WO2020219264A1 - Inhibiteurs de ferrochélatase et leurs procédés d'utilisation - Google Patents

Inhibiteurs de ferrochélatase et leurs procédés d'utilisation Download PDF

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WO2020219264A1
WO2020219264A1 PCT/US2020/027132 US2020027132W WO2020219264A1 WO 2020219264 A1 WO2020219264 A1 WO 2020219264A1 US 2020027132 W US2020027132 W US 2020027132W WO 2020219264 A1 WO2020219264 A1 WO 2020219264A1
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pharmaceutically acceptable
acceptable salt
retinal
ferrochelatase
inhibitor
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PCT/US2020/027132
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English (en)
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Timothy W. CORSON
Shiek Pran Babu SARDAR PASHA
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Indiana University Research And Technology Corporation
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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • 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

  • Angiogenesis typically occurs in the body during development and wound repair processes. However, during numerous pathological conditions, angiogenesis can occur, notably in ocular diseases such as retinopathy of prematurity and diabetic retinopathy. After pathological angiogenesis occurs, newly formed blood vessels are fragile, porous and not fully differentiated. The formation of such new blood vessels in the eye may lead to hemorrhage, rapid photoreceptor degeneration, and eventual fibrotic scarring, with rapid, permanent vision loss.
  • Diabetic retinopathy Clinical symptoms of diabetic retinopathy are seen in 75% of diabetic patients, with 10% of them eventually developing visual impairment. Diabetic retinopathy is currently the leading cause of blindness among working age adults and accounts for 8% of the legal blindness in the United States. The proliferative form of diabetic retinopathy associated with retinal neovascularization has particularly serious effects on vision.
  • retinopathy of prematurity 1.5% were bom with very low birth weight. Almost 70% of these very low birth weight infants were likely to develop retinopathy of prematurity. The disease is estimated to cause visual loss in 1300 children per year in the United States, and severe visual impairment in a further 500 children per year. Overall, between 6% and 18% of childhood blindness is attributable to retinopathy of prematurity. Moreover, as more and more children survive premature birth in middle income countries due to improvements in neonatal intensive care, retinopathy of prematurity is becoming more prevalent worldwide.
  • retinopathy of prematurity survivors are more likely than the general population to develop posterior segment pathology, retinal detachment, myopia, amblyopia, strabismus, early cataracts, and glaucoma.
  • ferrochelatase inhibitors such as griseofulvin and N-methylprotoporphyrin (NMPP)
  • NMPP N-methylprotoporphyrin
  • a method is provided of treating an ocular disease in a patient wherein the ocular disease involves retinal neovascularization.
  • the method comprises administering a ferrochelatase inhibitor, or a pharmaceutically acceptable salt thereof, to the patient, and ameliorating the ocular disease involving retinal neovascularization in the patient wherein the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, does not cause toxicity in the retina of the patient.
  • ferrochelatase inhibitor or the pharmaceutically acceptable salt thereof, is selected from the group consisting of N-methylprotoporphyrin (NMPP) or an analog thereof, griseofulvin or an analog thereof, antisense RNA targeting ferrochelatase RNA, an agent for RNA silencing or RNA interference (RNAi) targeting ferrochelatase RNA, and combinations thereof.
  • NMPP N-methylprotoporphyrin
  • RNAi RNA interference
  • anti-VEGF agent is selected from the group consisting of ranibizumab, bevacizumab, aflibercept, abicipar pegol, brolucizumab, faricimab, vorolanib, biosimilars to any of these VEGF agents, and combinations thereof.
  • ferrochelatase inhibitor or the pharmaceutically acceptable salt thereof, is selected from the group consisting of N-methylprotoporphyrin (NMPP) or an analog thereof, griseofulvin or an analog thereof, and combinations thereof.
  • NMPP N-methylprotoporphyrin
  • Fig. 1A shows phase contrast images showing that griseofulvin blocks retinal sprouting ex vivo.
  • Fig. 2 A shows confocal images from explanted P4 juvenile retina stained with
  • GS-IB4 (white; labels vasculature) showing that griseofulvin inhibits the VEGF-induced retinal filopodia formation and extensions compared to a VEGF-treated control.
  • Fig. 4B is an immunoblot showing the temporal FECH protein expression in mouse retina from OIR mouse eyes compared to littermate controls; b-actin is a loading control. Pooled eyes from three independent animals per condition.
