Patent Application
20180208616
The present disclosure in general relates to the treatment of nitric oxide (NO)-related diseases. More particularly, the present disclosure relates to two types of dinitrosyl iron complex (DNIC), and their uses as a NO-releasing reagent to treat NO-associated diseases and/or conditions.
Nitric oxide (NO, also known as nitrogen oxide or nitrogen monoxide) has a formula of .NO, in which the dot ● represents an unpaired electron on the nitrogen atom. .NO involves a variety of signal transduction pathways in vascular, immune, and neuronal system in multicellular organisms, whereas the nature of .NO and the location where .NO is generated dictate its physiological and pathological function. .NO interacts with the heme center of soluble guanylate cyclase (sGC) to promote the conversion of GTP into cGMP and to activate cGMP-dependent vascular relaxation of blood vessels, cGMP/MAPK-dependent angiogenesis, and cGMP-dependent release of neurotransmitter. Bacterial .NO modulates hsf-1/daf-16 related genes to enhance stress resistance and longevity in C. elegans. Through the interaction with nuclear receptor proteins UNF and E75, .NO provides a concentration-dependent switching mechanism between the neuronal degenerative and regenerative states of axon. In Alexander disease, a serious degenerative neurological disorder, .NO derived from glia triggers astrocyte-mediated neuronal degeneration and cell death.
Nitroxyl (NO−, also known as azanone) is a one-electron-reduction sibling of .NO. Similar to .NO, NO− has been shown to induce vasorelaxation via activating sGC. Besides, NO− also triggers a positive myocardial inotropic effect in the cardiovascular system that is independent from β-adrenergic signaling pathway, especially during a congestive heart failure condition. In addition to cardiovascular effects, it is reported that NO− exhibits an anti-cancer effect via inhibiting GAPDH activity.
Based on the biological functions, both .NO and NO− donors have emerged as a promising agent to treat these .NO and/or NO− related diseases. However, the current .NO/NO− donors have some drawbacks, such as low activity, low solubility, poor specificity, short half-life, and adverse side-effect, that remain obstacles to clinical application. In view of the foregoing, there exists in the related art a need for a novel compound that has .NO /NO− releasing activity and may be employed as a lead compound for preparing or manufacturing medicaments to treat .NO/NO−-associated diseases and conditions.
The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.
As embodied and broadly described herein, one aspect of the disclosure is directed to a compound of formula (I),
or its pharmaceutically acceptable salt, solvate, derivative or prodrug.
According to some embodiments of the present disclosure, R is C1-6 alkyl or phenyl optionally substituted with —OH, —COON, or —NH2; and x is an integral between −2 to +2. Suitable examples of R include, but are not limited to, —CH2CH2OH, —CH2CH2COOH, —CH2CH3, —C6H5COOH and —CH2CH2NH2. According to one preferred example, R is —CH2CH2COOH, and x is 0.
The second aspect of the present disclosure is directed to a compound of formula (II),
or its pharmaceutically acceptable salt, solvate, derivative or prodrug.
According to certain embodiments of the present disclosure, R′ is —NH2, —NO2, —CH3 or —C6H5, and y is an integral between −2 to +2. Preferably, R′ is —NH2, —NO2 or —C6H5, and y is 0.
The present disclosure also provides a pharmaceutical composition and a composite material for delivery of NO so as to delay senescence, extend the lifespan, trigger the vasodilation, induce myocardial relaxation and inotropy, or treat a NO-associated disease and/or condition. The present pharmaceutical composition comprises,
(a) an effective amount of the present compound of formula (I) or (II), its pharmaceutically acceptable salt, solvate, derivative or prodrug; and
(b) a pharmaceutically acceptable excipient.
The present composite material comprises an active agent, and a metal-organic framework (MOF) having a pore structure for encapsulating the active agent therein, in which the active agent may be the compound of formula (I) or (II), its pharmaceutically acceptable salt, solvate, derivative or prodrug.
Another aspect of the present disclosure is directed to a method for delivery of NO in the purpose of delaying senescence, extending the lifespan, triggering the vasodilation, inducing myocardial relaxation and inotropy, or treating a NO-associated disease and/or condition. According to certain embodiments of the present disclosure, the method comprises administering to the subject in need thereof an effective amount of the pharmaceutical composition or the composite material in according to any aspects and embodiments of the present disclosure.
Exemplary diseases treatable by the present method include, but are not limited to, cardiovascular disease, diabetes mellitus, asthma, sickle cell disease, impotence, cystic fibrosis, pyloric stenosis, chronic kidney disease, neurodegenerative disorder, cancer, erectile dysfunction, and infectious disease.
Many of the attendant features and advantages of the present disclosure will becomes better understood with reference to the following detailed description considered in connection with the accompanying drawings.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The present description will be better understood from the following detailed description read in light of the accompanying drawings, where:
The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.
For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art. Also, unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms “a” and “an” include the plural reference unless the context clearly indicates otherwise. Also, as used herein and in the claims, the terms “at least one” and “one or more” have the same meaning and include one, two, three, or more.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating/working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example “C1-6” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, and C3-4.
Unless otherwise indicated, the term “alkyl” means a straight chain, branched and/or cyclic (“cycloalkyl”) hydrocarbon having from 1 to 6 (e.g., 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) carbon atoms. Alkyl moieties having from 1 to 4 carbons (C1-4 alkyl) are referred to as “lower alkyl.” Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, 2-isopropyl-3-methyl butyl, pentyl, pentan-2-yl, hexyl, and isohexyl. Cycloalkyl moieties examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Unless otherwise specified, each instance of an alkyl group is independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents.
