Diagnosis, monitoring and treatment of conditions characterized by intracellular free radicals

Abstract

The invention features the use of coelenterazine or similar analogs as a real-time reporter for systemic administration of ROS or RNS levels in the animal cell and organ environment. This real-time measurement can then be used to assess the severity of free radical dependent disease states, such as neurodegenerative disease, ischemia-reperfusion injury after organ transplantation, acute radiation syndrome injury, and the like. Methods may include real-time measurement of therapeutic efficacy of PrC-210 as a radical scavenger capable of eliminating oxygen and nitrogen radicals.

Description

Background Technology

[0001] The present invention is characterized by the use of coelenterate or a similar analogue as a real-time reporter of systemic ROS or RNS levels in the cellular and organ environments of animals and humans. This real-time measurement can then be used to assess the severity of free radical-dependent disease states, such as neurodegenerative diseases, post-organ transplant ischemia-reperfusion injury, acute radiation syndrome injury, etc. The method may include a real-time measurement of the therapeutic efficacy of PrC-210 as a free radical scavenger capable of eliminating oxygen and nitrogen free radicals. Summary of the Invention

[0002] In a first aspect, the present invention is characterized by a method for measuring intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a subject, the method comprising the following steps:

[0003] (i) Administering the compound of formula (I) to the subject:

[0004] A-(OB)n(I),

[0005] Wherein A is a luciferin group, each O is an oxygen atom connecting A and B, each B is independently a cleavable protecting group, and n is an integer from 1 to 4; and (ii) after step (i), intracellular bioluminescence from said luciferin is measured in vitro. In some embodiments, A is a group of coelenterate, vargulin, prolumepurple, or furimazine. In a particular embodiment, A is a group of coelenterate (e.g., enduRen or viviRen). In the compound of formula (I), each cleavable protecting group B may be selected to be cleaved by an esterase or a lipase.

[0006] In some implementations, the method further includes correlating the results of step (ii) with the subject's intracellular ROS and RNS levels.

[0007] In a particular implementation, the method further includes using the results of step (ii) to monitor the onset or severity of the subject's disease or condition.

[0008] In one related aspect, the present invention is characterized by a method for treating a disease or condition mediated by intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a subject, the method comprising the following steps:

[0009] (a) Measuring the intracellular ROS and RNS levels of the subjects; and

[0010] (b) Based on the level measured in step (a), administer an effective amount of the compound of formula (II) to the subject:

[0011]

[0012] Or a pharmaceutically acceptable acid addition salt thereof, wherein (x)A is -CH2NHR' and B is -CH2NHR, or A = -NRR' and B = H; and (y) each of R and R' is independently selected from H, C1-C6 alkyl, and C1-C6 heteroalkyl, provided that if B = H, then R and R' cannot both be H. Step (a) may include performing the diagnostic method of the present invention to measure the intracellular ROS and RNS levels of a subject.

[0013] In a particular embodiment, the compound of formula (II) is

[0014]

[0015] Or its pharmaceutically acceptable salt.

[0016] In certain implementations of any of the methods described above, the subject has already received a solid organ transplant (SOT) or a vascularized composite allogeneic graft (VCA), and acute or chronic transplant rejection of the graft and treatment success rate can be monitored.

[0017] In some embodiments of any of the methods described above, the subject suffers from a neurodegenerative disease, including but not limited to Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

[0018] In a specific implementation of any of the methods described above, the subject suffers from a neuroinflammatory disease, including but not limited to multiple sclerosis or aseptic encephalitis and non-aseptic encephalitis.

[0019] In some embodiments of any of the methods described above, the subject suffers from acute traumatic brain injury, such as traumatic brain injury, spinal cord injury, or stroke.

[0020] In a specific implementation of any of the methods described above, the subject suffers from acute radiation syndrome or has been, is being, or is about to be exposed to low-energy and high-energy radiation from a nuclear explosion, nuclear reactor leak, or spaceflight.

[0021] In some implementations of any of the methods described above, the subject suffers from a neuropsychiatric disorder, including but not limited to bipolar disorder (BD), schizophrenia, depression, anxiety disorder, attention deficit disorder, addiction disorder, personality disorder, autism, and Asperger's syndrome.

[0022] In some embodiments of any of the above methods, the subject suffers from cardiovascular disease, including but not limited to conditions such as arteriosclerosis, myocardial infarction, angioplasty, valvular heart disease, angina pectoris, peripheral circulatory disease, dialysis-induced vascular injury, vascular dementia, and transient ischemic attack.

[0023] In a specific implementation of any of the methods described above, the subject suffers from myopathy.

[0024] In some embodiments of any of the methods described above, the subject suffers from an autoimmune disease, including but not limited to conditions such as rheumatoid arthritis, type 1 diabetes, Morbus Crohn's disease, ulcerative colitis, and psoriasis.

[0025] In some implementations of any of the methods described above, the subjects had type 2 diabetes, obesity, and metabolic syndrome.

[0026] In some implementations of any of the methods described above, the subjects suffer from aging and age-related diseases.

[0027] In some implementations of any of the methods described above, the subject has asthma and inflammatory lung disease (e.g., chronic obstructive pulmonary disease (COPD)).

[0028] In some implementations of any of the methods described above, the subject suffers from an acute injury, including but not limited to injuries such as major injuries, major surgery, and burns.

[0029] In some implementations of any of the methods described above, the subject suffers from infectious complications, including but not limited to post-COVID syndrome.

[0030] In some embodiments of any of the methods described above, the subject suffers from a disease that causes fibrosis, such as, but not limited to, hepatitis that causes liver fibrosis, glomerulonephritis that causes kidney fibrosis, and primary and secondary pulmonary fibrosis.

[0031] In a particular embodiment of any of the methods described above, the bioluminescence in step (ii) occurs in the absence of any luciferase.