  • Fig. 6A shows OCT images of vehicle and griseofulvin injected groups at day 5 and 14 after injection suggesting retinal structure is normal.
  • Fig. 6B shows Fundus and FA images of vehicle and griseofulvin injected groups at day 14 after injection suggesting normal fundus and no vascular leakage.
  • GCF Ganglion cell layer
  • INF Inner nuclear layer
  • ONE Outer nuclear layer.
  • Figs. 8A-C show the Haematoxylin and eosin (H&E) stained retinal cross section images from vehicle and griseofulvin treated groups at 14 days after injection (DAI) in vivo.
  • Fig. 8A shows no histological changes upon griseofulvin treatment compared to vehicle- injected and untouched control.
  • Fig. 8B shows no significant change of retinal thickness in either griseofulvin-injected group compared to DMSO vehicle injected group.
  • Fig. 8C shows the counts of number of cell nuclei per retinal layer in griseofulvin and vehicle injected groups suggesting no change in cell layer thickness.
  • Figs. 9A-9C show retinal functional analysis using ERG.
  • Fig. 9A shows representative ERG curves of vehicle (DMSO) injected (black) and 100 mM griseofulvin injected (red) animals, in scotopic conditions after 10 days post-injection.
  • Fig. 9B shows representative ERG curves of vehicle (DMSO) injected (black) and 100 pM griseofulvin injected (red) animals, in photopic conditions after 10 days post- injection.
  • Fig. 9C is a quantification showing no significant changes in either a- or b- waves between griseofulvin and vehicle; in each set of bars the topmost bar is griseofulvin (100 pM) and the bottommost bar is vehicle.
  • Figs. 6A-B, Figs. 7A-B, Figs. 8A-C and Figs. 9A-C show that, in normal adult mice, intravitreal griseofulvin treatment at therapeutic concentrations does not damage in vivo retinal morphology as assessed by optical coherence tomography, appearance of the fundus, and fluorescein angiography of the retinal vasculature. Also, griseofulvin does not affect retinal function as measured by
  • electroretinogram and griseofulvin treatment does not disrupt the histology of the retina or the thickness/number of cells in retinal layers, nor does it induce gliosis (GFAP staining), apoptosis (caspase 3 staining), or microglial activation (Iba-1 staining), and it does not disrupt the normal vasculature (GS-IB4 staining).
  • Figs. 10A-C show that a partial loss-of-function point mutation in the Fech gene, called Fech mlP:iS , can ameliorate the two pathological features of the OIR model:
  • Figs. 11 A and 1 IB show vascular proliferation in Fech mlp as mutant retina in
  • Fig.l lA shows the retinal flatmount z-stack image stained for GS-IB4 (green, labels vasculature) and EdU (magenta, labels cell proliferation in S-phase) in Fech mlP:iS mutants suggesting less pathological vascular cell proliferation compared to wild type controls.
  • Figs. 13 A and 13B show vascular proliferation in NMPP treated retinas in OIR mouse model.
  • Fig.13 A shows the retinal flatmount z-stack image stained for GS-IB4 (green, labels vasculature) and EdU (magenta, labels cell proliferation in S-phase) in NMPP-injected retinas suggesting reduced vascular cell proliferation compared to vehicle injected wild type controls.
  • Fig. 14 shows temporal qPCR analysis of FECH and VEGF expression in OIR mouse model.
  • the leftmost bar is P12
  • the middle bar is P15
  • the rightmost bar is P17.
  • Fig. 15 shows that FECH expression (magenta) does not colocalize with hypoxic regions (pimonidazole; white) in the OIR mouse model.
  • Fig. 16 shows FECH expression and vascular pathology in OIR retinal flatmounts.
  • Fig. 17 is a scheme showing the step wise analysis and quantification of NV and
  • Fig. 18 shows differential hypoxic expression (pimonidazole; red) and vascular pathology (GS-IB4; green) in Fech mlpas mutant mice in OIR retinal flatmounts.
  • Fig. 20 shows cell proliferation (EdU; red) and its analysis in Vehicle and
  • Figs. 21A and 21B show GFAP (magenta) and Caspase-3 (green) expression in vehicle- and griseofulvin-treated retinal sections in adult mouse at days 7 and 14 after injection (DAI).