Unless otherwise indicated, the term “substituted,” when used to describe a chemical structure or moiety, refers to a derivative of that structure or moiety wherein one or more of its hydrogen atoms is substituted with an atom, chemical moiety or functional group such as, but not limited to, —OH, —CHO, —COON, —NH2, alkoxy, alkanoyloxy (e.g., —OAc), alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), aryl, aryloxy, halo, or haloalkyl (e.g., —CCl3, —CF3, —C(CF3)3). In a particular embodiment, the term “substituted,” when used to describe a chemical structure or moiety, refers to a derivative of that structure or moiety wherein one or more of its hydrogen atoms is substituted with one or more of: hydroxyl, amino, or carboxyl.
Unless otherwise indicated, one or more adjectives immediately preceding a series of nouns is to be construed as applying to each of the nouns. For example, the phrase “optionally substituted alky or phenyl” has the same meaning as “optionally substituted alky, or optionally substituted phenyl.”
As used herein, the term “nitric oxide” or “NO” refers to the reactive forms of nitric oxide, in particular (1) uncharged nitric oxide (having the formula of .NO), (2) negatively charged nitric oxide (e.g., nitroxyl, which has the formula of NO−), and (3) positively charged nitric oxide (e.g., nitrosonium, which has the formula of NO+).
The term “prodrug” as used herein, refers to any compound that when administered to a biological system yields the “drug” substance either as a result of spontaneous chemical reaction(s) or by enzyme catalyzed or metabolic reaction(s).
The term “treating” encompasses partially or completely preventing, ameliorating, mitigating and/or managing a symptom, a secondary disorder or a condition associated with NO, in which increasing the level of NO provides a benefit to the subject having or suspected of having such symptom, disorder or condition. The term “treating” as used herein refers to application or administration of one or more compounds of the present disclosure to a subject, who has a symptom, a secondary disorder or a condition associated with NO, with the purpose to partially or completely alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms, secondary disorders or features associated with NO. Symptoms, secondary disorders, and/or conditions associated with NO include, but are not limited to, cardiovascular disease, diabetes mellitus, asthma, sickle cell disease, impotence, cystic fibrosis, pyloric stenosis, chronic kidney disease, neurodegenerative disorder, cancer, erectile dysfunction, and infectious disease. Treatment may be administered to a subject who exhibits only early signs of such symptoms, disorder, and/or condition for the purpose of decreasing the risk of developing the symptoms, secondary disorders, and/or conditions associated with NO. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced as that term is defined herein. Alternatively, a treatment is “effective” if the progression of a symptom, disorder or condition is reduced or halted.
The term “effective amount” as referred to herein designate the quantity of a component which is sufficient to yield a desired response. For therapeutic purposes, the effective amount is also one in which any toxic or detrimental effects of the component are outweighed by the therapeutically beneficial effects. The specific effective or sufficient amount will vary with such factors as the particular condition being treated, the physical condition of the patient (e.g., the patient's body mass, age, or gender), the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. Effective amount may be expressed, for example, in grams, milligrams or micrograms or as milligrams per kilogram of body weight (mg/Kg). Alternatively, the effective amount can be expressed in the concentration of the active component (e.g., the compound of the present disclosure), such as molar concentration, mass concentration, volume concentration, molality, mole fraction, mass fraction and mixing ratio. Specifically, the term “therapeutically effective amount” used in connection with the compound described herein refers to the quantity of compound, which is sufficient to alleviate or ameliorate the symptoms associated with the NO-associated disease in the subject. Persons having ordinary skills could calculate the human equivalent dose (HED) for the medicament (such as the present compound) based on the doses determined from animal models. For example, one may follow the guidance for industry published by US Food and Drug Administration (FDA) entitled “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” in estimating a maximum safe dosage for use in human subjects.
The term “subject” refers to an animal including the human species that is treatable with the compounds of the present disclosure. The term “subject” is intended to refer to both the male and female gender unless one gender is specifically indicated, and may be any age, e.g., a child or adult.
The present disclosure provides two types of compounds, each of which may serve as a NO-releasing agent so as to delay senescence, extend the lifespan, trigger the vasodilation, induce myocardial relaxation and inotropy, or treat a NO-associated disease and/or condition. The said NO comprises uncharged nitric oxide ( .NO) and charged nitric oxide species, particularly including nitroxyl (NO−) and nitrosonium (NO+). The first type of compound has the structure of formula (I):
wherein R is C1-6 alkyl or phenyl optionally substituted with —OH, —COON, or —NH2, and x is an integral between −2 to +2.
According to embodiments of the present disclosure, R may be —CH2CH2OH, —CH2CH2COOH, —CH2CH3, —C6H5COOH or —CH2CH2NH2. According to one example, R is —CH2CH2COOH, and x is 0.
According to one embodiment of the present disclosure, once administering to a subject, the first compound delays the aging process and prolongs the lifespan of the subject. According to another embodiment of the present disclosure, the first compound induces vasodilation and myocardial relaxation in the subject.
The second compound has the structure of formula (II):
wherein R′ is —NH2, —NO2, —CH3 or —C6H5, and y is an integral between −2 to +2. According to the preferred embodiment, R′ is —NH2, —NO2 or —C6H5, and y is 0.
