Patent Application
20240109843
The invention generally relates to compounds that exhibit enhanced activity and safety for the treatment of sickle cell disease (SCD). In particular, the compounds are aromatic aldehydes that exhibit highly potent polymer destabilizing effects.
Hemoglobin (Hb) functions to carry oxygen through the blood and release it to tissues by equilibrating between two allosteric states: a tense (T) state, which exhibits low-affinity for oxygen, and a relaxed (R) state, which exhibits high affinity for oxygen. Sickle cell disease (SCD) is an inherited hematologic disorder and occurs as a result of replacement of βGlu6 with βVal6 in Hb, forming sickle Hb (HbS). Under hypoxia or low oxygen (O2) tension, which leads to an increased concentration of the low-affinity deoxygenated (T-state) Hb, HbS polymerizes into long, rigid, and insoluble fibers resulting in sickling of red blood cells (RBCs). The polymer which is initiated by the primary interaction involving βVal6 is stabilized by several other secondary contacts between the HbS molecules. Hypoxia-induced sickling leads to secondary pathophysiological effects, e.g. adhesion of RBCs to tissue endothelium, oxidative stress/damage, hemolysis (rupture) of RBCs, inflammation, vaso-occlusion, impaired microvascular blood flow, and decreased vascular nitric oxide bioavailability, which eventually lead to severe morbidity, such as painful crises, stroke, kidney failure, pulmonary hypertension, and premature mortality. Prior to the recent FDA approval of Voxelotor (Oxbryta), the only oral drugs approved for treating SCD were hydroxyurea (HU) and L-glutamine (Endari™). HU induces γ-globin expression to form fetal Hb (HbF), which can directly inhibit HbS polymerization and RBC sickling. However, not all patients respond to HU and responses may be limited. HU can also cause myelosuppression, a life-threatening adverse effect. While L-glutamine may have anti-oxidant properties that can reduce the frequency of pain crises, efficacy is limited and the drug is seldom used. More effective oral drugs are urgently needed, especially in the face of the significant morbidity, mortality, healthcare disparities, and public health burden imposed by SCD.
Aromatic aldehydes, such as Voxelotor, bind to the α-cleft of Hb and shift the allosteric equilibrium to the high-affinity R-state of HbS, which does not polymerize. Allosteric modulators have a clinical benefit for patients with SCD. In the Phase III HOPE trial, Voxelotor reduced markers of hemolysis and improved anemia by increasing Hb concentrations by at least 1 g/dl in most patients. However, the disease-modifying clinical effects of Voxelotor were modest, with persistent signs of chronic inflammation and hemolysis. For example, a significant reduction in vaso-occlusive events was not observed. The major physiologic impact of allosteric modulation is HbS dilution; drug-bound oxygenated HbS tetramers cannot be incorporated into polymers and, thus, there are fewer Hb tetramers available to polymerize. However, the magnitude of clinical benefit from HbS dilution is limited, and patients on Voxelotor have significant residual disease sequalae that impacts quality of life and longevity. Furthermore, the on-target pharmacodynamic effects of the drug on Hb allosteric states are directly dose limiting, as further increases in Hb O2-affinity would limit release of O2 to tissues and, eventually, induce clinical signs of tissue hypoxia. Drugs that can directly inhibit HbS polymerization may have more disease-modifying benefits. For example, it is well known that individuals with a rare co-inherited condition in which HbF levels remain above 25% into adulthood have essentially no clinical disease. HbF interferes with important lateral contacts to directly destabilize polymerization Similarly, individuals with a rare co-inherited Hb variant, Hb Stanleyville, also have a benign clinical phenotype with essentially no disease due to a lysine mutation on the surface of αF-helix of HbS that interrupts polymer stabilizing contacts.
A number of aromatic aldehydes that act directly on HbS to inhibit polymerization have been discovered to date. These compounds generally have comprised a benzaldehyde with a second ring structure (pyridine or benzene) connected to the benzaldehyde via a methoxy bridge ortho to the aldehyde group. The methoxy bridge positioned ortho to the aldehyde group on the benzaldehyde allows the second ring structure to extend towards the mouth of the α-cleft to make interactions with surface-located residues on the αF-helix of HbS. Compounds that make strong interactions with key αF-helix residues can destabilize HbS polymers by perturbing important secondary stabilizing contacts. Examples of a previously discovered compound is INN-310 (SAJ-310), which contains a methoxy meta to the aldehyde on the benzaldehyde ring and no substituents on a pyridine ring. INN-310 showed only very weak polymer destabilizing effects in vitro, likely due to limited interactions with αF-helix residues, and moreover, exhibited suboptimal pharmacokinetic properties in vivo. Further examples of highly potent polymer destabilizing compounds with improved pharmacokinetic properties include VZHE-039 and PP-14. Instead of a meta methoxy as with INN-310, VZHE-039 and PP-14 contain an ortho hydroxyl on the benzaldehyde that forms an intramolecular interaction that stabilizes and protects the aldehyde from rapid oxidative metabolism. Both VZHE-039 and PP-14 each also incorporate an additional chemical substituent on the second ring that can make strong hydrogen-bonding and hydrophobic interactions with the αF-helix; as anticipated, these interactions significantly improved the potency of polymer destabilizing effects. However, up to this point, the addition of chemical substituents on the second ring has also introduced significant drawbacks that have prevented any compound from becoming a viable human drug candidate. Substituents on the second ring have either proven to be metabolically labile (carboxylester or carboxylamide), introduced charged species (carboxylic acid) that may have reduced membrane permeability, or have been hepatotoxic (methyl hydroxyl). Hundreds of compounds containing different classes of chemical substituents added to the second ring to target the αF-helix have been tested to date. Nevertheless, no compounds had yet been discovered that are both highly potent polymer destabilizers and can safely and reliably achieve therapeutic drug levels required for the chronic oral treatment of SCD.
Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.
It is an object of the disclosure to provide a compound having a generic formula
Also provided is a method of preventing or treating one or more symptoms or conditions of sickle cell disease (SCD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound having a generic formula
In some aspects, the one or more symptoms or conditions are selected from the group consisting of red blood cell (RBC) sickling, adhesion of RBCs to tissue endothelium, oxidative stress and/or damage, hemolysis of RBCs, inflammation, vaso-occlusion, impaired microvascular blood flow, stroke, pain, and death. In further aspects, the step of administering is performed orally.
In a first aspect (Aspect I), disclosed herein are vanillin-based compounds (
As noted in the Background section above, hypoxia or low oxygen (O2) tension leads to an increased concentration of low-affinity deoxygenated (T-state) Hb. The compounds disclosed herein bind to liganded HbS and hold the target protein in a high-affinity oxygenated (relaxed) state thereby decreasing the concentration of deoxygenated HbS, as well as preventing premature and fast release of the bound oxygen prior to liganded Hb reaching tissue beds.
Furthermore, deoxygenated HbS typically polymerizes into long, rigid, and insoluble fibers, resulting in sickling of red blood cells (RBCs). The F helix of HbS is very important in stabilizing these polymers through secondary interactions with adjacent HbS. Without being bound by theory, it is believed that the bulky ester or amide moieties, or the charged sulphate or phosphate groups of the compounds interact with the surface located F helix of Hb, and this interaction leads to stereospecific inhibition of polymer formation by HbS. In other words, binding of these ligands to the F helix induces a conformation change, occludes the F helix, and abrogates interactions between HbS, thereby weakening the polymer and preventing sickling.
