JP2016512821A - Compositions and methods for modification of hemoglobin - Google Patents

Abstract

The present invention relates to pharmaceutical compositions for allosteric modification of hemoglobin (S) and their use in the treatment of diseases mediated by hemoglobin (S) and diseases that would benefit from tissue and / or cell supply. Provide a method.

Description

  The present invention relates to pharmaceutical compositions for the allosteric modification of hemoglobin (S) and their use in the treatment of diseases mediated by hemoglobin (S) and diseases that would benefit from tissue and / or cellular oxygen supply. Provide usage.

  Sickle cell disease is a red blood cell disease that is particularly seen in African and Mediterranean people. The basis of sickle cell disease is sickle hemoglobin (HbS or hemoglobin (S)), which contains point mutations to the predominant peptide sequence of hemoglobin (Hb).

  Hemoglobin (Hb) carries oxygen molecules from the lungs to various tissues and organs throughout the body. Hemoglobin binds to oxygen or releases oxygen through conformational changes. Sickle hemoglobin (HbS) contains point mutations in which glutamic acid is replaced by valine, and HbS is susceptible to polymerization, giving red blood cells containing HbS with its characteristic sickle shape. Sickle cells are also stiffer than normal red blood cells and are less flexible and can lead to occlusion of blood vessels. US Pat. No. 7,160,910 discloses compounds that are allosteric modifiers of hemoglobin. However, there is a need for additional therapeutic agents that can treat abnormal Hb mediated diseases such as Hb or HbS.

  The present invention relates generally to compositions suitable as allosteric modifiers of hemoglobin (S). In some embodiments, the invention relates to methods of treating diseases mediated by hemoglobin (S) and diseases that would benefit from tissue and / or cellular oxygen supply.

  In another aspect, the invention provides about 1 mg to about 10 g of a compound selected from the group consisting of the compounds in Table 1 and at least one pharmaceutically acceptable excipient, carrier, or diluent. It is related with the pharmaceutical composition containing.

  In yet another aspect, the present invention relates to a blood composition comprising blood and one or more compounds selected from the group consisting of the compounds in Table 1, wherein the blood comprises red blood cells and plasma, At least 20%, preferably at least 30%, more preferably at least 50%, even more preferably at least 80%, and even more preferably at least 90% of said one or more compounds of ) Containing red blood cells.

  In another aspect of the present invention, there is provided a blood composition comprising the compound of Table 1 and blood, wherein the blood comprises erythrocytes containing hemoglobin, at least a portion of hemoglobin being hemoglobin (S), At least a part of (S) is present as an adduct with the compound.

  In another aspect of the invention, there is provided herein an adduct of hemoglobin (S) and a compound selected from the group consisting of the compounds in Table 1.

In a preferred embodiment, the compound selected from the group consisting of Table 1 present in the adduct with erythrocytes and Hb-S is such that at least a portion of the compound is in the vascular space under steady state conditions. A distribution volume of the vascular space and the extravascular space that remains as a part of the vascular space. In one aspect, at least 20%, preferably at least 40%, even more preferably at least 60%, even more preferably at least 80%, even more preferably at least 95% of the compound is one of the adducts in the vascular space. Remain as part.

  FIG. 1 graphically illustrates high oral bioavailability, sustained exposure, and dramatic RBC distribution after a single dose of GBT440. The relevant specific pharmacokinetic parameters are shown in the table below.

  In another aspect, the invention relates to a method of treating a subject in need of treatment, comprising administering to the subject an effective amount of a pharmaceutical composition of the invention. As used herein, a subject refers to a mammal, such as a primate, preferably a human.

  The foregoing and other aspects of the invention are described in detail below.

FIG. 6 is a graph showing high in vivo oral bioavailability, sustained exposure, and high RBC distribution after a single dose of GBT440. FIG. 6 is a graph showing high in vivo oral bioavailability, sustained exposure, and high RBC distribution after a single dose of GBT440. FIG. 6 is a graph showing high in vivo oral bioavailability, sustained exposure, and high RBC distribution after a single dose of GBT440.