[0032] definition

[0033] To facilitate understanding of this invention, several terms are defined below. The terms defined herein have meanings commonly understood by one of ordinary skill in the art related to this invention. Terms such as “an,” “a,” and “the” are not intended to refer to a single entity only, but rather to encompass general categories that can be illustrated using specific examples. The terms herein are used to describe specific embodiments of the invention, but their use does not limit the invention unless set forth in the claims.

[0034] As used herein, the term “about” refers to a value within 10% above or below the stated value.

[0035] As used in this article, any value provided in the range includes the upper and lower limits, as well as any value contained within the upper and lower limits.

[0036] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a described compound that, to the extent of reasonable medical judgment, is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, etc., and is proportionate to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (edited by P.H. Stahl and C. G. Germuth), Wiley-VCH, 2008. These salts can be acid addition salts involving inorganic or organic acids. These salts can be prepared in situ during the final isolation and purification of the compounds described herein, or prepared separately by reacting a free base group with a suitable acid.

[0037] As used herein, the term "effective amount" refers to an amount sufficient to achieve a beneficial or desired outcome (such as a diagnostic result determining disease activity), and therefore, the determination of a "therapeutic effective amount" depends on the context in which it is applied. For example, in the context of administering a luciferin prodrug to provide real-time reporting of intracellular ROS or RNS levels in a subject. Methods may include real-time measurement of the therapeutically effective amount of an aminothiol (e.g., PrC-210) required to eliminate both oxygen and nitrogen free radicals. Thus, an effective amount of an aminothiol compound is, for example, an amount sufficient to improve symptoms or progression of a disease characterized by intracellular ROS and RNS substances. Depending on the nature of the condition being treated, the methods of the present invention may include systemic (e.g., intravenous) or local (e.g., topical or local injection) administration of the aminothiol compound.

[0038] As used herein and as is well known in the art, “diagnosing” and “monitoring” disease activity, and “treating” a condition or disorder, or “treatment” of various diseases and disorders, are methods for obtaining beneficial or desired outcomes (such as clinical outcomes). Beneficial or desired outcomes may include, but are not limited to, relief of one or more symptoms or conditions; reduction of the severity of a disease, disorder, or condition; stabilization (i.e., non-worsening) of the state of a disease, disorder, or condition; delay or slowing the progression of a disease, disorder, or condition; improvement or relief of a disease, disorder, or condition; and relief (partial or complete), whether detectable or undetectable. “Relief” of a disease, disorder, or condition means a reduction in the severity and / or undesirable clinical manifestations and / or a slowing or prolonging of the progression of a disease, disorder, or condition compared to its severity or time course without treatment.

[0039] As used herein, the term "subject" can refer to humans, non-human primates, or other mammals, such as, but not limited to, dogs, cats, horses, cows, pigs, goats, monkeys, rats, mice, and sheep. In a preferred embodiment, the subject is a human.

[0040] As used herein, the term "pharmaceutical composition" refers to an active compound formulated with one or more pharmaceutically acceptable excipients. In some embodiments, the compounds of the present invention are present in unit doses suitable for administration in treatment regimens that, when administered to relevant populations, show a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including forms suitable for: oral administration, e.g., infusions (aqueous or non-aqueous solutions or suspensions), tablets, or capsules; and parenteral administration, e.g., via subcutaneous, intramuscular, or intravenous injection.

[0041] As used herein, the term "pharmaceuticalally acceptable excipient" refers to any inactive ingredient (e.g., a medium capable of suspending or dissolving an active compound) that is biocompatible and suitable for administration to a subject. Typical excipients include, for example: anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, softeners, emulsifiers, diluents, film-forming agents or coatings, flavorings, fragrances, flow aids, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, or hydrated water. Excipients include, but are not limited to: butylated optional substituted hydroxytoluene (e.g., BHT), calcium carbonate, calcium hydrogen phosphate, calcium stearate, croscarmellose, croscarmellose, citric acid, croscarmellose, cysteine, ethyl cellulose, gelatin, optional substituted hydroxypropyl cellulose, optional substituted hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch, stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. A variety of reagents and materials that can be used as excipients are familiar to those skilled in the art.

[0042] As used herein, the term "alkyl" refers to a branched or straight-chain monovalent saturated aliphatic group containing only C and H when unsubstituted. The monovalent nature of an alkyl group does not include optional substituents on the alkyl group. For example, if an alkyl group is attached to a compound, the monovalent nature of the alkyl group refers to its attachment to the compound and does not include any additional substituents that may be present on the alkyl group. In some embodiments, the alkyl group may contain, for example, 1 to 6, 1 to 4, or 1 to 2 carbon atoms (e.g., C1-C6, C1-C4, or C1-C2). Examples include, but are not limited to, methyl, ethyl, isobutyl, sec-butyl, tert-butyl, 2-methylpropyl, and 2,2-dimethylpropyl.

[0043] As used herein, the term “C1-C6 heteroalkyl” refers to a branched or straight-chain monovalent saturated aliphatic group containing C, H and one or three N atoms.