Disclosed herein in a second Aspect, Aspect II, are benzaldehyde compounds that, unexpectedly, demonstrate potent and sustained polymer destabilization, in addition to allosteric modulation to increase the affinity of HbS for oxygen, despite having no chemical substituent on the second ring (pyridine or benzene) to make direct interactions with the αF-helix residues of HbS. These compounds lack a carboxylester, carboxylamide, methyl hydroxyl, or similar pharmacophore on the second ring that can make hydrogen bond interactions with surface residues of the αF-helix. However, unexpectedly, these compounds make very strong interactions with the αF-helix residues that are either entirely water-mediated directly through a pyridine, or alternatively a pyrimidine or triazine nitrogen, or even entirely hydrophobic via a benzene ring. Even more surprisingly, the direct polymer destabilization effects of some compounds in this group are the most potent observed to date, with sustained inhibition of RBC sickling in vitro for greater than 150 minutes. The results are unexpected because previously discovered compounds, e.g. INN-310, with no additional chemical substituents on the second ring to interact with the αF-helix demonstrated only very weak polymer destabilization effects, which was presumed to be due to lack of hydrogen-bond interactions. Thus, it was surprising that compounds in the present invention were not only potent, but the most potent discovered to date. The unexpected result appears to be the consequence of changes in the orientation of the compounds in the Hb a cleft with the addition of the ortho hydroxyl on the benzaldehyde ring to replace the meta methoxy of INN-310. This, fortuitously, moved the second ring structure in position to make its own strong interactions with αF-helix residues without any chemical substituents required. This is supported by the fact that changing the ortho hydroxyl to a halogen completely eliminated the polymer destabilizing effects of the compound. In retrospect, this also explains the reduced in vitro potency of TD-7, an analogue of VZHE-039, with meta methoxy instead of ortho hydroxy on the benzaldehyde similar to INN-310. The surface residues of αF helix of HbS are important for stabilizing the polymers through secondary interactions with the adjacent HbS. It is well known and accepted that the HbS variant Stanleyville, with a co-inheritance mutation on the surface of the αF-helix (αAsn78↔αLsy78), has limited polymerization. Without being bound by theory, it is believed that the compounds of the present invention interact with the surface located αF helix of HbS, and this interaction leads to stereospecific inhibition of polymer formation by HbS. In other words, binding of these ligands to the αF helix induces a conformation change, perturbs the αF helix, and abrogates interactions between HbS, thereby weakening the polymer and preventing sickling. While it was previously believed that hydrogen-bonding moieties, such as an esters or amide, methyl hydroxyl, or a charged sulphate or phosphate group, on the compounds was necessary to make a strong interaction to produce the desired pharmacodynamic effect, the present invention discloses that highly potent interactions can be made with compounds through entirely water-mediated or hydrophobic interactions alone. In addition to the surprising potency, compounds in this group also lack the negative drawbacks of other previous compounds with an additional moiety on the second ring, such as VZHE-039 and PP-14; the compounds have been observed to have rapid RBC partitioning, good oral bioavailability, impressive pharmacokinetics, and no signs of toxicity. In some versions of these compounds, one or more halogens replace hydrogen on either the benzaldehyde or second ring structures; these halogens do not interact with the αF-helix residues but instead have the potential to modify the metabolic pathway of compounds by limiting interactions with metabolic enzymes and/or eliminate potentially toxic metabolic intermediates.
Compounds disclosed herein are based on or derived from vanillin. The compounds, or a functional group or substituent of the compounds (e.g., the ester, amide, sulphate or phosphate groups) binds to the F helix of hemoglobin (Hb) and prevents or decreases the interaction between HbS molecules and thus prevent sickling of red blood cells (RBCs).
The compounds have a generic Formula I:
R1 and R2 are the same or different and are H; hydroxyl; halogen; or substituted or unsubstituted: alkyl, alkoxy, aryl, O-aryl, cycloalkyl or heterocyclic;
R3 is an alkyl ester, aryl ester, an alkylamide, an arylamide, phosphate or sulfate;
M and Q are the same or different and are O or (CH2)n where n=0-6 (e.g. 0, 1, 2, 3, 4, 5, or 6);
X, Y, Z, W and V are the same or different and are (independently) H, C, N, S or O;
m=0-6 (e.g., 0, 1, 2, 3, 4, 5, or 6), and
P=CHO or a promoiety.
Exemplary halogens include but are not limited to: Cl, Br, I and F.
Exemplary alkyl groups include but are not limited to: linear or branched C1-C12 alkyl e.g. methyl, ethyl, propyl, isopropyl, butyl (e.g. n-butyl, secondary butyl, isobutyl, tertiary butyl), pentyl (e.g. n-pentyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3-pentyl), hexyl, etc.) each of which may be substituted or unsubstituted. The alkyl group may be optionally substituted with one, two, or three substitutents, or, in the case of alkyl groups of two carbons or more, four substituents e.g. alkoxy, alkylsulfayl, amino, azido, halo, heterocyclyle)oxy, hydroxyl, nitro, oxo, thioalkoxy, thiol, etc.
Alkoxy refers to an alkyl group singularly bonded to oxygen (R—O), i.e. an alkyl group having an oxygen radical attached thereto. In some aspects, the R group of the alkoxy is a (C1-C12) alkyl (see above for exemplary alkyls) having an oxygen radical attached thereto. Exemplary alkoxy groups include but are not limited to: methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like, each of which may be substituted or unsubstituted. In some embodiments, the alkyl group or cyclic alkyl ring can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein (e.g., O, hydroxyl, alkoxy, etc.).
As used herein, “aryl” refers to any functional group or substituent that is or is derived from an aromatic ring, e.g., an aryl is a radical derived from an aromatic hydrocarbon by removal of a hydrogen atom. The aromatic hydrocarbon may be heteroaromatic (one or more atoms in the ring is/are not carbon and are instead e.g., O, N, S, etc.); polycyclic (containing two or more aromatic rings); or substituted (aromatic rings having other functional groups attached, e.g. alkyl, alkenyl, phenyl, aldehyde, hydroxyl, sulfhydryl, carboxyl, carbonyl, amide, amine, nitrate, sulfate, phosphate, pyridyl, etc.). Examples of aryl groups include but are not limited to: an aromatic hydrocarbon such as phenyl, naphthyl, thienyl, indolyl, tolyl, furyl, pyridyl, anthracenyl, fluorenyl, indanyl, indenyl, etc. each of which may be heteroaromatic and/or substituted or unsubstituted. “O-aryl” refers to an oxygen atom bonded to an aryl group, as described above, i.e., a radical derived from an aromatic hydrocarbon by removal of a hydrogen atom. O-aryl groups which are used in the compounds described herein include but are not limited to those listed above for aryl, each of which may be substituted or unsubstituted.
A cycloalkane is a cyclic compound in which all atoms of the ring (or rings) are C and the bonds between C atoms are saturated. As used herein, cycloalkyl refers to the radical (form that has an unpaired valence electron) and can form a bond with another entity. Cycloalkyl compounds may be unicyclic or polycyclic (e.g., bicyclic, tricyclic, etc.); and polycyclic substituents comprising more (e.g., 4, 5, 6, etc.) rings are also encompassed. If the system is polycyclic, the rings in the systems may have the same number of atoms (e.g., a bicyclic ring comprising two 6-membered rings); or the rings of the system may have two different types of rings (e.g., a bicyclic ring comprising one 6-membered ring and one 5-membered ring), and may be e.g., fused or bridged. Examples of cycloalkyls include but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, butyl, cyclopentyl, hexyl, heptyl, octyl, etc., and polycyclic combinations of these such as: decalin, camphorquinone, polyquinane, and the like. The cycloalkyl may be substituted or unsubstituted.
A heterocyclic compound or ring structure is a cyclic compound (e.g., a cycloalkane or aryl compound) that has atoms of at least two different elements as members of its ring(s). Generally, each ring of the system has about 4, 5, 6, or 7 atoms, at least one of which is carbon, and at least one of which is not carbon. For example, 1 or 2 atoms may be non-carbon atoms while the rest are carbon atoms. The rings may or may not contain double bonds, e.g. one or more C═C bonds. The heteroatoms are, for example, N, O, P or S, but other heteroatoms are not excluded. Exemplary heterocyclic systems are unicyclic, bicyclic or tricyclic; however, systems comprising more rings are also encompassed. If the system is multicyclic, the rings in the systems may have the same number of atoms (e.g., a bicyclic ring comprising two 6-membered rings) and the two rings may be the same or different; or the rings of the system may have two different types of rings (e.g., a bicyclic ring comprising one 6-membered ring and one 5-membered ring). Examples of heterocyclic systems that may be present in the compounds disclosed herein include but are not limited to: oxazole, pyrazoline, imidazole, pyrazole, pyrazine, purine, indoline, quinolone, pteridine, indene, piperidine, tetrahydrofuran, pyridine, pyrimidine, thiophene, pyrrole, furan, quinoline, benzothiophene, indole, benzofuran, heterocycles comprising two substituted benzene rings such as acridine, dibenzothiophene, carbazole, dibenzofuran, etc.
Alkyl esters and aryl esters are esters (chemical compounds derived from an organic or inorganic acid in which at least one —OH (hydroxyl) group is replaced by an —O-alkyl (alkoxy) group or an —O-aryl group, with the alkyl and aryl portions being those defined above.
Alkylamides and arylamide are amides (acid derivatives with the general formula R′—CO—NH2, where R′ is an alkyl or aryl group as described above.
Exemplary compounds of the invention include but are not limited to:
Mechanistically, these compounds increase the hemoglobin oxygen affinity and concomitantly exhibit antisickling activity in part via a Schiff-base interaction between the aldehyde moiety and the N-terminal αVal1 nitrogen of relaxed state hemoglobin. Like all aldehydes, the aldehyde moiety of the compounds is susceptible to rapid and significant metabolism, e.g. by aldehyde dehydrogenase, into the inactive carboxylate derivative that could potentially shorten the compounds pharmacologic effect. Consistently, several aromatic aldehyde antisickling agents, e.g., 5-HMF have failed in human clinical studies because of such metabolic shortcomings. To overcome such potential disruptive metabolism, in some aspect, the aldehyde group of the compounds presented herein is protected, e.g., via a coupling reaction with L-cysteine to form the thiazolidine complex 795). The promoiety or prodrug compounds exhibit improved bioavailability and/or a longer half-life due to decreased aldehyde metabolism. This permits the drugs to be administered i) at lower doses and/or ii) less frequently, while still maintaining the beneficial therapeutic effects of the unprotected aldehyde, thereby minimizing side effects and/or increasing patient compliance with administration.