According to one embodiment of the present disclosure, after administering to a subject, the second compound exhibits a myocardial inotropic effect therein; according to the embodiment, the second compound enhances the fractional shortening (FS) and cardiac output of the subject. According to another embodiment of the present disclosure, the second compound induces vasodilation in the subject.
Additionally, salts, solvates, derivatives and prodrugs of the compounds of formula (I) or (II) also are included in the present disclosure and can be used in the pharmaceutical composition, composite material and/or methods disclosed herein. The present disclosure further includes all possible stereoisomers and geometric isomers of the compounds of formula (I) or (II). The present invention includes both racemic compounds and optically active isomers. When a compound of formula (I) or (II) is desired as a single enantiomer, it can be obtained either by resolution of the final product or by stereospecific synthesis from either isomerically pure starting material or use of a chiral auxiliary reagent. Resolution of the final product, an intermediate, or a starting material can be achieved by any suitable method known in the art. Additionally, in situations where tautomers of the compounds of formula (I) or (II) are possible, the present disclosure is intended to include all tautomeric forms of the compounds. Prodrugs of compounds of formula (I) or (II) also are included in the present disclosure. It is well established that a prodrug approach, wherein a compound is derivatized into a form suitable for formulation and/or administration, then released as a drug in vivo, has been successfully employed to transiently (e.g., bioreversibly) alter the physicochemical properties of the compound. Suitable prodrugs include, for example, acid derivatives, such as amides and esters.
Salts of compounds of formula (I) or (II) can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation. The pharmaceutically acceptable salts of compounds of formula (I) or (II) can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, tartaric, and citric. Non-limiting examples of salts of compounds of the invention include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene sulphonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds of formula (I) or (II) appeared herein is intended to include compounds of formula (I) or (II) as well as pharmaceutically acceptable salts, solvates, or prodrugs thereof.
The compounds (I) and (II) may serve as a platform for delivery of NO, hence are potential lead compounds for manufacturing a medicament for treating a NO-associated disease or condition. Applicable disease or condition includes, but is not limited to, cardiovascular disease, diabetes mellitus, asthma, sickle cell disease, impotence, cystic fibrosis, pyloric stenosis, chronic kidney disease, neurodegenerative disorder, cancer, erectile dysfunction and infectious disease.
The second aspect of the present disclosure is directed to a pharmaceutical composition comprising an effective amount of an active agent and a pharmaceutically acceptable excipient. According to some embodiments of the present disclosure, the active agent is the compound of formula (I), or the pharmaceutically acceptable salt, solvate, derivative or prodrug thereof. According to certain embodiments of the present disclosure, the active agent is the compound of formula (II), or the pharmaceutically acceptable salt, solvate, derivative or prodrug thereof.
Depending on the desired purpose, the present pharmaceutical composition may be formulated into solid, semi-solid, or liquid forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, and injections. As such, administration of the active compound can be achieved in various ways, including oral, enteral, nasal, topical, transmucosal, subcutaneous, intradermal, intramuscular, intravenous, and intraperitoneal administration. In pharmaceutical dosage forms, the active compound may be administered alone or in combination with other known pharmaceutically active agent to treat NO-associated diseases and conditions. One of skilled person in the art is familiar with the various dosage forms that are suitable for use in each route. It is to be noted that the most suitable route in any given case would depend on the nature or severity of the disease or condition being treated.
In some embodiments, the present pharmaceutical compositions are solid dosage forms for oral administration. Such solid dosage forms may be capsules, sachets, tablets, pills, lozengens, powders or granules. In such forms, the active ingredient such as any of the compounds described above is mixed with at least one pharmaceutically acceptable excipient. Any of the described solid dosage forms may optionally contain coatings and shells, such as enteric coatings, and coatings for modifying the release rate of any of the ingredients. Examples of such coatings are well known in the art. In one example, the pharmaceutical compositions of this disclosure are tablets such as quick-release tablets. In still another example, the pharmaceutical compositions of this disclosure are formulated into sustained release forms. In another example, the pharmaceutical compositions of this disclosure are powders that are encapsulated in soft and hard gelatin capsules.
In some embodiments, the present pharmaceutical compositions are liquid dosage forms for oral administration. The liquid formulation may further include a buffering agent to maintain a desired pH. The liquid dosage formulations may also be filled into soft gelatin capsules. For example, the liquid may include a solution, suspension, emulsion, micro-emulsion, precipitate or any desired liquid media carrying any of the compound as described above, or a pharmaceutically acceptable derivative, salt or solvate thereof, or a combination thereof. The liquid may be designed to improve the solubility of active compound as described above to form a drug-containing emulsion or disperse phase upon release.
In some embodiments, the pharmaceutical compositions of this disclosure are formulations suitable for parenteral administration, such as subcutaneous, intradermal, intramuscular, intraperitoneal and intravenous injection. The pharmaceutical compositions may be formulated as isotonic suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatoary agents such as suspending, stabilizing or dispersing agents. Alternatively, the compositions may be provided in dry form such as powders, crystallines or freeze-dried solids with sterile pyrogen-free water or isotonic saline before use. They may be presented in sterile ampoules or vials.
