The present invention relates to biomarkers for dry eye disease and use of the biomarkers for treatment of the disorder.
Dry eye disease is a complex disease that results in ocular discomfort, visual disturbance, and tear film instability, which create the potential for damage to the ocular surface. It is characterized by increased osmolarity of the tear film and inflammation of the ocular surface. Estimates of the prevalence of dry eye disease vary considerably, depending on the criteria used to define the disease, but in the United States (U.S.), it has been estimated that as many as 20 million adults in the U.S. have dry eye disease. It has been projected that there will be a 40% increase in number of patients affected by 2030 (Schaumberg, Advances in Experimental Medicine and Biology, 2002, 506:989-98; Schaumberg, American Journal of Ophthalmology, 2003, 136:318-26; Schaumberg, Archives of Ophthalmology, 2009, 127:763-8). With the aging population in the U.S. and other countries of the developed world, and increasing computer use, dry eye disease is expected to become more prevalent. Thus, finding effective treatments is becoming more important (Brewitt, Survey of Ophthalmology, 2001, 45 Suppl 2:S199-202).
Aldehydes are reactive organic molecules that bind to proteins, carbohydrates, lipids and nucleic acids (Esterbauer, Free Radical Biology and Medicine, 1991, 11(1):81-128). Free aldehydes—aldehydes not sequestered or otherwise protected in specific metabolic processes—can be toxic, and aldehyde binding to cellular constituents can lead to inflammation (Yadav, Oxidative Medicine and Cellular Longevity, 2013, Volume 2013, Article ID 690545), molecular dysfunction (O'Brien, Critical Reviews in Toxicology, 2005, 35(7):609-62), and the accumulation of indigestible metabolites, such as lipofuscin components in the retina (Boyer, J Biol Chem., 2012, 287:22276-86).
In biological systems, aldehydes are formed by a variety of processes, including the oxidation of alcohols, polyamine and glucose metabolism, and oxidative stress. In some disease states, aldehyde concentrations may be increased. Increases in aldehyde concentrations has been described in a variety of inflammatory ocular diseases, including pterygium, Behcet's Disease, Sjögren's Syndrome, anterior uveitis, and dry eye disease (Sandikci, Acta Dermato-Venereologica, 2003, 83(5): 342-6; Cejkova, Histology and Histopathology, 2007, 22(9):997-1003; Balci, Molecular Vision, 2011, 17: 443-7; Turk, Ocular Immunology and Inflammation, 2014, 22(2):127-32; Choi, Current Eye Research, 2016, 41(9): 1143-9).
In some aspects, the present disclosure relates to use of biomarkers for dry eye disease (DED), also referred to herein as dry eye syndrome (DES; the two terms are used interchangeably), for use in assessing efficacy of treatments for dry eye disease and in the treatment of dry eye disease. In some embodiments, the biomarkers are also useful in the selection of patients for treatment of dry eye disease.
In one aspect, the present disclosure provides a method of assessing the effectiveness of an aldehyde trapping agent in treating dry eye disease in a patient. In some embodiments, the method comprises administering an aldehyde trapping agent to a patient with dry eye disease; measuring the level of an aldehyde marker of oxidative stress present in the eye of the patient; and comparing the measured level of the aldehyde marker of oxidative stress to level of aldehyde marker of oxidative stress in an appropriate control, wherein a reduction in level of aldehyde marker of oxidative stress indicates effectiveness of the aldehyde trapping agent in treating dry eye disease.
In another aspect, the present disclosure provides a method of assessing effectiveness of an aldehyde trapping agent in treating ocular inflammation in a subject, comprising: administering an aldehyde trapping agent to a subject with ocular inflammation; measuring the level of an aldehyde marker of oxidative stress present in the eye of the subject; and comparing the measured level of the aldehyde marker of oxidative stress to level of aldehyde marker of oxidative stress in an appropriate control; wherein a reduction in level of aldehyde marker of oxidative stress indicates effectiveness of the aldehyde trapping agent in treating ocular inflammation.
In another aspect, the present disclosure provides a method of treating ocular inflammation in a subject comprising:
wherein the subject is treated with a lower dosing frequency of reproxalap for a reduction of greater than about 20% in the measured level of the aldehyde marker of oxidative stress, and wherein the subject is treated with the same or higher dosing frequency of reproxalap for a reduction of about 20% or less in the measured level of the aldehyde marker of oxidative stress.
In some embodiments, the ocular inflammation is associated with dry eye disease, allergic conjunctivitis, pterygium, Behcet's Disease, Sjögren's Syndrome, or uveitis (including, for example, anterior uveitis). In some embodiments, the ocular inflammation is associated with a corneal disease (e.g., dry eye syndrome, cataracts, keratoconus, bullous and other keratopathy, and Fuch's endothelial dystrophy), other ocular disorders or conditions (e.g., allergic conjunctivitis, ocular cicatricial pemphigoid, conditions associated with PRK healing and other corneal healing, and conditions associated with tear lipid degradation or lacrimal gland dysfunction), and other ocular conditions associated with high aldehyde levels as a result of inflammation (e.g., uveitis, scleritis, ocular Stevens Johnson Syndrome, ocular rosacea (with or without meibomian gland dysfunction)). In some embodiments, the ocular inflammation is associated with macular degeneration, such as age-related macular degeneration (“AMD”), or Stargardt's disease.
In another aspect, the present disclosure provides a method of using an aldehyde marker of oxidative stress for treating a patient with dry eye disease. In some embodiments, a method of treating dry eye disease in a patient comprises: measuring the level of an aldehyde marker of oxidative stress in the eye of a patient with dry eye disease prior to treatment; treating the patient with an aldehyde trapping agent, wherein the aldehyde trapping agent is reproxalap and wherein the reproxalap is administered topically to the eye; and measuring the level of the aldehyde marker of oxidative stress in the eye of the patient following treatment; wherein the patient is treated with a lower dosing frequency of reproxalap for a reduction of greater than about 20% in the measured level of the aldehyde marker of oxidative stress, and wherein the patient is treated with the same or higher dosing frequency of reproxalap for a reduction of about 20% or less in the measured level of the aldehyde marker of oxidative stress.
In another aspect, the present disclosure provides a method of selecting a subject for treatment of ocular inflammation, comprising: measuring the level of an aldehyde marker of oxidative stress in an eye of a subject suspected of having ocular inflammation, wherein a measured level of at least about 2 fold or greater level of the aldehyde marker of oxidative stress as compared to level of aldehyde marker of oxidative stress in subjects without ocular inflammation is indicated for treatment.
In another aspect, the present disclosure provides a method of identifying or selecting a patient with dry eye disease for treatment. In some embodiments, a method of selecting a patient for treatment of dry eye disease comprises: measuring the level of an aldehyde marker of oxidative stress in an eye (for example, in the tears) of a patient suspected of having dry eye disease, wherein a measured level of at least about 2-fold higher level of the aldehyde marker of oxidative stress as compared to the level of aldehyde marker of oxidative stress in patients without dry eye disease is indicated for treatment.
In some embodiments, the method further comprises the step of treating the dry eye disease if the level of the aldehyde marker of oxidative stress is indicated for treatment, wherein the treatment comprises administering to the patient an effective amount of reproxalap.
In some embodiments, the method further comprises the step of treating the dry eye disease by administering to the patient an effective amount of reproxalap if the patient exhibits in at least one of the patient's eyes the measured level of at least about 2.5-fold higher of the aldehyde marker of oxidative stress as compared to level of aldehyde marker of oxidative stress in patients without dry eye disease.
In some embodiments, the level of the aldehyde marker of oxidative stress is indicated for treatment if it is about 2-fold to 6-fold higher than the level of aldehyde marker of oxidative stress in patients without dry eye disease. In some embodiments, the level of the aldehyde marker of oxidative stress is indicated for treatment if it is about 2-fold to 5-fold, about 2.5-fold to 4.5-fold, about 3-fold to 4-fold, or about 3.5-fold to 4-fold higher than the level of aldehyde marker of oxidative stress in patients without dry eye disease.
In some embodiments, the level of the aldehyde marker of oxidative stress is indicated for treatment if it is about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, or about 6-fold higher than the level of aldehyde marker of oxidative stress in patients without dry eye disease.
In some embodiments, the method of selecting or identifying a patient for treatment of dry eye disease is for treatment with an aldehyde trapping agent. In some embodiments, the aldehyde trapping agent is reproxalap.
In some embodiments, the aldehyde marker of oxidative stress in the methods herein is malondialdehyde or 4-hydroxynonenal. In some embodiments, the level of the aldehyde marker of oxidative stress measured is in the form of adducts of the aldehyde marker of oxidative stress present in the eye. In some embodiments, the adducts comprise stable adducts formed with proteins in the eye. In some embodiments, the adducts comprise adducts formed with malondialdehyde. In some embodiments, the adducts comprise adducts formed with 4-hydroxynonenal. In some embodiments, a sample of tear obtained from a patient is used to measure the level of the adducts.
In some embodiments, where the methods refer to an aldehyde trapping agent, the aldehyde trapping agent is reproxalap. In some embodiments, the reproxalap is in a composition for topical administration to the eye, particularly an ophthalmic aqueous solution. In some embodiments, reproxalap is at a concentration of 0.1% to 0.5% w/v. In some embodiments, the reproxalap is at a concentration of 0.15% to 0.45% w/v, 0.2% to 0.4% w/v. In some embodiments the reproxalap is at a concentration of 0.1% w/v, 0.15% w/v, 0.2% w/v, 0.25% w/v, 0.3% w/v, 0.35% w/v, 0.4% w/v, 0.45% w/v, 0.5% w/v. In some embodiments, the reproxalap is at a concentration of 0.25% w/v. In some embodiments, the reproxalap is at a concentration of 0.1% w/v. In some embodiments, the reproxalap is at a concentration of 0.5% w/v.
In some embodiments, the reproxalap is in an admixture with a pharmaceutically acceptable excipient, wherein the excipient is a cyclodextrin selected from sulfobutylether-p-cyclodextrin and hydroxypropyl-β-cyclodextrin, or a pharmaceutically acceptable salt thereof. Preferably, the pharmaceutically acceptable excipient is sulfobutylether-β-cyclodextrin or a pharmaceutically acceptable salt thereof. In some embodiments, the cyclodextrin is present at 5% to 20% w/v, for example 6% to 15% w/v. In some embodiments, the cyclodextrin is present at about 7% w/v, 8% w/v, 9% w/v, 10% w/v, or 11% w/v. In some embodiments, the cyclodextrin is present at 7% w/v. In some embodiments, the cyclodextrin is present at 11% w/v.
In some embodiments, the reproxalap referenced in the methods is 0.25% w/v reproxalap and 7% w/v of cyclodextrin, particularly sulfobutylether-β-cyclodextrin. In some embodiments, the reproxalap referenced in the methods is 0.25% w/v reproxalap and 11% w/v of cyclodextrin, particularly sulfobutylether-β-cyclodextrin.
In another aspect, the present disclosure provides a kit for use in treating ocular inflammation in a subject, comprising: a container comprising an ophthalmic formulation comprising reproxalap as described herein; an assay kit for testing the levels of one or more aldehyde markers of ocular inflammation in the subject's tears as described herein; and, optionally, instructions for using the assay to test the levels of one or more aldehyde markers of ocular inflammation in the subject's tears.
Reactive aldehyde species (RASP) are reactive organic molecules that bind to proteins, carbohydrates, lipids, and nucleic acids. RASP that are not sequestered or otherwise protected in specific metabolic processes are toxic, and aldehyde binding to cellular constituents leads to inflammation via activation of NFNB and other pro-inflammatory mediators, molecular dysfunction, and the accumulation of indigestible metabolites, such as lipofuscin components in the retina.
Reproxalap topical ocular solution is being developed for treatment of ocular inflammation, ocular dryness, ocular irritation, ocular redness, ocular itching, and other symptoms of ocular discomfort. Ocular discomfort symptoms include dryness, itchiness, tearing, burning, stinging, grittiness, foreign body sensation, cloudy vision, sensitivity to environment, sensitivity to light (photophobia), and stringy ocular secretion. In some embodiments, treatment of ocular symptoms is measured by an assay selected from ocular vital staining, tear film break-up time, tear osmolarity, and tear volume. In some embodiments, treatment of ocular symptoms is measured by an assay selected from Visual analog scale eye dryness score assessed over 24 hours after a first dose of reproxalap, and over 90 minutes in CAE® (Controlled Adverse Environment) Ora Calibra® Ocular Discomfort Scale assessed over 24 hours after first dose of reproxalap, and over 90 minutes in CAE® Ocular Discomfort & 4-Symptom Questionnaire assessed over 24 hours after first dose of reproxalap, and before and after CAE®; Ora Calibra® Conjunctival Allergen Challenge Ocular Itching Scale assessed over 24 hours after first dose of reproxalap, and before and after CAE®; Schirmer's Test change from baseline before and after the final dose of reproxalap; Change in tear RASP levels before and after a dose of reproxalap; Conjunctival Redness over 24 hours after first dose of reproxalap. The drug product, in various strengths, has completed a Phase 2a clinical trial and a controlled, double-masked Phase 2b clinical trial in dry eye disease.
