CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Application Serial No. 63/320,789, filed March 17, 2022, the contents of which are incorporated in their entirety for all purposes.

FIELD OF THE INVENTION

[0002] Disclosed are stable pharmaceutical compositions comprising an inclusion complex of epinephrine and a pharmaceutically acceptable macromolecular excipient and methods of making and using same.

BACKGROUND

[0003] Epinephrine (EPI), also commonly referred to as adrenaline, is a hormone which acts as a neurotransmitter. It is secreted by the adrenal medulla, when released into the bloodstream epinephrine acts to increase heart rate and blood pressure and muscle contractions. Its ability to direct blood to tissues under stress conditions results in its ability to maintain cardiovascular homeostasis. Epinephrine is used as a stimulant in cardiac arrest, as a vasoconstrictor in a state of shock and as a bronchodilator. Due to its antispasmodic property in bronchial asthma, ability to combat low blood pressure during hemorrhagic episodes and treatment of anaphylactic shock categorized it as a very important drug in pharmacotherapy.

[0004] Anaphylaxis is a sudden, and sometimes unpredictable systemic allergic reaction that can be fatal if left untreated. Consequently, allergists recommend that person with anaphylaxis history be prepared to always receive immediate emergency treatment. Parenteral (IV and IM) epinephrine solutions represent valuable products in the treatment of cardiac arrests and bronchodilation situations. In addition, they have become the agents of choice for the treatment of sudden lifethreatening allergic reactions (anaphylactic shock). [0005] Epinephrine is chemically categorized as a catechol. Catechols are a class of compounds that possess two phenolic groups on adjacent carbon atoms (ortho position) of an aromatic ring as shown below.

R ® A substituent

[0006] Catechols are susceptible to oxidation. Epinephrine, being a catechol, in aqueous solution deteriorates rapidly upon exposure to oxygen (air), light or heat to produce a colored (pink) degradant (adrenochrome) which is a quinone-type product. Oxidation of epinephrine may occur in the solid-state. Factors such as crystal packing, morphology and surface groups contribute to the rate of oxidation, since oxygen must diffuse through the crystal lattice. Oxidation may occur more rapidly in amorphous systems due to irregular arrangement of molecules and increased molecular mobility. It should be noted that during the formulation process of epinephrine, a small quantity of amorphous material may be formed which could lead to the formation of sufficient quantities of degradation products to cause a failure to meet QC specifications. Thus, there is a need in the art for improved methods and formulations for the administration of epinephrine.

BRIEF SUMMARY

[0007] Disclosed are methods of enhancing the solubility and stability of epinephrine (EPI) comprising forming a complex or mixture of a compound using a novel supramolecular (cyclodextrin (CD), cucurbituril (CB) etc.) of making a stable clear EPI formulation for intramuscular use. The preparation method of the complex includes preparing a stable EPI: CD, CB inclusion complex that exhibits greater resistance towards degradation due to oxidation, sulfonation and epimerization, than its corresponding physical mixture or un-complexed EPT, when the inclusion complex was prepared by three special controlled evaporation methods under reduced pressure and temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0009] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[00010] FIG 1 depicts a comparison of the ¹³ C solid-state NMR spectra for formulations as disclosed herein.

DETAILED DESCRIPTION

[00011] DEFINITIONS

[00012] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The methods may comprise, consist of, or consist essentially of the elements of the compositions and/or methods as described herein, as well as any additional or optional element described herein or otherwise useful in the described compositions and methods.

[00013] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[00014] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[00015] As used herein, the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[00016] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. Tn some aspects, the terms refer to humans. Tn further aspects, the terms may refer to children.

[00017] The active agent may form salts, which are also within the scope of the disclosure. Reference to a compound of the active agent herein is understood to include reference to salts thereof, unless otherwise indicated. The term “salt(s)”, as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when an active agent contains both a basic moiety, such as, but not limited to an amine or a pyridine or imidazole ring, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the term “salt(s)” as used herein. Pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts may be used, although other salts are also useful, e g., in isolation or purification steps, which may be employed during preparation. Salts of the compounds of the active agent may be formed, for example, by reacting a compound of the active agent with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. When the compounds are in the forms of salts, they may comprise pharmaceutically acceptable salts. Such salts may include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedi sulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p- aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates, phosphates, perchlorates, borates, acetates, benzoates, hydroxynaphthoates, glycerophosphates, ketoglutarates and the like. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like. Examples of organic bases include lysine, arginine, guanidine, diethanolamine, choline and the like.