The third aspect of the present disclosure pertains to a composite material comprising an active agent, and a metal-organic framework (MOF) having a pore structure for encapsulating the active agent therein. According to some embodiments of the present disclosure, the active agent is the compound of formula (I), or the pharmaceutically acceptable salt, solvate, derivative or prodrug thereof. According to certain embodiments of the present disclosure, the active agent is the compound of formula (II), or the pharmaceutically acceptable salt, solvate, derivative or prodrug thereof.
In structure, MOF is a compound consisting of metal ions/clusters and organic ligands, in which the organic ligands server as linkers to link the metal ions/clusters. The thus forming network may have a one-, two-, or three-dimensional structure, and comprises a plurality of voids (such as pores and channels) therein. MOF serves as a promising platform for drug delivery, based on the advantages of high drug loadings, biodegradability and versatile functionality. The nature of MOF (e.g., the size or structure of MOF, and the number and shape of voids comprised therein) may vary with the type of metal ion, which is usually selected from the group consisting of, aluminum (Al), chromium (Cr), antimony (Sb), gallium (Ga), cobalt (Co), nickel (Ni), magnesium (Mg), manganese (Mn), iron (Fe), zinc (Zn), cadmium (Cd) and ruthenium (Ru). According to one embodiment of the present disclosure, the metal ion of the present MOF is Fe.
Then, an active agent (such as a therapeutic drug or the present compound) can be loaded into the voids of MOF by impregnation of MOF in a solution comprising the active agent. Upon exposure to a suitable nucleophile (for example, water, blood, protein, or enzyme) or a stimulation (for example, a temperature higher than room temperature (e.g., 37° C.) or an electromagnetic radiation (e.g., ultraviolet light)), the active agent is displaced from the MOF and released into the aqueous environment. According to one embodiment of the present disclosure, the present composite material is produced by incubation of the MOF MIL-88B(Fe) in the solution of the compound of formula (I).
Depending on the desired purpose, the present composite material can be produced by impregnating the MOF with different active agents, for example, the present compound (i.e., the compound of formula (I) or (II)) and an additional agent. The additional agent may be a targeting molecule (such as an antibody, an aptamer or a polypeptide exhibiting binding affinity to a tissue-specific antigen), a therapeutic drug, a prophylactic drug, or any agent that possesses some beneficial effects on delaying senescence or on treating NO-associated disease.
Another aspect of the present disclosure pertains to a method for delivery of NO in the purpose of delaying senescence, extending the lifespan, triggering the vasodilation, inducing myocardial relaxation and inotropy, or treating a NO-associated disease and/or condition in a subject. The method comprises administering to the subject an effective amount of a compound of formula (I) or (II). According to embodiments of the present disclosure, the NO released from the compound of formula (I) or (II) activates NO relevant signal pathway, which then induces an anti-aging response and/or a therapeutic effect in the subject. The methods of the present invention can be accomplished by administering a compound of formula (I) or (II) as the neat compound, as a pharmaceutical composition, or as a composite material.
Exemplary diseases treatable by the present method include, but are not limited to, cardiovascular disease, diabetes mellitus, asthma, sickle cell disease, impotence, cystic fibrosis, pyloric stenosis, chronic kidney disease, neurodegenerative disorder, cancer, erectile dysfunction, or infectious disease.
As would be appreciated, the present method can be applied to the subject, alone or in combination with additional treatment that have some beneficial effects on delaying senescence or on treating NO-associated disease. Depending on the therapeutic purpose, the present method can be applied to the subject before, during, or after the administration of the additional treatment.
The subject treatable by the present method may be a human or a non-human, for example, a worm, a fish, a mouse, a rat, a cat, a dog, a monkey, a chimpanzee, a sheep or a cow.
The compound of formula (I) or (II) can be administered to the subject via a route selected from the group consisting of oral, enteral, nasal, topical, transmucosal, and parenteral administration, in which the parenteral administration is any of subcutaneous, intradermal, intramuscular, intraarticular, intravenous, intraspinal, or intraperitoneal injection. According to some embodiments of the present disclosure, the compound of formula (I) or (II) is orally administered to the subject.
In one embodiment of the present disclosure, administration of the compound of formula (I) delays the aging process, and prolongs the lifespan of the subject. In another embodiment of the present disclosure, administration of the compound of formula (I) or (II) induces vasodilation. In still another embodiment of the present disclosure, administration of the compound of formula (I) induces myocardial relaxation. In still another embodiment of the present disclosure, administration of the compound of formula (II) induces myocardial inotropy. In further embodiment of the present disclosure, administration of the compound of formula (II) enhances fractional shortening (FS) and cardiac output.
As would be appreciated, the compound of formula (I) or (II) may be administered in combination with a targeting molecules (e.g., conjugated with the targeting molecule, or encapsulated in a vesicle having the targeting molecule linked therewith) so as to improve the therapeutic effect thereof. In general, the target molecule may be an antibody, an aptamer or a polypeptide exhibiting binding affinity to a tissue-specific antigen.
The following Examples are provided to elucidate certain aspects of the present invention and to aid those of skilled in the art in practicing this invention. These Examples are in no way to be considered to limit the scope of the invention in any manner. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.