The objective of the Phase 2a clinical trial in dry eye disease was to assess the safety, tolerability, and pharmacodynamic activity of Reproxalap Ophthalmic Solutions in subjects with dry eye disease (DED) for 28 days of QID dosing with one of three different formulations. The formulations used were 0.1% w/v Reproxalap Ophthalmic Solution, and 0.5% w/v Reproxalap Ophthalmic Solution, and 0.5% w/v Ophthalmic Lipid Solution. No serious adverse events (SAEs) were observed during the 28-day treatment with any of the three reproxalap formulations, and no clinically significant change in visual acuity (VA), intraocular pressure (IOP), slit lamp biomicroscopic findings, or undilated funduscopic findings were observed. Drop comfort was less well tolerated with the 0.5% w/v Reproxalap Ophthalmic Solution and Ophthalmic Lipid Solution than with the 0.1% w/v Reproxalap Ophthalmic Solution. Statistically significant efficacy in within-subject improvement was observed over a broad array of DED signs and symptoms assessed as exploratory pharmacodynamics endpoints.
In a Phase 2b clinical trial, the efficacy of Reproxalap Ophthalmic Solutions (0.25% and 0.1%) was evaluated in patients with dry eye disease (see the details in Example 1). Administration of Reproxalap Ophthalmic Solutions for 12 weeks resulted in statistically significant improvements in multiple clinical assessments, including tear quantity (Schirmer's Test), tear quality (Tear Film Breakup Time (TBUT) and tear osmolarity), and ocular surface staining. While this group of clinical assessments is used in assessing efficacy of treatment, desirable are biomarkers for dry eye disease that can be quantitated and used as a basis for assessing the efficacy of treatments for dry eye disease. The biomarkers can also be used to guide treatment regimen and select/identify patients for treatment of dry eye disease. Accordingly, the present disclosure provides aldehyde markers of oxidative stress for use as biomarkers. The present disclosure further provides assays for use in quantifying aldehyde markers of oxidative stress and for use in evaluating a patient response to treatment with an aldehyde trapping agent such as reproxalap.
These aldehyde markers include aldehydes such as formaldehyde, acetaldehyde, acrolein, glyoxal, methylglyoxal, hexadecanal, octadecanal, hexadecenal, succinic semi-aldehyde, malondialdehyde, 4-hydroxynonenal (4-HNE or HNE), 4-hydroxy-2E-hexenal, 4-hydroxy-2E,6Z-dodecadienal, retinaldehyde, leukotriene B4 aldehyde, and octadecenal, as particular aldehydes which form adducts and one or more of which are present in a patient's tear. In some embodiments, a useful aldehyde marker of oxidative stress for dry eye disease is malondialdehyde or 4-hydroxynonenal or their adducts, particularly adducts of malondialdehyde or 4-hydroxynonenal present in the tear of patients. Measuring the levels of the aldehyde markers of oxidative stress can be used to determine the efficacy of a drug for treating dry eye disease or used to guide treatment regimes, particularly for drugs acting as aldehyde trapping agents. These biomarkers can also be used to identify or select patients for treatment of dry eye disease, as further described herein.
The general terms used herein are defined with the following meanings, unless explicitly stated otherwise.
The term “comprising” and “including” are used herein in their open-ended and non-limiting sense unless otherwise noted. It is to be further understood that where descriptions of various embodiments use the term “comprising” or “including,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
The terms “a” and “an” and “the” and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Where the plural form is used for compounds, salts, and the like, this is taken to mean also a single compound, salt, or the like.
The term “pharmaceutically acceptable” is defined herein to refer to those compounds, biologic agents, materials, compositions and/or dosage forms, which are, within the scope of sound medical judgment, suitable for contact with the tissues a subject e.g., a mammal or human, without excessive toxicity, irritation allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.
The term “treating” or “treatment” as used herein comprises a treatment relieving, reducing or alleviating at least one symptom in a subject or affecting a delay of progression of a disease, condition and/or disorder. For example, treatment can be the diminishment of one or several signs or symptoms of a disorder or complete eradication of a disorder. Within the meaning of the present invention, the term “treat” also denotes to arrest, delay the onset (e.g., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.
The term “subject” as used herein includes animals, such as mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats and transgenic non-human animals. In some embodiments, the subject is a human, also referred to as patient. In some embodiments, a subject means a subject or patient which has dry eye disease.
The term “about” or “approximately” shall have the meaning of within 10% of a given value or range. In some embodiments, the term “about” refers to within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a given value.
The term “w/v” as used herein refers to “gram/mL” (weight over volume), which is a concentration unit. For example, 7% w/v is equivalent to 70 mg/mL.
“Aldehyde trapping agent” or “aldehyde conjugating agent” refers to an agent that is reactive with an aldehyde, for example malondialdehyde and 4-hydroxynonenal, to form an adduct or conjugate between the aldehyde trapping agent and the aldehyde.
Reproxalap, also referred to as ADX-102, is of formula
Without wishing to be bound by any particular theory, reproxalap functions as an aldehyde sequestering agent, or “trap,” which binds rapidly to aldehydes and forms a cyclic product.
In one aspect, the present disclosure provides use of biomarkers for determining the efficacy of a treatment for dry eye disease, particularly a treatment with an aldehyde trapping agent. As noted above, aldehyde trapping agents comprise compounds that react with an aldehyde to form an adduct or conjugate between the aldehyde and the aldehyde trapping agent. In some embodiments, the aldehyde trapping agent is a chemical compound with an amino group capable of reacting with an aldehyde. In some embodiments, the aldehyde trapping agent has the general structure:
wherein R1 and R2 form a cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or a fused bicyclic cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring; and each Ra is independently C1-4 aliphatic optionally substituted with 1, 2, or 3 deuterium or halogen atoms; or each Ra, taken together with the carbon atom to which they are attached, form a 3- to 8-membered cycloalkyl or heterocyclyl ring containing 1-2 heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, each Ra is a straight chain alkyl, for example methyl, ethyl, propyl, or butyl, preferably methyl.
Various aldehyde trapping agents are disclosed in U.S. Pat. Nos. 7,973,025; 9,604,997; 9,814,701; 10,111,862; 9,687,481; 10,414,732; 10,550,085; U.S. patent publication 2018/0250306; U.S. patent publication 2018/0050989; U.S. patent publication 2020/0246345; U.S. patent publication 2020/0121591; and International patent publication WO2018039197; all of which are incorporated herein by reference. In some embodiments, an exemplary aldehyde trapping agent for the methods herein is reproxalap.
In some embodiments, the present disclosure provides a method of assessing effectiveness of an aldehyde trapping agent in treating dry eye disease in a patient. In some embodiments, a method of assessing effectiveness of an aldehyde trapping agent in treating dry eye disease in a patient comprises:
(i) administering an aldehyde trapping agent to a patient with dry eye disease;
(ii) measuring the level of an aldehyde marker of oxidative stress present in the eye of the patient; and
(iii) comparing the measured level of the aldehyde marker of oxidative stress to level of aldehyde marker of oxidative stress in an appropriate control;
wherein a reduction in level of aldehyde marker of oxidative stress indicates effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, the initial administration of step (i) is a single administration of the aldehyde trapping agent. In some embodiments, the initial administration of step (i) is two, three, four, five, six, ten, or more doses of the aldehyde trapping agent; or is for a predetermined period of time, such as one, two, three, four, five, six, seven, or more days, or one week, two weeks, three weeks, one month, two months, or more. In some embodiments, the method further comprises the step of (iv) treating the dry eye disease by administering to the patient an effective amount of reproxalap if the patient exhibits in at least one of the patient's eyes a measured level of at least about 2 fold or greater of the aldehyde marker of oxidative stress relative to an appropriate control, such as a healthy human subject who does not have dry eye disease or any other ocular inflammation. In some embodiments, the treatment of dry eye disease is performed if a sufficient reduction in level of the aldehyde marker of oxidative stress is observed. Such sufficient reduction in level of the aldehyde marker of oxidative stress is selected from one of the parameters listed below. For example, in some embodiments an at least 15% or greater reduction in level of the aldehyde marker of oxidative stress compared to control level is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease.
If the level of the aldehyde marker is not reduced sufficiently, the dose of the aldehyde trapping agent and/or frequency of dosing may be increased. Alternatively, treatment is discontinued, and the patient is optionally treated with a different aldehyde trapping agent or a standard of care treatment for dry eye disease. In some embodiments, treatment is discontinued if a reduction of less than 10% in the level of the aldehyde marker of oxidative stress is obtained. In some embodiments, treatment is discontinued in a given patient or group of patients if the reduction in the level of the aldehyde marker of oxidative stress is below average; or more than one standard deviation worse (i.e., a smaller reduction) than the average.
In some embodiments, the treatment of dry eye disease is performed at an increased dose and/or dosing frequency of the aldehyde trapping agent relative to the initial administration of the aldehyde trapping agent to the patient with dry eye disease. In some embodiments, the treatment of dry eye disease comprises administering the aldehyde trapping agent an additional once, twice, thrice, or four times daily relative to the initial administration.
In some embodiments, as noted above, the aldehyde marker of oxidative stress in dry eye disease is formaldehyde, acetaldehyde, acrolein, glyoxal, methylglyoxal, hexadecanal, octadecanal, hexadecenal, succinic semi-aldehyde, malondialdehyde, 4-hydroxynonenal (4-HNE or HNE), 4-hydroxy-2E-hexenal, 4-hydroxy-2E,6Z-dodecadienal, retinaldehyde, leukotriene B4 aldehyde, or octadecenal. Particularly useful aldehyde markers of oxidative stress are those that form stable adducts, such as with proteins and other biomolecules. More preferably, the aldehyde markers of oxidative stress are those whose adducts can be detected in tears of a patient.
In some embodiments, the aldehyde marker of oxidative stress in dry eye disease is malondialdehyde or 4-hydroxynonenal. In some embodiments, the aldehyde marker of oxidative stress in dry eye disease is malondialdehyde.
In some embodiments, the measuring of the level of an aldehyde marker of oxidative stress is conducted on a sample of tears obtained from the patient, for example prior to and following treatment with an aldehyde trapping agent.
In some embodiments, an appropriate control for comparing the measured level of the aldehyde marker of oxidative stress in the eye of a patient is the level of the aldehyde marker of oxidative stress prior to administration of the aldehyde trapping agent or level of the aldehyde marker of oxidative stress in patients diagnosed with dry eye disease.
In some embodiments, the level of the aldehyde marker of oxidative stress measured is in the form of adducts of the aldehyde marker of oxidative stress present in the eye, particularly in the tear of the patient or a sample of tear obtained from the patient. In some embodiments, the adducts comprise stable adducts formed with biomolecules in the eye, such as nucleic acids and proteins, in particular adducts formed with proteins. In some embodiments, the adducts measured or detected comprise adducts formed with malondialdehyde. In some embodiments, the adducts measured or detected comprise adducts formed with 4-hydroxynonenal. These adducts can be detected and measured by various methods available in the art, as further discussed below.
In some embodiments of the method for assessing efficacy of treatment, at least 15% or greater reduction in level of the aldehyde marker of oxidative stress compared to control level is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, at least about 20% or greater reduction in level of the aldehyde marker of oxidative stress compared to control level is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, at least about 25% or greater reduction in level of the aldehyde marker of oxidative stress compared to control level is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease.
In some embodiments, an about 10-40% reduction in level of the aldehyde marker of oxidative stress compared to control level is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, an about 15-30% reduction in level of the aldehyde marker of oxidative stress compared to control level is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, an about 20-30% reduction in level of the aldehyde marker of oxidative stress compared to control level is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, an about 14-28%, or a 14-26%, 14-25%, 14-24%, 14-23%, 14-22%, 14-21%, 14-20%, 15-30%, 15-28%, 15-26%, 15-25%, 15-24%, 15-23%, 15-22%, 15-21%, 15-20%, 16-30%, 16-28%, 16-26%, 16-25%, 16-24%, 16-23%, 16-22%, 16-21%, 16-20%, 17-30%, 17-28%, 17-26%, 17-25%, 17-24%, 17-23%, 17-22%, 17-21%, 17-20%, 18-35%, 18-33%, 18-31%, 18-30%, 18-28%, 18-26%, 18-22%, 19-35%, 19-33%, 19-31%, 19-30%, 19-28%, 19-26%, 19-22%, 20-35%, 20-33%, 20-31%, 20-30%, 20-28%, 20-26%, 20-24%, 21-35%, 21-33%, 21-31%, 21-30%, 21-28%, 21-26%, 21-24%, 22-35%, 22-33%, 22-31%, 22-30%, 22-28%, 22-26%, 22-24%, 23-35%, 23-33%, 23-31%, 23-30%, 23-28%, 23-26%, 24-35%, 24-33%, 24-31%, 24-30%, 24-28%, 24-26%, 25-40%, 25-35%, 25-33%, 25-31%, 25-30%, 25-28%, or 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% reduction in level of the aldehyde marker of oxidative stress compared to control level is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease.
In some embodiments, the control level is a mean malondialdehyde adduct concentration of about 14,000 pmol/L to about 14,900 pmol/L as measured according to Example 2 in the tear of subjects with dry eye disease.