Definitions In this specification and the appended claims, the singular forms “a”, “an”, and “the” (“the”) unless the context clearly dictates otherwise. Note that “)” includes multiple referents. Thus, for example, reference to “a solvent” includes a plurality of such solvents.

As used herein, the term “comprising” or “comprises” is intended to mean that the composition and method include the recited elements, but do not exclude other elements. . As used in the definition of compositions and methods, “consisting essentially of” shall mean excluding other elements of essential importance to the combination for the stated purpose. As such, a composition or process consisting essentially of the elements defined herein should not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. Let's go. “Consisting of” shall mean excluding other components beyond the trace elements and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

  Unless otherwise stated, all numbers representing amounts of ingredients, reaction conditions, etc. used in the specification and claims should be understood to be modified in all cases by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations. Each numeric parameter should be interpreted at least by applying normal rounding techniques in light of the reported number of significant digits. The term “about” used before a temperature, time, amount, and concentration, including a numerical name, eg, a range, is an approximation that can vary by (+) or (−) 10%, 5%, or 1%. Indicates the value.

  The term “pharmaceutically acceptable” refers to being safe and non-toxic for administration in vivo, preferably humans.

  The term “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable salt.

The term “salt” refers to an ionic compound formed between an acid and a base. Where the compounds provided herein contain acidic functional groups, such salts include, but are not limited to, alkali metal salts, alkaline earth metal salts, and ammonium salts. As used herein, ammonium salts include salts containing protonated nitrogen bases and alkylated nitrogen bases. Pharmaceutically acceptable useful exemplary non-limiting cationic _{salt, Na, K, Rb, Cs} , NH 4, Ca, Ba, imidazolium, and ammonium cations based on natural amino acids. Where the compounds utilized herein contain basic functional groups, such salts include, but are not limited to, salts of organic acids such as carboxylic acids and sulfonic acids, as well as hydrogen halides, sulfuric acid, phosphoric acid. There are salts of mineral acids. Exemplary non-limiting anions useful for pharmaceutically acceptable salts include oxalate, maleate, acetate, propionate, succinate, tartrate, chloride, sulfate, bisulfate, monobasic, dibasic, and There are tribasic phosphates, mesylate, tosylate and the like.

  The term “whole blood” refers to blood that contains all its natural components, components, or elements, or a significant amount of natural components, components, or elements. For example, some components may be removed by a purification process before blood is administered to the subject.

  The terms “treat”, “treating” or “treatment” as used herein alleviate, reduce or ameliorate a disease or condition or one or more symptoms thereof. Preventing additional symptoms, improving or preventing the underlying metabolic cause of symptoms, inhibiting a disease or condition, eg, stopping or suppressing the progression of a disease or condition, a disease or It includes prophylaxis, including alleviating a disease state, causing a disease or a disease state to retreat, alleviating a condition caused by a disease or a disease state, or suppressing symptoms of a disease or a disease state. The term also includes alleviating the disease or condition, eg, causing regression of clinical symptoms. The term also includes achieving a therapeutic and / or prophylactic effect. By therapeutic effect is meant eradication or amelioration of the underlying disorder being treated. Also, the therapeutic effect is due to the eradication or improvement of one or more physiological symptoms associated with the underlying disease, such that the improvement is observed in the individual even though the individual is still suffering from the underlying disease. Achieved. For preventive effects, the composition has not yet been diagnosed with an individual at risk of developing a specific disease or with an individual reporting one or more physiological symptoms of the disease. Also administered.

  The term “preventing” or “prevention” refers to a reduction in the risk of obtaining a disease or disorder (ie, the disease may be exposed to or susceptible to the disease, but the disease Subjects who do not experience or exhibit symptoms of at least one of the clinical symptoms of the disease). The term further includes, for example, preventing clinical symptoms from occurring in a subject at risk of suffering from such a disease or disorder, thereby substantially avoiding the development of the disease or disorder.

  The term “effective amount” refers to an amount effective for the treatment of a condition or disease by intranasal administration of a compound or composition described herein. In some embodiments, an effective amount of any of the compositions or dosage forms described herein is a disease mediated by hemoglobin or a disease that would benefit from tissue and / or cellular oxygen supply Of any of the compositions or dosage forms described herein to the subject in need thereof.