Materials and Methods
Instruments
The reagents metmyoglobin (TCl), Griess reagent (Aldrich), 5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside (Aldrich), potassium ferrocyanide (Alfa Aesar), potassium ferriccyanide (ACROS), sodium nitroprusside (ACROS), epinephrine (Sigma) were used as received. De-ionized water was produced from Milli-Q reagent water purification system. Infrared spectra of the vNo stretching frequencies were recorded on a JASCO 4200 spectrometer with sealed solution cells (0.1 mm, CaF2 windows). UV-vis spectra were recorded on a JASCO V-630 spectrometer. Fluorescence spectra were recorded on a Hitachi F-7000 fluorescence spectrophotometer. Bright-field and fluorescence images were recorded under confocal microscope system (TCS-SP5-XAOBS, Leica, Germany; ECLIPSE TE2000-U, Nikon, Japan) and fluorescence microscope (SMZ1500, Nikon/HG Lamp MODEL LH-M100C-1/4000R, Qlamging).
Preparation of DNIC
[(NO)2Fe(μ-SEtOH)2Fe(NO)2] (DNIC-1a), [(NO)2Fe(μ-SEtCOOH)2Fe(NO)2] (DNIC-1b), [(NO)2Fe(μ-SEt)2Fe(NO)2] (DNIC-1c), [(NO)2Fe(μ-SPhCOOH)2Fe(NO)2] (DNIC-1d), and [(NO)2Fe(μ-SEtNH2)2Fe(NO)2] (DNIC-1e)
A THF solution of [Fe(CO)2(NO)2] (1.0 mmol), freshly generated by reaction of [Na-18-crown-6-ether][Fe(CO)3(NO)] (0.914g, 2 mmol) and [NO][BF4] (0.256g, 2.2 mmol), was transferred via cannula under positive N2 to a 20-mL Schlenk tube in which 4-mercaptobenzoic acid (0.2313 g, 1.5 mmol) was loaded. The reaction solution was stirred at ambient temperature for overnight, and then monitored by Fourier transform infrared spectroscopy (FTIR). The IR vNO and vCO spectrum showed three absorption peaks at 1787, 1759, 1720 cm−1 and indicates the formation of complex [(NO)2Fe(μ-SPhCOOH)2Fe(NO)2] (DNIC-1d). This THF solution of DNIC-1d was then filtered through Celite. Addition of hexane to this THF solution of DNIC-1d led to the precipitation of brown solid of [(NO)2Fe(μ-SPhCOOH)2Fe(NO)2] (DNIC-1d) (yield 0.137 g, 23%). IR vNO: 1787, 1759, 1720 cm−1 (THF). Absorption spectrum (THF): 396 (10200) nm.
DNIC-1a (yield 0.102 g, 53%), DNIC-1b (yield 0.143 g, 81%), DNIC-1c (yield 0.103 g, 58%) and DNIC-1e (yield 0.110 g, 57%) were synthesized in a similar fashion. DNIC-1a: IR vNO: 1779, 1752 cm−1 (THF). Absorption spectrum (H2O): 312 (9300), 362 (8500) nm. DNIC-1b: IR vNO: 1776, 1751 cm−1 (THF). Absorption spectrum (H2O): 311 (9950), 362 (9100) nm. DNIC-1c: IR vNO: 1773 (9800), 1748 (9000) cm−1 (THF). Absorption spectrum (DMSO): 306, 361 nm. DNIC-1e: IR vNO: 1773, 1748 cm−1 (THF).
(NO)2Fe(μ-NH2Pyr)2Fe(NO)2] (DNIC-2a), [(NO)2Fe(μ-NO2Pyr)2Fe(NO)2] (DNIC-2b), [(NO)2Fe(μ-MePyr)2Fe(NO)2] (DNIC-2c), and [(NO)2Fe(μ-Pyr)2Fe(NO)2] (DNIC-2d)
[Fe(CO)2(NO)2] (1.0 mmol) was freshly prepared by reaction of [Na-18-crown-6-ether][Fe(CO)3(NO)] (0.446 g, 1.0 mmol) and [NO][BF4] (0.116 g, 1.0 mmol) in THF at 4° C. This THF solution of [Fe(CO)2(NO)2] (1.0 mmol) was then transferred via cannula under positive N2 to a 20 -mL Schlenk tube loaded with 3-aminopyrazolate (0.082 g, 1.0 mmol). After the reaction solution was stirred at ambient temperature for overnight, appearance of IR vNO peaks at 1807, 1790, 1736, and 1722 cm−1 indicated the formation of complex [(NO)2Fe(μ-NH2Pyr)2Fe(NO)2] (DNIC-2a). Solvent was then removed under vacuum. The crude brown solid was redissolved in diethyl ether and then filtered through Celite. Solvent was then removed under vacuum to yield the dark-brown powder of [(NO)2Fe(μ-NH2Pyr)2Fe(NO)2] (DNIC-2a) (yield 0.182 g, 46%). IR vNO: 1807, 1791, 1736, and 1722 cm−1 (CH2Cl2); 1802, 1785, 1735, 1720 cm−1 (diethyl ether). Absorption spectrum (CH3OH): 339(3835).
DNIC-2b (yield 0.133 g, 67%), DNIC-2c (yield 0.124 g, 62%) and DNIC-2d (yield 0.148 g, 57%) were synthesized in a similar fashion. DNIC-2b: IR vNO: 1813 sh, 1797 s, 1743 sh, 1728 s cm−1 (THF); 1813 sh, 1802 s, 1745 s, 1737 sh (diethyl ether). Absorption spectrum (THF): 330(6900). DNIC-2c: IR vNO: 1807 sh, 1793 s, 1739 sh, 1724 s cm−1 (THF); 1809 sh, 1795 s, 1740 s, 1726 sh (CH2Cl2). Absorption spectrum (THF): 357(5870). DNIC-2d: IR vNO: 1810, 1797, 1743, 1728 cm−1 (THF); 1810, 1797, 1743, 1728 cm−1 (CH2Cl2); 1811, 1798, 1746, 1731 cm−1 (diethyl ether); 1810, 1801, 1745, 1736 cm−1 (hexane). Absorption spectrum (THF): 323 (3900).