In some embodiments, a measured level of malondialdehyde adduct concentration of about 12,000 pmol/L or lower by an appropriate assay, such as that described in Example 2, is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, a measured level of malondialdehyde adduct concentration of about 11,500 pmol/L or lower is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, a measured level of malondialdehyde adduct concentration of about 11,000 pmoUL or lower is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, a measured level of malondialdehyde adduct concentration of about 10,500 pmol/L or lower is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, a measured level of malondialdehyde adduct concentration of about 10,000 pmol/L or lower is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, a measured level of malondialdehyde adduct concentration of about 9,500 pmol/L or lower is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, a measured level of malondialdehyde adduct concentration of about 12,000 pmol/L or lower as measured according to Example 2 in human tears is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease.
In some embodiments, a decrease in HNE levels in a patient's eye of at least about 500 picograms/milliliter (pg/mL) is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, a decrease in HNE levels of at least about 600, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1500 pg/mL is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, a decrease in HNE levels of about 500-1500 pg/mL is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, a decrease in HNE levels of about 600-1450, 650-1400, 700-1350, 750-1300, 800-1250, 850-1200, 900-1150, 950-1100, or 1000-1050 pg/mL is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, a decrease in HNE levels of about 1018 pg/mL is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease. HNE levels may be measured by an appropriate assay, such as a modified version of that described in Example 2. In some embodiments, HNE levels are measured in the patient's tears. In some embodiments, HNE levels are measured by quantifying the concentration of an adduct of HNE with a protein.
In some embodiments, a measured level of malondialdehyde adduct concentration of about 12,000 pmol/L or lower (or another measured level recited above) and a decrease in HNE levels of about 600-1450, 650-1400, 700-1350, 750-1300, 800-1250, 850-1200, 900-1150, 950-1100, or 1000-1050 pg/mL is indicative of effectiveness of the aldehyde trapping agent in treating dry eye disease.
In some embodiments, the method is used to assess the effectiveness of aldehyde trapping compound reproxalap. In some embodiments, reproxalap is administered topically, such as in an ophthalmic aqueous solution. In some embodiments, reproxalap is administered in any form and concentration and dosing regimen described below.
One aspect of the present invention relates to the surprising discovery that, upon administration of an aldehyde trap such as reproxalap, specific (quantified) decreases in levels of aldehyde markers of oxidative stress, such as MDA and/or HNE, correlate with statistically significant improvement in one or more symptoms of dry eye disease in patients. By using the quantified decreases in levels of aldehyde markers of oxidative stress as biomarkers, it is now possible to provide more effective treatments for dry eye disease, monitor the effectiveness of reproxalap and other dry eye disease treatments, and select patients for treatment of dry eye disease, as described herein.
In some embodiments of the method, reproxalap is administered topically at a concentration of 0.1% to 0.5% w/v. In some embodiments, reproxalap is administered topically to the eye at a concentration of 0.25% w/v. In some embodiments, reproxalap is administered topically to the eye at a concentration of 0.1% w/v. In some embodiments, reproxalap is administered topically to the eye at a concentration of 0.5% w/v.
As further described herein, reproxalap is administered as an admixture with a pharmaceutically acceptable excipient, wherein the excipient is a cyclodextrin. In some embodiments, the cyclodextrin is selected from sulfobutylether-β-cyclodextrin and hydroxypropyl-β-cyclodextrin, preferably sulfobutylether-β-cyclodextrin. In some embodiments of the method, the cyclodextrin is present at 5% to 20% w/v. In some embodiments, the cyclodextrin is present at 7% to 11% w/v. In some embodiments, the cyclodextrin is present at 7% w/v. In some embodiments, the cyclodextrin is present at 11% w/v.
In some embodiments, reproxalap is at a concentration of 0.25% w/v and the cyclodextrin is present at 7% w/v, preferably sulfobutylether-β-cyclodextrin. In some embodiments, the reproxalap and cyclodextrin are present in the formulation at a ratio of reproxalap to sulfobutylether-β-cyclodextrin of about a mole of reproxalap per 3 moles of sulfobutylether-β-cyclodextrin.
In some embodiments, reproxalap is at a concentration of 0.25% w/v and the cyclodextrin is present at 11% w/v, preferably sulfobutylether-β-cyclodextrin. In some embodiments, the reproxalap and cyclodextrin is present in the formulation at a ratio of reproxalap to sulfobutylether-β-cyclodextrin of about a mole of reproxalap per 5 moles of sulfobutylether-β-cyclodextrin.
In some embodiments, the level of aldehyde marker of oxidative stress is measured after one or more of 6, 10, 14, 18, 24, or 28 days of treatment with the aldehyde trapping agent. In some embodiments, the level of the aldehyde marker of oxidative stress is measured after 28 days of treatment.
In some embodiments, various dosing regimens can be used in determining the effectiveness of the aldehyde trapping agent in treating dry eye disease. In some embodiments, the treatment comprises topically administering the aldehyde trapping agent up to six times a day. In some embodiments, the treatment comprises topically administering the aldehyde trapping agent four times a day (QID).
In some embodiments of assessing the effectiveness of treatment with an aldehyde trapping agent, the treatment comprises an initiation phase and/or exacerbation phase, followed by a maintenance phase, as further described herein. In some embodiments, the treatment in the initiation phase and/or exacerbation phase comprises topically administering the aldehyde trapping agent to the eye four times a day (QID), and the treatment in the maintenance phase comprises topically administering the aldehyde trapping agent to the eye 4 times a day (QID) or two times a day (BID).
In some embodiments where the aldehyde trapping agent is reproxalap, any of the treatment and dosing regimens described herein can be used.
In some embodiments of the method, measuring the levels of the aldehyde marker of oxidative stress is during and/or following the initiation and/or exacerbation phase. In some embodiments of the method, measuring the levels of the aldehyde marker of oxidative stress is in the maintenance phase, e.g., during the maintenance phase.
In another aspect, the present disclosure provides a method of treating dry eye disease in a patient using the biomarkers to guide the treatment regime, such as dosages and/or dosing frequency with an aldehyde trapping agent. In some embodiments, a method of treating dry eye disease in a patient comprises:
In some embodiments, as noted above, the aldehyde markers of oxidative stress in ocular inflammation are diverse and can include formaldehyde, acetaldehyde, acrolein, crotonaldehyde, glyoxal, methylglyoxal, pentanal, hexanal, hydroxyhexanal, hydroxyhexenal, hexadecanal, octadecanal, hexadecenal, succinic semi-aldehyde, malondialdehyde, 4-hydroxynonenal (4-HNE or HNE), 4-hydroxy-2E-hexenal, 4-hydroxy-2E,6Z-dodecadienal, retinaldehyde, leukotriene B4 aldehyde, malondialdehyde-acetaldehyde adducts (MAA) or octadecenal, and other aldehydes. Particularly useful aldehyde markers of oxidative are those that form stable adducts, such as with proteins and other biomolecules. More preferably, the aldehyde markers of oxidative stress are those whose adducts can be detected in tears of a patient.
In some embodiments, as noted above, the aldehyde marker of oxidative stress in dry eye disease is formaldehyde, acetaldehyde, acrolein, glyoxal, methylglyoxal, hexadecanal, octadecanal, hexadecenal, succinic semi-aldehyde, malondialdehyde, 4-hydroxynonenal (4-HNE or HNE), 4-hydroxy-2E-hexenal, 4-hydroxy-2E,6Z-dodecadienal, retinaldehyde, leukotriene B4 aldehyde, or octadecenal. Particularly useful aldehyde markers of oxidative stress are those that form stable adducts, such as with proteins and other biomolecules. More preferably, the aldehyde markers of oxidative stress are those whose adducts can be detected in tears of a patient.
In some embodiments, the aldehyde marker of oxidative stress in dry eye disease is malondialdehyde or 4-hydroxynonenal. In some embodiments, the aldehyde marker of oxidative stress in dry eye disease is malondialdehyde.
In some embodiments, the measuring of the level of an aldehyde marker of oxidative stress is conducted on a sample of tears obtained from the patient, for example prior to and following treatment with an aldehyde trapping agent.
In some embodiments, the level of the aldehyde marker of oxidative stress measured is in the form of adducts of the aldehyde marker of oxidative stress present in the eye, particularly in the tear of the patient or a sample of tear obtained from the patient. In some embodiments, the adducts comprise stable adducts formed with biomolecules in the eye, such as nucleic acids and proteins. In some embodiments, the adducts measured or detected comprise adducts formed with malondialdehyde. In some embodiments, the adducts measured or detected comprise adducts formed with 4-hydroxynonenal. These adducts can be detected and measured by various methods available in the art, as further discussed below.
In some embodiments of the method of treatment, reproxalap is administered topically at a concentration of 0.1% to 0.5% w/v. In some embodiments, reproxalap is administered topically to the eye at a concentration of 0.25% w/v. In some embodiments, reproxalap is administered topically to the eye at a concentration of 0.1% w/v. In some embodiments, reproxalap is administered topically to the eye at a concentration of 0.5% w/v.
As further described herein, reproxalap is administered as an admixture with a pharmaceutically acceptable excipient, wherein the excipient is a cyclodextrin. In some embodiments, the cyclodextrin is selected from sulfobutylether-β-cyclodextrin and hydroxypropyl-β-cyclodextrin, preferably sulfobutylether-β-cyclodextrin. In some embodiments of the method, the cyclodextrin is present at 5% to 20% w/v. In some embodiments, the cyclodextrin is present at 7% to 11% w/v. In some embodiments, the cyclodextrin is present at 7% w/v. In some embodiments, the cyclodextrin is present at 11% w/v.
In some embodiments, the reproxalap is at a concentration of 0.25% w/v and the cyclodextrin is present at 7% w/v, preferably sulfobutylether-β-cyclodextrin. In some embodiments, the reproxalap and cyclodextrin is present in the formulation at a ratio of reproxalap to sulfobutylether-β-cyclodextrin of about a mole of reproxalap per 3 moles of sulfobutylether-β-cyclodextrin.
In some embodiments, reproxalap is at a concentration of 0.25% w/v and the cyclodextrin is present at 11% w/v, preferably sulfobutylether-β-cyclodextrin. In some embodiments, the reproxalap and cyclodextrin is present in the formulation at a ratio of reproxalap to sulfobutylether-β-cyclodextrin of about a mole of reproxalap per 5 moles of sulfobutylether-β-cyclodextrin.
In some embodiments of the method of treatment, any of the dosages and dosing or treatment regimens described herein and below can be used for administering the reproxalap. In some embodiments, reproxalap is administered up to six times a day. In some embodiments, reproxalap is administered four times a day (QID).
In some embodiments of the method of treatment, the patient is treated with a lower dosing frequency of reproxalap for a reduction of about 25% or greater in the measured level of the aldehyde marker of oxidative stress.
In some embodiments of the method, the treatment for dry eye disease comprises an initiation and/or exacerbation phase, and a maintenance phase. In some embodiments, the measuring of the aldehyde markers of oxidative stress following treatment is done during the initiation phase. In some embodiments, the measuring is done during the exacerbation phase. In some embodiments, the measuring is done both during the initiation and the exacerbation phase. In some of these embodiments, reproxalap is administered four times a day in the initiation and/or exacerbation phase.
In some embodiments, the patient is treated with a lower dosing frequency of reproxalap in the maintenance phase for a reduction of greater than about 20% in the measured level of the aldehyde marker of oxidative stress in the initiation and/or exacerbation phase.
In some embodiments, the patient is treated with a lower dosing frequency of reproxalap in the maintenance phase for a reduction of about 25% or greater in the measured level of the aldehyde marker of oxidative stress in the initiation and/or exacerbation phase.
In some embodiments, the lower dosing frequency is two times a day (BID).
In another aspect, the present disclosure provides a method of selecting or identifying a patient for treatment of dry eye disease, particularly for treatments with aldehyde trapping agents. In some embodiments, a method of selecting a patient for treatment of dry eye disease comprises: measuring the level of an aldehyde marker of oxidative stress in an eye of a patient suspected of having dry eye disease, wherein a measured level of at least about 2 fold or greater level of the aldehyde marker of oxidative stress as compared to level of aldehyde marker of oxidative stress in patients without dry eye disease is indicated for treatment.
In some embodiments, the aldehyde marker of oxidative stress in dry eye disease is formaldehyde, acetaldehyde, acrolein, glyoxal, methylglyoxal, hexadecanal, octadecanal, hexadecenal, succinic semi-aldehyde, malondialdehyde, 4-hydroxynonenal (4-HNE or HNE), 4-hydroxy-2E-hexenal, 4-hydroxy-2E,6Z-dodecadienal, retinaldehyde, leukotriene B4 aldehyde, or octadecenal.
In some embodiments, the aldehyde marker of oxidative stress in dry eye disease is malondialdehyde or 4-hydroxynonenal. In some embodiments, the aldehyde marker of oxidative stress in dry eye disease is malondialdehyde.
In some embodiments, the measuring of the level of an aldehyde marker of oxidative stress is conducted on a sample of tears obtained from the patient, for example prior to and following treatment with an aldehyde trapping agent.