  As used herein, the term “carrier” refers to a relatively non-toxic chemical compound or agent that facilitates the incorporation of a compound into cells, eg, red blood cells or tissues.

Compounds The compounds used herein are selected from the following Table 1 or N-oxides thereof, or respective pharmaceutically acceptable salts thereof. The N-oxides of the compounds described below are considered novel, and the N-oxide compounds and their salts each form another embodiment of the present invention.

  The compounds in Table 1 are subject to one or more biological parameters such as, but not limited to, partitioning between red blood cells and plasma, volume of distribution, oxygen balance curve, oxygen affinity and polymerization activity. A compound that can meet one or more biological activity criteria measured on the basis of.

  In a preferred embodiment, the compound is Compound 12.

Pharmaceutical Compositions In a further aspect of the invention, there is provided a composition comprising any of the compounds described herein and at least one pharmaceutically acceptable excipient, wherein the compound of Table 1 is from 1 mg to Present in the composition in an amount of 10 g.

  In another aspect, the present invention provides a composition comprising any of the compounds described herein and a pharmaceutically acceptable excipient.

  Such compositions can be formulated for various administration routes. Compositions suitable for oral delivery will probably be used most often, but other routes available are transdermal, intravenous, intraarterial, lung, rectal, intranasal, intravaginal, tongue, There are intramuscular, intraperitoneal, intradermal, intracranial, and subcutaneous routes. Dosage forms suitable for administration of any of the compounds described herein include tablets, capsules, pills, powders, aerosols, suppositories, parenteral drugs, and suspensions, solutions, and emulsions. There are oral solutions containing. Sustained release dosage forms can also be used, for example, in the form of transdermal patches. All dosage forms can be prepared using methods that are standard in the art (see, for example, Remington's Pharmaceutical Sciences, 16th ed., A. Oslo editor, Easton Pa. 1980).

  Pharmaceutically acceptable excipients are non-toxic, aid administration, and do not adversely affect the therapeutic effect of the compounds of the invention. Such excipients may be any solid, liquid, semi-solid, and in the case of aerosol compositions, may be gaseous excipients that are generally available to those skilled in the art. The pharmaceutical compositions according to the present invention are prepared by conventional means utilizing methods known in the art.

  The compositions disclosed herein include vehicles and excipients commonly used in pharmaceutical preparations such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils , Paraffin derivatives, glycols and the like. Coloring and flavoring agents can also be added to the preparations, particularly to preparations for oral administration. Solutions can be prepared using water or physiologically compatible organic solvents such as ethanol, 1,2-propylene glycol, polyglycol, dimethyl sulfoxide, aliphatic alcohols, triglycerides, partial esters of glycerin and the like.

  Solid pharmaceutical excipients include starch, cellulose, hydroxypropyl cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, Examples include sodium chloride and dried skim milk. Liquid and semi-solid excipients include glycerol, propylene glycol, water, ethanol, and various oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, You can choose from sesame oil. In certain embodiments, the compositions provided herein comprise one or more of α-tocopherol, gum arabic, and / or hydroxypropylcellulose.

  In one embodiment, the present invention provides sustained release formulations such as drug depots or patches comprising an effective amount of a compound provided herein. In another embodiment, the patch further comprises gum arabic or hydroxypropylcellulose separately or in combination in the presence of α-tocopherol. Preferably, the hydroxypropyl cellulose has an average MW of 10,000 to 100,000. In a more preferred embodiment, the hydroxypropyl cellulose has an average MW of 5,000 to 50,000.

  The compounds and pharmaceutical compositions of the present invention can be used alone or in combination with other compounds. When administered with other drugs, co-administration can be any method in which both pharmacological effects are evident in the patient simultaneously. Thus, co-administration does not require that a single pharmaceutical composition, the same dosage form, or even the same route of administration be utilized for the administration of both the compound of the invention and the other drug, It need not be administered exactly at the same time. However, co-administration will most conveniently be achieved substantially simultaneously by the same dosage form and the same route of administration. Obviously, such administration proceeds most conveniently by delivering both active ingredients simultaneously in the novel pharmaceutical composition according to the present invention.

