and once with 70% ethanol, the nanoparticles were resuspended in chloroform containing 5.7 mg of Soy PC, 0.57 mg of cholesterol, and 0.33 mg of DSPE-mPEG2k. After chloroform was removed using a rotary evaporator, the resultant lipid film was hydrated with 1 mL of 10 mM Tris-HCl buffer (pH 7.4) to form the NAD+-NP. To produce empty nanoparticles (ENP), the same process was followed, without addition of NAD+.
Example 2 - Preparation of NADH-Lipid-Metal Organic Framework Nanoparticles (NADH-MOF)
[0082] The NADH-MOF NP may be prepared by mixing zinc nitrate, 2-methylimidazole, and NADH in water under ultrasonication to yield the ZIF-8 core, which may be subsequently stabilized by lipid coating via an extrusion process. NADH (6.6 mg) and zinc nitrate hexahydrate (18.6 mg) are dissolved in 35 mL DI water. 2-Methylimidazole (166 mg, dissolved in 10 mL DI water) are added to the NADH solution. The resultant mixture is vortexed for 10 s and is kept still for 5 min to allow MOF growth. The MOF nanoparticles are then pelleted through centrifugation at 10,000 g for 45 min, are redispersed in 1.5 mL water and are ultrasonicated for 30 times. The MOF nanoparticle are mixed with a liposome solution (composed of Soy PC and cholesterol, 10: 1 w/w, 40 mg/mL) and are extruded through a 0.4 pm polycarbonate porous membrane using an Avanti mini extruder to obtain NADH-MOF.
Example 3 -Characterization and Stability of NAD(H) NP
[0083] NAD+-NP prepared as in Example 1 resulted in nanoparticles with an average hydrodynamic diameter of approximately 150 nm (FIG. 3A) and zeta potential of about -6.5 mV. Under TEM, NAD+-NP exhibited a spherical shape with a diameter around 130 nm (FIG. 3B) The loading efficiency of NAD+-NP was quantitatively assessed to be approximately 55%, and the loading content of NAD+-NP was found to be around 12%. Stability studies indicated that NAD+-NP were stable at 4 °C for at least one week (FIG. 3C). In addition, lyophilized NAD+-NP retained stability for at least ten weeks stored at -20 °C, as demonstrated in FIG. 3D.
Example 4 - NAD+-NP Improved Renal Function
[0084] Animals that received NAD+-NP via direct kidney injection, in all three concentrations tested, demonstrated a significant decrease in blood creatinine after 24 hours compared to animals injected with ENP (FIG. 4; 2.5 and 5.0 mg/kg P < 0.0001, 20 mg/kg P < 0.001). In addition, animals treated with direct injection of 2.5 and 5.0 mg/kg NAD+-NP had significantly lower blood creatinine compared to control animals injected with normal saline (FIG. 4; 2.5 mg/kg P < 0.01, 5.0 mg/kg P < 0.05). Likewise, mice that received 20 mg/kg NAD+-NP intravenously also showed significantly reduced levels of creatinine compared to control mice injected intravenously with only free NAD+ (FIG. 4; P < 0.050). No protective effect of NAD+-NP was seen in the creatinine levels of IP treated animals.
[0085] Kidneys were retrieved for histological analysis 24 hours after IR injury. Kidney histology of the sham surgery animals was normal as expected (FIGS. 5A and 5B), however, substantial renal changes in control mice that received saline, ENP or free NAD+ were seen, including loss of tubule brush borders, tubule luminal congestion and tubule denuclearization (FIGS. 5C and 5D). In contrast, renal histology in NAD+-NP’s treated mice demonstrated near normal brush borders in slightly dilated tubules but no luminal congestion or denuclearization (FIGS. 5E and 5F).
[0086] Renal tubule histology was evaluated by a blinded, board-certified kidney pathologist and each kidney was lesion-scored on a scale of 0 - 5 (FIG. 6). Pathological evaluation determined mice that received NAD+-NP via direct kidney injection at concentrations of 2.5 and 5.0 mg/kg demonstrated a significant decrease in lesion score after 24 hours compared to animals injected with ENP (FIG. 4; 2.5 mg/kg P < 0.05, 5.0 mg/kg P < 0.01). Likewise, mice that received 20 mg/kg NAD+-NP intravenously also showed significantly reduced lesion scores compared to control mice injected intravenously with only free NAD+ (FIG. 4; P < 0.01). No protective effect of NAD+-NP was seen in the lesion scores of IP treated animals.