[000181 One of the great problems of catechol moiety containing products like epinephrine, is the susceptibility of their formulations to oxidation. Their catechol function is rapidly oxidized by oxygen (air) to form ortho-quinone type compounds (shown below) which can undergo further chemical alterations to produce mixtures of colored decomposition products. [00019] Oxidation of catechol derivative to its corresponding orthoquinone.

Catechol Ortho quinone

R = A substituent

[00020] The formation of the ortho-quinone can be easily monitored by its high absorbance in the visible region of its UV-visible spectrum (around 650 nm). Thus, the oxidative degradation of catechol-containing formulations is usually accompanied by the occurrence of a dark brown coloration denoting the formation of oxidative degradants such as ortho-quinone species.

[00021] In addition to its oxidation, epinephrine aqueous solutions are susceptible to degradation by heat (catalyzing the epimerization of its single asymmetric center). Because of its pH-induced instability. Low pH values, around pH 2.5 - 3.5, are required to keep the two phenolic hydroxyl groups protonated. It is known that the oxidative degradation of epinephrine proceeds through the formation of phenoxide anion. Thus, epinephrine aqueous solutions are formulated, preferably, at low pH values (around pH 3.0 or less).

[00022] In addition, such solutions usually contain an antioxidant to prevent or slow down the oxidation which converts the catechol to its orthoquinone form. The formation of the latter species can autocatalyze the further oxidation of the catechol and thus can accelerate the decomposition of the epinephrine product.

[00023] The use of sodium metabisulfite as an antioxidant to combat the susceptibility to oxidation provides an impediment in the design of formulations of the drug. More specifically, in the presence of heat, the bisulfite reacts with the secondary hydroxyl group function of its side chain to form a sulfonate derivative of epinephrine. The later, along the colored oxidation (quinone derivative) products of epinephrine are among the most common impurities of the drug’s aqueous solution formulations. [00024] The pharmacologically active L isomer of epinephrine possesses a single center of asymmetry (optically active) at the exocyclic aliphatic carbon bearing its secondary aliphatic hydroxyl group. Under acidic conditions, the hydroxyl function is eliminated in the form of water to produce an olefinic intermediate. This highly conjugated olefinic species can suffer attack by water on both sides of the plane to produce a racemic (50% L and 50% D isomer) mixture of epinephrine. Alternatively, the olefinic 3 can be attacked by a bisulfite species (HSO3) to form a sulphonated derivative of epinephrine. The bisulfite nucleophilic HSO3 species is generated from sodium metabisulphite in (Na2S2Ch) in acidic media. In summary, epinephrine when formulated in an acidic medium (usually at pH of 2.5-3.5), in the presence of oxygen and in the presence of sodium metabisulphite (antioxidant), it suffers an oxidation of its catechol moiety resulting in conversion to its DL form and undergoing sulfonation.

[00025] Epinephrine is known to be susceptible to photodecomposition. Thus, its aqueous formulations must be protected from light exposure.

[00026] As mentioned above, oxidation of epinephrine is a major problem in the stability of its aqueous parenteral formulations. In recent years a great number of publications have demonstrated that complexation with cyclodextrins (CD) can protect effectively oxidizable substances against atmospheric oxidations, increases the solubility of poorly soluble drugs and can cause reduction of the loss of highly volatile substances from their final products. (Szejtli, J, Past, present, and future of cyclodextrin, Pure, Appl. Chem; 2004; 76, 1825 - 1845).

[00027] Disclosed herein are novel formulations, and methods of making and using same, having improved chemical stability of epinephrine in an aqueous solution. The disclosed epinephrine formulations employ a cyclodextrin and/or cucurbituril derivative to form an inclusion complex with epinephrine which may be manufactured utilizing a method of slow evaporation under reduced pressure.

[00028] While not intending to be limited by theory, it is believed that cyclodextrin may be used to prevent decomposition of substances susceptible to oxidation. Disclosed is the use of sulfobutylether-P-cyclodextrin (SBEpCD) in one example and hydroxypropyl-P-cyclodextrin (HPpCD) in another, with a aqueous formulation of epinephrine. The epinephrine formulation solution utilized contained a 1 mg of the drug, 6 mg of sodium chloride, 1.67 mg of sodium metabisulfite (SMB), 10-25 mg of cyclodextrin (either SBEpCD or HP[3CD) or cucurbituril (< 3%), hydrochloric acid enough to adjust the pH to 2.9 and water for injection (WFI) to make 1.0 mL of EPI solution (See Table 1).