  • Figs. 22A and 22B show vasculature (GS-IB4; green) and microglia activation
  • Figs. 23A and 23B are an overview of H and E staining of vehicle- and griseofulvin-treated paraffin retinal sections in adult mouse at days 7 and 14 after injection (DAI).
  • “a” or“an” may mean one or more.
  • “about” in reference to a numeric value including, for example, whole numbers, fractions, and percentages, generally refers to a range of numerical values (e.g., +/- 5 % to 10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
  • the terms“treat,”“treating,”“treated,” or“treatment” refer to both therapeutic treatment and prophylactic or preventative treatment.
  • “inhibited” in reference to symptoms of retinal neovascularization mean reducing a detrimental effect, eliminating a detrimental effect, preventing progression of a detrimental effect, or any other effect on the retinal neovascularization that would be considered by a physician to be a therapeutic, prophylactic, or preventative treatment.
  • “does not cause toxicity” means that one or more types of toxicity that might normally occur in the retina does not occur.
  • ferrochelatase as an antiangiogenic therapy.
  • angiogenesis is inhibited by blocking ferrochelatase resulting in inhibition of retinal neovascularization, thereby treating neovascular eye diseases such as retinopathy of prematurity, diabetic retinopathy, hypertensive retinopathy, central retinal vein occlusion, branch retinal vein occlusion, neovascular glaucoma, retinopathy of prematurity,
  • the present invention relates to methods of treatment of a patient with ferrochelatase inhibitors, or pharmaceutically acceptable salts thereof.
  • the invention relates to methods of treatment of ocular diseases involving retinal neovascularization with ferrochelatase inhibitors, such as griseofulvin and N- methylprotoporphyrin (NMPP), or pharmaceutically acceptable salts thereof.
  • ferrochelatase inhibitors such as griseofulvin and N- methylprotoporphyrin (NMPP), or pharmaceutically acceptable salts thereof.
  • a method of treating an ocular disease in a patient wherein the ocular disease involves retinal neovascularization comprising administering a ferrochelatase inhibitor, or a pharmaceutically acceptable salt thereof, to the patient, and ameliorating the ocular disease involving retinal neovascularization in the patient wherein the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, does not cause toxicity in the retina of the patient.
  • ferrochelatase inhibitor or the pharmaceutically acceptable salt thereof, is selected from the group consisting of N-methylprotoporphyrin (NMPP) or an analog thereof, griseofulvin or an analog thereof, antisense RNA targeting ferrochelatase RNA, an agent for RNA silencing or RNA interference (RNAi) targeting ferrochelatase RNA, and combinations thereof.
  • NMPP N-methylprotoporphyrin
  • RNAi RNA interference
  • anti-VEGF agent is selected from the group consisting of ranibizumab, bevacizumab, aflibercept, abicipar pegol, brolucizumab, faricimab, vorolanib, biosimilars to any of these VEGF agents, and combinations thereof.
  • a method of inhibiting retinal neovascularization comprising contacting retinal tissue with a ferrochelatase inhibitor, or a pharmaceutically acceptable salt thereof, and inhibiting retinal neovascularization wherein the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, does not cause toxicity in the retinal tissue.
  • ferrochelatase inhibitor or the pharmaceutically acceptable salt thereof, is selected from the group consisting of N-methylprotoporphyrin (NMPP) or an analog thereof, griseofulvin or an analog thereof, and combinations thereof.
  • NMPP N-methylprotoporphyrin
  • Ferrochelatase is a nuclear-encoded, mitochondrial inner membrane- associated enzyme responsible for the final step of heme biosynthesis.
  • FECH catalyzes the insertion of ferrous ion (Fe 2+ ) into the center of protoporphyrin IX (PPIX) to complete the formation of heme.
  • Fe 2+ is supplied by the inner membrane iron transporter mitoferrin stabilized by the channel ABCB10, while PPIX is produced by a cascade of porphyrin synthetic enzymes ending with protoporphyrinogen oxidase, which likely complexes with FECH to deliver PPIX.
  • a method of inhibiting retinal neovascularization comprises contacting retinal tissue with a ferrochelatase inhibitor, or a pharmaceutically acceptable salt thereof, and inhibiting retinal neovascularization wherein the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, does not cause toxicity in the retinal tissue.
  • a“patient” can be a human or, in the case of veterinary applications, the patient can be a laboratory, an agricultural, a domestic, or a wild animal.