Thus, in some aspects, the compounds described and depicted above may be further rendered resistant to in vivo metabolism by replacing the CHO functional group with a protecting group. Examples of suitable promoieties (pharmacophores) include but are not limited to
Other means of protecting the aldehyde include but are not limited to conversion of the aldehyde to the corresponding imine, acetal, hemiacetal, ester, or alcohol.
Examples of compounds that comprise a promoiety include but are not limited to:
In a second aspect (Aspect II), compounds disclosed herein are benzaldehydes with a second ring (e.g., pyridine, pyrimidine, etc.) that interacts directly with the αF helix of sickle hemoglobin (HbS) without any direct hydrogen-bond donor/acceptor group or substituent (e.g., ester, amide, hydroxyl, sulphate or phosphate groups). Binding of the compound to the αF helix of HbS prevents or decreases the interaction between HbS molecules, and thus prevent sickling of red blood cells (RBCs).
wherein W, X, Y, and Z are the same or different and are independently C or N, with the proviso that at least one of W, X, Y, and Z is an N, and R2, R3, R4, and R5 are the same or different and independently F, Cl or H. Exemplary halogens include but are not limited to F and Cl.
Exemplary compounds include but are not limited to those having a formula
Additional exemplary compounds have the general formula
Exemplary compounds of the invention include but are not limited to:
These compounds rapidly partition into RBCs to bind to N-terminal valine amines in the HbS α-cleft via a Schiff-base interaction between the aldehyde moiety and the N-terminal αVal1 nitrogen of relaxed state hemoglobin. Mechanistically, the Schiff-base interaction stabilizes the relaxed state resulting in increased hemoglobin oxygen affinity and, consequently, anti-sickling activity. Like all aldehydes, the aldehyde moiety of the compounds is susceptible to rapid metabolism, e.g. by aldehyde oxidase, into the inactive carboxylate derivative that could potentially shorten the duration of the compounds pharmacologic effect. Consistently, the aromatic aldehyde anti-sickling agent 5-HMF was abandoned in human clinical studies because of its metabolic liability. In some aspect, the aldehyde group of the compounds presented herein is protected via an intramolecular bond with the adjacent ortho hydroxyl. The ortho hydroxyl also serendipitously positions the second ring to interact with the F helix.
Pharmaceutically acceptable salts of the compounds are also encompassed. Examples of such salts include, but are not limited to, salts formed with inorganic acids (for example, hydrochloric acid (HCl), hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, methanesulfonic acid, p-toluenesulfonic acid and polygalacturonic acid. Other salts include pharmaceutically acceptable quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula —NR+Z—, wherein R is hydrogen, alkyl, or benzyl, and Z is a counterion, including chloride, bromide, iodide, —O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate).
Oral prodrugs of the compounds are also encompassed. Oral prodrugs may be used, for example, to improve oral formulation and/or oral bioavailability or improve absorption characteristics and gastrointestinal tolerability. Examples of such prodrugs include, but are not limited to prodrugs formed by replacing the aldehyde group with a promoiety, such as thiazolidine, imine, acetal, hemiacetal, ester, or alcohol.
In some aspects, the compounds will be administered as prodrugs including but not limited to the following:
A typical synthesis will involve the condensation of the benzaldehyde, 1 with equimolar L-cysteine ethyl ester, 2 in the presence of N-ethyldiisopropylamine hydrochloride (EDA) or triethyl amine (NET3) to yield the ethyl ester prodrug, 3. Hydrolysis of the ethyl ester protecting group of 3 with sodium hydroxide will yield the acid prodrug, 4.
The compounds described herein exhibit improved pharmacologic activity, i.e., more potency and/or increased (lengthened, longer-lasting, etc.) half-lives, and/or improved bioavailability under physiological conditions (e.g., in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold or more greater potency circulation, in blood, etc.) compared to other benzaldehyde compounds. For example, the compounds exhibit in vivo half-lives of at least about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5 or 12.0 hours, or even longer, e.g. about 12 to 60 hours, i.e. about 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, or 60 hours. In some aspects, compared to vanillin or TD-7 (
The compounds described herein in Aspects I and II are used to treat or prophylactically treat diseases and/or conditions related to SCD and other diseases/conditions described below. As used herein, “prophylactically treat” (“prophylactic treatment”, “prophylactically treating” etc.) and “prevent” (“prevention”, “preventing” etc.) refer to warding off or averting the occurrence of at least one symptom of a disease or unwanted condition (such as at least one symptom of SCD), by prophylactic administration of a composition comprising at least one compound as described herein, to a subject in need thereof. Generally, “prophylactic” or “prophylaxis” relates to a reduction in the likelihood of the patient developing a disorder or a symptom of a disorder. Typically, the subject is considered by one of skill in the art to be at risk of or susceptible to developing at least one symptom of the disease or unwanted condition or is considered to be likely to develop at least one symptom of the disease/condition in the absence of medical intervention. In some aspects, for “prevention” or “prophylactic treatment”, administration occurs before the subject has, or is known or confirmed to have, symptoms of the disease (condition, disorder, syndrome, etc.; unless otherwise indicated, these terms are used interchangeably herein). In other words, symptoms may not yet be overt or observable, or may be very “early stage” symptoms. The subject may be considered at risk due to a variety of factors, including but not limited to: genetic predisposition; evidence of “early” symptoms; tests such as blood tests, etc. In such aspects, treatment of the subject may prevent the noxious or harmful effects or outcomes (results) of full blown disease. “Prevention” or “prophylactic treatment” of a disease or condition may involve completely preventing the occurrence of detectable symptoms, or, alternatively, may involve lessening or attenuating the degree, severity or duration of at least one symptom of the disease that would otherwise occur in the absence of the medical interventions provided herein.
“Treat” (treatment, treating, etc.) as used herein refers to administering at least one composition comprising a compound as described herein, to a subject that already exhibits at least one symptom of a disease such as SCD. In other words, at least one parameter that is known to be associated with the disease has been measured, detected, experienced or observed in the subject. For example, the symptom may be the primary pathophysiology of RBC sickling associated with sickle cell disease. In addition, the compounds disclosed herein ameliorate several of the cascading secondary adverse events of SCD, including adhesion of RBCs to tissue endothelium, oxidative stress, hemolysis of RBCs, decreased vascular NO bioavailability, vaso-occlusion, impaired microvascular blood flow, increased blood pressure, stroke, and painful crises, e.g., due to polymerization of RBCs. For example, the compounds generally do one or more of the following: increase O2-affinity of HbS; decrease fiber formation; reduce sickle cell mechanical fragility; reduce RBC hemolysis; attenuate hypoxia-induced cell necrosis and apoptosis; improve microvascular function (e.g. from sequelae of SCD or during recovery from hemorrhagic shock); results in hemodynamic and oxygenation benefits during hypoxia (e.g. maintenance of blood pressure and heart rate; preservation of microvascular blood flow; reduction in heart and brain hypoxia areas); reduce pain or the frequency of pain crises; decrease lactate dehydrogenase and/or RBC hemolysis; reduce diastolic blood pressure; increase blood oxygen levels (SpO2) during hypoxia challenge, etc.
“Treatment” of a disease involves the lessening or attenuation, or in some instances, the complete eradication, of at least one symptom of the disease that was present prior to or at the time of administration of the composition.
Provided herein are compositions comprising at least one compound as described herein, and methods of administering the same to treat e.g., SCD, hypoxia, etc. Implementation of the methods generally involves identifying patients suffering from or at risk of developing a disease or condition described herein (for example SCD or hypoxia), and administering a composition as described herein by an appropriate route. The exact dosage to be administered may vary depending on the age, gender, weight and overall health status of the individual patient, severity of disease symptoms, or on other treatments being received by the patient, as well as the extent or progression of the disease condition being treated and the precise etiology of the disease. However, in general for administration to mammals (e.g. humans), sufficient composition is administered to achieve dosages in the range of from about 0.1 to about 1000 mg or more per kg of body weight per 24 hr., e.g. from about 1 to about 500 mg, 5 to 100, or 10-50 mg per kg of body weight per 24 hr. Generally, a therapeutically effective dose is from about 20 to about 150 mg per kg of body weight per 24 hr. The dose will vary with the route of administration, the bioavailability, and the particular formulation that is administered, as well as according to the nature of the malady that is being prevented or treated.