In some embodiments, the level of the aldehyde marker of oxidative stress measured is in the form of adducts of the aldehyde marker of oxidative stress present in the eye, particularly in the tear of the patient or a sample of tear obtained from the patient. In some embodiments, the adducts comprise stable adducts formed with biomolecules in the eye, such as nucleic acids and proteins. In some embodiments, the adducts measured or detected comprise adducts formed with malondialdehyde. In some embodiments, the adducts measured or detected comprise adducts formed with 4-hydroxynonenal. These adducts can be detected and measured by various methods available in the art, as further discussed below.
In some embodiments, the method is used to identify or select patients with dry eye disease for treatment with an aldehyde trapping agent. In some embodiments, the aldehyde trapping agent is reproxalap. In some embodiments, any of the ophthalmic formulations, dosages and dosing regimens, as well as various treatment regimens described herein is considered for treatment of the patient.
In some embodiments, the patient is selected or identified for treatment with reproxalap, wherein the reproxalap is administered topically at a concentration of 0.1% to 0.5% w/v. In some embodiments, the patient is selected or identified for treatment with reproxalap, wherein reproxalap is administered topically to the eye at a concentration of 0.25% w/v. In some embodiments, the patient is selected or identified for treatment with reproxalap, wherein reproxalap is administered topically to the eye at a concentration of 0.1% w/v. In some embodiments, the patient is selected or identified for treatment with reproxalap, wherein reproxalap is administered topically to the eye at a concentration of 0.5% w/v.
In some embodiments, the reproxalap is administered as an admixture with a pharmaceutically acceptable excipient, wherein the excipient is a cyclodextrin. In some embodiments, the cyclodextrin is selected from sulfobutylether-β-cyclodextrin and hydroxypropyl-β-cyclodextrin, preferably sulfobutylether-β-cyclodextrin. In some embodiments of the method, the cyclodextrin is present at 5% to 20% w/v. In some embodiments the cyclodextrin is present at 7% to 11% w/v.
In some embodiments, reproxalap is at a concentration of 0.25% w/v and the cyclodextrin is present at 7% w/v, preferably sulfobutylether-β-cyclodextrin. In some embodiments, the reproxalap and cyclodextrin is present in the formulation at a ratio of reproxalap to sulfobutylether-β-cyclodextrin of about a mole of reproxalap per 3 moles of sulfobutylether-β-cyclodextrin.
In some embodiments, reproxalap is at a concentration of 0.25% w/v and the cyclodextrin is present at 11% w/v, preferably sulfobutylether-β-cyclodextrin. In some embodiments, the reproxalap and cyclodextrin is present in the formulation at a ratio of reproxalap to sulfobutylether-β-cyclodextrin of about a mole of reproxalap per 5 moles of sulfobutylether-β-cyclodextrin.
In some embodiments for selecting or identifying a patient for treatment, the aldehyde marker of oxidative stress is in the form of malondialdehyde adduct, wherein a measured level malondialdehyde adduct of at least about 3.4 fold or greater as compared to level of malondialdehyde adduct in patients without dry eye disease is indicated for treatment of the patient.
In some embodiments herein, the level of aldehyde marker of oxidative stress and/or level of the adducts formed by the aldehyde markers of oxidative stress can be measured by any number of techniques. These include, by way of example and not limitation, mass spectroscopy (MS), chromatography (e.g., HPLC), LC/MS, antibody reagents (e.g., enzyme linked immunosorbent assay—ELISA). In some embodiments, the aldehyde markers of oxidative stress are detected by LC-MS, ultraviolet spectrometry (UV), HPLC, mass spectrometry (MS), monoclonal antibody detection assay, gas chromatography (GC), GC/MS, GC/flame ionization detector (FID), capillary electrophoresis with amperometric detection (CE-AD), liquid chromatography/fluorescence detection, or a combination thereof. These and other techniques are described in, for example, Houglum et al., J Clin Invest., 1990; 86(6): 1991-1998; Ishi et al., Chem. Res. Toxicol. 2006, 19(1):122-129; and Soares et al., J Liquid Chrom. & Related Technol., 2004; 27(15):2357-2369. In some embodiments, the assay is substantially as described in Example 2, Example 4, or Example 5, below. In some embodiments, the level of aldehyde marker of oxidative stress (e.g., MDA and/or HNE) and/or level of the adducts formed by the aldehyde marker of oxidative stress is/are used as an endpoint for a treatment regimen for ocular inflammation, such as dry eye disease.
In various embodiments referring to reproxalap and its use in the methods described above, the reproxalap, or a pharmaceutically acceptable salt thereof, is formulated as an ophthalmic solution at a concentration suitable for treating dry eye disease, in particular without causing severe or intolerable adverse effects. In some embodiments, any of the ophthalmic solutions described herein can be used in the methods. In some embodiments, the ophthalmic solution comprises about 0.1% to 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the excipient comprises a cyclodextrin, such as sulfobutylether β-cyclodextrin (SBECD) or hydroxypropyl β-cyclodextrin.
In some embodiments, an ophthalmic solution comprises reproxalap and a cyclodextrin excipient in a ratio of less than 1:2.1 on a mole:mole basis. In some embodiments, the ratio of reproxalap and cyclodextrin is about 1:2.1 to about 1:25 ratio on a mole:mole basis. In some embodiments, the ratio is about 1:2.2 to 1:20, 1:2.5 to 1:20, 1:2.5 to 1:10, 1:2.75 to 1:10, 1:3 to 1:8, 1:3.5 to 1:7, 1:4 to 1:6, or 1:4 to 1:5 in a mole:mole basis. In some embodiments, the ratio is about 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4.0, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5.0, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, or 1:25 on a mole:mole basis.
In some embodiments, the cyclodextrin excipient is one of those described herein, such as sulfobutylether β-cyclodextrin (SBECD). The average degree of substitution of the SBECD is about 6.5.
In some embodiments, the ratio of reproxalap to the excipient is about 1:2.1 or less on a mole:mole basis.
In some embodiments, the excipient is a cyclodextrin and the ratio of reproxalap to the excipient is about 1:2.1 to about 1:25 on a mole:mole basis.
In some embodiments, the excipient is a cyclodextrin and the ratio of reproxalap to the excipient is about 1:2 to about 1:5 on a mole:mole basis.
In some embodiments, the present invention provides an ophthalmic solution comprising reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the concentration of reproxalap, or a pharmaceutically acceptable salt thereof, is about 0.5% w/v or less and about 0.1% w/v or greater. In some embodiments, the ophthalmic solution comprises about 0.15 to about 0.45% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the ophthalmic solution comprises about 0.2 to about 0.4% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the ophthalmic solution comprises about 0.21 to about 0.35% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the ophthalmic solution comprises about 0.22 to about 0.3% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the ophthalmic solution comprises about 0.22 to about 0.29% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the ophthalmic solution comprises about 0.25% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the ophthalmic solution comprises about 0.25% w/v reproxalap and a pharmaceutically acceptable excipient selected from a cyclodextrin. In some embodiments, the ophthalmic solution comprises about 0.5% w/v reproxalap and a pharmaceutically acceptable excipient selected from a cyclodextrin.
In some embodiments, the present invention provides an ophthalmic solution comprising less than 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the present invention provides an ophthalmic solution comprising at least 0.1% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In some embodiments, the present invention provides an ophthalmic solution comprising reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the concentration of reproxalap, or a pharmaceutically acceptable salt thereof, is less than 0.5% w/v and 0.1% w/v or greater.
In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of less than 0.45% w/v and at least 0.1% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of less than 0.4% w/v and at least 0.1% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of less than 0.35% w/v and at least 0.1% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of less than 0.3% w/v and at least 0.1% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of less than 0.25% w/v and more than 0.1% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of less than 0.2% w/v and at least 0.1% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of less than 0.15% w/v and at least 0.1% w/v.
In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of 0.5% w/v or less and at least 0.15% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of 0.5% w/v or less and at least 0.2% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of 0.5% w/v or less and at least 0.25% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of 0.5% w/v or less and at least 0.3% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of 0.5% w/v or less and at least 0.35% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of 0.5% w/v or less and at least 0.4% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of 0.5% w/v or less and at least 0.45% w/v.
In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of about 0.1% to 0.5%, 0.15% to 0.45% w/v, 0.15% to 0.4% w/v, 0.15% to 0.35% w/v, 0.15% to 0.3% w/v, 0.15% to 0.25% w/v, or 0.15% to 0.2% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of 0.2% to 0.45% w/v, 0.2% to 0.4% w/v, 0.2% to 0.35% w/v, 0.2% to 0.3% w/v, or 0.2% to 0.25% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of 0.25% to 0.45% w/v, 0.25% to 0.4% w/v, 0.25% to 0.35% w/v, or 0.25% to 0.3% w/v. In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of 0.3% to 0.45% w/v or 0.3% to 0.4% w/v.
In some embodiments, reproxalap, or a pharmaceutically acceptable salt thereof, in an ophthalmic solution of the invention is at a concentration of about 0.1% w/v, 0.15% w/v, about 0.2% w/v, about 0.25%, about 0.3% w/v, about 0.35% w/v, about 0.4% w/v, about 0.45% w/v, or about 0.5% w/v.
In some embodiments, as further described herein, the foregoing concentrations of reproxalap can be selected and applied to treatment regimen that includes an initiation phase, an exacerbation phase, and/or a maintenance phase.
In some embodiments, a pharmaceutically acceptable excipient in an ophthalmic solution of the invention is a cyclodextrin. In some embodiments, the cyclodextrin is α-, β- or γ-cyclodextrin. In some embodiments, a cyclodextrin is a pharmaceutically acceptable derivative of a cyclodextrin, including, but not limited to, the hydroxyalkyl derivatives of α-, β- and γ-cyclodextrin (especially the hydroxyethyl and hydroxypropyl derivatives of β-cyclodextrin and γ-cyclodextrin), randomly methylated β-cyclodextrin, sulfobutylether β-cyclodextrin, sulfobutylether γ-cyclodextrin, and the so-called branched β- and γ-cyclodextrin derivatives such as glucosyl-β-cyclodextrin and glucosyl-γ-cyclodextrin. The natural cyclodextrins are either used alone or in a mixture of two or more cyclodextrins, by way of non-limiting example, a mixture of the γ-cyclodextrin and the more water-soluble hydroxypropyl γ-cyclodextrin, or γ-cyclodextrin and sulfobutylether γ-cyclodextrin, or β-cyclodextrin and hydroxypropyl-β-cyclodextrin, or β-cyclodextrin and sulfobutylether β-cyclodextrin.
In some embodiments, a cyclodextrin in an ophthalmic solution of the invention is at a concentration of 0 to 20% w/v. In some embodiments, a cyclodextrin in an ophthalmic solution of the invention is at a concentration of 1 to 18% w/v, 1 to 16% w/v, 1 to 14% w/v, 2 to 12% w/v, 4 to 10% w/v, 5 to 9% w/v, or 6 to 8% w/v. In some embodiments, the cyclodextrin in an ophthalmic solution of the invention is at a concentration of 7% to 11% w/v. In some embodiments, a cyclodextrin in an ophthalmic solution of the invention is at a concentration of about 1% w/v, 2% w/v, 3% w/v, 4% w/v, 5% w/v, 6% w/v, 7% w/v, 8% w/v, 9% w/v, 10% w/v, 11% w/v, 12% w/v, 13% w/v, 14% w/v, 15% w/v, 16% w/v, 17% w/v, 18% w/v, 19% w/v, or 20% w/v.
In some embodiments, a pharmaceutically acceptable excipient in an ophthalmic solution of the invention is sulfobutylether-β-cyclodextrin, in particular at any of the specified concentrations and ranges of concentrations above, such as about 7% w/v. In some embodiments, a pharmaceutically acceptable excipient in an ophthalmic solution of the invention is hydroxypropyl-β-cyclodextrin, in particular at any of the specified concentrations and ranges of concentrations specified above, such as about 7% w/v.
In some embodiments, the ophthalmic solution comprises about 0.2% to 0.4% w/v reproxalap and about 7% to 25% w/v of a cyclodextrin excipient such as SBECD. In some embodiments, the ophthalmic solution comprises about 0.2%, 0.25%, 0.3%, 0.35%, or 0.4% w/v reproxalap and about 7% to 25% w/v of a cyclodextrin excipient such as SBECD.
In some embodiments, the ophthalmic solution comprises about 0.25% w/v reproxalap and about 4.7% to about 25% w/v of a cyclodextrin excipient such as SBECD.
In some embodiments, the ophthalmic solution comprises about 0.25% w/v reproxalap and about 7% to 25% w/v of a cyclodextrin excipient such as SBECD.
In some embodiments, the ophthalmic solution comprises about 0.25% w/v reproxalap and about 4.75% to about 11% w/v of a cyclodextrin excipient such as SBECD.
In some embodiments, the ophthalmic solution comprises about 0.5% w/v reproxalap and about 9.5% to about 11% w/v of a cyclodextrin excipient such as SBECD. In some embodiments, the ratio of reproxalap to SBECD is about a mole of reproxalap per 2 moles of SBECD.
In some embodiments, the ophthalmic solution comprises about 0.25% w/v reproxalap and about 7% w/v of a cyclodextrin excipient such as SBECD. In some embodiments, the ratio of reproxalap to SBECD is about a mole of reproxalap per 3 moles SBECD.