[0044] As used herein, the term "luciferin" refers to a class of bioluminescent pigments and their synthetic analogs or functionally equivalent chemical substances present in organisms with bioluminescent capabilities, which can be oxidized to produce oxidized luciferin and release energy in the form of light. D-luciferin, or 2-(6-hydroxybenzothiazolyl)-2-thiazoline-4-carboxylic acid, was first isolated from the firefly (Photinus pyralis). A variety of chemically diverse forms of luciferin have been discovered and studied in a wide range of organisms, primarily from the ocean, such as fish and squid; however, many luciferins have been found in terrestrial organisms (e.g., worms, beetles, and various other insects) (Day et al. (2004) Luminescence 19:8-20). As used herein, luciferin also includes luciferin derivatives or analogs. Besides fully synthetic luciferins, such as cycloalkylaminoluciferin (CycLucl), there are at least five other generally bioevolved luciferins, each chemically distinct and catalyzed by chemically and structurally different luciferases employing a variety of different cofactors. The first is firefly luciferin, a substrate of firefly luciferase, which requires ATP for catalysis (EC1.13.12.7). The second is bacterial luciferin, also found in some squid and fish, composed of long-chain aldehydes and reduced riboflavin phosphate. Bacterial luciferase is FMNH-dependent. The third is dinoflavin luciferin, a tetrapyrrole chlorophyll derivative found in dinoflavins (marine planktonic organisms) that produce nocturnal marine phosphorescence. Dinoflavin luciferase catalyzes the oxidation of dinoflavin and consists of three identical, catalytically active domains. The fourth is imidazopyrazine luciferin, which is found in some ostracods and deep-sea fish (e.g., Porichthys). The last one is coelenterate (imidazopyrazine), a luminescent agent of the protein jellyfish, found in radiolarians, comb jellies, cnidarians, squid, copepods, chaetognathians, fish, and shrimp.

[0045] As used herein, the term "coelenterin" refers to a class of luciferin that serves as a substrate and is found in cnidarians, copepods, chaetognaths, comb jellies, decapods, mysids, radiolarians, and some fish taxa (Greer and Szalay (2002) Luminescence 17:43-74). For example, cnenterin analogs / derivatives, using Renal luciferase, can produce light emission between 418 nm and 547 nm (Loening et al. (2007) Nature Methods 4:641-643). A cnenterin analog / derivative (400A, DeepBlueC) is described as producing light emission at 400 nm via Renal luciferase (WO 01 / 46691, which is incorporated herein by reference). Other examples of cnenterin luciferins are enduRen, Prum Violet, Prum Violet II, Prum Violet III, viviRen, and furazolidone. Other examples of coelenterin analogues / derivatives include, but are not limited to, the compounds disclosed in U.S. Patent 9,624,425 and U.S. Patent Publication 20140302539, each of which is incorporated herein by reference.

[0046] Other features and advantages of the present invention will become apparent from the following detailed description, drawings and claims. Attached Figure Description

[0047] Figure 1 The chemical reaction that caused the photons to be released from coelenterin was described.

[0048] Figure 2 A comparison of the chemical structures of coelenterin, enduRen, and vivRen was depicted.

[0049] Figure 3 The distribution of photons in mice was compared between injected coelenterate and ViviRen. When administered intravenously, coelenterate induced photon signals primarily within the blood vessels, rather than at specific organ sites. ViviRen resulted in a uniform distribution of photons throughout the body.

[0050] Figure 4(Plate a and Plate b) show the treatment of U373 MG human astrocytoma cells in tissue culture with phorbol myristate (PMA). Addition of PMA to the culture medium induced superoxide production (measured by its reaction with coelentrin), and in Plate b, we observed PrC-210 dose-dependent capture and inhibition of the generated superoxide (relative to background). Plate c shows PMA dose-dependent production of superoxide in previously irradiated mice (measured by its reaction with ViviRen), and the data also show that intraperitoneal injection of PrC-210 (0.5 MTD, 252 mg / kg bw) significantly reduced detectable levels of superoxide in mice prior to PMA administration.

[0051] Figure 5 The distribution of viviRen in mice was compared between intravenous and subcutaneous injections.

[0052] Figure 6 The imaging ROS levels following organ- and time-specific distribution patterns after irradiation with 8.68 Gy are shown.

[0053] Figure 7 (A) Shows the level of free radicals in mice after irradiation with 8.68 Gy; the level of free radicals was suppressed to background levels by systemic administration of PrC-210. (B, C) Levels of caspase 3 / 7 biomarkers in mouse plasma after irradiation with 8.68 Gy, which were elevated in the absence of PrC-210 treatment and suppressed to background levels by PrC-210, i.e., (B) administration of 0.5 MTD PrC-210 30 minutes before irradiation with 8.68 Gy or (C) administration of 0.3 MTD PrC-210 24 hours after irradiation with 8.68 Gy.

[0054] Figure 8 The levels of ROS (A), MDA (B), and caspase 1 (C) in the mouse brain after irradiation with 8.68 Gy were shown; both ROS and MDA levels were suppressed to background levels by systemic administration of PrC-210.

[0055] Figure 9 The levels of ROS, caspase 8, and caspase 3 / 7 biomarkers in the mouse brains after irradiation with 8.68 Gy are shown. These biomarkers were elevated in the absence of treatment and were suppressed to background levels by PrC-210. Caspase levels 'reflect' ROS levels.

[0056] Figure 10 (A) shows three types of SOD1 G93AQuantitative measurement of brain ROS levels in mutant mice and wild-type controls (measured by their response to venous ViviRen). (B) The same three SOD1 G93A Changes in mouse body weight over time. Body weight gradually decreased with the onset of ALS motor symptoms and hindlimb paralysis, and was associated with increased ROS levels in (A). Systemic administration of PrC-210 (0.1 MTD, intraperitoneal) was associated with an immediate plateau in body weight loss.

[0057] Figure 11 The study shows the whole-body free radical levels after irradiation with different doses of PrC-210, in cases where no PrC-210 was administered, or A) different doses of PrC-210 were administered intravenously 30 minutes before irradiation and free radical imaging was performed 30 minutes after irradiation, or B) different doses of PrC-210 were administered intravenously immediately before free radical imaging 30 minutes after irradiation. Detailed Implementation

[0058] The invention is characterized by the use of a luciferin prodrug administered as a real-time reporter of ROS and RNS levels in animal and human cellular and organ environments. This real-time measurement can then be used to assess the activity and severity of free radical-dependent disease states, such as neurodegenerative diseases, post-organ transplant ischemia-reperfusion injury, and acute radiation syndrome injury. The method may include a real-time measurement of the therapeutic efficacy of PrC-210 as a free radical scavenger capable of eliminating oxygen and nitrogen free radicals.