[00029] Table 1. Composition of Epinephrine IV formulation

[00030] The addition of either of the two cyclodextrins (SBEpCD or HPpCD) was found to increase considerably the resistance of the above solutions to oxidation and sulfonation. In fact, when an inclusion complex of EPI with either one of the CD in a 1 : 1 or 1 :2 molar ratio resulted in a solution which remained colorless (free of the existence of the orthoquinone oxidation product) when stressed at 40 °C for 6 (six) months.

[00031] It was found that the rate of oxidation of non-complexed epinephrine which was obtained in an amorphous state by evaporation under low temperature was considerably faster than when it existed in its crystalline state. Surprisingly, it was discovered, unexpectedly, that the epinephrine was oxidized at a very slower rate when it was allowed to be converted to an inclusion complex with a macromolecule like cyclodextrin via a slow evaporation of its solutions at a low temperature. These data suggested that the nature of the association of EPI with CD played a pivotal role in the oxidative resistance and in the chemical reactivity of the drug. It was evident that the physical state in which the EPI existed with the inclusion complex with CD was important. Therefore, an examination of the EPT -CD complex using ¹³ C solid-state nuclear magnetic resonance was merited. [00032] The ¹³ C SSNMR spectra showed that EPT macromolecular solid state inclusion complex existed in a different chemical form when its aqueous solution was evaporated to dryness by a one of the evaporation solidification methods under reduced pressure and temperature and its resulting ¹³ C SSNMR spectra were found to be different from those of its physical mixture with CD and/or CB. In the latter state all the solid state ¹³ C-SSNMR peaks of the EPI are clearly detected. In contrast, however, the ¹³ C-SSNMR peaks of EPI are completely absent when the drug is involved in an inclusion complex with CD which resulted from the treatment of slow evaporation under reduced pressure and temperature.

[00033] Literature ¹³ C SSNMR suggests that in the case of other drugs complexed with cyclodextrins, the API exists in an amorphous state exhibiting different physicochemical properties than if it were in a physical mixture where its crystallinity is intact (Sohivirat, S., et al., characterization of prednisone in controlled porosity osmotic pump pellets using solid-state NMR spectroscopy, J. Pharm. Sci. 2007; 96; 1008-1017 and Hong J., et al., Effect of cyclodextrin derivation and amorphous state of complex on accelerated degradation of ziprasidone, J. Pharm. Sci. 2011; 100; 2703-2716).

[00034] A solid-state NMR analysis of EPI and CD complexes was conducted at Kansas Analytical Services Laboratory (KAS) 4921 Eagle Lake Drive Fort Collins, CO 80524). A comparison of the ¹³ C solid-state NMR spectra of four samples was conducted by KAS. The samples examined comprised of a pure EPI (KAS 0522001), a physical mixture of the EPI with CD (1 :2) (KAS 0522002), an EPLCD complex 1 :2 (KAS 0522003), and EPI-CD complex that was stressed for three weeks at 40 °C (KAS 0522004).

[00035] FIG 1 shows a comparison of the ¹³ C solid-state NMR spectra for the above 4 samples. As seen from FIG 1, the signals corresponding to EPLCD complex (KAS 0522003) and that of the EPLCD stressed complex (KAS 0522004) are completely absent between 110 to 155 ppm suggesting that the EPI drug is found in an amorphous state when is associated in an inclusion complex with CD. These results are in agreement with literature findings. In contrast, in the case of EPI and the physical mixture of EPI with CD (1 :2) their signals belonging to the uncomplexed drug are clearly seen (FIG 1). [00036] The complexation of the EPT with CD was accomplished by slow evaporation of its aqueous solution in the presence of macromolecular species (cyclodextrin or cucurbitural) under reduced pressure. The resulting inclusion EPI-CD complex was found to exist in an amorphous state by ¹³ C solid-state NMR as described above.

[00037] Interestingly, the rate of oxidation of EPI present in the above produced inclusion EPI-CD complex was found to be slower than an equimolar solution of a physical mixture of the EPI and the macromolecule CD. The observed slower oxidation of the EPI in the complexed state was an unexpected observation since it was shown by ¹³ C solid-state NMR (see FIG 1) that the EPI existed in an amorphous state. It is known from general organic chemistry, that in an amorphous state, the rate of the chemical reactivity of a molecule like the oxidation or sulfonation of a compound is greater due to the greater movement of its molecules. In contrast to this principle, the present EPI-CD complex oxidizes at a slower rate despite that it is found to be in amorphous state. This unexpected behavior occurs due to the fact that the molecules of EPI are in constricted state (with low entropy) by its slow complexation with the CD (macromolecule).