In some embodiments, the ophthalmic solution comprises about 0.25% w/v reproxalap and about 11% w/v of a cyclodextrin excipient such as SBECD. In some embodiments, the ratio of reproxalap to SBECD is about a mole of reproxalap per 5 moles SBECD.
In some embodiments, an ophthalmic solution of the invention comprises a pharmaceutically acceptable buffering agent. In some embodiments, a pharmaceutically acceptable buffering agent is a phosphate buffer, citrate buffer, tris buffer, histidine buffer or acetate buffer.
In some embodiments, a pharmaceutically acceptable buffering agent is sodium phosphate, dibasic. In some embodiments, a pharmaceutically acceptable buffering agent is sodium phosphate, monobasic. In some embodiments, a pharmaceutically acceptable buffering agent is a mixture of sodium phosphate, dibasic, and sodium phosphate, monobasic. In some embodiments, an ophthalmic solution of the invention comprises about 0.083% w/v sodium phosphate, dibasic, and about 0.017% w/v sodium phosphate, monobasic.
In some embodiments, the ophthalmic solution of the invention is at an approximately neutral pH. In some embodiments, an ophthalmic solution of the invention is at a pH of 6.5 to 8. In some embodiments, an ophthalmic solution of the invention is at a pH of 6.9 to 7.7. In some embodiments, an ophthalmic solution of the invention is at a pH of 7.1 to 7.5. In some embodiments, an ophthalmic solution of the invention is at a pH of about 7.3. In some embodiments, an ophthalmic solution of the invention is at a pH of 7.3±0.01.
Pharmaceutically acceptable acids and/or bases may be used in the ophthalmic solution to adjust pH. In some embodiments, an ophthalmic solution of the invention comprises a pharmaceutically acceptable acid. In some embodiments, an ophthalmic solution of the invention comprises a pharmaceutically acceptable base. In some embodiments, an ophthalmic solution of the invention comprises a pharmaceutically acceptable acid and base. In some embodiments, a pharmaceutically acceptable acid is hydrochloric acid. In some embodiments, pharmaceutically acceptable base is sodium hydroxide.
In some embodiments, an ophthalmic solution of the invention comprises a tonicity agent. In some embodiments, a tonicity agent is selected from the group consisting of dextrose, potassium chloride, propylene glycol, and sodium chloride. In some embodiments, an ophthalmic solution of the invention comprises a tonicity agent at a concentration of less than about 0.5% w/v. In some embodiments, an ophthalmic solution of the invention comprises a tonicity agent at a concentration of about 0.45%, 0.4%, 0.35%, 0.3%, 0.25%, 0.2%, 0.15%, or 0.1% w/v. In some embodiments, a tonicity agent is sodium chloride.
In some embodiments, the ophthalmic solution comprises reproxalap at the specified concentrations, cyclodextrin, phosphate, and sodium chloride. In some embodiments. In some embodiments, the ophthalmic solution comprises reproxalap at the specified concentrations herein (e.g., 0.1% w/v, 0.25% w/v, 0.5% w/v, etc.), 5 to 9% w/v cyclodextrin (e.g., sulfobutylether-β-cyclodextrin or hydroxypropyl-β-cyclodextrin); 0.07% to 0.09% w/v sodium phosphate (dibasic), 0.015% to 0.19% w/v sodium phosphate (monobasic), and 0.2 to 0.3% w/v sodium chloride. In some embodiments, the ophthalmic solution comprises reproxalap at the specified concentrations herein (e.g., 0.1% w/v, 0.25% w/v, 0.5% w/v, etc.), about 7% w/v cyclodextrin (e.g., sulfobutylether-β-cyclodextrin or hydroxypropyl-β-cyclodextrin); 0.07% to 0.09% w/v sodium phosphate (dibasic), 0.015% to 0.019% w/v sodium phosphate (monobasic), and 0.2 to 0.3% w/v sodium chloride. In some embodiments, the ophthalmic solution is adjusted to an appropriate pH with sodium hydroxide or HCl.
In some embodiments, the ophthalmic solution comprises the following (0.5% Reproxalap Ophthalmic Solution A):
In some embodiments, the ophthalmic solution comprises the following (0.5% Reproxalap Ophthalmic Solution B)
In some embodiments, the ophthalmic solution comprises the following (0.25% Reproxalap Ophthalmic Solution A)
In some embodiments, the ophthalmic solution comprises the following (0.25% Reproxalap Ophthalmic Solution B)
In some embodiments, the ophthalmic solution comprises the following (0.1% Reproxalap Ophthalmic Solution A)
It is to be understood that variations of the ophthalmic solutions within the scope of the disclosure may be prepared given the guidance provided herein.
In one aspect, the present invention provides a method for treating dry eye disease in a subject, comprising topically administering to an eye of a subject in need thereof a therapeutically effective amount of an ophthalmic solution of the invention. In some embodiments, the concentration of reproxalap in the ophthalmic solution used in the method is as described above.
In some embodiments, an ophthalmic solution of the invention can be administered at different frequencies suitable for effectively treating dry eye disease, for example, without causing severe or intolerable adverse effects.
In some embodiments, an ophthalmic solution of the invention can be topically administered one to six times a day. In some embodiments, a method of the invention comprises topically administering an ophthalmic solution of the invention six times a day. In some embodiments, a method of the invention comprises topically administering an ophthalmic solution of the invention five times a day. In some embodiments, a method of the invention comprises topically administering an ophthalmic solution of the invention four times a day (QID). In some embodiments, a method of the invention comprises topically administering an ophthalmic solution of the invention three times a day (TID). In some embodiments, a method of the invention comprises topically administering an ophthalmic solution of the invention two times a day (BID). In some embodiments, a method of the invention comprises topically administering an ophthalmic solution of the invention once a day (QD). In some embodiments, a method of the invention comprises topically administering an ophthalmic solution of the invention as needed (PRN).
In some embodiments, a method of the invention comprises topically administering to an eye of a subject with dry eye disease a therapeutically effective amount of an ophthalmic solution of the invention six times a day, five times a day, four times a day (QID), three times a day (TID), two times a day (BID), once a day (QD), followed by administration as needed (PRN).
In some embodiments, a method of the invention comprises topically administering an ophthalmic solution of the invention at various strengths (for example, at different reproxalap concentrations and different administration frequencies, as described herein).
In some embodiments, a method of the invention comprises topically administering an ophthalmic solution comprising about 0.25% w/v reproxalap, or a pharmaceutically acceptable salt thereof, four times a day, three times a day, or two times a day.
In some embodiments, a method of the invention comprises topically administering an ophthalmic solution comprising about 0.30% w/v reproxalap, or a pharmaceutically acceptable salt thereof, four times a day, three times a day, or two times a day.
In some embodiments, a method of the invention comprises topically administering an ophthalmic solution comprising about 0.35% w/v reproxalap, or a pharmaceutically acceptable salt thereof, four times a day, three times a day, or two times a day.
In some embodiments, a method of the invention comprises topically administering an ophthalmic solution comprising about 0.4% w/v reproxalap, or a pharmaceutically acceptable salt thereof, four times a day, three times a day, or two times a day.
In some embodiments, a method of the invention comprises topically administering an ophthalmic solution comprising about 0.45% w/v reproxalap, or a pharmaceutically acceptable salt thereof, four times a day, three times a day, or two times a day.
In some embodiments, a method of the invention comprises topically administering an ophthalmic solution comprising about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, four times a day, three times a day, or two times a day.
In some embodiments, a method of the invention comprises topically administering an ophthalmic solution comprising 0.3% to 0.4% w/v reproxalap, or a pharmaceutically acceptable salt thereof, four times a day, three times a day, or two times a day.
In some embodiments, a method of the invention comprises topically administering an ophthalmic solution comprising 0.2% to 0.3% w/v reproxalap, or a pharmaceutically acceptable salt thereof, four times a day, three times a day, or two times a day.
In some embodiments, a method of the invention comprises topically administering an ophthalmic solution comprising 0.2% to 0.4% w/v reproxalap, or a pharmaceutically acceptable salt thereof, four times a day, three times a day, or two times a day.
In some embodiments, a method of the invention comprises two or more phases, wherein an ophthalmic solution of the invention is topically administering at different strengths in different phases. In some embodiments, a method of the invention comprises an initiation phase and a maintenance phase, wherein the ophthalmic solution is topically administered at a higher strength in the initiation phase than in the maintenance phase. In some embodiments, a treatment cycle of a method of the invention comprising multiple phases, including an exacerbation phase during which signs and/or symptoms become worse.
In some embodiments, the method of the invention comprises two or more phases, wherein an ophthalmic solution of the invention is topically administering at different strengths in different phases. In some embodiments, a method of the invention comprises an initiation phase, wherein the ophthalmic solution is topically administered at a high strength in the initiation phase, at a low strength in the maintenance phase, and at a high strength during an exacerbation of disease signs and/or symptoms.
In some embodiments, an ophthalmic solution administered in an initiation phase comprises a higher concentration of reproxalap, or a pharmaceutically acceptable salt thereof, than an ophthalmic solution administered in a maintenance phase. In some embodiments, the ophthalmic solution administered in an initiation phase or an exacerbation phase and the ophthalmic solution administered in a maintenance phase, comprises reproxalap, or a pharmaceutically acceptable salt, at a concentration selected from the group consisting of about 0.5% w/v, 0.45% w/v, 0.4% w/v, 0.35% w/v, 0.3% w/v, 0.25% w/v, 0.2% w/v, 0.15% w/v, and 0.1% w/v.
In some embodiments, an ophthalmic solution of about 0.5% w/v reproxalap is administered in an initiation phase or exacerbation phase, and less than 0.5% w/v reproxalap administered in a maintenance phase. In some embodiments, an ophthalmic solution of about 0.4% w/v, 0.35% w/v, 0.3% w/v, 0.25% w/v, 0.2% w/v, 0.15% w/v or 0.1% w/v reproxalap is administered in the maintenance phase.
In some embodiments, an ophthalmic solution of about 0.5% w/v to about 0.4% reproxalap is administered in an initiation phase or exacerbation phase, and less than 0.4% w/v reproxalap administered in a maintenance phase. In some embodiments, an ophthalmic solution of about 0.35% w/v, 0.3% w/v, 0.25% w/v, 0.2% w/v, 0.15% w/v or 0.1% w/v reproxalap is administered in the maintenance phase.
In some embodiments, an ophthalmic solution of about 0.5% w/v to about 0.3% reproxalap is administered in an initiation phase or exacerbation phase, and less than 0.3% w/v reproxalap administered in a maintenance phase. In some embodiments, an ophthalmic solution of about 0.25% w/v, 0.2% w/v, 0.15% w/v or 0.1% w/v reproxalap is administered in the maintenance phase.
In some embodiments, an ophthalmic solution of about 0.4% w/v to about 0.3% reproxalap is administered in an initiation phase or exacerbation phase, and less than 0.3% w/v reproxalap administered in a maintenance phase. In some embodiments, an ophthalmic solution of about 0.25% w/v, 0.2% w/v, 0.15% w/v or 0.1% w/v reproxalap is administered in the maintenance phase.
In some embodiments, an ophthalmic solution of about 0.3% w/v to about 0.2% reproxalap (e.g., 0.3%, 0.25%, or 0.2% w/v) is administered in an initiation phase or exacerbation phase, and 0.25% w/v or less reproxalap administered in a maintenance phase. In some embodiments, an ophthalmic solution of about 0.25% w/v, 0.2% w/v, 0.15% w/v or 0.1% w/v reproxalap is administered in the maintenance phase.
In some embodiments, an ophthalmic solution of the invention is topically administered more frequently per day in an initiation phase and an exacerbation phase than in a maintenance phase. In some embodiments, an ophthalmic solution of the invention is topically administered five times a day in an initiation phase, followed by four, three, two, or one times a day in a maintenance phase. In some embodiments, an ophthalmic solution of the invention is topically administering four times a day in an initiation phase or exacerbation phase, followed by three, two, or one times a day in a maintenance phase. In some embodiments, an ophthalmic solution of the invention is topically administering three times a day in an initiation phase or exacerbation phase, followed by two or one times a day in a maintenance phase. In some embodiments, an ophthalmic solution of the invention is topically administering two times a day in an initiation phase or exacerbation phase, followed by once daily in a maintenance phase.
In some embodiments, an ophthalmic solution administered in an initiation phase or exacerbation phase is at a higher reproxalap concentration and higher administration frequency than an ophthalmic solution administered in a maintenance phase.
In some embodiments, the present invention provides a method for treating dry eye disease in a subject, comprising topically administering to the subject an ophthalmic solution comprising about 0.4% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the ophthalmic solution is administered at a higher strength in an initiation phase or exacerbation phase followed by a lower strength in a maintenance phase, wherein each of the initiation phase, exacerbation phase, and maintenance phase is as described herein.
