US20190142915A1 - Ocular delivery of cell permeant therapeutics for the treatment of retinal edema - Google Patents

Ocular delivery of cell permeant therapeutics for the treatment of retinal edema Download PDF

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US20190142915A1
US20190142915A1 US16/243,884 US201916243884A US2019142915A1 US 20190142915 A1 US20190142915 A1 US 20190142915A1 US 201916243884 A US201916243884 A US 201916243884A US 2019142915 A1 US2019142915 A1 US 2019142915A1
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caspase
rvo
xbir3
signaling pathway
cell
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Carol M. Troy
Ying Y. Jean
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Columbia University in the City of New York
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Columbia University in the City of New York
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Priority to US17/538,924 priority patent/US11857609B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present disclosure relates to compositions and methods for the inhibition of diabetic macular edema (DME) and/or retinal vein occlusion (RVO).
  • DME diabetic macular edema
  • RVO retinal vein occlusion
  • DME diabetic retinal diseases
  • the other treatment is laser photocoagulation, which can reduce vision loss by 50%; the goal is to reduce progression of the disease, however significant improvement of vision is uncommon.
  • the present disclosure provides a method for treating DME and/or RVO by administering to the retina of a patient in need thereof an effective amount of a caspase-9 signaling pathway inhibitor.
  • the present disclosure also provides a pharmaceutical composition including an effective amount of a caspase-9 signaling pathway inhibitor and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is formulated for administration to a patient via eye drops.
  • the present disclosure further provides a pharmaceutical composition as described above for use in a method as described above.
  • the caspase-9 signaling pathway inhibitor may include a peptide caspase-9 inhibitor; ii) the peptide caspase-9 inhibitor may include XBIR3; iii) the caspase-9 signaling pathway inhibitor may be conjugated to a cell-penetrating peptide; iv) the cell-penetrating peptide may be selected from the group consisting of Penetratinl, transportan, pISl, Tat(48-60), pVEC, MAP, and MTS; v) the caspase-9 signaling pathway inhibitor may include XBIR3 and wherein the XBIR3 is conjugated to Penetratin 1; vi) the effective amount may treat DME and/or RVO by decreasing edema in the retina of the patient, as detected by OCT; vii) the effective amount may treat DME and/or RVO by decreasing retinal de
  • FIG. 1 is a schematic diagram of a caspase-9 mediated signaling pathway that leads to edema unless blocked, for example by Pen1-XBIR3.
  • FIG. 2 is a set of blots and corresponding bar graphs presenting results for uptake of Pen1-XBIR3 eyedrops by the retina in mice and rabbits.
  • FIG. 3 is a set of blots presenting results for presence of Pen1-XBIR3 in the plasma of rabbits after eyedrop treatment.
  • FIG. 4 is a series of brightfield and OCT images showing the effects of RVO or RVO and Pen1-XBIR3 on retinal edema.
  • FIG. 5 is a set of blots depicting results of assays for RVO activation of caspase-9 (left) and Pen1-XBIR3 binding of caspase-9 (right).
  • FIG. 6 is a series of brightfield and OCT images and a graph showing effects of RVO or RVO and Pen1-XBIR3 (caspase-9 inhibitor) on retina edema.
  • FIG. 7 is a representative OCT image and a graph showing effects of RVO or RVO and Pen1-XBIR3 (caspase-9 inhibitor) on retinal detachment.
  • FIG. 8 is a representative OCT image and a series of graphs showing the effects of RVO or RVO and Pen1-XBIR3 (caspase-9 inhibitor) on internal retinal layers.
  • FIG. 9 is a representative OCT image and a graph showing the effects of RVO or RVO and Pen1-XBIR3 (caspase-9 inhibitor) on the retinal outer plexiform layer.
  • FIG. 10 is a set of representative OCT images and a graph showing the effects of RVO or RVO and Pen1-XBIR3 (caspase-9 inhibitor) on the retinal photoreceptor layer.
  • FIG. 11 is a graph of the effects of RVO or RVO and Pen1-XBIR3 on retinal inner nuclear layer thickness.
  • FIG. 12 is a graph of the effects of RVO, Pen1-XBIR3, or RVO and Pen1-XBIR3 on B-wave amplitude, which relates to retinal function.