[0059] Luciferin and luciferin prodrug

[0060] The method of this invention is characterized by the use of the bioluminescent luciferin coelenterate (Shimomura O, Johnson, FH, "Chemical nature of bioluminescence systems in coelenterates," Proceedings of the National Academy of Sciences of the United States of America; April 1975), which emits a photon when oxidized by a free radical molecule. Figure 1The emitted photons can be quantitatively measured using bioluminescence monitors, such as Lago X, Spectral Instruments (https: / / spectralinvivo.com / imaging-systems / ). The oxidation reaction of luminescent coelentrin does not require the presence of any additional catalytic enzymes. Therefore, coelentrin can react anywhere, at any time, in close contact with free radicals, and then release photons. The oxidation of coelentrin to release photons is enzyme-independent, such as luciferase. This enzyme-independent reaction makes luciferin derivatives (such as coelentrin) independent diagnostic agents for in-situ free radical detection.

[0061] Coelentrins are a class of luciferins that serve as substrates and are found in cnidarians, copepods, chaetognaths, comb jellies, decapods, mysids, radiolarians, and some fish taxa (Greer and Szalay (2002) Luminescence 17:43-74). For example, coelentrin analogs / derivatives can produce light emission between 418 nm and 547 nm via renin luciferase (Loening et al. (2007) Nature Methods 4:641-643). A coelentrin analog / derivative (400A, DeepBlueC) has been described as producing light emission at 400 nm via renin luciferase (WO 01 / 46691, which is incorporated herein by reference). Other examples of coelentrin luciferins include enduRen, purm violet, purm violet II, purm violet III, viviRen, and furazolidone. Other examples of coelenterate analogs / derivatives used in the methods of this invention include, but are not limited to, compounds disclosed in U.S. Patent 9,624,425 and U.S. Patent Publication 20140302539, each of which is incorporated herein by reference. Other luciferin compounds that can replace coelenterate also exist, such as firefly luciferin, sea firefly luciferin, etc. This invention uses coelenterate because this compound emits blue light, thus having higher tissue penetration. This is important because this invention is used to measure photons emitted from tissue sites of living organisms. Tissues and organs (such as the brain, kidneys, heart, intestines) are located a few millimeters below the body surface. The emitted blue light, due to its shorter wavelength, can penetrate body tissue more effectively and is therefore detectable at the body surface. For example, sea firefly luciferin also emits blue light and can therefore replace coelenterate. Furthermore, the addition or removal of the benzyl ring can increase or decrease the rigidity of the compound or change its polarity. Therefore, the emission wavelength can be changed according to the detection needs, making the wavelength shorter (380 nm to 10 nm in the ultraviolet) or longer (500 nm to 2500 nm in the near-infrared).

[0062] Application of luciferin prodrug

[0063] The animal and human dosages of the coelenterin reporter compounds of this invention depend on several factors, including the route of administration, the disease to be diagnosed, monitored, and treated, and the physical characteristics of the subject, such as age, weight, and general health status, target organ, and target species (e.g., human, mouse, or pig). Typically, the amount of coelenterin reporter molecule (e.g., coelenterin) included in a single dose can be an amount that allows differentiation of free radical concentrations above the background level. The dosage will be adjusted by clinicians based on animal and human studies and according to routine factors such as the severity of the disease, the method and location of free radical measurement (e.g., by whole-body photometry, endoscopy, intra-organ sensors, or percutaneous or local measurement), and different physiological parameters of the subject, and also depends on the safety margin of the compound, which can be 100 times the concentration of currently used compounds. For example, the LD50 of viviRen administered orally in rats is 14,500 mg / kg, which is 100 times the dose we used in our experiments. As we have... Figure 4 As shown in studies of phorbol myristate (PMA)-treated U373 cells and irradiated ICR mice, a standard curve needs to be established to visualize the free radical background and therapeutic window of the species. In this model, PMA induces the production of superoxide radicals, which are free radicals with a very short half-life. In human U373 astrocytoma cells in tissue culture, phorbol myristate (PMA) induced the production of superoxide radicals (…). Figure 2 (Plate A) and the concentration-dependent inhibition (capture) of superoxide by PrC-210 (B) of the generated superoxide. U373 cells were grown in DMEM medium containing 10% FBS; PMA was added to the medium in 10% ethanol:90% saline. Coelentrin was added to the medium after 30 seconds, and coelentrin photon emission was continuously monitored in the IVIS imaging chamber. PrC-210 was added to the medium 10 minutes before PMA addition. In Plate C, PMA in 20% ethanol:80% saline solution was intravenously injected into the tail vein of ICR mice. ViviRen was similarly injected into the tail vein of mice 10 minutes later. The level of superoxide-ViviRen conjugate in mice was monitored by quantitative photon emission in the IVIS chamber. As shown in the figure, intraperitoneal injection of 0.5 MTD dose of PrC-210 (252 mg / kg) before intravenous administration of PMA to mice significantly reduced measurable superoxide-viviRen products.