[(NO)2Fe(μ-SEtCOOH)2Fe(NO)2]@MIL-88B (Fe)
To a 20-mL Schlenk flask loaded with 0.1 g of MIL-88B(Fe) was added 5-mL THF solution of DNIC-1 b (0.044 g, 0.1 mmol). The reaction solution was stirred at ambient temperature under N2 atmosphere for 24 hours. The reaction solution was then centrifuged at 6000 rpm for 10 min before the above THF solution was removed. The residual brown solid was extracted with 5 mL of THF two times and then dried under vacuum (0.088 g). The IR spectrum of the brown solid exhibited two IR vNO stretching frequencies at 1777 and 1751 cm−1, whereas the pXRD spectrum of the brown solid showed two strong peaks at 2θ=9.3° and 10.4°. These spectroscopic data demonstrated the successful synthesis of [(NO)2Fe(μ-SEtCOOH)2Fe(NO)2]@MIL-88B (Fe).
Crystallography
The crystals of DNIC-2b and DNIC-2d chosen for X-ray diffraction studies were mounted on a glass fiber and quickly coated in epoxy resin. Unit cell parameters were obtained by least-squares refinement. Diffraction measurement for DNIC-2b, and DNIC-2d was carried out on a SMART Apex CCD diffractometer with graphite-monochromated Mo Ka radiation (I=0.71073 A) and between 2.38 and 22.69° for DNIC-2b, between 1.59 and 25.04° for DNIC-2d, respectively. Least-squares refinement of the positional and anisotropic thermal parameters of all non-hydrogen atoms and fixed hydrogen atoms was based on F2. A SADABS absorption correction was made. The SHELXTL structure refinement program was employed.
Reaction of [(NO)2Fe(μ-RPyr)2Fe(NO)2] (DNIC-2) and metmyoglobin (metMb)
A typical procedure for nitroxyl-transfer reaction of DNIC-2 and metmyoglobin was described below using DNIC-2a as an example, whereas reaction of DNIC-2b/DNIC-2d and metMb follows the same procedure. 10 μM of metMb in 3.3 mL of 25 mM phosphate buffer (pH 7.4) was loaded in a 4 -mL quartz cuvette and sealed with rubber septum under anaerobic N2 atmosphere. 10 μM of DNIC-2a (13.0 μL of 2.5 mM stock solution of DNIC-2a in THF) was added into this aqueous solution, which is thermostated at 25° C. UV-vis spectra of this reaction solution were measured after this reaction solution was incubated for 10 minutes. Shift of UV-vis absorption bands from 407, 503 and 629 nm to 419, 543, and 577 nm indicated the nitroxyl transfer from DNIC-2a to metmyoglobin affording {Fe(NO)}7 species (MbNO). DNIC-2b and DNIC-2d displayed similar nitroxyl-transfer reactivity toward metmyoglobin.
Reaction of [(NO)2Fe(μ-SR)2Fe(NO)2] (DNIC-1) and metmyoglobin (metMb)
A typical procedure for reaction of DNIC-1 and metmyoglobin was described below. To a 4 -mL quartz cuvette containing 10 μM of metMb in 3.3 mL of 25 mM phosphate buffer (pH 7.4), 10 μM of DNIC-1, derived from 13.0 μL of 2.5 mM stock solution of DNIC-1 in THF was added via gas-tight syringe at room temperature. The reaction solution was incubated at ambient temperature for 8 hours. No change of UV-vis spectra for this reaction solution was observed.
In vitro release of .NO from DNIC-1 characterized by Griess reagent and fluorescence probe FA-OMe
A typical procedure for characterization of the total amount of nitric oxide released from DNIC-1a, DNIC-1b, and DNIC-1d using Griess reagent was described below. 15 μM DNIC-1 was prepared via addition of 300 μL of 0.75 mM stock solution of DNIC-1 in DMSO to 14.7 mL of 25 mM KPi buffer, pH 7.0. After this solution was incubated under aerobic condition for 48 hours, 1.5 mL of 10 mM Griess reagent in H2O was added. A540 was then measured to estimate the amount of nitric oxide released from DNIC-1 according to the calibration curve made of 0, 1, 2, 5, 10, 20, and 50 μM of NaNO2, respectively. Three independent experiments were executed to measure the average amount of .NO released from 15 μM of DNIC-1 a.
Release of nitric oxide from DNIC-1a, DNIC-1b, and DNIC-1d was also investigated using the fluorescence probe FA-OMe specific for .NO. 20 μM FA-OMe was prepared via addition of 1.0 μL of 100 mM stock solution of FA-OMe in DMSO to 5 mL of 25 mM KPi buffer, pH 7.0. After addition of 500 μM DNIC-1 (50.0 μL of 50 mM stock solution of DNIC-1 in DMSO) to 20 μM FA-OMe, the fluorescence intensities were then recorded at 524 nm with excitation at 460 nm to corroborate the release of nitric oxide from DNIC-1 and DEANONOate, respectively.