  • FIG. 13 is a series of photomicrographs stained to allow detection of cleaved caspase-7 in retinal blood vessels to show the effects of RVO or RVO and Pen1-XBIR3.
  • the scale bar is 20 ⁇ m.
  • FIG. 14 is a blot showing the effects of RVO or RVO and Pen1-XBIR3 on VEGF and ERK (top left), a graph showing the effects of RVO or RVO and Pen1-XBIR3 on VEGF expression (bottom left), and a series of photomicrographs showing the effects of RVO or RVO and Pen1-XBIR3 on HIF-1 ⁇ expression in the retina.
  • the present disclosure relates to compositions and method for treating DME and/or RVO in a patient.
  • the present disclosure relates to methods and compositions for inhibiting caspase-9 signaling activity associated with the induction and/or exacerbation of DME and/or RVO in a patient.
  • DME refers to clinically detectable diabetic macular edema. DME occurs in patients having clinically detectable diabetes mellitus (also referred to herein simply as diabetes), frequently in type 2 diabetes mellitus but also in type 1 diabetes mellitus. Clinical symptoms of DME include retinal edema and diabetic retinopathy with macular edema. DME may be detected using optical coherence tomography (OCT). DME is the major cause of blindness in working age adults (20-70 years old).
  • OCT optical coherence tomography
  • RVO refers to clinically detectable retinal vein occlusion.
  • RVO can occur in any patients, but is more common in those also having clinically detectable atherosclerosis, diabetes, hypertension, glaucoma, macular edema, or vitreous hemorrhage.
  • RVO is more common in elderly patients.
  • RVO can cause glaucoma and macular edema, including DME.
  • RVO may be detected using angiography and/or OCT.
  • RVO is the second leading cause of blindness in working age adults.
  • the term “patient” refers to any animal, including any mammal, including, but not limited to, humans, and non-human animals (including, but not limited to, non-human primates, dogs, cats, rodents, horses, cows, pigs, mice, rats, hamsters, rabbits, and the like.
  • the patient is a human.
  • an “effective amount” is an amount sufficient to cause a beneficial or desired clinical result in a patient.
  • An effective amount can be administered to a patient in one or more doses. It is typically administered to the retina of the patient.
  • an effective amount is an amount that is sufficient to ameliorate the impact of and/or inhibit the induction and/or exacerbation of DME and/or RVO in a patient, or otherwise reduce the pathological consequences of the disease(s).
  • the effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors may be taken into account when determining an appropriate dosage to achieve an effective amount.
  • composition also referred to herein as a “treatment,” “inhibitor,” or “conjugate”) being administered.
  • treat refers to of ameliorating the impact of and/or inhibiting the induction and/or exacerbation of DME and/or RVO in a patient.
  • the caspase-9 signaling pathway as discussed herein may include the pathway shown in FIG. 1 .
  • RVO induces activation of caspase-9 in blood vessels which leads to activation of caspase-7.
  • Caspase-7 cleaves the co-chaperone protein p23.
  • p23 is a negative regulator of HIF-1 ⁇ , thus cleavage of p23 leads to an increase in HIF-1 ⁇ , the rate-limiting step in the formation of the HIF-1 transcription factor, which increases VEGF levels leading to edema.
  • the caspase-9 signaling pathway inhibitor shown as Pen1-XBIR3 in FIG. 1 as an illustrative example, may act by blocking the action of caspase-9 on caspase-7.
  • the instant disclosure is directed to methods of or uses of treatments disclosed herein in ameliorating the impact of and/or inhibiting the induction and/or exacerbation of DME and/or RVO in a patient by administering an effective amount of a caspase-9 signaling pathway inhibitor, or conjugate thereof.
  • the methods of the present disclosure are directed to the administration of a caspase-9 signaling pathway inhibitor, or conjugate thereof, via eye drops in order to inhibit DME and/or RVO.
  • the treatment when used to treat the effects of DME and/or RVO, may be administered as a single dose or multiple doses.