  In another aspect of the invention, one or more adducts are conceivable in which the compound selected from Table 1 is bound to hemoglobin S. In one embodiment, the adduct is generated from compound 12 and hemoglobin S.

Therapeutic Methods The present invention provides methods for improving the red blood cell oxygen carrying capacity. In certain embodiments, the invention relates to red blood cells or whole blood in vivo, in vitro, in situ or ex.
A method of treatment with one or more compounds or pharmaceutical compositions of the invention in vivo, comprising administering said one or more compounds or pharmaceutical compositions, or treating them with blood, in particular hemoglobin (S) Relates to a method by contacting with blood containing.

  In some embodiments, methods of storing and / or using the compounds and pharmaceutical compositions of the invention ex vivo are contemplated, including but not limited to autologous blood transfusion or non-blood transfusion. The compound and / or pharmaceutical composition is mixed with whole blood used for procedures such as autologous blood transfusion, coronary artery bypass surgery, and any extracorporeal procedure including perfusion and / or reperfusion of blood to a subject. In certain embodiments, the compound and / or pharmaceutical composition can be mixed with whole blood for storage.

  In another aspect of the method, the invention relates to a method of treating a subject in need of treatment (eg sickle cell anemia) by administering to the subject an effective amount of a pharmaceutical composition of the invention. In one preferred aspect, the pharmaceutical composition comprises from about 0.1 mg / kg to about 1 g / kg, more preferably from about 1 mg / kg / day to about 100 mg / kg / day of one or more compounds of Table 1. Including the day.

  In aspects of the present invention, a method for increasing the oxygen affinity of hemoglobin S in a subject's body is provided, the method comprising a pharmaceutical composition of the present invention or one of Table 1 for a subject in need of treatment. Administration of a therapeutically effective amount of a blood composition comprising the above compound. In a preferred embodiment, the blood composition does not include hemoglobin (S).

  In embodiments of the present invention, a method of treating a condition associated with hypoxia is provided, the method comprising administering to the subject in need of treatment a therapeutically effective amount of the aforementioned pharmaceutical composition or blood composition. .

  In another aspect of the invention, a method for treating hypoxia associated with sickle cell anemia is provided, wherein the method administers a therapeutically effective amount of the aforementioned pharmaceutical or blood composition to a subject in need of treatment. Including doing.

  Furthermore, the compounds and pharmaceutical compositions of the present invention can be added to whole blood or packed cells, preferably upon storage or at the time of transfusion. In some embodiments, the compound and pharmaceutical composition can be added to the whole blood or red blood cell fraction in a closed system using a suitable reservoir, in which the compound or pharmaceutical composition is placed prior to storage. Or it is present in the anticoagulant solution in the blood collection bag.

Synthetic Methods The synthesis of the compounds in Table 1 is described in US Patent Application No. 61 / 661,320 and US Patent Application No. 61 / 581,053, each of which is a synthesis of these compounds. For purposes of illustration only, the entire contents of which are incorporated herein by reference.

  The following examples are set forth in order to illustrate various embodiments of the invention. They in no way limit the invention. The present invention is well suited to accomplishing its objectives, achieving the aforementioned goals, and obtaining benefits, and also performing its unique objectives, achieving its goals, and obtaining benefits. Those skilled in the art will recognize that this is the case. This example (as well as the method described herein) is presently representative of the preferred embodiment. They are exemplary and do not limit the scope of the invention. Modifications and other uses are encompassed within the spirit of the invention as defined by the claims, and those skilled in the art will envision them.

Example 1
The compounds provided in the present invention are allosteric modifiers of hemoglobin. As such, these compounds do not modify erythrocytes alone. Instead, Table 1
In the presence of the compound increases the red blood cell response to hemoglobin concentration. The compounds in Table 1 are expected to be effective against red blood cells because they can improve the function of hemoglobin.

  This experiment was established and used to evaluate the pharmacokinetic (PK) properties of the compounds.

  Sampling and data analysis: Rats (Sprague-Dawley, male, 8-12 weeks old) were administered one of three compounds corresponding to Compound 12, Compound 22 or Compound 23. Rats received an oral (10 mg / kg) or intravenous (1 mg / kg) dose of compound. Rats were fasted overnight prior to the experiment and fed after a 2 hour sampling time point.