[00038] After parenteral administration of drug-CD complex with Kri below 10' ⁴ p' ¹ the in-vivo drug release is enhanced by drug protein binding, and other type of competitive displacement in vivo along with the excessive dilution of the released drug in the systemic circulation. As a result, complexed drugs with CD are known to rapidly and quantitatively released from their CD complexes upon parenteral and oral administration (see, e.g., Stella, V.J. et al., 1999, Mechanism of drug release from cyclodextrin complexes, Adv. Drug Deliv. Rev. 36; 3-16 and Loftsson, T., et al., 2016, pharmacokinetics of cyclodextrins and drugs after oral and parenteral administration of drug cyclodextrin complexes, J. Pham. Pharmacol., 68; 544-555).

[00039] The rapid release in-vivo of a drug from a CD-drug complex is further substantiated by the fact that over a dozen drugs have been approved to date by the FDA and are marketed in cyclodextrin-complexed parenteral form (IV as well as IM aqueous formulations). A good example of the above is the well-studied and confirmed in-vivo release of ziprasidone (a product marketed in a solution form for the treatment of schizophrenia) from its complexed form with sulfobutylether-P-cyclodextrin (SBEpCD) (Hong, J., et al., 2011, effect of cyclodextrin derivation and amorphous state of complex on accelerated degradation of ziprasidone, J. Pharm. Sci. 100; 2703-2716). The active ingredient in this product (APT) has been shown to be in an amorphous state complexed with the cyclodextrin (SBEpCD).

[00040] Compounds which are susceptible to oxidation, like EPI, can be protected by complexation with a cyclodextrin. This is illustrated by a disclosure that was made in 2017 claiming that complexation of epinephrine with a cyclodextrin enhances its resistance to oxidation and extends the shelf-life of one of the parenteral aqueous products (Surakitbanharn Y., stabilization of epinephrine formulations, 2017; W02017/218918).

[00041] While studying the ¹³ C-solid state NMR ( ¹³ C-SSNMR) spectra of EPI inclusion complex, Applicant found that when EPI was complexed by slow evaporation of its aqueous solution with a macromolecule such as CD or CB under reduced pressure and low temperature, it existed in a different physical form (probably in amorphous and low entropic state) than when complexed as a physical mixture (PM) with the macromolecule (cyclodextrin and/or cucurbituril. In addition, and most importantly, how the EPI was complexed with the CD had a profound effect on its physical and chemical properties. Simple mixing of the EPI with a macromolecule like CD does not produce protracted stability of its solutions (see Examples # 2 to 4). In contract, it was found that an aqueous formulation incorporating EPI-CD macromolecular resistance towards degradation which includes oxidation, sulfonation and epimerization when compared with a corresponding physical mixture of EPT with CD.

[00042] These results were opposite in nature to what was expected from knowledge of general chemistry. Compounds existing in an amorphous state are known to decompose (oxidize) at a higher rate due to the greater movement of their molecules. The EPI molecules in the inclusion complex with a macromolecule (described in the present invention) oxidize at a slower rate despite their existence in an amorphous state as shown by ¹³ C solid-state NMR. The EPI molecules appear to exist in a low entropy state (highly constricted in their movement) when complexed with cyclodextrin or cucurbituril under slow evaporation and low temperature conditions, as described by the present invention.

[00043] More specifically it was found that an aqueous formulation incorporating EPI-CD macromolecular inclusion complex prepared through a slow evaporative solidification methods exhibits greater resistance towards degradation which includes oxidation, sulfonation and epimerization when compared with a corresponding physical mixture of EPI with CD.

[00044] A well-known problem of aqueous solutions of Epinephrine is stability, since the compound is highly susceptible to oxidation. To improve the stability of Epinephrine due to oxidation in an aqueous solution, the use of suitable antioxidants (for example, sodium metabisulfite) was thought to be essential. Solutions of EPI containing no antioxidant are unstable (see Examples # 11 to 13). Approved and marketed solutions of Epinephrine, generally contain the antioxidant sodium metabisulfite, which is thought to stabilize the pharmaceutical product. While improving the stability of Epinephrine formulations, antioxidant agents such as sodium metabisulfite may cause the formation of undesirable products.