The compositions are generally administered in a pharmaceutically acceptable formulation which includes suitable excipients, elixirs, binders, and the like (generally referred to as “pharmaceutically and physiologically acceptable carriers”), which are pharmaceutically acceptable and compatible with the active ingredients. The prodrugs or derivatives may be present in the formulation as pharmaceutically acceptable salts (e.g. alkali metal salts such as sodium, potassium, calcium or lithium salts, ammonium, etc.) or as other complexes. It should be understood that the pharmaceutically acceptable formulations include solid, semi-solid, and liquid materials conventionally utilized to prepare solid, semi-solid and liquid dosage forms such as tablets, capsules, liquids, aerosolized dosage forms, and various injectable forms (e.g., forms for intravenous administration), etc. Suitable pharmaceutical carriers include but are not limited to inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers (diluents, excipients) include lactose, starch, conventional disintegrating agents, coatings, lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers include but are not limited to various aqueous or oil based vehicles, saline, dextrose, glycerol, ethanol, isopropanol, phosphate buffer, syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, isopropyl myristate, ethyl cocoate, octyl cocoate, polyoxyethylenated hydrogenated castor oil, paraffin, liquid paraffin, propylene glycol, celluloses, parabens, stearyl alcohol, polyethylene glycol, isopropyl myristate, phenoxyethanol, and the like, or combinations thereof. Water may be used as the carrier for the preparation of compositions which may also include conventional buffers and agents to render the composition isotonic. Oral dosage forms may include various thickeners, flavorings, diluents, emulsifiers, dispersing aids, binders, coatings and the like. The composition of the present disclosure may contain any such additional ingredients so as to provide the composition in a form suitable for the intended route of administration. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glycerol monostearate or glycerol distearate, alone or mixed with wax. Other potential additives and other materials (preferably those which are generally regarded as safe [GRAS]) include: colorants; flavorings; surfactants (TWEEN®, oleic acid, etc.); and solvents, stabilizers, binders or encapsulants (lactose, liposomes, etc.). Preservatives such as methyl paraben or benzalkium chloride may also be used. Depending on the formulation, it is expected that the active components (e.g. at least one prodrug or derivative) will be present at about 1% to about 99% of the composition and the vehicular “carrier” will constitute about 1% to about 99% of the composition. The pharmaceutical compositions of the present disclosure may include any suitable pharmaceutically acceptable additives or adjuncts to the extent that they do not hinder or interfere with the therapeutic effect(s) of the composition. Still other suitable formulations for use in the present disclosure can be found, for example in Remington's Pharmaceutical Sciences 22nd edition, Allen, Loyd V., Jr editor (September 2012); and Akers, Michael J. Sterile Drug Products: Formulation, Packaging, Manufacturing and Quality; publisher Informa Healthcare (2010).
The compositions (preparations) of the present disclosure are formulated for administration by any of the many suitable means which are known to those of skill in the art, including but not limited to: orally, by injection, rectally, by inhalation, intravaginally, intranasally, topically, as eye drops, via sprays, transdermally, sublingually, by rectal and buccal delivery, by inhalation of an aerosol, by microneedle delivery, etc. In some aspects, the mode of administration is oral, by injection or intravenously, preferably via an orally administered pill.
The administration of the compound of the present disclosure may be intermittent, or at a gradual or continuous, constant or controlled rate (e.g. in a sustained release formulation which further extends the time of bioavailability). In addition, the time of day and the number of times per day that the pharmaceutical formulation is administered vary and are best determined by a skilled practitioner such as a physician. Generally, the compounds are administered at least once a day, and may be administered e.g., 2, 3, 4, or more times per day. During a crisis, administration may be more frequent.
Administration of the compound by any means may be carried out as a single mode of therapy, or in conjunction with other therapies and treatment modalities, e.g., antibiotics, pain medication, hydroxyurea, vaccinations, blood transfusions, provision of supplemental oxygen, gene therapy, nitric oxide, drugs to boost fetal hemoglobin production, statins, etc. In addition, if disease sequalae includes other morbidities, e.g. a heart condition, then additional treatments for heart disease may be provided, including surgery. Other treatment options include various neutraceuticals, diet regimens, exercise, etc. “In conjunction with” refers to both administration of a separate preparation of the one or more additional agents, and to inclusion of the one or more additional agents in a composition of the present disclosure.
The subject to whom the composition is administered is generally a mammal, frequently a human, but this is not always the case. Veterinary applications of this technology are also contemplated, e.g., for companion pets (cats, dogs, etc.), or for livestock and farm animals, for horses, and even for “wild” animals that have special value or that are under the care of a veterinarian, e.g., animals in preserves or zoos, injured animals that are being rehabilitated, etc.
In some aspects, the disease or condition that is prevented or treated as described herein is sickle cell disease and pathophysiologies associated with SCD, such as RBC sickling. In addition, the compounds ameliorate cascading secondary adverse events, including adhesion of RBCs to tissue endothelium, oxidative stress, hemolysis of RBCs, decreased vascular NO bioavailability, vaso-occlusion, impaired microvascular blood flow, increased blood pressure, stroke, and painful crises. In addition, the compounds increase O2-affinity of HbS, decrease fiber formation, reduce sickle cell mechanical fragility, and reduce RBC hemolysis.
In other aspects, the compounds are used to treat or prevent symptoms of hypoxia that is or is not related to SCD. As used herein hypoxia (also known as hypoxiation) is a condition in which the body or a region of the body is deprived of adequate oxygen supply at the tissue level. Hypoxia is classified as either generalized, affecting the whole body, or local, affecting a region of the body. There are four types of hypoxia: (1) the hypoxemic type, in which the oxygen pressure in the blood going to the tissues is too low to saturate the hemoglobin; (2) the anemic type, in which the amount of functional hemoglobin is too small, and hence the capacity of the blood to carry oxygen is too low; (3) the stagnant type, in which the blood is or may be normal but the flow of blood to the tissues is reduced or unevenly distributed; and (4) the histotoxic type, in which the tissue and/or cells are poisoned and are therefore unable to make proper use of oxygen. Diseases of the blood, the heart and circulation, and the lungs may all produce some form of hypoxia.
Generalized hypoxia occurs, for example, in healthy people when they ascend to high altitude, where it causes altitude sickness leading to potentially fatal complications such as high-altitude pulmonary edema (HAPE) and high altitude cerebral edema (HACE). Hypoxia also occurs in healthy individuals when breathing mixtures of gases with a low oxygen content, e.g. while diving underwater or when in outerspace, and especially when using closed-circuit rebreather systems that control the amount of oxygen in the supplied air. Hypoxia also occurs as a consequence of preterm birth in the neonate due to immature lung development. Hypoxia resulting from ischemia (insufficient blood flow to a tissue or organ), is referred to as ‘ischemic hypoxia’ and is caused by e.g. an embolic event, a heart attack that decreases overall blood flow, or trauma to a tissue that results in damage, or may be purposefully induced in some medical procedures, e.g. implantation of a stent, application of a tourniquet, etc. Diseases such as peripheral vascular disease can cause local hypoxia. Other causes include alterations in respiratory drive, such as in respiratory alkalosis, physiological or pathological shunting of blood, diseases interfering in lung function resulting in a ventilation-perfusion mismatch, such as a pulmonary embolus, or alterations in the partial pressure of oxygen in the environment or lung alveoli. When hemoglobin is deficient, anemia can result and can cause ‘anemic hypoxia’ if tissue perfusion is decreased. Carbon monoxide poisoning can cause hypoxia, either acutely, as with smoke intoxication, or over a period of time, as with cigarette smoking or exposure to smog. Certain odorless asphyxiant gases (e.g. nitrogen, methane, etc.) induce hypoxia as does cyanide poisoning and the formation of methemoglobin e.g. by ingesting sodium nitrite or certain other drugs and chemicals. The compounds described herein are used to prevent or treat symptoms of one or more of any of these hypoxia-related conditions. In addition, the compounds attenuate hypoxia-induced cell necrosis and apoptosis, improve microvascular function during resuscitation from hemorrhagic shock, result in hemodynamic and oxygenation benefits during hypoxia (e.g. maintenance of blood pressure and heart rate; preservation of microvascular blood flow; reduction in heart and brain hypoxia areas, etc.), and provide improvement in several clinical symptoms, including reduced pain, decreased lactate dehydrogenase and/or RBC hemolysis, reduction in diastolic blood pressure, and an increase in blood oxygen levels (SpO2) during hypoxia challenge.
A generic scheme for making the compounds of the first aspect (Aspect I) disclosed herein is depicted below in Scheme I:
In this exemplary reaction, step (a) is conducted in the presence of N-bromosuccinimide (NBS), α,α′-Azoisobutyronitrile (AIBN), and CCl4, at a temperature in the range of from about 45-75° C., for about 5 h. (e.g. from about 1-10, 2-9, 3-8, 4-8, or 5 or 6 hours); and step (b) is conducted in the presence of K2CO3 and anhydrous DMF, room temperature, for about 5-10 h.