Blood samples were collected at various time points. The blood was anticoagulated with 3.2% TSC (trisodium citrate) and a portion was separated into plasma fractions by centrifugation and removal of blood cells. The drug concentration of plasma and lysed blood samples was analyzed using LC-MS / MS. PK parameters were calculated by non-compartmental analysis of concentration-time characteristics using WinNonLin software (Pharsight, Mountain View, CA). The apparent elimination half-life (t _1/2 ) value was calculated as ln (2) / k. The area under the blood concentration time curve (AUC) value was estimated using the linear trapezoidal method. The AUC _last value was calculated from the administration time to the final measurable concentration. The AUC _inf value is calculated as the ratio of the sum of the corresponding AUC _last and the detectable final concentration divided by k. Plasma clearance (Cl) is calculated from dose / AUC _inf . The distribution volume (V _ss ) in the steady state is calculated from the average residence time _inf × Cl _ss . The highest concentration (C _max ) and C _max arrival time (T _max ) were recorded as observed values. The blood / plasma distribution ratio at the time of each experiment was calculated.

  Results: Table 2 summarizes selected PK parameters for the compounds described below.

The distribution volume of Compound 12 is 0.14 L / kg, which indicates that Compound 12 is not well distributed in the extravascular space in rats (control normal Vz = 0.1 L / kg). Two related compounds have relatively high V _ss (compound 22 and compound 23, 3.1 and 3.15, respectively), which are more likely to be distributed in extravascular space and other compartments than compound 12. It shows that.

However, considering the red blood cell compartment, compound 12 is surprisingly much more distributed in the blood than compound 22 or compound 23. When the compound is administered orally, the relative ratio in blood at the peak concentration (C _max ) (when compared to plasma) is that compound 12 (21 times) is compound 22 (5 times) or compound 23 (3 times). It was much higher. When the red blood cell / plasma ratio was measured at the peak concentration, compound 12 was distributed in the red blood cells in a 70 to 1 ratio, demonstrating preferential distribution to the compartment containing the drug target hemoglobin. Supporting data were reported in an in vitro system that measures the binding of Compound 12 to hemoglobin and human serum albumin. This functional assay showed that Compound 12 binds preferentially to hemoglobin when both proteins are present at their respective physiological ratios.

  Another surprising and surprising finding was found when all exposures were followed in animals dosed orally with Compound 12. Compound 12 had 55 times higher levels of compound in blood than the level of compound in plasma compared to the blood / plasma ratio of compound 22 (5-fold) or compound 23 (1-fold).

  Compound 12 can preferentially partition into erythrocytes, but this was also confirmed in mice treated intravenously. It was observed that the blood / plasma ratio in mice was 15.4 (at in vivo peak concentration) and 30 (at all exposures). Similar to the measurements in rats, the distribution volume (Vss) was also low in mice (0.10). Therefore, compound 12 is not expected to be widely distributed in extravascular space in mice.

  In conclusion, the results shown in Table 2 indicate that compound 12 is not distributed in extravascular tissue (low Vss) and is selectively distributed to the target compartment (red blood cells), so that compound 12 may have low toxicity. I understand.

  Accordingly, provided herein is a blood composition comprising one or more compounds selected from Table 1 and blood, wherein at least 30% of the one or more compounds in the blood are present in the blood. Red blood cells are bound.

Example 2
To evaluate other pharmacokinetic (PK) properties of the compound of Example 1, another series of assays was performed.

Inverse Hex assay Whole blood oxygen balance curves (OEC) before and after treatment with various concentrations of compounds 12, 22 and 23 were obtained using a HEMOX analyzer (TCS Scientific, New Hope, PA). Went like that. Blood samples from homozygous sickle cell patients were obtained through the Hemoglobin Pathology Center at Children's Hospital Oakland Research Institute (CHORI) with the approval of the institutional review board. Hematocrit was adjusted to 20% using autologous plasma and blood samples were incubated for 1 hour at 37 ° C. in the absence or presence of compound. 100 μl of these samples were added to 5 mL of Hemox buffer (30 mM TES, 130 mM NaCl, 5 mM KCl, pH = 7.4) at 37 ° C. and then transferred to the Hemox sample chamber. The sample was saturated with oxygen by flushing with compressed air for 10 minutes. The sample is then flushed with pure nitrogen and the respective absorbances of oxygenated Hb and reduced Hb with solution pO2 are recorded. The oxygen equilibrium data is then fitted to the Hill model to obtain a value for p50. Both whole blood alone (control) deoxygenation curves and whole blood deoxygenation curves in the presence of compound were collected using TCS software.