REFERENCES
1. Ye M, Zhao Y, Wang Y, et al. NAD(H)-loaded nanoparticles for efficient sepsis therapy via modulating immune and vascular homeostasis. Nat Nanotechnol 2022; 17(8): 880-90.
EQUIVALENTS
[0087] While certain embodiments have been illustrated and described, a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the nanoparticles of the present technology or derivatives, prodrugs, or pharmaceutical compositions thereof as set forth herein. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed in regard to any or all of the other aspects and embodiments.
[0088] The present technology is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to particular methods, conjugates, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof. No language in the specification should be construed as indicating any non-claimed element as essential.
[0089] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified. The terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. More specifically, it will be understood that each use of terms such as “comprising,” “consisting essentially of,” or “consisting of’, discloses and provides written description and support for the use any of the other terms with the same or any other embodiment described herein.
[0090] 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. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the technology. This includes the generic description of the technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
[0091] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member, and each separate value is incorporated into the specification as if it were individually recited herein.
[0092] All publications, patent applications, issued patents, and other documents (for example, journals, articles and/or textbooks) referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.
[0093] The present technology may include, but is not limited to, the features and combinations of features recited in the following lettered paragraphs, it being understood that the following paragraphs should not be interpreted as limiting the scope of the claims as appended hereto or mandating that all such features must necessarily be included in such claims:
A. A method of reducing ischemia-reperfusion (IR) injury in a kidney to be transplanted comprising administering an effective amount of a bioavailable nanoparticle comprising NAD+ and/or NADH to the kidney prior to surgical transplantation of the kidney into a subject. B. The method of Paragraph A, wherein administering an effective amount of the bioavailable nanoparticle comprises injecting an aqueous solution comprising the effective amount of the bioavailable nanoparticle into a renal blood vessel and/or storing the kidney in the aqueous solution prior to transplantation of the kidney in the subject.
C. The method of Paragraph A, wherein the bioavailable nanoparticle comprises an inorganic core, NAD+ and/or NADH, and a coating comprising a lipid bilayer, wherein the inorganic core is selected from calcium phosphate or a metal organic framework (MOF); the MOF comprises a transition metal ion coordinated to a coordinating ligand, wherein the transition metal ion is selected from the group consisting of zinc, iron, zirconium, copper, and cobalt ions, and the coordinating ligand is selected from an imidazolate ligand or a carboxylate ligand.
D. The method of Paragraphs B or Paragraph C, wherein the aqueous solution comprises saline and/or one or more additives selected from the group consisting of potassium lactobionate, potassium hydrogen phosphate, magnesium sulfate, raffinose, denosine, glutathione, allopurinol, and hydroxyethyl starch.
E. The method of any one of Paragraphs B-D, wherein the aqueous solution is at a temperature from 0°C to about 25°C.
F. The method of any one of Paragraphs B-E, wherein the aqueous solution is at a temperature from 0°C to 5 °C.
G. The method of any one of Paragraphs A-F, wherein the effective amount of NAD+ and/or
NADH is about 1 pM to about 10 mM.
H. The method of any one of Paragraphs A-G, wherein the effective amount of NAD+ and/or
NADH is about 2 pM to about 100 pM.
I. The method of any one of Paragraphs A-H, wherein the effective amount of NAD+ and/or
NADH is about 5 pM to about 20 pM. J. A method of reducing ischemia-reperfusion (IR) injury to a transplanted kidney in a subject comprising administering an effective amount of a bioavailable nanoparticle comprising NAD+ and/or NADH to the subj ect after transplantation of the kidney into the subject.
K. The method of Paragraph J, wherein the bioavailable nanoparticle is administered intravenously to the subject.
L. The method of Paragraph J or Paragraph K, wherein the bioavailable nanoparticle comprises an inorganic core and NAD+ and/or NADH, and a coating comprising a lipid bilayer, wherein the inorganic core is selected from calcium phosphate or a metal organic framework (MOF); the MOF comprises a transition metal ion coordinated to a coordinating ligand, wherein the transition metal ion is selected from the group consisting of zinc, iron, zirconium, copper, and cobalt ions, and the coordinating ligand is selected from an imidazolate ligand or a carboxylate ligand.
M. The method of any one of Paragraphs J-L, wherein the effective amount of NAD+ and/or
NADH is 0.1 mg/kg to 200 mg/kg.
N. The method of any one of Paragraphs J-M, wherein the effective amount of NAD+ and/or
NADH is 1 mg/kg to 100 mg/kg.
O. The method of any one of Paragraphs J-N, wherein the effective amount of NAD+ and/or
NADH is 2.5 mg/kg to 40 mg/kg.