A generic scheme for making the promoiety-containing compounds of Aspect I disclosed herein is depicted below in Scheme II:
A typical synthesis will involve the condensation of the aldehydes, 2 with equimolar of L-cysteine ethyl ester, 2 in the presence of N-ethyldiisopropylamine hydrochloride (EDA) or triethyl amine (NET3) to afford the ethyl ester prodrug, 3. Hydrolysis of the ethyl ester protecting group of 3 with sodium hydroxide will yield the acid prodrug, 4.
In some exemplary aspects, the compounds include the “PP” series of compounds and are made by the following synthetic Scheme III:
Scheme IV describes a generic scheme for making the compounds disclosed herein is depicted below. Briefly, substituted hydroxybenzaldehyde and either substituted chloromethylpyridine or substituted bromomethylpyridine were reacted together under basic conditions at room temperature for 2 hours, and then at 60° C. for 4-6 hours to give Compounds 1-13 in 9-47% yields.
It is to be understood that this invention is not limited to particular embodiments described herein above and below, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range (to a tenth of the unit of the lower limit) is included in the range and encompassed within the invention, unless the context or description clearly dictates otherwise. In addition, smaller ranges between any two values in the range are encompassed, unless the context or description clearly indicates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Representative illustrative methods and materials are herein described; methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual dates of public availability and may need to be independently confirmed.
It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as support for the recitation in the claims of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitations, such as “wherein [a particular feature or element] is absent”, or “except for [a particular feature or element]”, or “wherein [a particular feature or element] is not present (included, etc.) . . . ”.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
We tested whether derivatizing the alcohol moiety of vanillin into aryl or alkyl amides and esters or inorganic phosphate esters would improve the pharmacologic properties of these compounds. First the substitutions would slow or make the compounds resistant to metabolism and lead to significantly sustained and enhanced pharmacologic activities. In addition, bulky ester or amide moieties would increase interactions with Hb with a concomitant increase in Hb oxygen affinity and lead to increased antisickling potency. The increased interactions would also slow down dissociation of the compound from the Hb and limit extensive metabolism of the free aromatic aldehyde. The increased hydrophobicity of the compound would also lead to increased partitioning of the compound into red blood cells and reduce off-target toxicity. Importantly, the bulky moiety on the pyridine ring would increase the interaction with the F helix subunit and lead to stereospecific inhibition of polymer formation, resulting in the compounds exhibiting a second antisickling mechanism of action that is independent of the primary mechanism of increasing Hb affinity for oxygen. The F helix is very important in stabilizing the polymer through secondary interaction between adjacent sickle Hb molecules, therefore any compound that binds to the F helix and affect its conformation is expected to abrogate this interaction and weaken the polymer with concomitant antisickling activity. Consistently, the Hb variant Stanleyville (αAsn78↔αLsy78) inhibits polymerization.
A mixture of 2-hydroxyl-5-methoxybenzaldehyde (1 eq) and methyl 2-(bromomethyl)isonicotinate (1 eq) was dissolved in anhydrous N,N-Dimethylformamide (DMF). Anhydrous potassium carbonate (K2CO3) (1.2 eq) was added to this mixture and the reaction was stirred at room temperature for 8-10 hours. The solvent was then evaporated and the reaction mixture extracted with ethyl acetate and water. The organic layer was dried over sodium sulfate, filtered and the solvent evaporated. The crude product was purified using SiO2 column chromatography and eluted with the solvent system EtOAc: hexanes=3:2 to obtain pure product as white powder with a yield of 82%. IR (Diamond, cm−1): 2923, 1729, 1686, 1608, 1566, 1450, 1382, 1292, 1276, 1216, 1190, 1171; 1H-NMR (400 MHz, DMSO-d6): δ10.43 (s, 1H), 8.81 (dd, J=5, 0.56 Hz, 1H), 8.02 (s, J=1.44 Hz, 1H), 7.80 (dd, J=5.04, 1.56 Hz, 1H), 7.24 (m, 3H), 5.41 (s, 2H), 3.91 (s, 3H), 3.76(s, 3H); 13C-NMR (100 MHz, DMSO-d6): δ188.81, 165.02, 157.80, 154.78, 153.63, 150.44, 137.79, 125.09, 122.92, 121.82, 120.33, 116.25, 110.73, 71.27, 55.58, 52.81. HRMS (ESI) m/z found 302.10 (M+H)+, 324.08 (M+Na)+, Calculated 301.2940 [M]+. The purity of the compound was checked by HPLC and was found to be 98% pure.
Methyl 6-((2-formyl-3-hydroxyphenoxy)methyl)nicotinate (PP5)
Human blood samples were obtained from healthy volunteers at Virginia Commonwealth University. The use of the biological samples was reviewed and approved by the IRB, in accordance of institutional regulations. Leftover blood samples from patients with homozygous SS were obtained and utilized, based on an approved IRB protocol at the Children's Hospital of Philadelphia, with informed consent. Hemoglobin was purified from normal human blood samples according to the literature procedure.
Oxygen equilibrium curve (OEC) studies to determine the effect of the compounds on Hb affinity for oxygen were conducted following standard procedure. Whole blood (30% hematocrit) was incubated in the absence or presence of compounds (0.5 mM, 1.0 mM and 2.0 mM) solubilized in DMSO. The blood-compound reaction samples were then incubated in an IL 237 tonometer (Instrumentation Laboratories, Inc., Lexington, MA) for about 7 min at 37° C. under a gas mixture containing O2 at concentrations of 0.804%, 2.935% and 5.528% and allowed to equilibrate at O2 tensions of 6, 20 and 40 mmHg. After equilibration, the sample was removed via syringe and aspirated into an ABL 700 series table top automated blood gas analyzer (Radiometer America, Inc., Westlake, OH) to determine total hemoglobin (tHb), hematocrit (Hct), pH, pCO2, partial pressure of oxygen (pO2), and the Hb-O2 saturation (sO2) values. The measured values of pO2 and sO2 at each oxygen saturation level were then subjected to nonlinear regression analysis using the software Scientist (Micromath, Salt Lake City UT) with the following equation:
Time dependent and concentration dependent Hb-compound adduct formation studies were performed with whole blood using standard procedures. In a 96-well deep well plate (Thermo Scientific), 0.5 mM, 1.0 mM and 2.0 mM concentrations of the compounds were added to 600 μL of whole blood (30% hematocrit) and/or Hb (1.56 mM). These were incubated at 37° C. for 24 h with shaking (at 140 rpm). At 1, 4, 8, 12 and 24 h time intervals, 50 μL aliquots of this mixture were removed from each well using a multichannel pipette and added to respective tubes containing 50 μL of sodium cyanoborohydride (NaBH3CN) and sodium borohydride (NaBH4) mixture (1:1 v/v 50 mM stock) to terminate the Schiff-base reaction, fix the Schiff-base adducts and reduce the free reactive aldehyde. After mixing, the tubes were stored immediately at −80° C. until ready for batch analysis by cation-exchange HPLC (Hitachi D-700 Series, Hitachi Instruments, Inc. San Jose, CA). The observed Hb adducts, calculated as percentages (%) of Hb modification were plotted as a function of time (h); peak areas were quantitated using the HPLC software.
Blood suspensions from subjects with homozygous SCD (resuspended in Hemox™ buffer to a final hematocrit 20%) were incubated under air in the absence or presence of the compounds (0.5 mM, 1 mM and 2 mM) at 37° C. for 1 h to ensure that binding had attained equilibrium. The suspensions were then incubated under hypoxic conditions (2.5% oxygen/97.5% nitrogen) at 37° C. for 1 h. Aliquot samples were fixed with 2% glutaraldehyde solution without exposure to air, and then subjected to microscopic morphological analysis of bright field images (at 40× magnification) of single layer cells on an Olympus BX40 microscope fitted with an Infinity Lite B camera (Olympus), and the coupled Image Capture software. The residual samples were washed in phosphate-buffer saline, and hemolyzed in hypotonic lysis buffer for subsequent Hb-modification and oxygen equilibrium analyses.
For oxygen equilibrium studies, approximately 100 μL aliquot samples from the above clarified lysate were added to 4 mL of 0.1M potassium phosphate buffer, pH 7.0, in cuvettes and subjected to hemoximetry analysis using a Hemox™ Analyzer (TCS Scientific Corp.) to assess P50 shifts. Degree of P50 shift (ΔP50) was expressed as percentage fractions of control DMSO-treated samples.