[00045] It would therefore be desirable to have a stable epinephrine solution that does not have the potential for producing adverse effects upon administration. The current application relates to novel epinephrine compositions that maintain the stability of the pharmaceutical formulation in an aqueous solution with minimal amounts of additives.

[00046] It has been surprisingly found that an aqueous composition containing epinephrine, sodium chloride, sterilized water, and a pH-adjusting agent, is stable with minimal addition of sodium metabisulfite. The formulation pH of the stable solution was in the acidic range and the solution was prepared under inert gas like nitrogen. (See Examples # 5 to 7). Solutions of EPI prepared in the absence of an inert gas (like nitrogen) are unstable (See Examples # 8 to 10).

[00047] In one aspect, a composition comprising EPI as the active pharmaceutical ingredient (API) complexed with CD/CB is disclosed. The API may be in the form of a salt or the free base. In one aspect, the EPI is in a salt form. In one aspect, disclosed is a pharmaceutical composition wherein EPI as the active ingredient is present in an amount of from about 0.5 to about 2 mg/mL.

[00048] In a one aspect, the amount of cyclodextrin is between 1 : 1 to 1 :3 molar ratio of the composition and more preferably 1:2. In one aspect, the antioxidant is sodium metabisulfite with the ratio EPI: antioxidant of 1 : 1 (w: w). [00049] In one aspect, the disclosed compositions may comprise a pH modifier. In one aspect, a pH modifier is optionally present in the composition. The pH modifier may be selected for instance from sodium hydroxide and hydrochloric acid. The EPI solutions are optimally formulated at a pH <3, or 2.6-2.8. Higher pH values for the EPI formulations (like pH 5.8) lead to unstable products (See Examples # 14 to 16).

[00050] In one aspect, a composition comprising from about 0.5 to about 2 mg/ml EPI as the active pharmaceutical ingredient (API), in a form of free base or salt form is disclosed. In one aspect, the composition may comprise a supramolecule, for example, a water-soluble material selected from cyclodextrin derivatives such hydroxypropyl-3 -cyclodextrin (HPpCD) or sulfobutyl-P-cyclodextrin (SBPCD) and/or cucurbituril derivatives or a mixture thereof. (See Examples 5-7) In one aspect, the water soluble supramolecule cyclodextrin derivative such as HPpCD SBEpCD, or a mixture thereof can be used. In one aspect, the amount of cyclodextrin used is 1 : 1 to 1 :3, or 1:2 molar ratio.

[00051] In one aspect, a method for preparing an epinephrine-macromolecule inclusion complex is disclosed. The method may comprise: a. evaporating at a temperature of less than 18°C and at a pressure between 2 - 10 mbar over a period of about 24-72 hours; b. manifold vacuum drying at a temperature of less than about -20 °C and a pressure between 0.1 - 1 mbar over a period of about 24 to about 48 hrs; and c. lyophilizing, wherein said lyophilizing comprises: i. starting with a shelf temperature of from about 5 to about 10 °C for loading; ii. cooling a mixture to freezing at a temperature of from about -20 to about -40 ° C for about 2 to about 4 hours to form a frozen solution; iii. increasing the temperature of said mixture to about 10 to about 20 °C, for about 3 to about 5 hours; iv. holding said mixture at a temperature of about 10 to about 20 °C for about 10 to about 20 hours; v. ramping said mixture to a temperature of about 30 to about 40 °C for about 5 to about 10 hours; wherein a vacuum pressure of from about 100 to about 200 microns is maintained throughout said manifold vacuum drying and from about 200 to about 400 microns throughout said lyophilizing.

[00052] In one aspect, the macromolecule may be selected from one or both of cyclodextrin (CD) and cucurbituril (CB). The epinephrine-macromolecule complex may be characterized in that it exhibits greater resistance to degradation due to oxidation, sulfonation and epimerization than its corresponding physical mixture or un-complexed epinephrine. In one aspect, the epinephrine may be complexed with the macromolecule in a molar ratio of 1 : Ito 1 : 10. The cucurbituril (CB) compound may comprise at least one compound selected from CB homologues containing 5 to 10 glycoluril residues (CBn, n=5- 10) joined by two methylene bridges.

[00053] In one aspect, the method may further comprise adjusting the pH of said mixture to about 2.5 to about 3.5.