  • multiple doses may be administered at intervals of 6 times per 24 hours or 4 times per 24 hours or 3 times per 24 hours or 2 times per 24 hours or 1 time per 24 hours or 1 time every other day or 1 time every 3 days or 1 time every 4 days or 1 time per week, or 2 times per week, or 3 times per week.
  • the initial dose may be greater than subsequent doses or all doses may be the same.
  • the inhibitor used in connection with the methods and uses of the instant disclosure is a Pen1-XBIR3 conjugate as disclosed herein.
  • the Pen1-XBIR3 conjugate is administered to a patient suffering from DME and/or RVO either as a single dose or in multiple doses.
  • the concentration of the Pen1-XBIR3 composition administered is, in certain embodiments: 0.1 ⁇ M to 1,000 ⁇ M; 1 ⁇ M to 500 ⁇ M; 10 ⁇ M to 100 ⁇ M; or 20 ⁇ M to 60 ⁇ M.
  • a specific human equivalent dosage can be calculated from animal studies via body surface area comparisons, as outlined in Reagan-Shaw et al., FASEB J., 22; 659-661 (2007).
  • eye size comparisons can be employed to calculate a specific human equivalent dosage.
  • the caspase-9 signaling pathway inhibitor is administered in conjunction with one or more additional therapeutics.
  • the additional therapeutics include, but are not limited to an anti-VEGF therapeutic and/or a steroidal therapeutic.
  • the method involves the administration of one or more additional caspase-9 signaling pathway inhibitors either alone or in the context of a membrane-permeable conjugate.
  • the caspase-9 signaling pathway inhibitors of the present disclosure are peptide inhibitors of caspase-9.
  • the caspase-9 signaling pathway inhibitors include, but are not limited to the class of protein inhibitors identified as Inhibitors of Apoptosis (“IAPs”).
  • IAPs generally contain one to three BIR (baculovirus IAP repeats) domains, each consisting of approximately 70 amino acid residues.
  • BIR baculovirus IAP repeats
  • certain IAPB also have a RING finger domain, defined by seven cysteines and one histidine (C 3 HC 4 ) that can coordinate two zinc atoms.
  • Exemplary mammalian IAPB suitable for use herein include, but are not limited to c-IAP1 (Accession No. Q13490.2), cIAP2 (Accession No. Q13489.2), and XIAP (Accession No. P98170.2), each of which have three BIRs in the N-terminal portion of the molecule and a RING finger at the C-terminus.
  • NAIP Accession No. Q13075.3
  • another suitable mammalian IAP contains three BIRs without RING, and survivin (Accession No. 015392.2) and BRUCE (Accession No. Q9H8B7), which are two additional suitable IAPB, both of which contain just one BIR.
  • the peptide inhibitor of caspase-9 is XBIR3 having the sequence
  • the peptide inhibitor of caspase-9 is XBIR3 having the sequence
  • the peptide inhibitors of caspase-9 include, but are not limited to EG Z-VEID-FMK (“VEID” disclosed as SEQ ID NO: 1) (WO 2006056487); Z-VAD-FMK, CrmA, and Z-VAD-(2, 6-dichlorobenzoyloxopentanoic acid) (Garcia-Calvo, et al., J. Biol. Chem., 273, 32608-32613 (1998)).
  • Peptide inhibitors of caspase-9 include those amino acid sequences that retain certain structural and functional features of the identified caspase-9 inhibitor peptides, yet differ from the identified inhibitors' amino acid sequences at one or more positions. Such variants can be prepared by substituting, deleting, or adding amino acid residues from the original sequences via methods known in the art.
  • such substantially similar sequences include sequences that incorporate conservative amino acid substitutions.
  • a “conservative amino acid substitution” is intended to include a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including: basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); ⁇ -branched side chains (e.g., threonine, valine, isoleucine); and aromatic side chains (e.g.,
  • a peptide inhibitor of caspase-9 of the present disclosure is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the original peptide inhibitor of caspase-9, such as an IAP, and is capable of caspase-9 inhibition.
  • the percent homology between two amino acid sequences may be determined using standard software such as BLAST or FASTA. The effect of the amino acid substitutions on the ability of the synthesized peptide inhibitor of caspase-9 to inhibit caspase-9 can be tested using the methods disclosed in Examples section, below.