In some embodiments, a multiple phase treatment cycle can include an initiation phase or exacerbation phase of up to 12 weeks with an ophthalmic solution comprising about 0.5%, 0.4% or 0.35% w/v (e.g., 0.5% to 0.35% w/v) reproxalap, or a pharmaceutically acceptable salt thereof, is up to 12 weeks, followed by a maintenance phase. In some embodiments, an ophthalmic solution comprising about 0.5%, 0.4% or 0.35% w/v (e.g., 0.5% to 0.35% w/v) reproxalap, or a pharmaceutically acceptable salt thereof, is administered four times a day in an initiation phase or exacerbation phase followed by three, two, or one times a day in the maintenance phase. In some embodiments, an ophthalmic solution comprising about 0.5%, 0.4% or 0.35% w/v (e.g., 0.5% to 0.35% w/v) reproxalap, or a pharmaceutically acceptable salt thereof, is administered three times a day in an initiation phase or exacerbation phase followed by two or one times a day in the maintenance phase.
In some embodiments, an ophthalmic solution comprising about 0.4%, 0.35% or 0.3% w/v (e.g., 0.4% to 0.3% w/v) reproxalap, or a pharmaceutically acceptable salt thereof, is administered four times a day in an initiation phase or exacerbation phase followed by three, two, or one times a day in the maintenance phase. In some embodiments, an ophthalmic solution comprising about 0.4%, 0.35% or 0.3% w/v (e.g., 0.4% to 0.3% w/v) reproxalap, or a pharmaceutically acceptable salt thereof, is administered three times a day in an initiation phase or exacerbation phase followed by two or one times a day in the maintenance phase.
In some embodiments, an ophthalmic solution comprising about 0.3%, 0.25% or 0.2% w/v (e.g., 0.3% to 0.2% w/v) reproxalap, or a pharmaceutically acceptable salt thereof, is administered four times a day in an initiation phase or exacerbation phase followed by three, two, or one times a day in the maintenance phase. In some embodiments, an ophthalmic solution comprising about 0.3%, 0.25% or 0.2% w/v (e.g., 0.3% to 0.2% w/v) reproxalap, or a pharmaceutically acceptable salt thereof, is administered three times a day in an initiation phase or exacerbation phase followed by two or one times a day in the maintenance phase.
In some embodiments, the present invention provides a method for treating dry eye disease in a subject, comprising topically administering to the subject an ophthalmic solution comprising 0.35% to 0.45% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the ophthalmic solution is administered at a higher strength in an initiation phase or exacerbation phase followed by a lower strength in a maintenance phase, wherein each of the initiation phase, exacerbation phase and maintenance phase is as described herein. In some embodiments, a multiple phase treatment cycle of an ophthalmic solution comprising 0.35% to 0.45% w/v reproxalap, or a pharmaceutically acceptable salt thereof, is up to 12 weeks. In some embodiments, an ophthalmic solution comprising 0.35% to 0.45% w/v reproxalap, or a pharmaceutically acceptable salt thereof, is administered four times a day in an initiation phase or exacerbation phase followed by three, two, or one times a day in maintenance phase. In some embodiments, an ophthalmic solution comprising 0.35% to 0.45% w/v reproxalap, or a pharmaceutically acceptable salt thereof, is administered three times a day in an initiation phase or exacerbation phase followed by two or one times a day in maintenance phase.
In some embodiments, an ophthalmic solution is administered QID for about 10 to 14 weeks, preferably about 12 weeks. In some embodiments, an ophthalmic solution is administration QID for about 2 to 6 weeks, preferably about 4 weeks followed by administration BID for about 6 to 10 weeks, preferably about 8 weeks. In some embodiments, the ophthalmic solution for the foregoing treatment regimen is 0.25% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and about 7% w/v SBECD.
In some embodiments, an ophthalmic solution is administered QID for about 2 to 6 weeks, preferably about 4 weeks, followed by administration BID for about 6 to 10 weeks, preferably about 8 weeks. In some embodiments, the ophthalmic solution for the foregoing treatment regimen is 0.25% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and about 11% w/v SBECD.
In some embodiments, the present invention provides a method for treating certain subjects with dry eye disease. In some embodiments, a subject with dry eye disease is 18 years or older. In some embodiments, a subject with dry eye disease has a history of dry eye for at least six months prior to receiving the treatment of the invention. In some embodiments, a subject with dry eye disease has a history of use or desire to use eye drops for dry eye symptoms within six months prior to receiving the treatment of the invention.
In some embodiments, the present invention provides a method for treating a subject with dry eye disease, in particular moderate-to-severe dry-eye disease, comprising identifying subjects satisfying one or more of the following criteria for at least one eye, prior to receiving the treatment of the invention (for example, a screening performed at about one and/or two weeks before receiving the treatment):
In some embodiments, a subject with dry eye disease is not a female patient who is pregnant, nursing, or planning a pregnancy. In some embodiments, a subject with dry eye disease has not previously used reproxalap ophthalmic solution.
In some embodiments, the present invention provides a method for treating a subject with dry eye disease comprising a screening to exclude subjects having one or more of the following conditions for at least one eye, prior to receiving the treatment of the invention:
As described herein, an ophthalmic solution of the invention can achieve an early onset of effect in subjects with dry eye disease. As used herein, an “early onset effect” refers to early efficacy (e.g., within 1 to 2 weeks of initiation of treatment—in initiation or exacerbation phase) in ameliorating symptoms of dry eye disease. In some embodiments, the “early onset effect” is for the same dose and frequency of administration in the initiation or exacerbation phase. Accordingly, in some embodiments, the present invention provides a method for treating a subject with dry eye disease comprising topically administering to the subject an ophthalmic solution of the invention, wherein the ophthalmic solution is administered at a dose strength which can achieve an early onset profile. In some embodiments, an early onset profile comprises early onset of effect for symptoms (e.g., ocular discomfort including dryness, itchiness, tearing, burning, stinging, grittiness, cloudy vision, sensitivity to environment, stringy ocular secretion). In some embodiments, an early onset profile comprises early onset of effect for signs (e.g., ocular vital staining, tear film break-up time, tear osmolarity, tear volume).
In some embodiments, a dose strength which can achieve an early onset of effect comprises topically administering an ophthalmic solution comprising reproxalap, or a pharmaceutically acceptable salt thereof, at a concentration as described herein. In some embodiments, a dose strength which can achieve an early onset of effect comprises topically administering an ophthalmic solution comprising reproxalap, or a pharmaceutically acceptable salt thereof, at a frequency at described herein. In some embodiments, a dose strength which can achieve an early onset of effect comprises topically administering an ophthalmic solution comprising reproxalap, or a pharmaceutically acceptable salt thereof, at a concentration and a frequency at described herein.
In some embodiments, a method of the invention can achieve an onset of effect in about two weeks. At different dose strengths (for example, different concentration and administering frequency), a method of the invention can achieve an onset in fewer than about two weeks. For example, in some embodiments, a method of the invention can achieve an onset in about 14, 13, 12, 11, ten, nine, or eight days. At a certain dose strength, a method of the invention can achieve an onset in about one week or less. In some embodiments, a method of the invention can achieve an onset in about seven, six, five, four, three, two, or one days. In some embodiments, the early onset is accompanied by a reduction in an aldehyde marker of oxidative stress, for example about a 15%-30% reduction in level of the aldehyde marker of oxidative stress compared to control level. In some embodiments, the reduction is by about 20%.
In some embodiments, the present invention provides a method for treating dry eye disease in a subject, comprising topically administering to the subject an ophthalmic solution comprising about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the ophthalmic solution is administered three, two, or one times a day. In some embodiments, an ophthalmic solution comprising about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, is administered three times a day. In some embodiments, an ophthalmic solution comprising about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, is administered two times a day. In some embodiments, an ophthalmic solution comprising about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, is administered once daily.
In some embodiments, the present invention provides a method for treating dry eye disease in a subject, comprising topically administering to the subject an ophthalmic solution comprising about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the ophthalmic solution is administered at a higher strength in an initiation phase or exacerbation phase, followed by a lower strength in a maintenance phase, wherein each of the initiation phase, exacerbation phase, and maintenance phase is as described herein. In some embodiments, an ophthalmic solution comprising about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, is administered four times a day in an initiation phase or exacerbation phase followed by three, two, or one times a day in a maintenance phase. In some embodiments, an ophthalmic solution comprising about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, is administered three times a day in an initiation phase or exacerbation phase followed by two or one times a day in a maintenance phase. In some embodiments, an ophthalmic solution comprising about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, is administered two times a day in an initiation phase followed by one time a day in a maintenance phase. In some embodiments, an ophthalmic solution comprising about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, is topically administered in an initiation phase or exacerbation phase, followed by topical administration of an ophthalmic solution comprising less than about 0.5% w/v reproxalap, or a pharmaceutically acceptable salt thereof, in a maintenance phase, wherein the administration frequency of each ophthalmic solution is selected from those as described above.
In another aspect, the present disclosure provides a kit or patent pack for use in treating ocular inflammation or a disease that causes ocular inflammation or other symptoms described above that produce elevated RASP levels in the eye of a subject. Such a kit comprises: a container comprising an ophthalmic formulation comprising reproxalap as described herein; an assay kit for testing the levels of one or more aldehyde markers of ocular inflammation in the subject's tears as described herein; and, optionally, instructions for using the assay to test the levels of one or more aldehyde markers of ocular inflammation in the subject's tears.
The formulation may be packaged in any suitable device or container, such as a flask, bottle, glass or plastic unit-dose container such as a bottle with an eye-dropper integrated into its lid, stick-pack, tube, ampoule, etc., typically under sterile conditions. More commonly the pharmaceutical formulations of the invention are prescribed to the patient in “patient packs” containing a number of dosing units or other means for administration of metered unit doses for use during a distinct treatment period in a single package. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions. The patient packs encompass at least one container containing the suitable amount of the liquid ophthalmic formulation as disclosed herein.
Alternatively, they can be supplied in a larger container as a bottle, then the invention also relates to pharmaceutical kit of parts comprising a bottle containing any liquid formulation as disclosed herein, a cap and/or an eye dropper or a dropper-cap system, and optionally instructions.
The bottle can be made of any material convenient with the storage and the use requirements comprising polymers, metal and glass and so on. It is of importance that the bottle material does not interfere with the components of the liquid formulation as disclosed herein. In an embodiment it is made of plastic.
The following examples are intended to illustrate the invention and are not to be construed as being limitations thereon. Reproxalap can be synthesized as reported previously, for example, in WO 2006/127945, the entire content of which is incorporated herein by reference.
Objectives:
Investigational Product:
In the Phase 2b study, reproxalap was formulated as an ophthalmic solution as described in the specification.
Duration: A subject's participation was estimated to be approximately 14 weeks (98 days).
Dosage/Dose Regimen/Instillation/Application/Use: Screening: Between Visits 1 and 2, all subjects received 14 consecutive days (±2) of Run-in (vehicle) ocular drops self-administered QID in both eyes.
Treatment: During the 12-week (84±3 days) treatment period, Reproxalap Ophthalmic Solution at concentrations of 0.1%, 0.25%, or vehicle ophthalmic solution was administered QID by bilateral topical ocular dosing. Subjects were randomized to one of three treatment groups (1:1:1) to receive study drug after the Post-CAE® assessments at Visit 2.
Summary of Visit Schedule: Six visits over the course of approximately 14 weeks
Condition/Disease: Dry Eye Disease (DED)
Inclusion Criteria: Subjects for treatment were based on the following criteria:
Exclusion Criteria: Subject were excluded based on the following criteria:
The following efficacy measures and endpoints were used in the study:
Safety Measures:
General Statistical Methods and Types of Analyses
Sample Size: The study sample size of 100 per group was selected based on prior Phase 2 and 3 clinical trial results using the DED Hybrid CAE study design with other development programs and the effect size seen in Phase 2a with reproxalap on change from baseline after four weeks of treatment. This sample size was deemed sufficient to assess the effect size on the DED sign and symptom endpoints with reproxalap vs vehicle, to confirm the endpoint selection and sample size needed for Phase 3 studies with reproxalap. A sample size of 100 per group provided 90% power at α=0.05 to detect an effect size of 0.26 for inferior Lissamine green staining (Ora Calibra® scale), assuming a common standard deviation of 0.56 and an effect size of 0.44 for ocular discomfort assessed with the Ora Calibra® Ocular Discomfort Scale assuming a common standard deviation of 0.97.
Efficacy Analysis
The phase 2b data are shown in
Key Observations from Phase 2b Clinical Trial
The study was to assess levels of malondialdehyde (MDA) adducts in tears collected from subjects with dry eye disease enrolled in a Phase 2a, randomized, double-masked, clinical study to assess the safety, tolerability, and pharmacodynamic activity of ADX-102 ophthalmic solution in subjects with dry eye diseases, also referred to herein as dry eye syndrome (DES).
Tears were collected during Visit 1, prior to treatment, and during Visit 3, following four weeks of treatment. Treatment groups consisted of ADX-102 Ophthalmic Solution (0.5%), ADX-102 Ophthalmic Solution (0.1%), and ADX-102 Ophthalmic Lipid Solution (0.5%).
Based on results from a prior study in which normal human tear (NHT) were diluted 1:20, resulting in readings in the linear range of a standard curve, 1:20 and 1:80 dilutions in D/C subject tear samples and NHT were tested to provide reference points for MDA adduct concentrations in tears from DES and normal subjects. In the prior study, the 1:80 dilution of the high concentration spiked control sample (20,000 pmol/mL) resulted in an OD value within the standard curve (data not shown). It was anticipated that tears from DES subjects would contain similar concentrations of MDA adducts, and therefore a 1:80 dilution would result in readings in the linear range of the standard curve.