[00054] In one aspect, the composition may have an osmolarity of from about 200 to about 400 Osm/kg.

[00055] In one aspect, the composition may comprise from about 1 to about 2 mg/mL sodium metabisulfite.

[00056] In one aspect, the disclosed compositions made according to the preceding method may have an appearance that is clear and free of precipitate after 6 months at 40 °C. In one aspect, the clear, stable epinephrine solution made according to the disclosed methods may comprise the inclusion complex as described herein and one or more excipient.

[00057] In one aspect, the disclosed compositions may comprise a cyclodextrin compound selected from hydroxypropyl derivatives of P- and y-cyclodextrin, a randomly methylated P- cyclodextrin, sulfobutyl ether P-cyclodextrin, glucosyl-P-cyclodextrins, and combinations thereof. In one aspect, the cucurbituril (CB) compound may comprise at least one compound selected from CB homologues containing 5 to 10 glycoluril residues (CBn, n=5-10) joined by two methylene bridges. The epinephrine may be complexed with the macromolecule in a molar ratio of 1 : 1-10. [00058] The solution of any of claims 10 through 13, wherein, when subjected to a temperature of 40 °C, said solution has a shelf life longer than equimolar solutions containing a mixture of epinephrine-CD or epinephrine-CB complex. The solution may have an appearance that is clear and free of precipitate after 6 months at 40 °C, and may contain total impurities of not more than 22 % after 6 months at 40 °C. The disclosed solution may have a pH of from about 2.5 to about 3.5, and an osmolarity of from about 200 to about 400 Osm/kg, and may comprise sodium metabisulfite as an antioxidant from 1-2 mg/ml.

[00059] In a further aspect, disclosed is a method of treating an individual for anaphylactic shock, comprising administering a composition (solution) as disclosed herein. The administration may be via intramuscular injection.

EXAMPLES

[00060] The effects of different methods of preparation of the macromolecular complex on its chemical and physical stability have been evaluated.

[00061] At each time point, samples were randomly selected for evaluation. Initially, a visual inspection was made, and any observation was noted regarding coloration or formed precipitates. According to the United States Pharmacopoeias (USP) stability guidelines, a product has chemical stability if each of the active ingredients retains its labeled potency overtime.

[00062] Epinephrine content and impurities were analyzed using an HPLC method based on the USP method. USP requirements are as follow: epinephrine injection is formulated to contain 90 - 115% of the labeled amount of epinephrine, the pH of the injections should be in the range of 2.5 - 5.0. There are no compound requirements for degradation products (such as nor-epinephrine). Discolored solutions of epinephrine are considered unusable, regardless of potency.

[00063] Also disclosed are preparation methods of this inclusion complex. Specifically, the said pharmaceutical composition comprising EP: CD, CB inclusion complex is prepared as follow:

[00064] The proposed EPI complex is prepared by mixing cyclodextrins and/or cucurbituril water for injection (WFI) adjusted to pH of about 2.6 (using a 0. IN hydrochloric acid) prior to slowly adding EPI until completely dissolved. The solution is then slowly evaporated until dryness under one of the prescribed evaporative coprecipitation methods to obtain a solid complex material.

[00065] The final weight of each solution is adjusted to target weight. The solution is finally filtered through 0.22 pm hydrophilic polyvinylidene fluoride (PVDF) membrane.

[00066] The described examples are in connection with EPI formulations that exhibit various degrees of resistance to degradation (oxidation, sulfonation and epimerization) in a solution or in solid state. The EPI solutions are designed to aid the creation of parenteral IV or IM products with longer shelf lives.

[00067] The compositions of various formulations are presented below. The following examples are provided to illustrate certain aspects of the present disclosure, and are not intended to limit the scope of the invention in any manner.

[00068] Example 1. Epinephrine formulation is prepared by mixing sodium chloride, sodium metabisulphite, into water for injection (WFI) adjusted to pH of 2.9 (using a 0.1N hydrochloric acid) prior to adding EPI. When the formulation was tested under an accelerated stability condition at 40 °C for 6 months, it was found that the solution is unstable. Epinephrine potency was approximately 75%. D-EPT (inactive epimer of EPI) was determined to be over 10% and the sulfonic acid impurity was above 18%. Discoloration with brownish precipitates were visually present.