  • the caspase-9 signaling pathway inhibitor is conjugated to a cell penetrating peptide to form an inhibitor-cell penetrating peptide conjugate.
  • a “cell-penetrating peptide” is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non-endocytotic) translocation properties associated with transport of the membrane-permeable complex across the plasma and/or nuclear membranes of a cell.
  • the cell-penetrating peptide used in the membrane-permeable complex of the present disclosure preferably comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with the caspase-9 signaling pathway inhibitor, which has been modified for such linkage.
  • Representative amino acid motifs conferring such properties are listed in U.S. Pat. No.
  • the cell-penetrating peptides of the present disclosure may include, but are not limited to, Penetratinl, transportan, pIsl, TAT(48-60), pVEC, MTS, and MAP.
  • the cell-penetrating peptides of the present disclosure include those sequences that retain certain structural and functional features of the identified cell-penetrating peptides, yet differ from the identified peptides' amino acid sequences at one or more positions.
  • Such polypeptide variants can be prepared by substituting, deleting, or adding amino acid residues from the original sequences via methods known in the art.
  • such substantially similar sequences include sequences that incorporate conservative amino acid substitutions, as described above in connection with peptide caspase-9 inhibitors.
  • a cell-penetrating peptide of the present disclosure is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the identified peptide and is capable of mediating cell penetration.
  • the effect of the amino acid substitutions on the ability of the synthesized peptide to mediate cell penetration can be tested using the methods disclosed in Examples section, below.
  • the cell-penetrating peptide of the membrane-permeable complex is Penetratinl, comprising the peptide sequence C(NPys)-RQIKIWFQNRRMKWKK (SEQ ID NO: 2), or a conservative variant thereof.
  • a “conservative variant” is a peptide having one or more amino acid substitutions, wherein the substitutions do not adversely affect the shape—or, therefore, the biological activity (i.e., transport activity) or membrane toxicity—of the cell-penetrating peptide.
  • Penetratinl is a 16-amino-acid polypeptide derived from the third alpha-helix of the homeodomain of Drosophila antennapedia. Its structure and function have been well studied and characterized: Derossi et al., Trends Cell Biol., 8(2):84-87, 1998; Dunican et al., Biopolymers, 60(1):45-60, 2001; Hallbrink et al., Biochim. Biophys. Acta, 1515(2):101-09, 2001; Bolton et al., Eur. J. Neurosci., 12(8):2847-55, 2000; Kilk et al., Bioconjug.
  • Penetratinl efficiently carries avidin, a 63-kDa protein, into human Bowes melanoma cells (Kilk et al., Bioconjug. Chem., 12(6):911-16, 2001). Additionally, it has been shown that the transportation of Penetratinl and its cargo is non-endocytotic and energy-independent, and does not depend upon receptor molecules or transporter molecules. Furthermore, it is known that Penetratinl is able to cross a pure lipid bilayer (Thoren et al., FEBS Lett., 482(3):265-68, 2000). This feature enables Penetratinl to transport its cargo, free from the limitation of cell-surface-receptor/-transporter availability.
  • the delivery vector previously has been shown to enter all cell types (Derossi et al., Trends Cell Biol., 8(2):84-87, 1998), and effectively to deliver peptides (Troy et al., Proc. Natl. Acad. Sci. USA, 93:5635-40, 1996) or antisense oligonucleotides (Troy et al., J. Neurosci., 16:253-61, 1996; Troy et al., J. Neurosci., 17:1911-18, 1997).
  • RL16 H-RRLRRLLRRLLRRLRR-OH
  • RLRRRRRR-OH a sequence derived from Penetratinl with slightly different physical properties
  • RVG-RRRRRRRRRRR a rabies virus sequence which targets neurons see P. Kumar, H. Wu, J. L. McBride, K. E. Jung, M. H. Kim, B. L. Davidson, S. K. Lee, P. Shankar and N. Manjunath, Transvascular delivery of small interfering RNA to the central nervous system, Nature 448 (2007), pp. 39-43.
  • the cell-penetrating peptide of the membrane-permeable complex is a cell-penetrating peptide selected from the group consisting of: transportan, pISl, Tat(48-60), pVEC, MAP, and MTS.