P. The method of any one of Paragraphs J-O, wherein the bioavailable nanoparticle comprises a human cell membrane selected from the group consisting of a human platelet membrane, a human red blood cell membrane, a human white blood cell membrane and a human macrophage cell membrane.
Q. The method of Paragraph P, wherein the human cell membrane is the human platelet membrane. R. The method of Paragraph P or Paragraph Q, wherein the weight ratio of the human cell membrane coating to the remainder of the bioavailable nanoparticle is 2: 1 to 1 :2.
S. The method of any one of Paragraphs P-R, wherein the weight ratio of the human cell membrane coating to the remainder of the bioavailable nanoparticle is about 1 : 1.
T. The method of any one of Paragraphs J-S, wherein the bioavailable nanoparticles are administered to the subject by injection.
U. The method of any one of Paragraphs A-T, wherein the subject is a human.
V. The method of any one of Paragraphs A-U, wherein the bioavailable nanoparticle comprises 1 wt%-50 wt% NAD+ or NADH.
W. The method of any one of Paragraphs A-V, wherein the bioavailable nanoparticle comprises 1 wt% to 25 wt% NAD+ or NADH.
X. The method of any one of Paragraphs A-W, wherein the bioavailable nanoparticle comprises 10 wt%-75 wt% lipid bilayer.
Y. The method of any one of Paragraphs A-W, wherein the bioavailable nanoparticle comprises 30 wt%-75 wt% human cell membrane.
Z. The method of any one of Paragraphs C-I or L-Y, wherein the lipid bilayer comprises lipids selected from the group consisting of of L-a-phosphatidylcholine (PC), 1,2- dioleoyl-sn-glycero-3 -phosphate (DOPA), l,2-dioleoyl-3-trimethylammonium- propane (DOTAP), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phospho-L- serine (DOPS), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)] (DSPE-PEG), cholesterol, a cell membrane extracted from a red blood cell, macrophage, neutrophil or platelet, and combinations of two or more thereof.
AA. The method of any one of Paragraphs C-I or L-Z, wherein the lipid bilayer comprises lipids conjugated to polyethylene glycol) (PEG). AB. The method of any one of Paragraphs C-I or L-AA, wherein up to 100 mol% of the lipids in the lipid bilayer are conjugated to PEG.
AC. The method of any one of Paragraphs C-I or L-AB, wherein the lipids of the lipid bilayer comprise a combination of PC and DOPA and/or cholesterol.
AD. The method of any one of Paragraphs C-I or L-AC, wherein the lipid bilayer comprises
DSPE-PEG wherein: the PEG has a free terminus selected from the group consisting of OH, O-C1-4 alkyl ether, NH2, NHR, COOH, COOR, wherein R is an alkyl or alkenyl group, a dye, a targeting ligand, and a metal chelating ligand, and the PEG has a number average molecular weight ranging from 300 to 10,000 Da.
AE. The method of any one of Paragraphs C-AD, comprising 10 - 90 wt% inorganic core.
AF. The method of any one of Paragraphs C-AE, wherein the inorganic core is calcium phosphate.
AG. The method of any one of Paragraphs C-AF, wherein the inorganic core is MOF.
AH. The method of any one of Paragraphs C-AG, where the coordinating ligand is selected from the group consisting of imidazole, 2-methyl-imidazole, benzimidazole, 5- methylbenzimidazole, terephthalic acid, 2-methyl-pterphthalic acid, 2-hydroxy- terphthalic acid, and 2-amino-terphthalic acid, benzene-l,3,5-tricarboxylic acid, l,3,5-tris(4-carboxyphenyl)benzene, 2,6-naphthalenedicarboxylic acid, 4,4',4"-s- triazine-2,4,6-triyl-tribenzoic acid, and 2,5-dihydroxyterephthalic acid.
Al. The method of any one of Paragraphs C-AH, wherein the MOF comprises zinc ions and imidazolate ligands.
AJ. The method of any one of Paragraphs C-AI, wherein the imidazolate ligand is selected from imidazole, 2-methyl-imidazole, benzimidazole, or 5 -methylbenzimidazole. AK. The method of any one of Paragraphs C-AJ, wherein the imidiazolate ligand is selected from 2-methyl-imidazole.
AL. The method of any one of Paragraphs A-AK, wherein the bioavailable nanoparticle has an average hydrodynamic diameter of from 60 to less than 1000 nm.
AM. The method of any one of Paragraphs A-AL, wherein bioavailable nanoparticles are administered as a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
[0094] Other embodiments are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled.