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WO2025155929A1 - Nanoparticules chargées en nad(h) et méthodes de réduction de lésions d'ischémie-reperfusion dans des greffes de reins - Google Patents

Nanoparticules chargées en nad(h) et méthodes de réduction de lésions d'ischémie-reperfusion dans des greffes de reins

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Publication number
WO2025155929A1
WO2025155929A1 PCT/US2025/012213 US2025012213W WO2025155929A1 WO 2025155929 A1 WO2025155929 A1 WO 2025155929A1 US 2025012213 W US2025012213 W US 2025012213W WO 2025155929 A1 WO2025155929 A1 WO 2025155929A1
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Prior art keywords
nad
bioavailable
nanoparticle
nadh
kidney
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PCT/US2025/012213
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English (en)
Inventor
David AL-ADRA
Shaoqin Gong
Bret VERHOVEN
Yao TONG
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Wisconsin Alumni Research Foundation
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Wisconsin Alumni Research Foundation
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Publication of WO2025155929A1 publication Critical patent/WO2025155929A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a bioavailable nanoparticle comprising NAD + and/or NADH
  • the effective amount of bioavailable nanoparticles may be administered by injection, e.g., intravenously, to the subject.
  • FIG. 1 is a schematic illustration of an illustrative embodiment of a bioavailable nanoparticle structure.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 + .
  • 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.
  • 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.
  • prevent is not an absolute term.
  • the term “prevent” is not an absolute term.
  • 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.
  • a “cell penetrating peptide” 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.
  • 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.
  • 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).
  • cyanine dyes e.g., Cy2, Cy3, Cy5, Cy5.5, Cy7, and sulfonated versions thereof
  • FITC fluorescein isothiocyanate
  • ALEXA FLUOR® dyes e.g., ALEXA FLUOR® 488, 546, or 633
  • DYLIGHT® dyes e.g., DY
  • a “metal chelating ligand” as used herein refers to ligands that chelate metal isotopes for use in imaging.
  • 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).
  • the metal chelating ligand may be one for use in PET or MRI.
  • 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.
  • the aqueous solution may be used at a temperature from 0°C to about 5 °C.
  • the effective amount of NAD + and/or NADH may be about 1 pM to about 10 mM.
  • 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.
  • 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
  • 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.
  • IR ischemiareperfusion
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ligands are suitable for use with a particular type of metal to form a metal organic framework component.
  • zinc may be used with imidazolate ligands and iron may be used with carboxylate ligands, especially dicarboxylate ligands.
  • the present nanoparticles may have a hydrodynamic diameter ranging from at least 50 nm to less than 1000 nm.
  • 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.
  • the present technology provides NPs with a median or average hydrodynamic diameter, also selected from any of the foregoing values or ranges.
  • X 1 and X 2 are independently absent or selected from unsubstituted Ci-6 alkylene or C2-6 alkenylene;
  • Y 1 and Y 2 are each independently absent, C(O)O, or C(O)NH;
  • Y 3 is absent, C(O), C(O)O, or C(O)NH;
  • 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.
  • 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.
  • 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.
  • 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.
  • the PAO may be linear. In any embodiments, the PAO may be branched.
  • the PAO may be a linear polyamine oligomer.
  • the PAO may be a branched polyamine oligomer, e.g., a polyethyleneimine (PEI) oligomer.
  • 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.
  • the PAO may have a number average molecular weight of about 400 Da to about 800 Da.
  • the PAO may comprise a poly(amidoamine) oligomer (PAMAM), e.g., a PAMAM dendrimer with an ethylenediamine core.
  • PAMAM dendrimer may be a generation 0 or 1.
  • the PAMAM dendrimer may be capped with a C2-4 alkylenediamine, e.g., ethylenediamine.
  • the PAO may have the structure of Formula V:
  • the PAO may or may not be polydisperse.
  • the PAO may have a poly dispersity of about 1 to about 2.
  • 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.
  • 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.
  • the compound of Formula IV may include various amounts of different R groups.
  • 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.
  • 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.
  • about 50% of R groups may be Ce-20 alkyl groups, e.g., Cio-18 alkyl groups or any of those disclosed herein.
  • the present technology provides lipid nanoparticles (LNP) that include a compound of Formula IV as disclosed herein and a PEG-lipid.
  • 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.
  • 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.
  • LNPs of the present technology may include a variety of PEG-lipids.
  • the LNP may include DMG-PEG, DSPE-PEG and/or ceramide-PEG.
  • the PEG-lipid may include DMG-mPEG.
  • the PEG-lipid may further include CPP-DMG- mPEG and/or glucose-DMG-mPEG.
  • a pharmaceutical composition comprising bioavailable nanoparticle as described herein and a pharmaceutically acceptable carrier and/or excipient.
  • the bioavailable nanoparticles may be administered to the subject, e.g., a human subject, as a pharmaceutical composition.
  • 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.
  • 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.
  • 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),
  • compositions include ionizable components, salts such as pharmaceutically acceptable salts of such components may also be used.
  • 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.
  • CaC12 Calcium chloride
  • IGEPAL CO-520 polyoxyethylene nonylphenyl ether, a non-ionic surfactant
  • Ca2HPO4 Disodium hydrogen phosphate
  • Soy PC Soy PC
  • DOPA dioleoylphosphatydic acid
  • 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).
  • DLS Dynamic light scattering
  • TEM Transmission electron microscopy
  • 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).
  • IP intraperitoneal injection
  • IV intravenously
  • VC vena cava
  • 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.
  • 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.
  • 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.
  • Statistical Analysis were performed via Tukey’s multiple comparisons test using GraphPad Prism v.10.0.03 software. Data are expressed as mean ⁇ SEM.
  • 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 + .
  • 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 + .
  • NAD + empty nanoparticles
  • NADH-MOF NADH-Lipid-Metal Organic Framework Nanoparticles
  • 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
  • zinc nitrate hexahydrate 18.6 mg
  • 2-Methylimidazole 166 mg, dissolved in 10 mL DI water
  • 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.
  • 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.
  • NAD + -NP 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).
  • lyophilized NAD + -NP retained stability for at least ten weeks stored at -20 °C, as demonstrated in FIG. 3D.
  • 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.
  • 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).
  • 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).
  • 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.

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Abstract

La présente technologie concerne des méthodes permettant de réduire des lésions d'ischémie-reperfusion (IR) dans un rein à greffer, consistant à administrer une quantité efficace d'une nanoparticule biodisponible contenant du NAD+ et/ou du NADH au rein avant la greffe chirurgicale du rein dans un sujet. De plus, la présente technologie concerne des méthodes permettant de réduire des lésions d'ischémie-reperfusion (IR) d'un rein greffé dans un sujet, consistant à administrer une quantité efficace d'une nanoparticule biodisponible contenant du NAD+ et/ou du NADH au sujet après la greffe du rein chez le sujet et concerne également des nanoparticules biodisponibles contenant du NAD+ et/ou du NADH destinées à être utilisées dans de telles méthodes.
PCT/US2025/012213 2024-01-19 2025-01-17 Nanoparticules chargées en nad(h) et méthodes de réduction de lésions d'ischémie-reperfusion dans des greffes de reins Pending WO2025155929A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4829984A (en) * 1983-12-15 1989-05-16 Gordon Robert T Method for the improvement of transplantation techniques and for the preservation of tissue
US20190070211A1 (en) * 2016-02-26 2019-03-07 Beth Israel Deaconess Medical Center, Inc. Niacinamide (nam) in ischemic tissue injury
CN114128893A (zh) * 2021-11-30 2022-03-04 北京姿美堂生物技术有限公司 一种含烟酰胺和谷胱甘肽的组合物及其应用
US20230032473A1 (en) * 2021-07-23 2023-02-02 Wisconsin Alumni Research Foundation Nad(h) nanoparticles and methods of use
US20230257795A1 (en) * 2020-07-09 2023-08-17 Helsingin Yliopisto Method for determining amounts of nad metabolites from sample and methods and uses related thereto

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4829984A (en) * 1983-12-15 1989-05-16 Gordon Robert T Method for the improvement of transplantation techniques and for the preservation of tissue
US20190070211A1 (en) * 2016-02-26 2019-03-07 Beth Israel Deaconess Medical Center, Inc. Niacinamide (nam) in ischemic tissue injury
US20230257795A1 (en) * 2020-07-09 2023-08-17 Helsingin Yliopisto Method for determining amounts of nad metabolites from sample and methods and uses related thereto
US20230032473A1 (en) * 2021-07-23 2023-02-02 Wisconsin Alumni Research Foundation Nad(h) nanoparticles and methods of use
CN114128893A (zh) * 2021-11-30 2022-03-04 北京姿美堂生物技术有限公司 一种含烟酰胺和谷胱甘肽的组合物及其应用

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