  • Transportan is a 27-amino-acid long peptide containing 12 functional amino acids from the amino terminus of the neuropeptide galanin, and the 14-residue sequence of mastoparan in the carboxyl terminus, connected by a lysine (Pooga et al., FASEB J., 12(1):67-77, 1998). It includes the amino acid sequence GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 5), or a conservative variant thereof.
  • pIsl is derived from the third helix of the homeodomain of the rat insulin 1 gene enhancer protein (Magzoub et al., Biochim. Biophys. Acta, 1512(1):77-89, 2001; Kilk et al., Bioconjug. Chem., 12(6):911-16, 2001).
  • pIsl includes the amino acid sequence PVIRVW FQNKRCKDKK (SEQ ID NO: 6), or a conservative variant thereof.
  • Tat is a transcription activating factor, of 86-102 amino acids, that allows translocation across the plasma membrane of an HIV-infected cell, to transactivate the viral genome (Hallbrink et al., Biochem. Biophys. Acta., 1515(2):101-09, 2001; Suzuki et al., J. Biol. Chem., 277(4):2437-43, 2002; Futaki et al., J. Biol. Chem., 276(8):5836-40, 2001).
  • a small Tat fragment extending from residues 48-60, has been determined to be responsible for nuclear import (Vives et al., J. Biol.
  • pVEC is an 18-amino-acid-long peptide derived from the murine sequence of the cell-adhesion molecule, vascular endothelial cadherin, extending from amino acid 615-632 (Elmquist et al., Exp. Cell Res., 269(2):237-44, 2001).
  • pVEC includes the amino acid sequence LLIILRRRIRKQAHAH (SEQ ID NO: 8), or a conservative variant thereof.
  • MTSs or membrane translocating sequences
  • MTSs are those portions of certain peptides which are recognized by the acceptor proteins that are responsible for directing nascent translation products into the appropriate cellular organelles for further processing (Lindgren et al., Trends in Pharmacological Sciences, 21(3):99-103, 2000; Brodsky, J. L., Int. Rev. Cyt., 178:277-328, 1998; Zhao et al., J. Immunol. Methods, 254(1-2):137-45, 2001).
  • MPS MPS peptide, a chimera of the hydrophobic terminal domain of the viral gp41 protein and the nuclear localization signal from simian virus 40 large antigen; it represents one combination of a nuclear localization signal and a membrane translocation sequence that is internalized independent of temperature, and functions as a carrier for oligonucleotides (Lindgren et al., Trends in Pharmacological Sciences, 21(3):99-103, 2000; Morris et al., Nucleic Acids Res., 25:2730-36, 1997).
  • MPS includes the amino acid sequence GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 9), or a conservative variant thereof.
  • Model amphipathic peptides, or MAPs form a group of peptides that have, as their essential features, helical amphipathicity and a length of at least four complete helical turns (Scheller et al., J. Peptide Science, 5(4):185-94, 1999; Hallbrink et al., Biochim. Biophys. Acta., 1515(2):101-09, 2001).
  • An exemplary MAP comprises the amino acid sequence KLALKLALKALKAALKLA (SEQ ID NO: 10)-amide, or a conservative variant thereof.
  • the cell-penetrating peptides and the caspase-9 signaling pathway inhibitors described above are covalently bound to form conjugates.
  • the cell-penetrating peptide is operably linked to a peptide caspase-9 inhibitor via recombinant DNA technology.
  • a nucleic acid sequence encoding that peptide caspase-9 inhibitor can be introduced either upstream (for linkage to the amino terminus of the cell-penetrating peptide) or downstream (for linkage to the carboxy terminus of the cell-penetrating peptide), or both, of a nucleic acid sequence encoding the peptide caspase-9 inhibitor of interest.
  • Such fusion sequences including both the peptide caspase-9 inhibitor encoding nucleic acid sequence and the cell-penetrating peptide encoding nucleic acid sequence can be expressed using techniques well known in the art.
  • the caspase-9 signaling pathway inhibitor can be operably linked to the cell-penetrating peptide via a non-covalent linkage.
  • non-covalent linkage is mediated by ionic interactions, hydrophobic interactions, hydrogen bonds, or van der Waals forces.
  • the caspase-9 signaling pathway inhibitor is operably linked to the cell penetrating peptide via a chemical linker.
  • linkages typically incorporate 1-30 nonhydrogen atoms selected from the group consisting of C, N, O, S and P.
  • Exemplary linkers include, but are not limited to, a substituted alkyl or a substituted cycloalkyl.
  • the heterologous moiety may be directly attached (where the linker is a single bond) to the amino or carboxy terminus of the cell-penetrating peptide.
  • the linker may be any combination of stable chemical bonds, optionally including, single, double, triple or aromatic carbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds.
  • the linker incorporates less than 20 nonhydrogen atoms and are composed of any combination of ether, thioether, urea, thiourea, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.
  • the linker is a combination of single carbon-carbon bonds and carboxamide, sulfonamide or thioether bonds.
  • a general strategy for conjugation involves preparing the cell-penetrating peptide and the caspase-9 signaling pathway inhibitor components separately, wherein each is modified or derivatized with appropriate reactive groups to allow for linkage between the two.
  • the modified caspase-9 signaling pathway inhibitor is then incubated together with a cell-penetrating peptide that is prepared for linkage, for a sufficient time (and under such appropriate conditions of temperature, pH, molar ratio, etc.) as to generate a covalent bond between the cell-penetrating peptide and the caspase-9 signaling pathway inhibitor molecule.
  • the caspase-9 signaling pathway inhibitor molecule when generating a disulfide bond between the caspase-9 signaling pathway inhibitor molecule and the cell-penetrating peptide of the present disclosure, can be modified to contain a thiol group, and a nitropyridyl leaving group can be manufactured on a cysteine residue of the cell-penetrating peptide.
  • Any suitable bond e.g., thioester bonds, thioether bonds, carbamate bonds, etc.
  • Both the derivatized or modified cell-penetrating peptide, and the modified caspase-9 signaling pathway inhibitor are reconstituted in RNase/DNase sterile water, and then added to each other in amounts appropriate for conjugation (e.g., equimolar amounts). The conjugation mixture is then incubated for 60 min at 37° C., and then stored at 4° C. Linkage can be checked by running the vector-linked caspase-9 signaling pathway inhibitor molecule, and an aliquot that has been reduced with DTT, on a 15% non-denaturing PAGE. Caspase-9 signaling pathway inhibitor molecules can then be visualized with the appropriate stain.
  • the present disclosure is directed to a Penetratinl 1-XBIR3 (Pen1-XBIR3) conjugate.
  • the sequence of the Pen-1-XBIR3 is: C(NPys)-RQIKIWFQNRRMKWKK-s-s-MGSSHHHHHHSSGLVPRGSHMSTNTCLPRNPSMADYEARIFTFGTWIYSVNK EQLARAGFYTDWALGEGDKVKCFHCGGGLRPSEDPWEQHARWYPGCRYLL EQRGQEYINNIHLTHS (SEQ ID NO 2 and 11, respectively, linked by a disulfide bond).
  • the sequence of the Pen1-XBIR3 is: C(NPys)-RQIKIWFQNRRMKWKK-s-s-MGSSHHHHHHSSGLVPRGSHMSTNTLPRNPSMADYEARIFTFGTWIYSVNKE QLARAGFYTDWALGEGDKVKCFHCGGGLRPSEDPWEQHARWYPGCRYLLE QRGQEYINNIHLTHS (SEQ ID NO 2 and 12, respectively, linked by a disulfide bond).
  • the caspase-9 signaling pathway inhibitors or conjugates of the present disclosure are formulated for retinal administration.
  • a solution or suspension containing the caspase-9 signaling pathway inhibitor or conjugate can be formulated for direct application to the retina by conventional means, for example with a dropper, pipette or spray.
  • the caspase-9 signaling pathway inhibitor or conjugate of the present disclosure is formulated in isotonic saline.
  • the caspase-9 signaling pathway inhibitor or conjugate of the present disclosure is formulated in isotonic saline at or about pH 7.4.
  • the caspase-9 signaling pathway inhibitor, or conjugate thereof, of the present disclosure may, in various compositions, be formulated with a pharmaceutically-acceptable carrier, excipient, or diluent.
  • a pharmaceutically-acceptable carrier excipient, or diluent.
  • pharmaceutically-acceptable means that the carrier, excipient, or diluent of choice does not adversely affect either the biological activity of the caspase-9 signaling pathway inhibitor or conjugate or the biological activity of the recipient of the composition.
  • Suitable pharmaceutical carriers, excipients, and/or diluents for use in the present disclosure include, but are not limited to, lactose, sucrose, starch powder, talc powder, cellulose esters of alkonoic acids, magnesium stearate, magnesium oxide, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, gelatin, glycerin, sodium alginate, gum arabic, acacia gum, sodium and calcium salts of phosphoric and sulfuric acids, polyvinylpyrrolidone and/or polyvinyl alcohol, saline, and water.
  • Specific formulations of compounds for therapeutic treatment are discussed in Hoover, J. E., Remington's Pharmaceutical Sciences (Easton, Pa.: Mack Publishing Co., 1975) and Liberman and Lachman, eds. Pharmaceutical Dosage Forms (New York, N.Y.: Marcel Decker Publishers, 1980).
  • the quantity of the caspase-9 signaling pathway inhibitor or conjugate thereof that is administered to a cell, tissue, or subject should be an effective amount.
  • mice Pen1-XBIR3 (10 ⁇ g) eyedrops were applied, then the animals were sacrificed at the indicated times.
  • 200 ⁇ g Pen1-XBIR3 eyedrops or a saline vehicle were administered BID for 4.5 days. The final dose given 5 h prior to harvest of retinas. Plasma from rabbits obtained at baseline and harvest.
  • XBIR3 contains a His tag, so uptake of XBIR3 is detectable using anti-His. Blots for the mouse and rabbit samples, along with graphs quantifying the results, are presented in FIG. 2 .
  • XBIT3 uptake was observed in both mouse and rabbit samples. Uptake in the mouse samples was detected by 1 h and maintained through 24 h. In rabbit there was significant XBIR3 in retina at 5 d.
  • Baseline and post-treatment plasma from rabbits was analyzed by immunoprecipitation with XIAP followed by western blot with anti-His. A Ponceau protein stain was used to show input protein amounts. XBIR3 was not detected in rabbit plasma ( FIG. 3 ), indicating that it remains localized in the eye.
  • RVO is the model that was used for testing anti-VEGF therapies for DME. Brown et al., Ophthalmology 117, 1124-1133 e1121 (2010); and Campochiaro et al., Ophthalmology 117, 1102-1112 e1101 (2010).
  • Rose Bengal a photoactivatable dye, is injected into the tail veins of adult C57B16 mice and photoactivated by laser of retinal veins around the optic nerve head.
  • a clot is formed and edema or increased retinal thickness develops rapidly. Inflammation, also seen in diabetes, also develops.
  • Fluorescein leakage and maximal retinal edema measured by fluorescein angiography and optical coherence tomography (OCT), respectively, using the Phoenix Micron IV, is observed 24 h after RVO.
  • Retinal edema is maintained over the first 3 days RVO.
  • OCT optical coherence tomography
  • mice were anesthetized with intra-peritoneal (IP) injection of ketamine and xylazine.
  • IP intra-peritoneal
  • One drop of 0.5% alcaine was added to the eye as topical anesthetic.
  • the retina was imaged with the Phoenix Micron IV to choose veins for laser ablation using the Phoenix Micron IV image guided laser.
  • One to four veins around the optic nerve head were ablated by delivering a laser pulse (power 50 mW, spot size 50 ⁇ m, duration 3 seconds) to each vein.
  • Pen1-XBIR3 eyedrops were delivered immediately after RVO and at 24 h. At 48 h, the eyes were imaged via OCT.
  • FIG. 4 presents images from individual animals (2 control, 2 RVO, 4 RVO+Pen1-XBIR3). For each animal there are three sets of OCT and brightfield images.
  • the brightfield image has a horizontal line showing the level of the OCT.
  • mouse retinas were harvested for western blot to detect activated caspase-9 (clCasp9) ( FIG. 5 , left panel).