The pilot assay was conducted using 1:20 and 1:80 dilution of samples, in duplicate. Data from the pilot study showed that OD values from the D/C subject tears diluted 1:20 and 1:80 were within the linear range of the standard curve.
The pilot assay was conducted using 1:20 and 1:80 dilution of samples, in duplicate. Based on the pilot assay data, the DES subject study samples were diluted 1:60 to maximize the likelihood of the OD values falling within the linear portion of the standard curve, and thus provide the most accurate results.
Human tears were collected from subjects with DES during the Phase 2a clinical trial, according to the schedule in the table below. Tears (up to 10 μL) from both eyes of each subject were collected and pooled at Visit 1. At Visit 3, after 28 days of treatment with ADX-102 Ophthalmic Solution, human tears were collected from both eyes of each subject still enrolled. The Visit 3 tear samples from both eyes of each subject were not pooled at the time of collection, which was not in accordance with the Tear Collection Procedure Manual. Instead, right eye and left eye samples from each subject were pooled at the time of analysis for MDA adducts. Out of the 51 enrolled subjects, 12 subjects dropped out before Visit 3 tear collection and were considered D/C subjects. Tears collected at Visit 1 from D/C subjects were used in MDA adduct assay method development.
Fifty-one subjects were enrolled, for a total of 17 subjects per trial arm. Subjects were randomized 1:1:1 to receive ADX-102 Ophthalmic Solution (0.1%), ADX-102 Ophthalmic Solution (0.5%), or ADX-102 Ophthalmic Lipid Solution (0.5%). A vehicle control was not included in the clinical trial.
Thirty-nine subjects completed the trial: 16 subjects in the ADX-102 Ophthalmic Solution (0.1%) group; 12 subjects in the ADX-102 Ophthalmic Solution (0.5%) group; and 11 subjects in the ADX-102 Ophthalmic Lipid Solution (0.5%) group. Twelve subjects did not complete the study (D/C subjects).
Subjects self-administered ADX-102 Ophthalmic Solution four times per day (morning, noon, afternoon, and before bed) throughout the study. Subjects did not use study drug prior to Study Visits.
MDA adduct ELISA. Normal human tears, pooled from three individuals (two males and one female), were purchased from Bioreclamation IVT (catalog number hmtears). The MDA adduct ELISA kit is commercially available and was purchased from Cell Biolabs, Inc., San Diego, Calif. (OxiSelect MDA Adduct Competitive ELISA, catalog number STA-832).
The assay is a competitive ELISA. An MDA conjugate is adsorbed onto an ELISA plate. Samples containing unknown amounts of MDA adducts or MDA-BSA standards are then added to the plate and incubated. An MDA antibody is then added to the plate, followed by an HRP-labelled secondary antibody. The plate is washed and an HRP detection agent is added. The plate is read in a microplate reader at 450 nm. The assay OD reading decreases with increasing MDA adducts in the samples, as the adsorbed MDA competes for binding to the MDA antibody with MDA adducts in the test sample.
A standard curve for the assay was generated using 0, 0.025, 0.05, 0.10, 0.20, 0.39, 0.78, 1.56, 3.13, and 6.25 μg/mL of MDA-BSA. Standard and unknown samples volumes in the assay were 50 μL each.
Pilot Assay. In addition to the standard curve, neat NHT samples and NHT samples spiked with MDA-BSA were measured. NHT spiked with 12,000 pmol/mL concentration of the internal standard, MDA-BSA, was used to determine dilutional integrity. D/C samples and NHT spiked samples were diluted 20- and 80-fold in PBS buffer containing 0.1% BSA prior to assay. Neat NHT samples were diluted 20-fold to serve as a baseline reference.
DES Study Samples. Based on the results of the pilot assay, a 1:60 dilution was determined to maximize the likelihood of the OD values falling within the linear portion of the standard curve, and thus provide the most accurate results. All tear samples from subjects (0.5% ADX-102 Ophthalmic Solution) and (0.1% ADX-102 Ophthalmic Solution) were analyzed, but results from some tear samples were excluded from the data analysis due to contamination with ocular staining dye in the Visit 3 samples. The Visit 3 tear sample from some subjects (0.5% ADX-102 Ophthalmic Solution) was excluded from the data analysis due to insufficient volume.
Results—Pilot assay of D/C subject and NHT. The detection range of the assay was 6 to 1500 nM, and the linear range of the assay was approximately 10 to 110 nM. Normal human tears diluted 20-fold had an average calculated MDA adduct concentration of 2,266 pmol/mL (2,266 nM). Approximately 100% of the spiked MDA-BSA adduct (12,000 pmol/mL) was recovered in NHT. Tears from D/C DES subjects had a mean MDA adduct concentration of 7,798 pmol/mL, a 3.4-fold increase relative to NHT; this difference was statistically significant (
Assay of tears from subjects who completed the trial.
Although individually all treatment groups had lower MDA adduct concentrations on Visit 3 compared to Visit 1, the differences were not statistically significant.
Discussion MDA adducts were detected in tear samples collected from all DES subjects at Visit 1 and at Visit 3. MDA adduct concentrations were significantly lower on Visit 3 after a 4-week treatment with ADX-102 Ophthalmic Solution, relative to pre-treatment values on Visit 1, as shown in
The data show that MDA adduct levels are significantly higher in subjects with DES and are consistent with literature describing elevated MDA levels in tears of DES patients, compared to normal subjects. Furthermore, the data suggest that treatment with ADX-102 Ophthalmic Solution decreases MDA adducts in tears from subjects with DES.
General Statistical Methods and Types of Analyses:
Changes over 24 hours after the first dose on Day 1 and changes over 90 minutes in the CAE will be assessed via mixed effect model for repeated measures (MMRM) of change from baseline (pre-dose Day 1), with baseline score as a covariate, and time and test article group as factors. For assessments that include both eyes, eye will be added as a factor.
Schirmer's Test will be assessed via MMRM of change from baseline (screening), with baseline score as a covariate, and pre/post dose, eye, and test article group as factors.
Tear RASP levels will be assessed via MMRM of change from pre to post dose on Day 1 and Day 2, with baseline (pre-dose) scores as a covariate, and dose and test article group as factors.
Safety endpoints will be summarized using descriptive statistics.
Statistical analyses will be detailed in the Statistical Analysis Plan (SAP), which will dominate any statistical language herein.
The clinical objectives for this Phase 2/3 study are to evaluate the efficacy of reproxalap, as assessed by conjunctival redness, tear RASP levels, Schirmer's Test, and symptoms after dosing prior to and during exposure to the Controlled Adverse Environment® (CAE) in subjects with dry eye disease.
Overall Study Design
This study is a Phase 2/3, multi-center, randomized, double-masked, parallel design, vehicle-controlled trial designed to evaluate the efficacy and safety of 0.25% Reproxalap Ophthalmic Solution compared to vehicle in subjects with dry eye disease. Approximately twenty subjects will be enrolled in the Initial Cohort, and approximately 300 subjects will be enrolled in the Main Cohort. Male and female subjects at least 18 years of age with a subject-reported history of dry eye disease in both eyes and meeting all other eligibility criteria will be randomized to receive reproxalap or vehicle in a 1:1 ratio (approximately 150 subjects in each treatment group).
This study uses a challenge-model of dry eye disease known as a “dry eye chamber.” Challenge-model trials utilizing a controlled chamber are an FDA accepted design for pivotal endpoints. See, for example, FDA published “Dry Eye: Developing Drugs for Treatment” draft guidance; December 2020.
Dry eye chambers control relative humidity, temperature, airflow, and visual tasking in order to stress the ocular surface. Chambers simulate a “bad day” scenario in the life of a dry eye disease sufferer. Trial designs utilizing chambers are able to confirm the utility of drugs with rapid onset of action during an acute ocular surface challenge.
Order of Events:
Study Parameters
Primary Endpoint: Conjunctival redness assessed via digital photography over 90 minutes in CAE®
Safety Endpoints:
Secondary Endpoints:
Regarding the use of tear RASP levels as a clinical endpoint, it is important to note that this is (to date) unprecedented. The FDA has not agreed to the use of RASP before as a clinical endpoint. In this regard, we have reached agreement with the US Food and Drug Administration (FDA) for the use of RASP (reactive aldehyde species) as an objective sign for the treatment of dry eye disease. RASP are pre-cytokine pro-inflammatory mediators that are elevated in the tears of patients with dry eye disease, and correlate with dry eye disease symptoms and signs. In a Phase 2a dry eye disease clinical trial, reproxalap demonstrated reduction in tear RASP levels following 28 days of treatment. In in vitro studies, RASP were eliminated within 60 to 90 minutes when exposed to reproxalap at equimolar concentrations. Reproxalap, when administered topically to the eye, is thought to be more than 500-fold in excess of tear RASP levels, and has demonstrated consistent statistically significant and clinically relevant activity in dry eye disease, allergic conjunctivitis, and other forms of ocular inflammation across numerous Phase 2 and Phase 3 clinical trials. The use of RASP as a trial endpoint represents the first novel objective sign for the treatment in dry eye disease in over a decade.
Tear RASP levels from the TRANQUILITY run-in cohort were reduced after single doses of the novel RASP inhibitor reproxalap, as assessed by enzyme-linked immunosorbent assay (ELISA) of 4-hydroxynonenal protein adducts (HNE), a RASP selected based on results from a natural history study of dry eye patients conducted by Aldeyra. For subjects with sufficient tear volumes for analysis, across the two doses where tear RASP levels were assessed before and after drug administration, HNE levels declined by an average of 1018 picograms/milliliter (pg/mL) in reproxalap-treated patients (n=9) versus an increase of 32 pg/mL in vehicle-treated patients (n=7). Accordingly, tear RASP levels have been selected as a secondary endpoint for confirmation of mechanism of action. HNE is well-characterized in the scientific literature as a critical pro-inflammatory RASP,1 and ocular levels of HNE correlate with the signs and symptoms of dry eye disease.
Detailed Study Procedures
Examination Procedures
Procedures to be Performed at the Study Visit with Regard to Study Objective(s)
Visit 1 (Day −14 −16/+2, Screening)
Visit 2 (Day 1, Baseline and In-Office Dosing)
Statistical Hypotheses
The following hypothesis will be tested comparing reproxalap to vehicle. The null hypotheses must be rejected for the dosing regimen to claim efficacy.
H01: There is no difference between reproxalap and vehicle in the overall mean change from baseline in conjunctival redness.
H11: The overall mean improvement from baseline in conjunctival redness levels is larger with reproxalap than with vehicle.
Sample Size
Based on the conjunctival redness results from the Initial Cohort, 150 subjects per arm are required for 90% power to detect a statistically significant difference between arms, assuming a difference between groups of approximately 0.1 units and a standard deviation of the difference of 0.3 units.
Statistical Analysis
General Considerations:
Quantitative variables will be summarized descriptively using number of subjects (n), mean, standard deviation, median, minimum, and maximum. Qualitative variables will be summarized using counts and percentages.
All summaries will be presented by treatment group Summaries will be provided for demographics, baseline medical history, concurrent therapies, and subject disposition.
For the purpose of summarization, medical history, concurrent therapies, and adverse events will be coded to MedDRA and WHODrug dictionaries, as appropriate.
Baseline measures are defined as the last non-missing measure prior to the initiation of randomized study treatment at Day 1. Change from baseline will be calculated as follow-up visit value minus baseline value. Treatment comparisons between active and vehicle will be matched by dosing regimen and calculated as active minus vehicle.
All analyses will be 2-sided at a significance level of 0.05. 95% confidence intervals will be provided where appropriate.
The statistical analysis plan (SAP) will detail the statistical procedures, and will dominate any text herein.
Unit of Analysis:
Safety endpoints will be analyzed for both eyes. RASP assessments will be made on tear samples pooled across both eyes; redness, Schirmer's Test, and Ocular Discomfort Scores will be collected and analyzed for each eye; and VAS dryness and Ocular Discomfort & 4-Symptom Questionnaire scores will be collected for both eyes in aggregate. Assessment scales are detailed in the Appendices.
Missing Data:
As sensitivity measures, efficacy analyses may be conducted with multiple imputation under missing at random (MAR) and missing not at random (MNAR) assumptions. Per-protocol population analysis may also be conducted to assess sensitivity. Further detail will be described in the SAP.
Multiplicity Considerations:
No multiplicity corrections will be required.
Primary Efficacy Analysis:
The primary efficacy analysis of change from baseline in conjunctival redness over all time points in the CAE will be analyzed via MMRM, with baseline score as a covariate, and eye, time, and test article group as factors.
Secondary Efficacy Analyses:
Changes over 24 hours after the first dose on Day 1 and changes over 90 minutes in the CAE will be assessed via mixed effect model for repeated measures (MMRM) of change from baseline (pre-dose Day 1), with baseline score as a covariate, and time and test article group as factors. For assessments that include both eyes, eye will be added as a factor. Schirmer's Test will be assessed via MMRM of change from baseline (screening), with baseline score as a covariate, and pre/post dose, eye, and test article group as factors. Tear RASP levels will be assessed via MMRM of change from pre to post dose on Day 1 and Day 2, with baseline (pre-dose) scores as a covariate, and dose and test article group as factors.