Finally, for the Hb adduct formation studies, the above clarified lysates were subjected to cation-exchange HPLC (Hitachi D-7000 Series, Hitachi Instruments, Inc., San Jose, CA), using a weak cation-exchange column (Poly CAT A: 30 mm×4.6 mm, Poly LC, Inc., Columbia, MD). A commercial standard consisting of approximately equal amounts of composite HbF, HbA, HbS and HbC (Helena Laboratories, Beaumont, TX), was utilized as the reference for isotypes. The areas of new peaks, representing HbS adducts, were obtained, calculated as percentage fractions of total Hb area, and reported as levels of modified Hb.
To demonstrate potential non Hb-O2 affinity-dependent activities of the molecules, we tested the antisickling properties of selected compounds (PP9 and PP14) under complete deoxygenated conditions. We used TD7 and GBT440 as reference controls; the latter is also an aromatic aldehyde with the most potent reported in vitro p50 shifts and antisickling properties, and is currently in phase III clinical studies for the treatment of SCD. Briefly, antisickling studies were conducted as previously described, using 2 mM concentrations of the test molecules. After 1 h, aliquot samples were fixed with 2% glutaraldehyde without exposure to air. Then the incubation chamber was opened and exposed to air for 15 minutes to ensure complete re-oxygenation and reversal of the sickled cells to normal round cells. Reversal was confirmed by microscopy. The incubation chamber was then closed and the assay was repeated under 100% nitrogen gas for 30 minutes, at which point aliquots were obtained and fixed. Aliquot samples were then subjected to microscopic morphological analysis of bright field images (at 40× magnification) of single layer cells on an Olympus BX40 microscope fitted with an Infinity Lite B camera (Olympus), and the coupled Image Capture software. Resulting sickled cells (percentages) were compared across samples, and between aliquots of the same samples that had been obtained either under 2.5% oxygen or 100% nitrogen. This experimental design utilized aliquots of the same samples under different gas conditions, and thereby ensured that the assay was free of any potential errors associated with variability in hematocrit and accuracy in adding the molecules.
Freshly made solutions of compounds in DMSO were added to deoxygenated (deoxy) Hb (30 mg/mL protein) at an Hb tetramer-compound ratio of 1:10. Then, the complex mixture was saturated with carbon monoxide and allowed to incubate for 2 h to form COHb-compound complexes. Sodium cyanoborohydride (NaBH3CN) was then added to this mixture to reduce the Schiff-base adduct formed between the protein and compound to the corresponding irreversible alkylamine covalent bond. The resulting solution was crystalized using 10-20% PEG 6000, 100 mM HEPES buffer, pH 7.4 using the batch method as previously published. Single cherry red needle crystals were formed in 1-3 days and were used to collect x-ray diffraction data at 100 K using Rigaku MicroMax™ 007HF X-ray Generator, Eiger R 4M Detector and Oxford Cobra Cryo-system (The Woodlands, TX). The crystals were first cryoprotected with 80 μL mother liquor mixed with 62 μL of 50% PEG6000. The dataset was processed with the d*trek software (Rigaku) and the CCP4 suite of programs. The crystal structures of the COHb-compound complexes were determined by a molecular replacement method with Phenix program, using the native R2-state crystal structure (PDB ID 1BBB) as a search model. The structures were refined using both Phenix and CNS while model building and correction was carried out using COOT.
We tested in vivo pharmacologic (pharmacodynamics) effects of select PP compounds in C57BL/6 mice. The animals were treated with a single intraperitoneal (IP) (150 mg/kg body weight) dose of PP6, PP10, PP14, and TD7, which were formulated with 30% PEG300. Blood samples were obtained prior to treatment (0) and 1, 3 and 6 h post-treatment, via submandibular bleeding, and subjected to hemolysis using standard methods. Clarified lysates, free of cell debris and red blood cell ghosts were analyzed by cation-exchange HPLC to determine the levels of drug-modified hemoglobin (adducts); and to conduct oxygen equilibrium studies to determine degrees of shift in p50 values using methodologies described earlier for in vitro studies on human samples. In a subsequent follow-up experiment, a new excipient obtained from Catalent Pharma Solutions was also used to formulate PP14 and used for a single intraperitoneal (IP) and oral (150 mg/kg body weight) dose. Blood samples were obtained prior to treatment (0) and 1, 3 or 5 h post-treatment, via submandibular bleeding, and used to determine the levels of drug-modified hemoglobin (adducts); and to conduct oxygen equilibrium studies to determine degrees of shift in p50 values as described above.
A representative general scheme for synthesizing “PP” compounds is shown in Scheme III (above) and representative compounds PP1, PP2, PP3, PP4, PP5, PP6, PP7, PP8, PP9, PP10, PP11, PP12, PP13, and PP14 are depicted in
Based on crystallographic binding of vanillin and TD7 to Hb, we structurally modified the TD compounds into PP compounds to increase interactions with Hb, as well as make closer contacts with the F helix. Several PP compounds were crystallized with Hb and with CO-liganded Hb and the crystal structures of several of these complexes in the R2-state conformation were successfully determined. The structure of PP9 which has been refined to 1.9 Å is described here, noting that the other complexes show similar binding, although differences also occur that might explain differences in their functional activities.
The overall tetrameric structure of PP9 is indistinguishable (rmsd ˜0.4 Å) from the native R2 structure (1BBB) or the R2 structure in complex with 5-HMF (PDB code 1QXE) or in complex with TD7. As observed with these compounds, a pair of PP9 covalently bound to the N-terminal αVal1 amines (Schiff-base interaction) in a symmetry-related fashion is seen at the α-cleft as shown in
In summary, replacing the hydroxyl group with an ester leads to novel compounds that (1) bind strongly to Hb and exhibit increased functional and biological effects; (2) increase strong interactions with the F helix that translate into strong perturbation of the helix and lead to polymer destabilization, leading to a second mechanism of antisickling; and (3) reduce dissociation of the bound compound, thus reducing metabolism. These observations are consistent with functional and biological data.
Aromatic aldehydes are known to prevent hypoxia-induced Hb S polymerization by increasing the Hb affinity for oxygen. We therefore tested the PP compounds in a dose-dependent manner (0.5, 1, 2 mM) for their effect on Hb oxygen binding property. Vanillin and TD7 were tested as controls (
Aromatic aldehydes that increase Hb affinity for oxygen with a concomitant antisickling effect do so by using the aldehyde moiety to bind to the N-terminal αVal1 nitrogens of Hb, forming Schiff-base adducts. Schiff-base adducts can be accurately quantified by HPLC (Hb modification or Hb adduct). To test the degree and duration of Hb modification, we incubated 2 mM concentrations of PP compounds, and the controls vanillin and TD7 with normal adult blood, hematocrit adjusted to 30% at 37° C. for 24 h. At defined time points (1, 4, 8, 12 and 24 h) aliquot samples were drawn, mixed with a solution containing sodium borohydride/cyanoborohydride to fix the Schiff-base, and subsequently analyzed by cation-exchange HPLC. The results in
As noted above, a Schiff-base interaction is an equilibrium between bound and unbound complex, and when unbound the compound can potentially be metabolized into a pharmacologically inactive non-aldehyde. Therefore, for formation of a stable Schiff-base complex and thus an effective pharmacologic outcome, aromatic aldehydes would make strong interactions to Hb, and exhibit a slow rate of dissociation. Our structural studies clearly show that binding between PP9 and Hb is stronger than binding between TD7 and Hb, suggesting that PP9 dissociates slowly from the bound protein when compared to TD7, and consistent with the results of the time-dependent Hb modification studies. Similar strong interactions with the protein, and particularly with the F helix, are also observed for several of the PP compounds. These findings validate the novelty of these compounds as they clearly suggest an improvement in the metabolic profile of the PP compounds when compared to TD7. As discussed below, these in vitro findings translated into improved PD/PK properties in vivo.
The studies described above were conducted using normal whole blood. Next, we investigated whether the improvement in Hb oxygen affinity and the compounds sustained activity also translated to similar effects in SS blood from SCD patients. The PP compounds with the control vanillin and TD7 were therefore tested using sickle RBCs for their effect on Hb modification, Hb oxygen affinity and sickling at 0.5, 1 and 2 mM. The results are summarized in
Also noteworthy and significant is the observation that while TD7 and some of the PP compounds demonstrated an almost linear correlation between their ability to increase Hb oxygen affinity and antisickling activity, others, such as PP2, PP3, PP6, PP8, PP9 and PP14 showed a weak correlation between these parameters. In fact, these compounds demonstrated the most potent antisickling effect despite only marginally increasing Hb affinity for oxygen. This observation is consistent with these compounds exhibiting multiple mechanisms of antisickling activity that include increasing the oxygen affinity of Hb, and direct stereospecific polymer destabilization; the latter is validated by the crystal structures that show that PP9 and several of the PP compounds make significant hydrophobic and hydrogen-bond interactions with the F helix. As discussed below, F helix perturbation leads to these compounds inhibiting RBC sickling even at 100% nitrogen, which is independent of the primary antisickling mechanism of increasing Hb oxygen affinity.