[00069] Example 1 [00070] Example 2, 3 and 4. The formulation procedure is the same as Example 1, but wherein 1 : 1 equimolar ratio of EPI: HPpCD, EPI: SBEpCD and EPI:CB is added, respectively. It was found that EPI forms a stable binary complex with the three host molecules: HPpCD, SBEpCD and CB7. After 6 (six) months of thermal stress at 40 °C under nitrogen, formulations 2 to 4 were determined to be unstable since total impurities exceed the USP requirements of not more than 22% total impurities. (See Examples 2-4.) No signs of EPI formulation solution discoloration or presence of precipitates. Approximately 10% loss in potency was recorded. Detected impurities including epinephrine sulfonic acid were about 22%. Examples 2 through 4 show that simple mixing of EPI with one of the macromolecules excipients like CD or CB does not impart stability to a formulation to pass a stress period of six (6) months at 40 °C.

[00071] Example 2

[00072] Example 3

[00073] Example 4

[00074] Example 5, 6 and 7

[00075] The formulation procedure is the same as example 2-4, but wherein, the molar ratio of EPI: CD and EPI: CB is 1 :2. After 6 months of stress stability at 40 °C under nitrogen, formulations 5-7 were determined to be stable. No signs of EPI formulation solution discoloration or presence of precipitates and less than 10% loss in potency was recorded. Detected impurities including epinephrine sulfonic acid were less than 22%. Examples 5 to 7 established that a molar ratio of 1:2 in EPICD results in greater stability of the EPI formulation, compared to 1 :1 molar ration, when stressed for six (6) months at 40 °C.

[00076] Example 5

[00077] Example 6

[00078] Example 7

[00079] Example 8, 9 and 10

[00080] The formulation procedure is the same as example 2-4, but no nitrogen protection or blanketing during formulation or storage was provided. After 6 (six) months of stress at 40 °C, formulations 8-10 were determined to be unstable Signs of EPI formulation solution discoloration or presence of precipitates and less than 10% loss in potency were observed. Detected impurities including epinephrine sulfonic acid were less than 22%. Examples 8 to 10 established that nitrogen protection is needed to achieve greater stability for six (6) months at 40 °C when the EPI: CD, CB prepared from simple physical mixture.

[00082] Example 9

[00083] Example 10

[000841 Example 11, 12 and 13

[00085] Compositions of Examples 11 to 13 show the destabilizing effects caused by the absence of SMB. Examples 11-13 provide the compositions of 1.0 mg/mL epinephrine formulations at pH 2.9, 6 mg/mL of sodium chloride, no SMB and 1 : 1 equimolar ratio of EPI: CD and EPI: CB with nitrogen protection and/or blanketing during formulation and storage. After 6 (six) months of stress stability at 40 °C, formulations 11-13 were determined to be unstable. Signs of EPI formulation solution discoloration or presence of precipitates. Over 10% loss in potency. Total Detected impurities including epinephrine sulfonic acid were over 22%. Example 11 to 13 suggest that presence of SMB is necessary to achieve greater stability in EPI formulations that contain EPI: CD, CB in a physical mixture form.

[00086] Example 11

[00087] Example 12

[00088] Example 13

[00089] Example 14, 15 and 16 provides the compositions of 1.0 mg/mL epinephrine formulations at pH 5.5, 6.0 mg/mL of sodium chloride, 1.67 mg/ml of sodium metabisulfite and 1 :2 molar ratio of EPI: CD and EPI: CB with nitrogen protection and/or blanketing during formulation or storage. After 6 months of stress stability at 40 °C, formulations 14-16 were determined to be unstable with signs of formulation solution discoloration or presence of precipitates. Over 10% loss in potency. Total Detected impurities including epinephrine sulfonic acid were over 22%. Higher shift in pH value does have a detrimental effect on stability. Example 14 to 16 establish that acidic conditions are necessary to achieve stability for a stress period of 6 months at 40 °C. Examples 1-16 provided different scenarios for limited epinephrine stabilization including resistance to oxidation, heat, and epimerization. Stabilization of EPI solutions toward degradative processes (like thermal decomposition, sulfonation and epimerization) as illustrated by Examples 1 to 16, was significantly improved using macromolecular inclusion complex of EPI: CD, CB. The latter complexes are prepared by several slow complexation of a solution containing the EPl under reduced pressure and low temperatures and/or controlled freeze drying. The macromolecular inclusion complexes with CD or CB are subsequently introduced into aqueous mixture containing other excipients of which an antioxidant like sodium metabisulphite, adds to the oxidative resistance of the products.