  • the blot showed a 10-fold induction of clCasp9.
  • mice were given Pen1-XBIR3 eyedrops and retinas were harvested and immunoprecipitated with anti-His followed by western blot for clCasp9 ( FIG. 5 , right panel). There was a 21-fold increase in binding of XBIR3 and clCasp9 by 4 h and a 45-fold increase by 24 h.
  • Pen1-XBIR3 eyedrops were given immediately after RVO and at 24 h. At 48 h OCT images showed significant protection against RVO ( FIG. 6 ) with less increase in retinal thickness and abrogation of retinal detachment (**P ⁇ 0.01).
  • Retinal layers include the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), the outer nuclear layer (ONL), the inner segments (IS), the outer segments (OS), and the retinal pigment epithelium (RPE), which is located next to the choroid.
  • Pen1-XBIR3 decreased retinal detachment ( FIG. 7 , **P ⁇ 0.01), protected the inner retinal layers ( FIG. 8 , **P ⁇ 0.01), decreased swelling of the outer retina layers, such as the outer plexiform layer ( FIG. 9 , **P ⁇ 0.01) and protected the photoreceptors ( FIG. 10 , *P ⁇ 0.05, **P ⁇ 0.01).
  • TUNEL staining is a marker of cell death.
  • RVO induces TUNEL staining by 24 h in the INL.
  • Retinas were harvested at 48 h from mice treated with Pen1-XBIR3 or untreated mice, then processed for immunohistochemistry. Analysis of samples showed that TUNEL positive cells were decreased by Pen1-XBIR3 eyedrops and that the eyedrops maintained INL thickness ( FIG. 11 ).
  • RVO induces a decrease in A and B waves on an electroretinogram (ERG).
  • ERG electroretinogram
  • RVO induces activation of caspase-7, a target of active caspase-9, in blood vessels.
  • Pen1-XBIR3 prevents this increase at 24 h as shown by a comparison of retinal section obtained 24 h post-RVO and stained as indicated ( FIG. 13 .)
  • Example 8 Pen1-XBIR3 Prevented Induction of VEGF and HIF-1 ⁇ by RVO
  • RVO leads to induction of vascular endothelial growth factor (VEGF) and Hypoxia-inducible factor 1-alpha (HIF-1 ⁇ ) within 4 h of induction of RVO.
  • VEGF vascular endothelial growth factor
  • HIF-1 ⁇ Hypoxia-inducible factor 1-alpha
  • Pent-XBIR3 after RVO abrogated the increase in VEGF and HIF-1 ⁇ .
  • Retinas were harvested at 4 h post-RVO and analyzed by western blot for VEGF expression ( FIG. 14 , left) and by immunohistochemistry for HIF-1 ⁇ expression ( FIG. 14 , right).

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WO2022140118A1 (fr) * 2020-12-22 2022-06-30 The Trustees Of Columbia University In The City Of New York Compositions et méthodes pour traiter des patients présentant une déficience en complexe mitochondrial i avec des inhibiteurs de la voie de signalisation de la caspase-9

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US20130137642A1 (en) * 2010-04-23 2013-05-30 Demetrios Vavvas Methods and compositions for preserving photoreceptor and retinal pigment epithelial cells
WO2013052498A2 (fr) * 2011-10-04 2013-04-11 Albert Einstein College Of Medicine Of Yeshiva University Inhibition de la caspase 9 et peptides bri2 dans le traitement de la démence
WO2013188676A2 (fr) * 2012-06-13 2013-12-19 The Trustees Of Columbia University In The City Of New York Administration intranasale d'agents thérapeutiques augmentant la perméabilité cellulaire pour le traitement de l'œdème

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WO2021091881A1 (fr) * 2019-11-04 2021-05-14 The Trustees Of Columbia University In The City Of New York Compositions de conjugués d'inhibiteurs de caspase de pénétration cellulaire à concentration élevée et procédés associés
WO2022140118A1 (fr) * 2020-12-22 2022-06-30 The Trustees Of Columbia University In The City Of New York Compositions et méthodes pour traiter des patients présentant une déficience en complexe mitochondrial i avec des inhibiteurs de la voie de signalisation de la caspase-9

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