Results: Table 3 below lists Δp50% values, “+” indicates that Δp50% is 0-29, “++” indicates that Δp50% is 30-50, and “++++” indicates It shows that Δp50% is 50 or more. A positive Δp50 value corresponds to the curve shifting to the left and a lower p50 value compared to the control, and the compound acts to modify Hb (S) and increase its affinity for oxygen. Indicates.

R / T Assay Compounds 12, 22 that maintain a high oxygen affinity relaxed (R) state of hemoglobin under deoxygenated conditions using a relaxed to tensioned transition assay (“R / T assay”). And 23 abilities were examined. This ability can be expressed as a “ΔR” value (ie, the change in time of the R state after treatment of hemoglobin with the compound compared to the time without treatment with the compound). ΔR is a comparison of% R after compound treatment with% R without treatment (for example, R% without treatment is 8% and 48% R when treated with 30 μM target compound). Sometimes the% R of the compound is 40%.

The HbS / A mixture was purified from blood obtained from sickle cell patients with the approval of the institutional review board through the Hemoglobinpathy Center at Children's Hospital Oakland Research Institute (CHORI). HbS / A (final concentration 3 μM) in 96-well plates in the presence or absence of compounds, 50 μM potassium phosphate buffer, pH = 7.4 and 30 μM
Incubated in 2,3 diphosphoglyceric acid (DPG) for 1 hour at 37 ° C. in a final volume of 160 μl. Compounds were added to various concentrations (final concentrations 3 μM to 100 μM). The plate was coated with Mylar film. After incubation, the Mylar cover was removed and the plate was placed in a Spectrostar Nano plate reader preheated at 37 ° C. After 5 minutes, N ₂ (flow rate = 20 L / min) was passed through the spectrophotometer. Spectroscopic measurements (300 nm to 700 nm) were taken every 5 minutes for 2 hours. Data analysis was performed using linear regression from data acquired at all wavelengths.

  Results: Table 3 below lists the ΔR values, “+” indicates that ΔR is 0-30, “++” indicates that ΔR is 30-50, and “++” indicates that ΔR is 50. It is shown above.

Polymerization Assay The polymerization assay is performed in vitro using purified HbS exchanged for 1.8 M potassium phosphate buffer at pH 7.4. Using a slightly modified method (Antonini and Brunori, 1971), HbS was homozygous with the approval of the investigative committee from the red blood cell through the Hemoglobinopathic Center at Children's Hospital Oakland Research Institute (CHORI). Purify with CRO VIRUSYS from the obtained blood. Compounds are prepared in 100% DMSO and the desired amount is added to 50 μM purified HbS to a final DMSO concentration of 0.3%. Adjust the final potassium phosphate concentration to 1.8M using a mixture of 2.5M potassium phosphate stock solution and water at pH 7.4. The reaction mixture is incubated for 1 hour at 37 ° C. and then transferred to a 24-well plate for deoxygenation in a glove box containing 99.5% nitrogen and 0.5% oxygen. Incubate for 24 hours at 4 ° C. on a plate cooler in the glove box without covering the 24-well plate. 50 μl of the reaction mixture is transferred to a 96 well plate and the absorbance at 700 nm is measured with a plate reader in a glove box at 37 ° C. for 1 hour every minute. A plot of absorbance against time is fitted using a Boltzman sigmoid fitting and the lag time (from 0 to half Vmax time) is measured. To compare and rank compounds, the delay time is expressed as a delay rate (% DT), which is the difference between the delay time of HbS / compound and HbS alone multiplied by 100 and divided by the delay time of HbS alone. Is defined as

Results: The following compounds were tested in a polymerization assay. The active range is defined by the number of dagger (†) symbols displayed. † indicates activity ≧ 40% and ≦ 80%; †† indicates activity> 80% and ≦ 120%; ††† indicates activity> 120% and ≦ 140% †††† indicates activity> 160%.