  • the patient can be a laboratory animal such as a rodent (e.g., mouse, rat, hamster, etc.), a rabbit, a monkey, a chimpanzee, a domestic animal such as a dog, a cat, or a rabbit, an agricultural animal such as a cow, a horse, a pig, a sheep, a goat, or a wild animal in captivity such as a bear, a panda, a lion, a tiger, a leopard, an elephant, a zebra, a giraffe, a gorilla, a dolphin, or a whale.
  • a rodent e.g., mouse, rat, hamster, etc.
  • a rabbit, a monkey, a chimpanzee a domestic animal such as a dog, a
  • ferrochelatase inhibitors include, but are not limited to, antisense RNA targeting ferrochelatase RNA, an agent for RNA silencing or RNA interference (RNAi) targeting ferrochelatase RNA, an agent for CRISPR/Cas9-mediated genetic ablation of ferrochelatase (FECH) DNA, an agent for Zinc-finger nuclease-mediated genetic ablation of ferrochelatase (FECH) DNA, and combinations thereof, or, pharmaceutically acceptable salts thereof for the small molecule inhibitors described in this paragraph.
  • RNAi RNA silencing or RNA interference
  • the ferrochelatase inhibitor can inhibit neovascularization in the retina of the eye of the patient, can inhibit vaso-obliteration in the retina of the eye of the patient, or can inhibit vascular cell proliferation in the retina of the eye of the patient, or combinations thereof.
  • the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof does not cause toxicity in the retina of the patient.
  • the toxicity can be selected from the group consisting of retinal structural damage, retinal morphological damage, retinal vascular leakage, retinal functional damage, retinal cell death, and inflammation of the retina.
  • a suitable dosage of the ferrochelatase inhibitor, or a pharmaceutically acceptable salt thereof may achieve a final intravitreal concentration of the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, of from about 0.5 mM to about 10 pM, about 0.5 pM to about 20 pM, about 0.5 pM to about 30 pM, about 0.5 pM to about 40 pM, about 0.5 pM to about 50 pM, about 0.5 pM to about 60 pM, about 0.5 pM to about 70 pM, about 0.5 pM to about 80 pM, about 0.5 pM to about 90 pM, about 0.5 pM to about 100 pM, about 0.5 pM to about 150 pM, about 0.5 pM to about 200 pM, about 0.5 pM to about 300 pM, about 0.5 pM to about 400 pM, about 0.5 pM to about 500
  • amounts to be administered to the patient for parenteral administration can range, for example, from about 0.05 mg to about 500 mg, from about 0.05 mg to about 400 mg, from about 0.05 mg to about 300 mg, from about 0.05 mg to about 200 mg from about 0.05 mg to about 100 mg, from about 0.05 mg to about 50 mg, from about 0.05 mg to about 30 mg, 0.05 mg to about 25.0 mg, about 0.05 mg to about 20.0 mg, about 0.05 mg to about 15.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 9.0 mg, about 0.05 mg to about 8.0 mg, about 0.05 mg to about 7.0 mg, about 0.05 mg to about 6.0 mg, about 0.05 mg to about 5.0 mg, about 0.05 mg to about 4.0 mg, about 0.05 mg to about 3.0 mg, about 0.05 mg to about 2.0 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.4 mg, about 0.05 mg to about 0.3 mg, about
  • the ferrochelatase inhibitor can be in the form of a “pharmaceutically acceptable salt” of the ferrochelatase inhibitor.
  • pharmaceutically acceptable salt refers to those salts whose counter ions may be used in pharmaceuticals.
  • such salts include, but are not limited to 1) acid addition salts, which can be obtained by reaction of the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methane sulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid and the like; or 2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethamine,
  • suitable acid addition salts are formed from acids which form non-toxic salts.
  • Illustrative examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate,
  • suitable base salts are formed from bases which form non- toxic salts.
  • bases which form non- toxic salts.
  • Illustrative examples include the arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
  • Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.
  • the ferrochelatase inhibitor, or a pharmaceutically acceptable salt thereof, described herein may contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers.
  • the ferrochelatase inhibitor, or a pharmaceutically acceptable salt thereof may be capable of existing as geometric isomers. Accordingly, various embodiments may include pure geometric isomers or mixtures of geometric isomers.