[0064] Measuring intracellular ROS / RNS levels

[0065] The invention is further characterized by a method for measuring organ and “disease-related” free radical concentrations. Therefore, it may sometimes be necessary to ensure that only intracellular free radicals are measured. To ensure this, luciferin reporter molecules (such as coenula) need to be linked to cleavable blocking groups that, once the blocking group is linked to the luciferin, block any oxidation and associated photon emission. These blocking groups need to be selected so that they do not inhibit cellular uptake of the luciferin reporter or blood-brain barrier penetration. Good examples of these combined luciferins (“blocking luciferins”) are viviRen and enduRen (Otto-Duessel M, Khankaldyyan V, Gonzalez-Gomez I, Jensen MC, Laug WE, Rosol M., “InVivo Testing of Renilla Luciferase Substrate Analogs in an Orthotopic Murine Model of Human Glioblastoma,” Molecular Imaging, 5(2), 2006), which are analogs of coenula. Figure 2 Once the compound enters the cell, the attached blocking group is cleaved by cellular lipases and esterases. Any endogenously present enzyme in all cells can be used as a cleavage mechanism for the blocking group. This cleavage then activates coelentrin or its analogues, allowing coelentrin to be oxidized and release photons. Coelentrin without a blocking group is mostly oxidized at the point of application or within the bloodstream before reaching the target organ (see [link to relevant documentation]). Figure 3 (Example of coelenterate in the present invention). This invention has demonstrated that intracellular release results in the uniform distribution of luciferin throughout the body (see [example]). Figure 3 (viviRen in the middle).

[0066] The photon signal emitted by a luciferin substrate (such as coelentrin or its analogues) is measured in a bioluminescence apparatus (such as an IVIS machine from PerkinElmer). Other methods for measuring photons include, for example, charge-coupled devices (CCDs) or complementary metal-oxide-semiconductor (CMOS) devices. Because this invention does not rely on catalytic enzymes (such as luciferase), the amount of luminescent signal measured is significantly lower than that measured from mutant cells expressing such catalytic enzymes, and therefore cannot be measured using standard measurement protocols. To receive a sufficient photon signal for distinguishing analysis, a longer acquisition time is typically required for the exposure time. The acquisition time can be reduced if: i) the photon sensitivity of the measurement-type bioluminescence apparatus is increased, ii) the amount of bioluminescent compound is increased, and / or iii) the wavelength is optimized for a specific organ or measurement mode.

[0067] Potential imaging applications include:

[0068] Photon detection, whether external or internal to the body, is performed by cameras (such as digital cameras, including DSLRs, mirrorless cameras, and smartphone cameras) capable of converting captured photons into electrons. These cameras have CCD (charge-coupled device) or CMOS (complementary metal-oxide-semiconductor) sensors that capture images. Both CCD and CMOS sensors are used to detect light and convert it into electrical signals, which are then processed into images.

[0069] Skin measurements: Wearable devices, including CCD (charge-coupled device) cameras or CMOS (complementary metal-oxide-semiconductor) sensors from smartphone cameras, such as those that image through a thin layer of dermis (e.g., wrist or finger), to determine the overall state of inflammation (e.g., monitoring Alzheimer's disease, multiple sclerosis (MS), rheumatoid arthritis, lupus, or vasculitis).

[0070] Subcutaneous measurements: Similar to those used in continuous glucose monitoring devices (such as optical sensors or transcutaneous optical fibers connected to a CCD camera). This can be used to evaluate the overall inflammatory status of autoimmune diseases such as multiple sclerosis (MS), rheumatoid arthritis, arteriosclerosis, aging, and / or neurodegeneration (such as aging and / or neurodegeneration in Alzheimer's disease, ALS, or Parkinson's disease).

[0071] Intramuscular measurements: Intramuscular imaging needles for evaluating muscle degeneration (potentially reduced signal) or inflammation (increased signal) (e.g., ALS, MS) are known in the art (see, for example, Sanchez, Gabriel N. et al., Neuron 88.6 (2015):1109-1120).

[0072] Intra-organ measurements can be performed using, for example, optical fibers placed inside the organ (connected to a CCD camera to measure inflammatory activity in organs such as the brain, liver, kidneys, and pancreas).

[0073] Imaging of hollow organs, such as the digestive tract, respiratory system, nasal cavity, and kidneys, can be performed using CCD endoscopy (for example, routine or intraoperative laparoscopic imaging of the digestive tract, used for example, the detection of tumors or inflammatory bowel disease); the respiratory system (bronchodilator imaging, used for example, inflammation); the nasal cavity (including deep brain imaging (e.g., pituitary gland), and the possibility of imaging the corpus callosum from the nasal cavity (used for example, Alzheimer's disease, ALS)); the kidneys (ureteroscopy, used for example, the detection of kidney disease); or arthroscopy (to determine the state of arthritis).

[0074] Arterial imaging is used to image arteries to determine the state of arteriosclerosis, or to image the heart to determine the state of myocarditis.

[0075] aminothiols

[0076] The aminothiol used in the treatment methods of the present invention can be synthesized, for example, as described in U.S. Patent 7,314,959.

[0077] aminothiol administration

[0078] The dosage of the disclosed compounds depends on a variety of factors, including the route of administration, the disease to be treated, and the subject's physical characteristics, such as age, weight, and overall health. Typically, the amount of the disclosed compound (e.g., PrC-210) contained in a single dose or in multiple doses over a longer period can be an amount that effectively treats the disease without inducing significant toxicity. Clinicians can adjust the dosage based on routine factors such as the severity of the disease and different parameters of the subject. Typically, the pharmaceutical compositions of this disclosure can be administered in amounts ranging from about 0.001 mg / kg / day to up to about 500 mg / kg / day of aminothiols (such as PrC-210).

[0079] For each species, a 'normal' range of free radicals can be established in relation to age, skin pigmentation, and body weight, serving as a comparison to determine deviations of free radicals from the normal range (see, for example, Curtis et al., Temporal variations of skin pigmentation in C57BL / 6 mice affect optical bioluminescence quantitation. Mol Imaging Biol. 2011; 13(6):1114-1123.). Animal and human studies can be used to establish the dosage and administration regimen of PrC-210 to maintain free radicals within this normal range for each species and disease.