Placing a bulkier group e.g. ester or amide moiety on the pyridine moiety results in compounds that behave differently than previous aromatic antisickling aldehydes. The resulting molecules make closer interactions with the surface located αF helix and lead to stereospecific inhibition of polymer formation. Thus, the compounds exhibit a second antisickling mechanism of action (an Hb-O2 affinity independent antisickling mechanism), and this can be followed by conducting the antisickling assay at 100% nitrogen. Selected PP compounds were therefore tested using sickle RBCs for their antisickling effect at 100% nitrogen. TD7 and GBT440 were used as positive controls. GBT440 is also an antisickling aromatic aldehyde that is currently in phase III clinical studies for the treatment of SCD (clinicaltrials.gov, NCT03036813). GBT440 is highly potent in preventing hypoxia-induced polymerization and concomitant RBC sickling, and has shown proof-of-concept in animal models and humans. As indicated earlier, for internal control purposes, the same samples were used for testing the antisickling effect of these compounds with 2.5% oxygen prior to testing under nitrogen gas, therefore helping elucidate the antisickling effects due to increased Hb affinity for oxygen. The results are summarized in
Three of the PP compounds (PP6, PP10 and PP14) with high and sustained functional/biological functional/biological activities were selected for an assessment of their in vivo pharmacologic (pharmacodynamic) effects using wild-type C57BL/6 mice. Excipients for formulating the compounds were optimized.
In an earlier experiment, using 30% PEG300 to formulate the test compounds, the animals were treated with a single intraperitoneal (IP) 150mg/kg body weight) dose of PP6, PP10, PP14. All three selected PP compounds demonstrated significant in vivo modification of intracellular Hb in mice after IP administration, with increasing levels from 1 h to the 6 h l experimental period (n=2 mice per compound,
Using the 30% PEG300 excipient did not give a uniform dispersion and this could affect bioavailability and the PD effect. We therefore experimented with several different excipients for formulating one of the compounds, PP14, for testing. The optimal excipient, which gave uniform compound dispersions, was obtained from Catalent Pharma Solutions. Consistent with the optimized formulation, we observed a significant increase in PD effect. At 150 mg/kg, mice treated with PP14 via IP injection showed significantly higher Hb modifications of 21.8±1.6% (1 hr), 37.7±5.0% (3hr) and 51.1±4.3% (5 hr), respectively (
The study demonstrates the superiority of the PP compounds compared to TD7 and vanillin, which is likely attributable to the structural modifications described herein.
We have developed new compounds to overcome the disadvantages of predecessor molecules. The compounds exhibit several advantageous attributes: first, the structural modifications allow the compounds to make very strong interactions with the surface located αF helix of Hb, which is known to be important in stabilizing the polymer. The interactions with the F helix destabilize the polymer, resulting in the compounds exhibiting an independent and novel Hb-O2 antisickling mechanism of action, in addition to the primary Hb-O2 dependent antisickling mechanism. This dual mechanism translated into very potent and significantly enhanced antisickling activities. Moreover, the Hb-O2 independent antisickling mechanism permits the prevention of sickling without drastically changing oxygen delivery capabilities, making the compounds more useful in treating SCD compared to other related aromatic aldehydes. This is critical for a disease that is characterized by severe hypoxia.
Second, due to the structural modifications, the compounds are resistant to metabolism due to enhanced binding to hemoglobin, which stabilizes the Schiff-base adduct and leads to significantly sustained and improved pharmacologic activities in vitro and in vivo. These attributes are critical properties for a regularly administered agent to treat SCD—as it is chronic condition.
To a stifling solution of L-cysteine ethyl ester hydrochloride (15.9 mmol) and ethyl-diisopropyl amine (15.9 mmol) in anhydrous ethanol (30 mL) at room temperature is added dropwise an equimolar amount of the aldehyde compound (15.9 mM) in anhydrous ethanol (20 mL). The reaction mixture is stirred at the same temperature for 2 hrs or overnight. The mixture is then diluted with water (100 mL) and the product extracted with ethyl acetate (3×50 mL). The organic phase is dried, evaporated, and the product purified through a column with EtOAc:Hexanes as the eluent.
Recently, we, serendipitously, discovered a set of benzaldehydes that exhibit highly potent polymer destabilizing effects, but without any chemical substituent on the second ring (pyridine or benzene) that would be expected to be needed to make strong interactions with the αF-helix residues, other than possibly the replacement of a hydrogen with a halogen (e.g., F or Cl). Initially, these compounds were synthesized and tested on the basis that they represented a chemical scaffold for the discovery of new polymer destabilizing benzaldehydes by screening new chemical substituents to add to the second ring, but it was presumed that they would have very weak polymer destabilizing effects, similar to INN-310, since they lack a chemical substituent to make strong interactions with the αF helix, as with VZHE-039 and PP-14. However, unexpectedly, these compounds can make very strong interactions with the αF-helix residues that are either entirely water-mediated directly through a pyridine, or alternatively a pyrimidine or triazine nitrogen, or even entirely hydrophobic via a benzene ring. Although in some aspects, halogens replace hydrogen on the ring structures, these halogens do not interact with the αF-helix residues, but instead are intended to possibly limit or modify interactions with metabolic enzymes and/or eliminate possibly toxic metabolic intermediates. The unexpected result appears to be the consequence of slight changes in the orientation of the compounds in the Hb a cleft with the addition of the ortho hydroxyl on the benzaldehyde ring to replace the meta methoxy of INN-310. This, fortuitously, moved the second ring structure in position to make its own strong interactions with αF-helix residues without any hydrogen-bond donor/acceptor substituent required. On the contrary, replacement of the ortho hydroxyl on the benzaldehyde with a fluorine (Compound 9 and 13) completely negates the polymer destabilization effects of the compounds. Also, addition of a meta fluorine next to the ortho hydroxyl on the benzaldehyde (Compound 12) significantly reduced the potency of polymer inhibition.
Therefore, a series of novel analogues with unexpected polymer destabilizing effects and surprising potency has been prepared. In fact, Compounds 1 and 2 (Table 1) are the most potent polymer destabilizers discovered to date, consistently demonstrating rapid partitioning into RBCs and robust and sustained inhibition of RBC sickling in complete anoxia (
Synthesis of 2-hydroxy-6-(pyridin-3-ylmethoxy)benzaldehyde (Comp 1)
99%; 1H NMR (80 MHz, CDCl3) δ12.02 (s, 1H), 10.55 (s, 1H), 8.82 (d, J=4.9 Hz, 2H), 7.53-7.09 (m, 2H), 6.50 (dd, J=11.4, 8.4 Hz, 2H), 5.42 (s, 2H).
Synthesis of 3-fluoro-2-hydroxy-6-(pyridin-3-ylmethoxy)benzaldehyde (Comp 12)
All compounds (Comp 1-13) and comparator molecules (VZHE-039, PP-14, INN-310, and Voxelotor) were evaluated following established procedures. Blood samples from individual donors with homozygous SCD (obtained with informed consent) were diluted using Hemox buffer solution with the addition of glucose (10 mM) and BSA (0.2%) to adjust hematocrit of the blood suspensions to 20%, which standardizes the procedure to normalize the ratio of RBCs to drug, for assay consistency and reproducibility. Briefly, blood suspensions were incubated under room air in the presence of various concentrations of test compounds (0-2 mM) at 37° C. for 1 hour in 96-well round bottom plates. Subsequently, the suspensions were incubated with a hypoxic gas mixture (2.5% Oxygen gas/balance Nitrogen gas) at 37° C. for up to 2 hours prior to fixation with 2% glutaraldehyde solution without exposure to air. The fixated blood aliquots were evaluated by microscopic morphological analysis (image analysis on Image J software) to identify the proportion of sickled and non-sickled RBCs, and sickle inhibition calculated based on the % change from negative control (DMSO).
To establish oxygen-independent sickle inhibition, the incubation chamber was subsequently opened and exposed to room air for 15 minutes to ensure complete re-oxygenation and reversal of the sickled cells to normal round cells. Reversal was confirmed by microscopy. The incubation chamber was then closed, and the assay was repeated with incubation in 100% nitrogen gas for 1 hour (for screening), or up to 2.5 h (for time-course studies), with collection and fixation of blood aliquots at defined intervals. Aliquot samples were again subjected to microscopic morphological analysis of bright field images to identify the proportion of sickled and non-sickled cells, and sickle inhibition calculated.