  • ferrochelatase inhibitor, or a pharmaceutically acceptable salt thereof, described herein may exist in unsolvated forms as well as solvated forms, including hydrated forms.
  • the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention.
  • the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, described herein can be administered to the patient using any suitable method known in the art.
  • the term“administering” or“administered” includes all means of introducing the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, to the patient, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, intravitreal, intraocular, topical, and the like.
  • the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, described herein may be administered in a unit dosage form and/or in formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • pharmaceutically acceptable salt thereof can be administered by injection, in the form of eye drops, in the form of an eye ointment, by parenteral administration, or the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, can be administered orally, or by any other suitable means of administration.
  • the preparation under sterile conditions, by lyophilization, to produce a sterile lyophilized powder for a parenteral formulation may readily be accomplished using standard pharmaceutical techniques well-known to those skilled in the art.
  • the solubility of the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, used in the preparation of a parenteral formulation may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.
  • the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof may be administered to the patient, for example using a regimen of daily or weekly administration, such as once a day, two times a day, three times a day, every day, every other day, one time weekly, two times weekly, three times weekly, once monthly, once every two, three, four, five, or six months, or any other suitable regimen that would be considered a suitable regimen by a person skilled in the art for administration of the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof.
  • a regimen of daily or weekly administration such as once a day, two times a day, three times a day, every day, every other day, one time weekly, two times weekly, three times weekly, once monthly, once every two, three, four, five, or six months, or any other suitable regimen that would be considered a suitable regimen by a person skilled in the art for administration of the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof.
  • the methods can further comprise administering an anti-VEGF agent.
  • the anti-VEGR agents may include, for example, anti-VEGF biologies such as ranibizumab, bevacizumab, or aflibercept, abicipar pegol, brolucizumab, faricimab, vorolanib, biosimilars to any of these VEGF agents, and combinations thereof.
  • an anti-VEGF agent is administered in combination with the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof.
  • the anti-VEGF agent can be administered to the patient before the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof.
  • the anti- VEGF agent can be administered to the patient at the same time as the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof, but in different formulations, or in the same formulation.
  • the anti-VEGF agent can be administered to the patient after the ferrochelatase inhibitor, or the pharmaceutically acceptable salt thereof.
  • mice were housed under standard conditions in the Indiana University Laboratory Animal Resource Center (LARC).
  • Fech mlp as mutant mice (Tutois et ak, 1991) were purchased from Jackson Laboratory on a BALB/c background and backcrossed into C57BL/6J; mixed-sex littermates from an FI generation were used for experiments at 6-8 weeks of age. Sample sizes for experiments were based on power analyses, and treatments were randomly assigned by cage and animals. The experimenter was masked to genotype or treatment during analyses.
  • mice were subjected to oxygen-induced retinopathy (OIR) as described previously (Smith et ak, 1994; Stahl et ak, 2010). Briefly, postnatal day 7 (P7) mice, with their nursing mothers, were exposed to 75% 02 (hyperoxia) in a Coy hyperoxia chamber for 5 days (P7-P12) to initiate retinal vascular obliteration and returned to room air (normoxia) at PI 2. When the mice are returned to room air on PI 2, the regions of vascular obliteration in the retina are rendered hypoxic.
  • OIR oxygen-induced retinopathy
  • a hypoxic condition stimulates the development of pathological neovascular tufts within the vitreous body and maximal neovascularization occurs at P17 in the OIR model.
  • P12, P15 and P17 pups were euthanized at various time points.
  • S-phase marker and thymidine analogue 5-ethynyl-2'-deoxyuridine, EdU (0.5 pg/g body weight) (Invitrogen, A10044) was injected subcutaneously at different time points in the OIR mouse model.
  • pimonidazole hydrochloride 60 mg/kg body weight
  • Hypoxyprobe, Inc was injected intraperitoneally at different time points in OIR mouse pups.
  • hydrochloride (1 mg/kg body weight; Antisedan). All instruments were sterilized before starting the surgical procedures.
  • OIR experiments for P12 pups, the eye lid was opened using tweezers to gain access to the globe. Pupils were dilated using 1% Tropicamide and 2.5 % phenylephrine. After dilation, a small incision was made at the nasal-temporal ora serrata (approximately 4 mm below the iris) to get access the posterior cavity (vitreous chamber) of the eye with the use of a 30-G insulin syringe needle. High precision sterile Hamilton syringes (0.5- 5 pL volume) with a sharp tip were used for injections.