[00090] Example 14

[00091] Example 15

[00092] Example 16

[00093] Examples 17 to 24 illustrate the three different methods for the preparation and use of the macromolecular EPI: CD, CB complexes in the design of aqueous parenteral EPI solutions.

[00094] Example 17. The solution containing epinephrine was first complexed with stabilizing CD and/or CB, at different molar ratios, via slow evaporation until dryness in a vacuum oven at temperature less than 18 °C under reduced pressure (2-10 mbar) to obtain a solid and stable inclusion complex of EPI-CD, CB.

[00095] Example 17

[00096] Example 18. The solution containing epinephrine was first complexed with stabilizing CD and/or CB, at different molar ratios, via Manifold drying wherein the temperature is less than -10 °C and a reduced pressure between 0.1 - 1 mbar in about 6 to about 12 hours to obtain a solid and stable inclusion complex material of EPI-CD, CB. Stabilization of EPI solutions toward degradative processes (like thermal decomposition, sulfonation and epimerization) [00097] Example 18

[00098] Example 19. Freeze-dried inclusion complex was prepared by dissolving the CD and/or CB in water and adding the stoichiometric amount of the EPI. The solution was stirred for 30 min at room temperature. Filtration (0.45 p filter) was performed, and the resulting solution was then freeze-dried via controlled lyophilization recipe over 24-72 hours. The obtained solid was grounded and preserved in a tight closed container until used.

[00099] Example 19

[000100] Example 20. EPI, cyclodextrin and/or cucurbituril were mixed with all other excipients (such as NaCl and MBS) in water for injection until completely dissolved, then followed evaporation until dryness per Example 18-20 to obtain a solid macromolecular inclusion complex material. [000101] Example 21 . Cyclodextrin and/or cucurbituril is mixed in acidified water, at about pH 2.6, to which the EPI is added while continue mixing until fully dissolved. The resulting mixture was filtered through a 0.45 pm filter and its water is also removed under a vacuum per Example 18-20. The resulting dried and stable solid inclusion complex is compounded with the other excipients (such as NaCl and MBS) to prepare the EPI aqueous pharmaceutical composition.

[000102] Example 22. Epinephrine formulation solutions containing different concentrations of sodium metabisulfite are formulated per example 18-20 and examined for stability. For example, reparation of an Epinephrine formulation with half the amount of antioxidant (0.8 mg/ml sodium metabisulfite vs. 1.67 mg/ml in the marketed formulation).

[000103] Example 23. Epinephrine solution containing EPI and cyclodextrin and/or cucurbituril were compounded according to Example 18-20 at ambient conditions, with no nitrogen or light protection for about 5 hours, followed by nitrogen purging and nitrogen protection during the filling of EPI formulation into containers under oxygen free environment.

[000104] Example 24. An inclusion complex powder prepared via physical mixture is compared to another inclusion complex prepared by examples 18-20, shows that the latter is superior in stability (slower degradation due to oxidation, sulfonation and epimerization) to the former and to un-complexed EPT. Tn summary, the results obtained indicated that Examples 18-24 prepared per the described complexation methods, found to be advantageous in stability compared to Examples 1-17. After 6 (six) months of thermal stress at 40 °C under full or partial nitrogen protection, formulations of Examples 18 to 24 were determined to meet the following stability specifications: 1) appearance of EPI formulation solution was clear with no discoloration or precipitates; 2) not more that 10% loss in potency was recorded; 3) not more than 10% of D- Epinephrine; and 4) total impurities not exceeding 22%.

[000105] REFERENCES

[000106] 1. Stella, V.J. et al., 1999, Mechanism of drug release from cyclodextrin complexes, Adv. Drug Deliv. Rev. 36; 3-16. [000107] 2 Loftsson, T., et al., 2016, Pharmacokinetics of cyclodextrins and drugs after oral and parenteral administration of drug cyclodextrin complexes, J. Pham. Pharmacol., 68; 544- 555).

[000108] 3. Sohivirat, S., et al., Characterization of prednisone in controlled porosity osmotic pump pellets using solid-state NMR spectroscopy, J. Pharm. Sci. 2007; 96; 1008-1017.

[000109] 4. Hong J., et al., Effect of cyclodextrin derivation and amorphous state of complex on accelerated degradation of ziprasidone, J. Pharm. Sci. 2011; 100; 2703-2716.

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[000112] All percentages and ratios are calculated by weight unless otherwise indicated.

[000113] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[000114] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[000115] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.” [000116] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[000117] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.