Example 3
Another set of assays was performed to examine the effects of the compounds of the present invention on the oxygen affinity of hemoglobin and the rheological properties of blood.

Oxygen dissociation assay In the 96-well format oxygen dissociation assay (ODA), compounds 5, 9 and 12 are all from 5-hydroxyfurfural (5-HMF), a drug currently being tested in sickle cell disease clinical trials. Was also potent in increasing the oxygen affinity of HbS.

  Results: Table 4 below lists the change in oxygen affinity (Δ oxygenation state). After 2 hours of passive deoxygenation, Hb oxygen affinity was increased 6-fold by equimolar concentration of Compound 12 with Hb. Even when compound 12 was present at a sub-stoichiometric concentration (ratio of compound 12 to Hb of 1: 3), the oxygen affinity of Hb improved by a factor of two, indicating that 16% more oxygenated Hb was present. It will be.

  The drug was then assayed with 25 μM purified Hb on a TCS Hemox analyzer. When the ratio of Compound 12: Hb was 1: 3, the oxygen affinity was improved by 15% compared to the Hb control, but the oxygen affinity improvement was greater than 70% at the stoichiometric concentration.

Reverse Hemox Assay Reverse Hemox assay was performed using washed red blood cells or whole blood substantially similar to that described in Example 2 above.

  Results: Table 4 below shows the rate of change of p50 (Δp50%) for each test compound. In washed red blood cells (RBC), compared to control red blood cells (Δp50 = 30 mmHg), 5-HMF, compound 5 and compound 12 (1 mM compound) resulted in p50 of 20 mmHg, 9 mmHg and 7 mmHg, respectively. To examine the effect of plasma proteins on compound activity, OEC in whole blood from sickle cell disease patients was measured. The p50 of control blood was 30 mmHg, whereas 5-HMF, Compound 5, Compound 9, and Compound 12 resulted in p50 of 27 mmHg, 18 mmHg, 11 mmHg, and 6 mmHg, respectively.

Viscosity Assay Sickle cell disease patients develop anemia as a means by which the circulatory system compensates for the increased blood viscosity caused by non-deformable sickle cell erythrocytes (ssRBC). Hypoxic ssRBC
In order to investigate whether these compounds reduce the viscosity of these, the effect of 5-HMF, Compound 5, Compound 9, or Compound 12 on sickle cell disease rheology was tested. Using blood from a sickle cell disease patient, whole blood (hematocrit 30%, approximately 1.5 mM Hb) together with 5-HMF, compound 5, compound 9, or compound 12 is hypoxic (2.4). % O ₂ ). The viscosity was measured using a conical plate viscometer at a shear rate ranging from 60 s ^{−1 to} 415 s ⁻¹ .

  Results: Table 4 below lists the change in centipoise (ΔcP) value for each compound. The compounds of the present invention dramatically improved blood viscosity. For example, Compound 12 improved (reduced) viscosity from 6.33 cP (no Compound 12) to 4.32 cP (equimolar Compound 12). The average viscosity of normotic sickle cell blood is 3.69 cP. This improvement in blood viscosity can reduce the residence time of ssRBC in hypoxic tissues and can reduce the level of polymerization in individual erythrocytes when transported through hypoxic tissues. Become. Furthermore, Compound 12 has also been shown to delay the polymerization and sickling of erythrocytes from sickle cell disease patients. All of these properties indicate that Compound 12 can cause a dramatic decrease in HbS polymer concentration and reduce the possibility of forming hard red blood cells that cause blood flow obstruction in patients with sickle cell disease.

Example 4
Another set of assays was performed to examine the effectiveness of Compound 12 and 5-HMF in delaying in vitro polymerization and preventing sickle formation of erythrocytes.