  • 0.5 pL of solvent with or without compound of interest were injected intravitreally without damaging the lens at the angle of about 45-60 degrees towards the plane of injection.
  • Successful injection led to mild perturbation in the anterior chamber and instant back flushing indicate a failed injection into a scleral pocket (e.g. between sclera and choroid) instead of injecting into vitreous space.
  • a scleral pocket e.g. between sclera and choroid
  • DMSO vehicle
  • NMPP FECH inhibitor N-methyl protoporphyrin
  • mice injecting 0.5 pL of a 100 pM aqueous solution of NMPP or griseofulvin, we estimate a final intravitreal concentration of 25 pM compound in young mice (based on the average vitreous volume of ⁇ 2 pL) and concentration of 10 pM compound in adult mice (based on the average vitreous volume of ⁇ 5 pL).
  • cryoslides were washed 3x15 minutes in PBS. After washing, cryoslides were incubated for 2 hr at room temperature with secondary antibodies (AlexaFluor 488-, 555-, 594-, 647 or Cy3 conjugated anti-rabbit, rat, mouse, goat IgG) (1:400, Life Technologies and Jackson Immunoresearch), or isolectin GS-IB4 conjugated streptavidin DyLight 488 (1:200, Invitrogen). After the incubation, cryoslides were washed 3 times in PBS and then slides were
  • retinal flatmounts were fixed overnight at 4°C. After fixation, retinal flatmounts were washed twice in PBS and then permeabilized for 2 hours in blocking buffer containing 0.5 % Triton X-100 in 10 % BSA prepared in PBS. After incubation, retinal flatmounts were stained for isolectin GS-IB4 (1:200) and FECH (1:200) in flatmount staining solution: 0.5 % Triton X-100 in 1 % BSA prepared in PBS for 2 to 3 days at 4°C in shaker.
  • retinal flatmounts were washed 4x15 minutes in PBS and respective secondary antibodies were added in a dilution of 1:400 in diluted flatmount staining solution was added to each retina and incubated overnight at room temperature protected from light.
  • retinal flatmounts were combined with EdU and
  • RNA extraction dissected retinas were either flash frozen (in liquid nitrogen) or collected in RLT (Qiagen RNeasy kit). Whole retinas were homogenized with Qiashredder (Qiagen). Total RNA was extracted according to the manufacturer’s instructions (Qiagen RNeasy mini kit). The isolated RNA was eluted in 30 pL RNase-free water, quantified using a NanoDrop and stored at -80 ° C until further use. DNase digestion was performed to prevent genomic DNA contamination ⁇ cDNA synthesis was performed with iScript cDNA synthesis kit (BioRad) according to the manufacturer’s protocol and 0.5-1 pg RNA was used for the reverse transcription.
  • iScript cDNA synthesis kit BioRad
  • PCR cycling conditions were as follows: 50°C for 2 minutes, 95 °C for 10 minutes, 40 cycles at 95°C for 15 s and annealing temperature at 60°C for 1 minute 30 sec, followed by 95 °C for 2 s.
  • Gene expression was analyzed with the ViiA7 Version 1.2 software. qPCR was performed in 10 pL volumes in a 384-well plate.
  • Primer/probe sets used were as follows: Vegfa (Mm00437306_ml), Fech (Mm00500394_ml) and housekeeping controls Hprt (Hs03024075_ml) and Tbp (Mm01277042_ml).
  • the data were analyzed using the AAC t method.
  • qPCR reactions were ran with at least 3 biological replicates (N) and as technical duplicates.
  • the expression levels of genes were normalized to the two housekeeping genes and calibrated to the age matched, untouched sample.
  • Retinas and choroids were snap frozen in liquid nitrogen immediately after dissection, and stored at -80°C.
  • Pooled retina and choroid were lysed in lxRIPA sample buffer (Thermo Scientific) containing 3% b-mercaptoethanol (Sigma), protease inhibitors (cOmplete mini, Roche), and phosphatase inhibitors (PhosSTOP, Roche) on ice for 20 minutes, homogenized, and clarified by centrifugation at 12,000 x g for 15 min at 4°C. Supernatants were collected and protein concentration was determined using a Bradford assay.