Worm and Zebrafish Strains and Maintenance
All experiments were performed in accordance with animal protection standards and were approved by the Animal Investigation Committee of National Tsing Hua University (permit No. 00990001). WT C. elegans (N2) and CX3553 strain were obtained from C. elegans core facility Taiwan (CECF). Both strains were grown at 20° C. on nematode growth medium (NGM) plates with OP50 bacteria as food. AB (wild type) and Tg (kdr: EGFP) strains of zebrafish adults and embryos were used and maintained according to the guidelines described in the Zebrafish Book.
Uptake and export of DNIC-1a in C. elegans
The uptake and export of DNIC-1a in C. elegans were evaluated based on the change of Fe content. 140 N2 worms were incubated with 500 μM of DNIC-1a for 15 minutes, washed with M9 buffer for three times, and transferred back to the agar plate containing E. coli OP50. These worms were then collected 0, 0.25, 0.5, 1, 2, and 6 hours later, respectively, and washed with M9 buffer for three times. Incubation of these worms with 80 μL household bleach and 25 μL of 10 M NaOH for 1 hour were then achieved to digest these worms. This solution was acidified via addition of 5% HNO3 and diluted to 2.5 mL before the quantitation of Fe content by ICP-MS. Three independent experiments were conducted to measure the average values of Fe content in C. elegans after treatment of DNIC-1a. The background amount of Fe in the worms was measured in the similar fashion, except the exclusion of the step for incubation with 500 μM of DNIC-1a for 15 minutes.
In vivo release of .NO from DNIC-1a in C. elegans
Ten N2 worms were incubated with 500 μM of DNIC-1a for 15 minutes and washed with M9 buffer for three times. After further incubation with 20 μM of FA-OMe for 0.5, 1, 2, and 4 hours, respectively, these worms were washed with M9 buffer for three times before the bright field and fluorescence images were taken. The fluorescence image was taken using the fluorescence microscope. For control experiments, N2 worms were incubated with either 500 μM of DNIC-1a for 15 minutes or 20 μM of FA-OMe for 30 minutes before the fluorescence images were taken.
Analysis of Lifespan in C. elegans
All experiments were executed at 20° C. and repeated at least three times. The worms at stage L4 were identified as day 0 for the analysis of lifespan. For the exposure of worms with DNIC-1a, worms were first moved to NGM plates without OP50 bacteria and then treated with 15 μM of DNIC-1a, prepared with M9 buffer, for 15 minutes on day 1, 4, and 7. Treatment steps for the control group were all the same, except that DNIC-1a was replaced with M9 buffer only. Worms were rinsed three times with M9 buffer after incubation and placed back to NGM plates with OP50 bacteria. Worms were considered dead when they stopped pharyngeal pumping or did not respond to pokes by a platinum wire. Worms died within seven days or with internal hatching were removed from the plates and excluded from lifespan calculation.
Analysis of Cell Senescence in C. elegans
Formation of cell senescence was evaluated based on the detection of senescence-associated beta-galactosidase (SA-βgal) activity using 5-bromo-4-chloro-3-indolyl-β-D-galactosiase (X-gal) as the substrate. All solutions and reagents used for β-Galactosidase cell staining were prepared according to the protocol. For the two treated groups, worms were transferred to NGM plates without OP50 bacteria. 10 μM of DNIC-1a and M9 buffer, respectively, were then provided on day 1, 4 and 7. Three-time wash with M9 buffer was executed after incubation. Worms were then placed back to NGM plates with OP50 bacteria and cultured until day 13. Worms on day 13 were collected and rinsed one time with 1X PBS before soaked with 1× fixative solution for 15 minutes. Worms were rinsed two times with 1× PBS after 1× fixative solution was removed. Worms were further incubated with the β-Galactosidase staining solution at 37° C. overnight. The intensity and area of the dark blue stain, which is indicative of SA-βgal activity, of each worm was quantified using ImageJ. All experiments were repeated at least three times. Control groups of worms on day 1, 7, and 13 were chosen individually for β-Galactosidase cell staining.
Activation of sGC in CX3553 Worms
The CX3553 (str-1::GFP) strain was selected to evaluate the activation of sGC using its intrinsic cGMP-dependent expression of GFP. 10 Worms on day 1 were treated with M9 buffer, 400 μM of DNIC-1a, and 400 μM of DNIC-2a, respectively, for 30 minutes and rinsed three times with M9 buffer. These worms were anesthetized via incubation with 0.1% tricane/0.01% tetramisole before the fluorescence images were taken. The fluorescence intensity of each worm was quantified using ImageJ.
Toxicity Test of DNIC in Zebrafish Embryos
WT-AB embryos at 3 days post fertilization (dpf) were soaked in 50 μM of DNIC-1a, 50 μM of DNIC-1b, 50 μM of DNIC-2a, and 200 μM of SNP for 30 minutes, respectively. The embryos were then rinsed three times and incubated at 28° C. The survival rate of embryos was tracked for the following 48 hours after the treatment. All experiments were triplicated.
Analysis of Vasodilation in Zebrafish Embryos
50 Tg (kdr:EGFP) embryos at 3 days post-fertilization (dpf) were incubated with fresh water, 50 μM of DNIC-1b, 50 μM of DNIC-2a, and 200 μM of sodium nitroprusside (SNP), respectively, for 30 minutes. The embryos were rinsed three times with fresh water after the treatment. Bright field and fluorescence images were then taken to measure the dorsal aorta diameter. All experiments were repeated three times.