[0080] Pharmaceutical Composition

[0081] The pharmaceutical compositions of the present invention contain one or more of the compounds disclosed herein as therapeutic compounds (e.g., one or more of the compounds of formula (I), formula (IA), and Table 1). In addition to a therapeutically effective amount of the compound, the pharmaceutical composition also contains a pharmaceutically acceptable excipient, and the pharmaceutical composition can be formulated by methods known to those skilled in the art. In some embodiments, the pharmaceutical composition for treating a disease contains one or more of the compounds disclosed herein (e.g., one or more of the compounds of formula (I), formula (IA), and Table 1), and can be formulated and / or administered with or without other therapeutic agents for a particular condition. Examples of such therapeutic agents (second therapeutic agents) are described herein.

[0082] The compounds disclosed herein (e.g., compounds of formula (I), formula (IA), and those in Table 1) may be used as free bases, as salts, or as solvates. All forms are within the scope of this disclosure.

[0083] Exemplary routes of administration for a pharmaceutical composition (or a compound of the composition) include oral, sublingual, oral, transdermal, intradermal, intramuscular, parenteral, intravenous, intra-arterial, intracranial, subcutaneous, intraorbital, intravenous, intraspinal, intraperitoneal, intranasal, inhalation, and topical application.

[0084] Treatment

[0085] In some embodiments, the invention includes correlating intracellular bioluminescence with intracellular ROS and RNS levels in a subject. The measured levels can be used to monitor the onset or severity of a subject's disease or condition. The measured levels can also be used to determine the effective amount of aminothiol (a compound of formula (II), such as PrC-210) required to treat the subject's ROS and RNS.

[0086] The method can be used to treat, monitor, and evaluate subjects who have received solid organ transplants (SOT) or vascularized composite allogeneic transplants (VCA), where acute or chronic transplant rejection of the graft and treatment success rates can be monitored.

[0087] The method can be used to treat, monitor, and evaluate subjects with neurodegenerative diseases, including but not limited to Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

[0088] The method can be used to treat, monitor, and evaluate subjects with neuropsychiatric disorders, including but not limited to bipolar disorder (BD), schizophrenia, depression, anxiety disorders, attention deficit disorder, addiction disorders, personality disorders, autism, and Asperger's syndrome.

[0089] The method can be used to treat, monitor, and evaluate subjects with cardiovascular diseases, including but not limited to conditions such as arteriosclerosis, myocardial infarction, angioplasty, valvular heart disease, angina pectoris, peripheral circulatory disease, dialysis-induced vascular injury, vascular dementia, and transient ischemic attack.

[0090] The method can be used to treat, monitor, and evaluate subjects with neuroinflammatory diseases, including but not limited to multiple sclerosis or aseptic and non-aseptic encephalitis.

[0091] The method can be used to treat, monitor, and evaluate subjects suffering from acute traumatic brain injury, such as traumatic brain injury, spinal cord injury, or stroke.

[0092] The method can be used to treat, monitor, and evaluate subjects with myopathy.

[0093] The method can be used to treat, monitor, and evaluate subjects with autoimmune diseases, including but not limited to rheumatoid arthritis, type 1 diabetes, Mobs Crohn's disease, ulcerative colitis, and psoriasis.

[0094] The method can be used to treat, monitor, and evaluate subjects with type 2 diabetes, obesity, and metabolic syndrome.

[0095] The method can be used to treat, monitor, and evaluate subjects suffering from aging and age-related diseases.

[0096] The method can be used to treat, monitor, and evaluate subjects with asthma and inflammatory lung diseases (e.g., chronic obstructive pulmonary disease (COPD)).

[0097] The method can be used to treat, monitor, and assess subjects suffering from acute injuries, including but not limited to major injuries, major surgeries, and burns.

[0098] The method can be used to treat, monitor, and evaluate subjects with infectious complications, including but not limited to post-COVID syndrome.

[0099] The method can be used to treat, monitor, and evaluate subjects suffering from diseases that cause fibrosis, such as, but not limited to, hepatitis that causes liver fibrosis, glomerulonephritis that causes kidney fibrosis, and primary and secondary pulmonary fibrosis.

[0100] The method can be used to treat, monitor, and evaluate subjects suffering from acute radiation syndrome, or those who have been, are being, or are about to be exposed to low-energy and high-energy radiation from nuclear explosions, nuclear reactor leaks, or spaceflight.

[0101] The following examples are intended to illustrate the present invention. They are not intended to limit the invention in any way.

[0102] Example

[0103] Example 1. Continuous monitoring of free radical levels in vivo.

[0104] ICR mice received 8.68 Gy of whole-body irradiation at time "0". The times indicated below the images (2 hours to 196 hours post-irradiation) are... Figure 6In this study, individual mice were anesthetized with 3% isoflurane, and a large pellet of viviRen was intravenously injected into the tail vein. Photons generated by whole-body ROS-ViviRen in the mice were then continuously monitored and quantified in an IVIS chamber. As can be seen from the image panel, changes in free radical levels over time followed an organ-specific pattern. Interestingly, this pattern reflects the time-dependent development of organ-specific post-irradiation toxicity reported by the CDC (see, for example, https: / / www.cdc.gov / nceh / radiation / emergencies / arsphysicianfactsheet.htm).

[0105] Example 2. Monitoring of free radical levels after irradiation.

[0106] ICR mice received 8.68 Gy of whole-body irradiation at time "0" ( Figure 7 At the time indicated on the X-axis, individual mice were anesthetized with 3% isoflurane, and a large dose of ViviRen was intravenously injected into the tail vein. The photons generated by ViviRen throughout the mice were then continuously monitored and quantified in the IVIS chamber. Treatment of mice with PrC-210 at 0.5 MTD (252 mg / kg) 30 minutes before irradiation, or ii) at 0.3 MTD (151 mg / kg) 24 hours after irradiation, significantly suppressed ROS signal intensity in uninjured mice. Interestingly, plasma caspase 3 / 7 reflected the free radical concentration observed in the IVIS chamber within the first 48 hours after irradiation; within 6 days after PrC-210 treatment, the marker remained completely suppressed at background levels. Free radical levels followed the same pattern but peaked after six days, according to… Figure 6 The organ pattern in the image appears to be an initial sign of multi-organ failure involving the liver and lungs.