  • Equal amounts of total protein (30-50 pg) from each sample were resolved by 4-20% NuSeP Tris- Glycine gels (nUview Precast gel, NB 10-420) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Proteins were immunoblotted with antibodies against FECH (LS Biotech) at 1:400 dilution, and b-actin (AC40) (Sigma- Aldrich) at 1:5000. Secondary antibodies (Thermo Fisher Scientific) were used at 1:10,000 dilutions. All of the dilutions were made in Tris Buffered Saline-0.05% (v/v) Tween-20 buffer containing 2% (w/v) bovine serum albumin (BSA). Immunoreactive bands were detected using Amersham ECL prime immunoblotting detection reagents (GE healthcare) on an Azure c600 Chemiluminescent imager (Azure Biosystems).
  • OCT optical coherence tomography
  • OCT optical coherence tomography
  • mice were then placed on a custom heated stage that moved freely to position the mouse eye for imaging. All imaging was done by a single experimenter (S.P.B.). Several horizontal and vertical OCT images were taken in untouched, vehicle and griseofulvin-injected mice as indicated at various time points.
  • mice were anesthetized as described above after dark adaptation overnight. Pupils were dilated and a drop of proparacaine hydrochloride (0.5%; Alcon) was applied on cornea for topical anesthesia. ERG recordings were obtained simultaneously from both eyes with gold wire loop mouse corneal electrodes (STelesSR, LKC Technologies), with the reference electrode placed under the skin at the skull and the ground subdermal electrode at the tail. Flash ERGs were obtained from vehicle control and griseofulvin (100 mM) treated animals on day 10 post intravitreal injection.
  • scotopic rod recordings were performed on overnight dark-adapted mice, with 10 increasing light intensities of white light, and responses were recorded with a visual ERG stimulus presented at intensities of 0.025, 0.25, and 2.5 log cd-s/m 2 at 10-, 20-, and 30-second intervals, respectively. Ten responses were recorded and averaged at each light intensity.
  • Photopic cone recordings were undertaken after mice were light adapted under the rod-saturating white background light of 100 cd-s/m 2 for 8- 10 minutes. Recordings were performed with four increasing flash intensities from 0, 5, 10 and 25 log cd-s/m 2 in the presence of a constant 100 mcd-s/m 2 rod suppressing background light.
  • the a-wave amplitude was measured from the baseline to the negative peak and the b-wave amplitude was measured from the a-wave trough to the maximum positive b-wave peak, behind the last prominent oscillatory potential.
  • the values of a- wave and b-wave amplitudes from scotopic 2.5 log cd-s/m 2 ERG (rod-driven response) and photopic 25 log cd-s/m 2 ERG (cone-driven response) were averaged.
  • tile scan images were acquired on an LSM700 confocal microscope (Plan-Apochromat lOx objective; Zeiss) and stitched using Zeiss Zen black or blue software.
  • H & E stained retinal overview images approximately 10 images were taken for each retina on an EVOS XL digital imaging system (4x objective) and all of these images were stitched using photomerge automation in Adobe Photoshop CS7. Retinal vasculature area, vasoobliteration and neovascularization area was quantified as described previously (Connor et ak, 2009, Stahl et ak, 2010).

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Abstract

L'invention concerne des méthodes de traitement d'un patient au moyen d'inhibiteurs de la ferrochélatase. En particulier, l'invention concerne des procédés de traitement de maladies oculaires impliquant une néovascularisation rétinienne avec des inhibiteurs de la ferrochélatase, tels que la griséofulvine et la N-méthylprotoporphyrine (NMPP), ou des sels pharmaceutiquement acceptables de celles-ci.
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US20160222388A1 (en) * 2015-02-03 2016-08-04 Indiana University Research And Technology Corporation Chemical inhibition of ferrochelatase as an antiangiogenic therapy

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160222388A1 (en) * 2015-02-03 2016-08-04 Indiana University Research And Technology Corporation Chemical inhibition of ferrochelatase as an antiangiogenic therapy

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Title
PASHA ET AL.: "Repurposing Griseofulvin as a Non-Toxic Angiogenesis Inhibitor", THE FASEB JOURNAL, vol. 32, no. 1, 20 April 2018 (2018-04-20), Retrieved from the Internet <URL:https://www.fasebj.org/doi/abs/10.1096/fasebj.2018.32.1-supplement.829.3> [retrieved on 20200528] *

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