The residual samples were then washed in phosphate-buffered saline, and hemolyzed in hypotonic lysis buffer, with or without sodium borohydride (50 mM). Aliquots obtained from the lysates with or without sodium borohydride (to reduce Schiff base adducts) were also assessed for level of Hb modification (drug-adduct formation) by cation-exchange HPLC (Hitachi D-7000 Series, Hitachi Instruments, Inc., San Jose, CA) with a weak cation-exchange column (Poly CAT A: 50 mm×4.6 mm, Poly LC, Inc., Columbia, MD). Hemoglobin isotype peaks are eluted with a linear gradient of phase B from 0% to 80% at A410 nm (Mobile Phase A: 40 mM Bis-Tris, 2 mM KCN, pH 6.9; Phase B: 40 mM Bis-Tris, 2 mM KCN, 0.2 M sodium chloride, pH 6.55). Clarified lysate from normal human adult Fib (Hb A), as well as commercial standards containing approximately equal amounts of Hb F, A, S and C (Helena Laboratories, Beaumont, TX), were utilized as reference isotypes. Reduced samples were used to assess “fixed adduct” formation (dependent on RBC membrane permeability), whereas non-reduced samples assessed drug-adduct formation independent of drug permeability.
RBC partitioning studies were conducted both for SS RBCs following sickling studies above, or as separate independent studies with non-sickle blood. Briefly, aliquots of blood samples, which had been incubated with test molecules, were lysed either in deionized water (un-fixed), or in water containing 50 mM sodium borohydride (fixed). Samples were subjected to cation exchange HPLC analyses, as described above. Modified Fib (Hb adducts) was determined as a proportion of total Hb in each sample, and partitioning ratio was determined as the relative fraction of Hb modification of fixed samples compared to corresponding non-fixed samples. The partitioning ratio is the relative fraction of Hb that was modified intracellularly by molecules that permeated the RBC membrane, as compared to unfixed adducts which reflect Hb binding without RBC membrane permeation.
Non-sickling human blood was incubated with test compounds at 0.25-2 mM concentrations at 37° C. for 1 hour in 96-well round bottom plates. At conclusion, RBCs were washed once with PBS, followed by lysis with deionized water. Approximately 100 μl aliquots of individual lysates were added to 3 mL of 0.1M potassium phosphate buffer, pH 7.0 in a cuvette and subjected to hemoximetry analysis using a Hemox™ Analyzer (TCS Scientific Corp.) as previously described. Change in p50 values (Δp50) was calculated by subtracting the p50 values of treated samples from the value of the control sample and expressing the difference as a percentage of control p50 values.
Single doses of each compound were administered as oral suspensions by gavage in 0.5% carboxy methylcellulose at 100 mg/kg. Whole blood levels were collected for at least 72 hours after dose administration. Bioanalyses were performed with liquid chromatography-tandem mass spectrometry (LS-MS).
Repeat doses of each compound were administered once daily as oral suspensions by gavage in 0.5% carboxy methylcellulose for 14 days at 100 mg/kg. Whole blood was collected at 7 time-points for up to 24 hours during the final dosing interval on Day 14. Clinical observations, body weights, and food consumption were recorded throughout the 14 day study. Clinical chemistry and hematologic parameters were assessed, and histopathology was performed on organs after 14 days of exposure to study drug.
Co-crystals of liganded Hb with compounds 1, 2, 5, and 8 were obtained following published methods. Briefly, 40-50 mg/mL of Hb in a round bottom flask was vacuumed for ˜1 hour to remove oxygen and form deoxygenated Hb. Subsequently, carbon monoxide was bubbled into the deoxygenated Hb to form CO-ligated Hb (COHb). COHb was then incubated with a 10-20 molar excess of compounds 1, 2, 5, or 8 for about 1 hour to form COHb-compound complexes. Sodium cyanoborohydride (NaCNBH4) was added at a 10 molar excess to reduce the reversible Schiff base adduct to the corresponding irreversible alkylamine covalent bond. COHb-compound complex solutions were then crystalized with 10-20% PEG 6000, 100 mM HEPES buffer, pH 7.4 using the batch method. X-ray diffraction data was collected at 100 K using Rigaku MicroMax™ 007HF X-ray Generator, Eiger R 4M Detector and Oxford Cobra Cryo-system (The Woodlands, TX). The crystals were first cryoprotected with 80 μL mother liquor mixed with 62 μL of 50% PEG6000. The diffraction data was processed using CrysAlisPro software (Rigaku) and the CCP4 suite of programs. The crystal structure of the complex was solved by the molecular replacement method with the Phenix program, using the native R2-state crystal structure (PDB ID 1BBB) as a search model. The structure was refined using Phenix and COOT.
While the invention has been described in terms of its several exemplary embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.
Compounds 1-13 in Table 1 were synthesized as shown in scheme 4. Briefly, substituted hydroxybenzaldehyde and either substituted chloromethylpyridine or substituted bromomethylpyridine were reacted together under basic conditions at room temperature for 2 hours, and then at 60° C. for 4-6 hours to give Compounds 1-13 in 9-47% yields. A detailed description of the synthesis of each compound is reported in the experimental section. The synthesis of comparator molecules in Table 2, such as TD-7, INN-310, PP-14, and Voxelotor, have been reported in the literature.
Compounds 1-13 (Table 1) were studied with comparator molecules INN-310, TD-7, VZHE-039, PP-14, and Voxelotor (Table 2). The syntheses of the comparator molecules have been reported in the literature. All comparator molecules are aromatic aldehydes and are known to increase Hb affinity for oxygen with concomitant inhibition of hypoxia-induced RBC sickling. While PP-14 and VZHE-039 exhibit potent anti-sickling effects through direct polymer destabilization, Voxelotor, INN-310, and TD-7 show limited to no such effect.
Compounds 1-13 formed adducts with Hb and exhibited potent sickle inhibition following incubation in hypoxia, and most compounds in the class, unexpectedly, also demonstrated potent polymer destabilizing effects with sickle inhibition in anoxia (Table 3;
In partitioning studies, all compounds exhibited high partitioning ratios above 90% (Table 4), indicating that the compounds rapidly permeated the RBC membrane. Therefore, differences in fixed drug-Hb adducts reflect largely only differences in Hb binding affinity of each compound. The most potent compounds, Comp 1, 2, and 5, demonstrated greater than 90% Hb adducts after incubation at a 2 mM concentration in AA blood.
Compounds 1-13 were co-crystallized with liganded Hb (carbonmonoxy Hb or COHb) using low-salt condition. Only Compounds 1, 2, 5 and 8 resulted in X-ray diffraction quality crystals with Hb, leading to their structure determination at 1.9 Å to 2.1 Å resolutions (
Most importantly, and consistent with the compounds' oxygen-independent anti-sickling effects in completely anoxic conditions, Compounds 1, 2, 5, and 8, like VZHE-039 and PP-14 but not INN-310 and Voxelotor, also make hydrogen-bond interactions with the surface-located αF-helix residues (
Single-dose pharmacokinetics (PK) in male Sprague Dawley (SD) rats (n=4) were assessed for each compound, and the maximum or peak whole blood concentration and area under the curve are shown in Table 5. Compounds 1, 2, and 5 exhibited the greatest oral exposure levels observed to date for polymer destabilizing benzaldehydes, especially on a molar equivalent basis. While not shown, PP-14 rapidly metabolizes into its carboxylic acid metabolite upon oral administration.
Repeat oral doses of each compound were administered by oral gavage for 14 days to male and female SD rats (n=3). Steady state peak drug concentrations and area under the curve for each compound is shown in Table 6. Compounds 2 and 5 exhibited the greatest oral exposure levels observed to date, especially on a molar equivalent basis. Oral exposure levels were of Compound 1 were similar to VZHE-039 and greater than Voxelotor, especially on a molar equivalent basis. Steady state exposures for the final dosing interval on Day 14 were similar to total compound exposure from single-dose PK. No clinical or histopathologic changes were observed after 14 days of exposure to Compounds 1, 2, or 5. Both clinical and histopathologic changes in the liver, including hepatocyte necrosis, was observed for animals administered VZHE-039 for 14 days.
This application is a continuation-in-part (CIP) application of U.S. Ser. No. 17/846,101 filed on Jun. 22, 2022, which was a divisional application of U.S. Ser. No. 16/982,645 filed Sep. 21, 2020, which was a national stage filing of PCT/US2019/022678 filed on Mar. 18, 2019, which claimed priority to U.S. Ser. No. 62/646,434 filed Mar. 22, 2018.This application also claims priority to U.S. Ser. No. 63/423,167 filed Nov. 7, 2022, and to U.S. Ser. No. 63/442,824 filed Feb. 2, 2023, and the complete contents of these is herein incorporated by reference.
This invention was made with government support under contract numbers HL156158, HL154864, and MD009124 awarded by the National Institutes of Health. The United States government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62646434 | Mar 2018 | US | |
| 63423167 | Nov 2022 | US | |
| 63442824 | Feb 2023 | US |
| Number | Date | Country | |
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| Parent | 16982745 | Sep 2020 | US |
| Child | 17846101 | US |
| Number | Date | Country | |
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| Parent | 17846101 | Jun 2022 | US |
| Child | 18501244 | US |