Polymerization Assay As described in Example 2 above, the ability of 5-HMF and Compound 12 to retard HbS polymerization was evaluated. Purified HbS (50 μM) was preincubated with 25 μM, 50 μM, or 100 μM 5-HMF or Compound 12, followed by passive deoxygenation at 4 ° C. in 1.8 M potassium phosphate. Polymerization was induced by a temperature jump from 4 ° C to 37 ° C. Polymerization was quantified based on the turbidity of the HbS solution under sustained hypoxia.

Results: Table 5 below lists the polymerization delay (minutes) for each test compound. Table 6 describes the polymerization delay by carbon monoxide (CO) coordinated HbA, a well-characterized HbS polymerization inhibitor, in both intracellular and in vitro assays. DT: Delay time. Compound 12 delayed HbS polymerization in a dose-dependent manner. The polymerization delay of the untreated HbS control was relatively longer than that observed by active deoxygenation using dithionite or laser treatment. However, Compound 12 delayed HbS polymerization to the same extent as CO-coordinated HbA.

Sickleization assay In sickle experiments, erythrocytes were preincubated with compound 12 or 5-HMF and then hypoxic (pO ₂ ~ 30 mmHg) for 0.5 hours in a humidified chamber at 37 ° C, followed by light microscopy. Was imaged using. The percentage of sickled erythrocytes in each image was calculated using CellVigene software.

  Results: Table 5 below lists the effect of each compound on the percent of sickled erythrocytes. HCT: hematocrit. Compound 12 prevented sickle formation of RBCs under hypoxia, suggesting that compound 12 has the ability to prevent intracellular HbS polymerization. In this sickle formation assay, red blood cells were exposed to hypoxia for much longer than the typical red blood cell transport time (less than 1 minute) through the microcirculation, thus preventing sickle formation under physiological conditions. The amount of compound required may be much smaller.

  From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without departing from the spirit and scope of the invention. I will.

  Throughout the description of the invention, reference is made to various patent applications and publications, each of which is incorporated herein by reference in its entirety.

Claims (18)

  A composition comprising about 1 mg to about 10 g of a compound selected from the group consisting of the compounds in Table 1 and at least one pharmaceutically acceptable excipient, carrier or diluent.   The composition of claim 1, wherein the compound is Compound 12 in Table 1. A blood composition comprising blood and one or more compounds selected from the group consisting of the compounds in Table 1,
The blood consists of red blood cells and plasma,
The composition wherein at least 20% of the one or more compounds in the blood are bound to the red blood cells under physiological conditions.   4. The blood composition of claim 3, wherein at least 30% of the one or more compounds are bound to the red blood cells.   4. The blood composition of claim 3, wherein at least 50% of the one or more compounds are bound to the red blood cells.   4. The blood composition of claim 3, wherein at least 80% of the one or more compounds are bound to the red blood cells.   4. The blood composition of claim 3, wherein at least 90% of the one or more compounds are bound to the red blood cells.   The blood composition of claim 3, wherein the composition is Compound 12 in Table 1.   The blood composition according to claim 3, wherein at least part of the red blood cells is sickled and at least part of the hemoglobin is bound to the compound.   4. The blood composition according to claim 3, wherein the blood is free or substantially free of hemoglobin. A blood composition comprising an adduct formed from blood and one or more compounds selected from the group consisting of the compounds in Table 1,
The blood consists of red blood cells and plasma,
The adduct is distributed between the vascular space and the extravascular space in vivo under steady state conditions;
At least some of the one or more compounds remain as part of the adduct in the vascular space;
Blood composition.   12. The blood composition of claim 11, wherein at least 20% of the one or more compounds remain as part of the adduct in the vascular space.   The blood composition of claim 11, wherein at least 40% of the one or more compounds remain as part of the adduct in the vascular space.   12. The blood composition of claim 11, wherein at least 60% of the one or more compounds remain as part of the adduct in the vascular space.   12. The blood composition of claim 11, wherein at least 80% of the one or more compounds remain as part of the adduct in the vascular space.   12. The blood composition of claim 11, wherein at least 95% of the one or more compounds remain as part of the adduct in the vascular space.   The blood composition of claim 11, wherein the composition is Compound 12 in Table 1.   A method of treating a subject in need of treatment comprising administering to said subject an effective amount of the pharmaceutical composition of claim 1.

Images