Results
Data for the run-in cohort was obtained and analyzed.
Regarding symptoms of DED, statistical significance was achieved for both sign and symptoms in dry eye chamber, as shown in Table 3 below:
Notable Results:
In addition, we have observed in previous Phase 2a studies that tear levels of MDA adduct were statistically lower after treatment (
We developed assays as described below to determine levels of RASP in biological samples such as patient tears.
Notable Results:
This report describes exploratory experiments conducted to evaluate aldehyde trapping as an analytical approach to determining free aldehyde levels in human tears. Several aldehydes and trapping agents, including reproxalap, were utilized in these studies.
Exploratory Trapping Studies in PBS and ACN/H2O
Semicarbazide (SCZ), glutathione (GSH), cysteine, and N-acetyl cysteine were used as trapping agents with the aldehydes 4-hydroxynonenal (4-HNE), malondialdehyde, acetaldehyde, hexanal, pentanal, and decanal. 10 μM of aldehyde was mixed with 1 mM of trapping reagent in either PBS or 50/50 ACN/H2O. As judged by MS, only semicarbazide and glutathione appeared to trap aldehydes (Decanal, Pentanal, Hexanal, and 4-HNE). SCZ was more effective than GSH.
Exploratory Trapping Studies in Tears
Semicarbazide (SCZ) and glutathione (GSH) were used as trapping agents with the aldehydes 4-hydroxynonenal (4-HNE), malondialdehyde, acetaldehyde, hexanal, pentanal, decanal, and acrolein. 50 μL of tears was spiked with trapping agent (final concentration 1 mM), incubated for 1 hour, and ACN was added (final percentage of ACN was 25%). Three aldehydes were detected by MS (hexanal, pentanal, and decanal).
Exploratory Trapping Studies in Tears Repeat
Semicarbazide (SCZ) was used as trapping agent with the aldehydes 4-hydroxynonenal (4-HNE), malondialdehyde, acetaldehyde, hexanal, pentanal, decanal, and acrolein. 50 μL of tears was spiked with trapping agent (final concentration 1 mM), incubated for 1 hour, and ACN was added (final percentage of ACN was 25%). Three aldehydes were detected using a longer gradient (hexanal, pentanal, and decanal). A putative endogenous butanal related aldehyde was also detected in the study at m/z=144.0773.
Aldehyde Stability Studies in Tears
The aldehydes hexanal, pentanal, and decanal were spiked into tears and trapped with d6-reproxalap (which is fully deuterated on both methyl groups of reproxalap) to test for trapping stability. A comparison with vehicle control samples indicated that hexanal, decanal, and pentanal were observed in the vehicle control samples at higher levels than in the samples where these aldehydes were spiked.
Aldehyde Presence in Plastic and Glass
A comparison of glass vs. plastic (as the source of aldehydes) was made using d6-reproxalap solution. No apparent differences in aldehyde levels were detected.
Aldehyde Presence in Solvents
Water, ACN, and PBS were “cleaned” of aldehydes by addition of 0.5 mM semicarbazide (SCZ) and allowing 1 hour to react. 0.7 mM acetaldehyde was added to quench any remaining SCZ and allowed to react for 15 minutes.
A new solution of d6-reproxalap (10 mM) was prepared using “clean” 1:1 ACN:Water. Aldehyde (Pentanal, Hexanal, and Decanal) conjugates of d6-reproxalap were monitored in glass HPLC vials, with the use of no lid, foil, or plastic lids. The results were nearly identical for each, with pentanal, hexanal, and decanal detectable at similar levels.
Aldehyde (Pentanal, Hexanal, and Decanal) conjugates of d6-reproxalap were monitored in “clean” PBS and water. “Clean” PBS and water without addition of d6-reproxalap were also monitored. d6-reproxalap showed detectable hexanal and decanal peaks down to a 50 uM concentration of d6-reproxalap by MS. Reproxalap showed a detectable peak for pentanal and decanal at 1000 uM and a detectable peak for decanal down to 100 uM.
Aldehyde Presence in API and Drug Product
Serial dilution of d6-reproxalap and reproxalap using “clean” water was also performed. Serial dilution of Drug Product using “clean” water/vehicle showed detectable amounts of pentanal and decanal at 2- and 20-fold dilution and decanal only at 200-fold dilution. Serial dilution of Drug Product using “clean” water showed detectable amounts of pentanal and decanal at 10-fold dilution and decanal only at 100-fold dilution.
Aldehyde Presence in Vehicle, Tears
Detecting semicarbazide covalent conjugates of aldehydes (Pentanal, Hexanal, 4-HNE, and Decanal) was performed in a sample of the vehicle and showed detectable levels of each. Incubation of semicarbazide with commercial tears also yielded detectable levels of all four conjugates.
Aldehyde Scavenging Using SCZ and d6-Reproxalap
Formation of a conjugate with 4-HNE was tested using ADX-102, d6-reproxalap and SCZ. Addition of SCZ to ADX-102-4-HNE conjugate results in conversion of ADX-102-4-HNE conjugate to SCZ-4-HNE conjugate. Addition of d6-reproxalap to ADX-102-4-HNE conjugate results in conversion of ADX-102-4-HNE conjugate to d6-reproxalap-4-HNE conjugate.
Addition of ADX-102 and d6-reproxalap to vehicle did not afford additional aldehyde conjugates. Addition of SCZ to emotional tears generated SCZ aldehyde conjugates.
Presence of SCZ Aldehyde Conjugates in PBS
Addition of SCZ to PBS did not initially afford aldehyde conjugates. After 5 days SCZ Hexanal and SCZ Pentanal were detected. Using 50% ACN in extraction of the 5-day sample afforded similar results.
Presence of SCZ Aldehyde Conjugates in Tears
Addition of SCZ to untreated Clinical Tears did not initially afford aldehyde conjugates. After 5 days SCZ Hexanal and SCZ Pentanal were detected. Addition of SCZ to treated Clinical Tears did not initially afford aldehyde conjugates. After 5 days, SCZ Hexanal and SCZ Pentanal were detected.
Presence of ADX-102 Aldehyde Conjugates in PBS
Mining for the presence of ADX-102 aldehyde conjugates in PBS did not afford aldehyde conjugates. Using 50% ACN in extraction of the sample afforded similar results.
Presence of ADX-102 Aldehyde Conjugates in Tears
Mining for the presence of ADX-102 aldehyde conjugates in untreated clinical tears did not afford aldehyde conjugates. Using 50% ACN in extraction of the sample afforded similar results. Mining for the presence of ADX-102 aldehyde conjugates in treated clinical tears afforded ADX-102-Hexanal and ADX-102-Pentanal aldehyde conjugates. Using 50% ACN in extraction of the sample afforded similar results. Leaving these samples in the autosampler for 5 days resulted in elevated levels of ADX-102 aldehyde conjugates.
Aldehyde Scavenging of Drug Product Using SCZ
ADX-102-Hexanal and ADX-102-Pentanal levels did not appear to alter immediately after addition of SCZ, 2 hours after addition of SCZ or incubating overnight. Similar results were obtained using 50% ACN in the sample extraction.
Determining LOQ for SCZ and ADX-102 4HNE
Serial dilution of ADX-102-4HNE conjugate were made to determine the limit of quantitation to be 0.1 μM. Serial dilution of SCZ-4HNE conjugate were made to determine the limit of quantitation to be between 0.1 μM and 1 μM. Serial dilution of ADX-102-4HNE conjugate in 1:1 water:ACN afforded an LOQ of 5 nM or 1.8 ng/mL.
Detection of Unknown Aldehyde Conjugates
Several aldehyde conjugates of unknown structure were detected in treated clinical tears. A table of these aldehyde conjugates is presented below.
Tear protein aldehyde level in the presence of absence of reproxalap was tested with both HABA/avidin/biotin method and streptavidin plate/lysozyme antibody method. In the HABA/avidin/biotin assay, tear protein is treated with Rx drug or placebo, conjugated with amine-PEG2-biotin and mixed with HABA/Avidin Premix. Unexpectedly, signal from the tear only control was as low as the PBS control. However, as expected, the placebo and drug groups were both higher than the PBS control. In the streptavidin plate/lysozyme antibody assay, tear protein was treated with reproxalap or placebo, conjugated with amine-PEG2-biotin, bound to a streptavidin-coated plate, and detected with lysozyme antibody-HRP. The tear only control was higher than the PBS control as expected, but the placebo and drug groups were both lower than the tear only control, indicating the interference of placebo/drug formulation component(s) to this assay. Due to these technical issues, no conclusion for the drug effect can be drawn. Further investigation is required to understand the possible interference of formulation components to aldehyde detection. However, the below procedures represent useful starting points for antibody-based assays for detecting RASP conjugates in a sample.
Reagent List:
Sample List:
Day 1:
1. Preincubate 40 uL drug (Drug Product) or vehicle (Placebo) with 10 uL tear (containing ˜100 uM lysozyme plus other protein) for 2 hours at 30 C. Incubate with No HNE in both drug and placebo
2. Wash Away unreacted drug: dialyze in 1 L of PBS+0.1% Triton (7 KD filters) for 1 h. Note: Use separate reservoirs for placebo vs drug!
3. Wash Away unreacted drug and surfactant: dialyze in 1 L of Sigma PBS (7 KD filters) overnight.
Day 2:
4. Transfer retentate (˜100 uL) to new labeled tubes.
5. Bring retentate to 0.1M Acetic Acid (10 uL), incubate 60 minutes (cyclize the drug conjugates)
6. Spike the sample with 2 uL of sodium cyanoborohydride solution (5M in 0.1M acetic acid) (quench imines to amines), incubate 120 minutes.
7. Spike the samples with 2 uL of 5 mM Amine-PEG2-Biotin (100 uM biotin final).
8. Incubate 2 hours at 30 C.
9. Wash away unreacted biotin: dialyze in 1 L of PBS+0.1% Triton (7 KD filters) for 1 h.
10. Wash away unreacted biotin and surfactant: dialyze in 1 L of Sigma PBS (7 KD filters) at 4 C overnight.
Day 3
11. Transfer this biotinylated sample (retentate) to Step 15.
12. Equilibrate the HABA/Avidin Premix to room temperature.
13. Add 100 μL of ultrapure water to one microtube of the HABA/Avidin Premix. Mix with pipette tip.
14. Pipette 140 μL of PBS into a microplate well.
15. Add 20 μL of the HABA/Avidin Premix solution from step 12 to the PBS in the well. Place microplate on an orbital shaker or equivalent to mix.
16. Measure the absorbance of the solution in the well at 500 nm and record the value as A500 HABA/avidin. (high)
17. Add all ˜100 μL of biotinylated sample or 40 uL of Biotinylated HRP (positive control) to the well containing the HABA/avidin reaction mixture. Mix as described above.
18. Measure the absorbance of the solution in the well at 500 nm and record the value as A500 HABA/avidin/biotin sample once the value remains constant for at least 15 seconds. (low)
19. The relative level of aldehyde is determined by subtracting A500 HABA/avidin/biotin value from A500 HABA/avidin value.
Results: See
Reagent List:
Sample List:
Day 1:
1. Preincubate 40 uL drug (Drug Product) or vehicle (Placebo) with 10 uL tear (containing ˜100 uM lysozyme plus other protein) for 1.5 hour at 30 C. Incubate with No HNE in both drug and placebo.
2. Wash away unreacted drug: dialyze 1× in PBS with 0.2% Tween 80 for 3 h.
3. Wash away unreacted drug and surfactant: dialyze 1× in PBS overnight.
Day 2:
4. Bring retentate (˜100 uL) to 0.1M Acetic Acid, incubate 30 minutes (cyclize the drug conjugates) (+10 uL)
5. Spike the sample with 2 uL of sodium cyanoborohydride solution (freshly dissolved, 5M in 0.1M acetic acid).
6. Spike the samples with 1 uL of 5 mM Amine-PEG2-Biotin (50 uM biotin final).
7. Incubate 2 hours at 30 C.
8. Wash away unreacted biotin: dialyze 1× in PBS with 0.2% Tween 80 for 2-3 h.
9. Wash away unreacted biotin and surfactant: dialyze 1× with PBS overnight.
Day 3:
10. Transfer samples to a streptavidin-coated plate. Incubate at r.t. for 1-2 h.
11. Wash each well 3× with PBS (200 uL).
12. Add 100 ul of Anti-Lysozyme antibody-HRP (ab197705) (1:1000 in 1% BSA/PBS).
13. Incubate at room temperature for 2 h.
14. Wash each well 3× with PBS with 0.1% Triton.
15. Add 1004, of 1-Step Turbo TMB-ELISA to each well.
16. Incubate for 5-30 minutes at room temperature. (10 min)
17. Add 1004, of Stop Solution (1M H2SO4) to each well.
18. Transfer to a clear bottom plate.
19. Measure the absorbance at 450 nm.
While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.
All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.
This application claims the benefit of U.S. Provisional Patent Application No. 63/034,935, filed Jun. 4, 2020; the entirety of which is hereby incorporated by reference.
Filing Document | Filing Date | Country | Kind |
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PCT/US2021/035948 | 6/4/2021 | WO |
Number | Date | Country | |
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63034935 | Jun 2020 | US |