[0107] Example 3. Monitoring of the decrease in free radical levels after irradiation and PrC-210 administration.

[0108] To observe the ROS response after irradiation, ICR mice were given 8.68 Gy of whole-body irradiation at time "0". Figure 8 At the time indicated on the X-axis, individual mice were anesthetized with 3% isoflurane, and a large dose of ViviRen was intravenously injected into the tail vein. Photons generated by ROS-ViviRen in the mouse brain were then continuously monitored and quantified in an IVIS chamber. Treatment of mice with PrC-210 at 0.5 MTD (252 mg / kg) 30 minutes before irradiation, or ii) at 0.3 MTD (151 mg / kg) 24 hours after irradiation, significantly suppressed ROS signal intensity in uninjured mice.

[0109] To measure the effects of irradiation-induced ROS on the mouse brain, ICR mice were subjected to whole-body irradiation of 8.68 Gy ( Figure 7 Mice were euthanized at a specified time after irradiation, and their brains were removed and homogenized in Tris buffer (pH 7.4). The homogenates were analyzed fresh or stored at -80°C prior to analysis. The levels of malondialdehyde (MDA, ROS-oxidized lipids) and caspase 1 (inflammasomes induced by free radicals) in the brain homogenates were measured as described in our previous study (Verhoven, BM, Fahl, WE et al., Transplantation Direct 6:8e578-586, 2020). The levels of MDA and caspase 1 in the brain homogenates were normalized to 100 μg of protein. Mice received an intraperitoneal injection of saline (control) or PrC-210 (0.5 MTD, 252 mg / kg bw) 30 minutes before irradiation, or an intraperitoneal injection of PrC-210 (0.3 MTD, 151 mg / kg) 24 hours after irradiation. Three replicates of MDA and caspase-1 analyses were performed. P-values ​​are shown for PrC-210-treated mice compared to control mice. Brain ROS levels in mice treated with and without PrC-210 were almost perfectly correlated with MDA and caspase-1 levels throughout the experimental time span. Malondialdehyde (MDA) (Stefan) is a marker of lipid peroxidation. Maria Wardas, Elzbieta Niedworok, Piotr WardasWiad Lek, 2004; 57(9-10): 453-5.) and the increase in caspase 1 in secondary reactions in response to increased free radicals (Fabio Martinon, Signaling by ROS drives inflammasome activation, European Journal of Immunology, Vol. 40, No. 3, March 2010, pp. 616-61) are the result of increased free radical levels and oxidative stress.

[0110] Example 4. Caspase level tracking of free radical levels after irradiation and PrC-210 treatment.

[0111] As shown in Example 3, 4ICR mice received 8.68 Gy of whole-body irradiation at time "0" ( Figure 8At the time points indicated on the X-axis, individual mice were anesthetized with 3% isoflurane, and a large dose of ViviRen was intravenously injected into the tail vein. Photons generated by ROS-ViviRen in the mouse brain were then continuously monitored and quantified in an IVIS chamber. Treatment of mice with PrC-210 at 0.5 MTD (252 mg / kg) 30 minutes before irradiation, or ii) at 0.3 MTD (151 mg / kg) 24 hours after irradiation, significantly suppressed ROS signal intensity in uninjured mice. Unlike Example 3, caspase 8 and caspase 3 / 7 in brain samples collected at different time points indicated on the X-axis were analyzed. This experiment yielded two main findings: i) Caspase 8 and caspase 3 / 7 activation reflected ROS levels, albeit with a slight delay, which is reasonable given that the apoptosis pathway requires activation in response to free radical-induced oxidative stress; ii) During the experiment, free radical levels were inhibited in the same manner as PrC-210 treatment, and in the same way that caspase 8 and caspase 3 / 7 activities were inhibited.

[0112] Example 5. Continuous monitoring of free radical levels in brain cells in vivo.

[0113] This experiment was designed to elucidate any relationship between brain free radical levels and the onset and progression of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Figure 10 In the diagram, it can be observed from panel A that there are three types of SOD1 G93A Quantitative measurement of ROS levels in the brains of mutant mice and wild-type controls (measured by their response to intravenous ViviRen). Figure 10 Chart B shows the same three charts A, SOD1. G93A Changes in mouse body weight over time. Body weight gradually decreases and declines exponentially with the onset of ALS motor symptoms.

[0114] This experiment yielded two main findings: i) The time it took for the three mice to develop motor symptoms was directly correlated with the increase in free radical levels in their bodies. ii) Systemic administration of PrC-210 (0.1 MTD, intraperitoneal) was associated with an immediate plateau in body weight gain, and weight loss ceased.

[0115] Example 6. Relationship between irradiation dose and free radical levels and measurement of free radical scavenging using PrC-210.

[0116] This experiment was designed to elucidate (i) the relationship between different irradiation doses administered to mice and the levels of free radicals produced in mouse organs by irradiation, and (ii) the ability of PrC-210 to inhibit irradiation-induced levels of free radicals in mouse organs through its scavenging capabilities.

[0117] Figure 11 A and Figure 11 B shows the linear relationship between the irradiation dose and the free radical levels measured by viviRen 30 minutes after irradiation.

[0118] Figure 11 A showed that intravenous injection of PrC-210 30 minutes before irradiation reduced free radical levels in a PrC-210 dose-dependent manner.

[0119] Figures A and B both show that as the irradiation dose increases, increasing the PrC-210 dose can suppress free radicals to background levels. This means that for any given irradiation dose, it should be possible to define a PrC-210 dose at which irradiation-induced free radicals are suppressed to background levels, regardless of whether PrC-210 is applied before or after irradiation.