Analysis of Myocardial Contractility and Cardiac Output in Zebrafish Embryos
Treatment of 3-dpf AB (wild type) embryos with fresh water, 50 μM of DNIC-1b, 50 μM of DNIC-2a, 200 μM of SNP, and 5 μg/mL epinephrine, respectively, for 30 minutes was conducted, which is followed by a three-time rinse with fresh water. 50 consecutive images recording at least 8 sequential cardiac cycles were taken to measure the short axis of the ventricular diameter during ventricular end systole (ES) and end diastole (ED). Fractional shortening was calculated from the formula:
Ventricular volume was calculated from the formula:
assuming the ventricle as a prolate spheroid, whereas l is the long-axis radius and s is the short-axis radius of the ventricle. Heart rate, beats per minutes, was measured by counting the heart beats in 20 seconds manually. Cardiac output was calculated from the formula: cardiac output=heart ratex (ED volume−ES volume). All experiments were repeated at least three times.
Statistics
All data were analyzed using Microsoft Excel and displayed in means±SD. Data comparisons were made using Student's t-test or Log-Rank test. Differences were judged to be statistically significant at a p-value <0.05. Statistical significance was specialized as * for p-value <0.05, ** for p-value <0.01, and *** for p-value <0.001.
In example 1, the reaction of a variety of DNICs with metmyoglobin was investigated so as to verify the universal role of bridging ligands, thiolate vs. pyrazolate, on dictating the NO−-transfer reactivity. As shown in
Regarding the reported .NO electronic structure in DNIC-1c and the null NO−-transfer reactivity, the .NO-release reactivity of DNIC-1 was evaluated by colorimetric Griess reagent and specific fluorescence probe FA-OMe. In contrast to the stability of DNIC-1a under anaerobic condition, steady decay of the UV-vis absorption band at 362 nm under aerobic condition revealed the O2-triggered decomposition of DNIC-1a (
aThe concentration of DNICs are 15 μM.
2.1 Effect on Aging and Lifespan
Extensive study on the mechanism for aging process using the transparent and primitive C. elegans with short lifespan was pursued to identify the aging-related genes and biological active compounds for modulation of aging. Trafficking of DNIC-1a in C. elegans and, in the meanwhile, the function of released .NO from DNIC-1a on the aging process were investigated in Example 2.1.
To explore the biological function of .NO released from DNIC-1a in C. elegans, influence of DNIC-1a on the lifespan and on the formation of cell senescence, which is indicative of biological aging, was further evaluated. Lifespan analysis on the C. elegans (n=200) respectively incubated with 0 μM and 10 μM of DNIC-1a for 15 minutes on Day 1, 4, and 7 was achieved. Tri-incubation of C. elegans with 10 μM of DNIC-1a resulted in a 10.7% extension of lifespan from 16.9±0.5 days to 18.7±0.4 days (
2.2 Effect on Vascular and Myocardial System
In addition to the critical function of .NO-release reactivity of DNIC-1a on long-term physiological regulation of aging process, the role of .NO-release reactivity of DNIC-1a and efficient NO−-transfer reactivity of DNIC-2a in vascular and myocardial system was investigated. Soluble guanylate cyclase (sGC) was evolved to sense both .NO and NO− for accelerated conversion of GTP into cGMP and for .NO/cGMP-dependent physiology. Using the heme-containing sGC in C. elegans as an in vivo sensor for NO, CX3553 worms featuring cGMP-dependent expression of green fluorescence protein (GFP) in AWB neurons were adopted to screen the nitrosylation reactivity of DNIC-1a and DNIC-2a. As illustrated in
aZebrafish was incubated with 50 μM of DNIC-1b, 50 μM of DNIC-2a, 200 μM of SNP, and 5 μg/mL epinephrine, respectively, for 30 min and washed three times with fresh water before the measurement was executed.
bED = end diastole, ES = end systole, Dd = short axis diameter at end diastole, Ds = short axis diameter at end systole.
eCardiac output was calculated from the formula: cardiac output = heart rate × (ED volume − ES volume).
2.3 Inotropic Effect
Observation of efficient nitroxyl-transfer reactivity of DNIC-2 toward FeIII-heme center as well as the potent efficacy on activation of sGC for vasodilation inspired us to characterize its role in myocardial system using the transparent zebrafish embryo. DNIC-1b and SNP were utilized as a dedicated .NO-donor to contrast the dual function of .NO/NO− in myocardial system, whereas epinephrine (epi) was used as a representative compound for activation of β-adrenergic dependent signaling pathway. After incubation of 3-dpf zebrafish with water, 50 μM DNIC-1b, 50 μM DNIC-2a, 200 μM SNP, and 5 pg/mL epi, respectively, for 30 minutes, 50 consecutive images of the heart area were taken to record 8 sequential cardiac cycles. Table 3 summarized the short-axis diameter at end diastole (ED) and end systole (ES), fractional shortening, ventricular volume, heart rate, and cardiac output. Of interest, a significant increase of the short-axis diameter of the heart and ventricular volume at end diastole (ED) was observed in the zebrafish treated with either DNIC-1b or SNP. This increase of ED short axis demonstrated that the .NO-releasing Fe-NO complexes triggered the myocardial relaxation at ED, while no effect was observed at end systole (Table 3,
It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.