[0120] Figure B shows that PrC-210 reduces irradiation-induced free radicals in a dose-dependent manner.

[0121] Figure 11 Both boards show a linear relationship between the applied irradiation dose and organ free radical production as measured by viviRen. The boards also show a dose-dependent relationship between irradiation-induced free radical levels and the inhibitory effect of PrC-210 doses on them. These data can be used to: i) measure the biological effects of a given irradiation dose on cells / organs by measuring cellular / organ free radical levels using viviRen; and ii) measure the free radical scavenging efficacy of PrC-210 in inhibiting free radical levels, regardless of whether PrC-210 is administered before or after irradiation.

[0122] Other implementation plans

[0123] Various modifications and variations to the compositions, methods, and uses of the invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the method has been described in conjunction with specific embodiments, it should be understood that the claimed method should not be unduly limited to such specific embodiments. In fact, the various modifications to the manner of carrying out the invention that are obvious to those skilled in the art are intended to be included within the scope of the invention.

[0124] Other embodiments are within the scope of the claims.

Claims

1. A method for measuring intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a subject, the method comprising the following steps: (i) Administering the compound of formula (I) to the subject: A-(OB)n(I), Where A is a fluorescein group, each O is an oxygen atom connecting A and B, each B is an independently cleavable protecting group, and n is an integer from 1 to 4; and (ii) Following step (i), intracellular bioluminescence from the luciferin is measured in vitro.

2. The method of claim 1, wherein A is a group of coelenterin, fluorescein, purum violet, or furazolidone.

3. The method of claim 2, wherein A is a group of coelenterin.

4. The method of claim 3, wherein the group of the coelenterin is enduRen or viviRen.

5. The method of any one of claims 1 to 4, wherein each cleavable protecting group B may be selected to be cleaved by an esterase or a lipase.

6. The method of any one of claims 1 to 5, further comprising correlating the result of step (ii) with the intracellular ROS and RNS levels of the subject.

7. The method of any one of claims 1 to 5, further comprising using the results of step (ii) to monitor the onset or severity of the subject's disease or condition.

8. A method for treating a disease or condition mediated by intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a subject, the method comprising the following steps: (a) Measuring the intracellular ROS and RNS levels of the subjects; and (b) Based on the level measured in step (a), administer an effective amount of the compound of formula (II) to the subject: Or its pharmaceutically acceptable acid addition salt, and wherein (x) A is -CH2NHR' and B is -CH2NHR, or A = -NRR' and B = H; and Each of (y)R and R' is independently selected from H, C1-C6 alkyl, and C1-C6 heteroalkyl. The prerequisite is that if B = H, then R and R' cannot both be H.

9. The method of claim 8, wherein step (a) comprises performing the method of any one of claims 1 to 7.

10. The method of claim 8, wherein the compound of formula (II) is Or its pharmaceutically acceptable salt.

11. The method of any one of claims 1 to 10, wherein the subject has received solid organ transplantation (SOT) or vascularized composite allogeneic transplantation (VCA), and acute or chronic transplant rejection of the graft and treatment success rate can be monitored.

12. The method of any one of claims 1 to 10, wherein the subject suffers from a neurodegenerative disease, including but not limited to Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

13. The method of any one of claims 1 to 10, wherein the subject suffers from a neuroinflammatory disease, including but not limited to multiple sclerosis or aseptic encephalitis and non-aseptic encephalitis.

14. The method of any one of claims 1 to 10, wherein the subject suffers from acute traumatic brain injury, such as traumatic brain injury, spinal cord injury, or stroke.

15. The method of any one of claims 1 to 10, wherein the subject suffers from acute radiation syndrome or has been, is being, or is about to be exposed to low-energy and high-energy radiation from a nuclear explosion, nuclear reactor leak, or spaceflight.

16. The method of any one of claims 1 to 10, wherein the subject suffers from a neuropsychiatric disorder, including but not limited to bipolar disorder (BD), schizophrenia, depression, anxiety disorder, attention deficit disorder, addiction disorder, personality disorder, autism, and Asperger's syndrome.

17. The method of any one of claims 1 to 10, wherein the subject suffers from cardiovascular disease, including but not limited to conditions such as arteriosclerosis, myocardial infarction, angioplasty, valvular heart disease, angina pectoris, peripheral circulatory disease, dialysis-induced vascular injury, vascular dementia, and transient ischemic attack.

18. The method of any one of claims 1 to 10, wherein the subject suffers from myopathy.

19. The method of any one of claims 1 to 10, wherein the subject suffers from an autoimmune disease, including but not limited to rheumatoid arthritis, type 1 diabetes, Mobs Crohn's disease, ulcerative colitis, and psoriasis.

20. The method of any one of claims 1 to 10, wherein the subject suffers from type II diabetes, obesity, and metabolic syndrome.

21. The method of any one of claims 1 to 10, wherein the subject suffers from aging and age-related diseases.

22. The method of any one of claims 1 to 10, wherein the subject suffers from asthma and inflammatory lung disease (e.g., chronic obstructive pulmonary disease (COPD)).

23. The method of any one of claims 1 to 10, wherein the subject suffers from an acute injury, including but not limited to injuries such as major injuries, major surgery, and burns.

24. The method of any one of claims 1 to 10, wherein the subject suffers from infectious complications, including but not limited to post-COVID-19 syndrome.

25. The method of any one of claims 1 to 10, wherein the subject suffers from a disease that causes fibrosis, such as, but not limited to, hepatitis that causes liver fibrosis, glomerulonephritis that causes kidney fibrosis, and primary and secondary pulmonary fibrosis.

26. The method of any one of claims 1 to 10, wherein the bioluminescence in step (ii) occurs in the absence of any luciferase.

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