MXPA06008032A - Antimicrobial preservatives to achieve multi-dose formulation using beta-cyclodextrins for liquid dosage forms - Google Patents

Abstract

The present invention is directed to pharmaceutical compositions containing a therapeutically effective amount of an Active Pharmaceutical Ingredient ("API"), a pharmaceutically acceptable cyclodextrin and a pharmaceutically acceptable preservative. The invention is also directed to pharmaceutical compositions of the compounds of Formula (I) wherein R2 is selected from the group consisting of methyl, ethyl, isopropyl, sec-butyl and tert-butyl and a pharmaceutically acceptable cyclodextrin and preservative. Formula (I):In particular, the invention is directed to pharmaceutical compositions of the compound of Formula la, and a pharmaceutically acceptable cyclodextrin and a preservative.

Description

ANTIMICROBIAL PRESERVATIVES TO ACHIEVE A FORMULATION OF MULTIPLE DOSE USING CICLODEXTRINS ß FOR LIQUID DOSAGE FORMS FIELD OF THE INVENTION The present invention relates to pharmaceutical compositions containing a therapeutically effective amount of a pharmaceutically active ingredient ("API"), a pharmaceutically acceptable cyclodextrin and a pharmaceutically acceptable preservative. The invention also relates to pharmaceutical compositions of the compounds of formula I, wherein R 2 is selected from the group consisting of methyl, ethyl, isopropyl, sec-butyl, and fer-butyl and a pharmaceutically acceptable cyclodextrin and preservative.

In particular, the invention relates to pharmaceutical compositions of the compound of formula la, and a pharmaceutically acceptable cyclodextrin and preservative.

The invention further relates to the improvement of the tolerance at the injection site of aqueous injectable solutions comprising the compound of formula I, or its pharmaceutically acceptable salts, a β-cyclodextrin and a preservative. The invention also relates to a method of developing a composition of a conserved API.

BACKGROUND OF THE INVENTION The administration of neurokinin receptor antagonists, including the compounds of formulas I and II, presents various problems with regard to tolerance at the site of injection (eg, irritability of the subject, irritation, inflammation, swelling). , and / or redness of the site). However, although there have been numerous studies regarding the improvement of tolerance at the site of injection through the use of various substances, none of these studies has focused on the administration of neurokinin receptor antagonists. The compounds of formula I or A are the subject of U.S. 5,807,867, U.S. 6,222,038 and U.S. 6,255,320. The preparation of the compounds of formula I or the one described in those documents. The compound of formula I can also be prepared as described in the co-pending United States Provisional Application No. 60 / 541,323, owned and assigned herewith to Pfizer, Inc. US Pat. No. 5,393,762. also discloses pharmaceutical compositions and treatment of emesis using NK-1 receptor antagonists. Provisional United States Provisional Application No. 60 / 540,697, owned and assigned herewith to Pfizer, Inc. discloses a procedure of improving the recovery of anesthesia in patients by administering the compound of formula I or la. The text of the aforementioned applications, patents and all other references cited in this specification are incorporated herein by reference in their entirety. The compound of formula la is a basic drug with two amine functional groups, a secondary amine with a pKa of 4.43 and a tertiary amine with a pKa of 9.31. The citrate salt of the compound of formula la has a solubility of 2.7 mg / ml at a pH of 4.2 in 0.02 M phosphate buffered solution / 0.02 M acetate. The desired solubility of 10 mgA / ml is it could be obtained by the addition of salts (for example, NaCl, CaCl2 or sodium acetate), using a partially aqueous, oleaginous, or micellar vehicle, or by adding a modified, parenterally acceptable cyclodextrin. However, in general, it was observed that formulations containing cyclodextrins provided better tolerance at the injection site over other approaches that increase solubility. The assurance of an adequate solubility of a pharmaceutical drug in parenteral formulations is crucial, especially when the drug has a low aqueous solubility. The modification of the pH of the solution, the selection of the salt form of the drug, and the use of co-solvents are common approaches used to achieve an adequate solubility. Atypical approaches involve excipients, such as complex formation agents. The cyclodextrin can enhance solubility by forming an inclusion complex with the drug molecule in which the insoluble / hydrophobic drug is inserted into the hydrophobic cavity of the cyclodextrin. Therefore, the outer hydrophilic shell of the cyclodextrin molecule enhances the solubility of the entire complex. The usual terminology for the formation of cyclodextrin complexes identifies the cyclodextrin as a "host" molecule and the drug as a "host" molecule. Unfortunately, the cyclodextrin used to form the inclusion complex can also bind to preservatives, inactivating a large amount of sparingly water-soluble preservatives. Sulfobutyl ether-β-cyclodextrin (hereinafter "SBE-CD") was found to be effective both by increasing the solubility of the formula compound la and by improving the reactions at the injection site. Unfortunately, the investigation determined that the SBE-CD formed complexes with both the antimicrobial preservative (eg, meta-cresol) and the compound of the formula la, resulting in competitive binding interactions and, in general, antimicrobial inefficiency. Accordingly, it was necessary to obtain an optimum balance between a sufficient concentration of cyclodextrin (e.g., SBE-CD) and antimicrobial preservative (e.g., meta-cresol). Although a lower concentration of SBS-CD would increase the efficacy of the antimicrobial preservative, however, this advantage would be compensated for by an acceptable decrease in tolerance at the injection site ("IST"). These competitive performance characteristics needed to balance the antimicrobial preservative efficacy (criterion A) and tolerance at the injection site acceptable to the product.

The interim United States Provisional Application with this No. 60 / 540,644, filed concurrently with the present application and assignment and ownership of Pfizer Inc, describes a procedure for improving tolerance at the injection site during parenteral administration of a composition containing the compound of formula I and cyclodextrin. A preservative compatible with cyclodextrin was also identified, providing desirable multiple use dosing options. Preferably, meta-cresol is used in the formulation to prevent bacterial and fungal development in the formulation during the proposed period of extended use.

SUMMARY OF THE INVENTION In one aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically active ingredient (API), a β-cyclodextrin, a pharmaceutically acceptable preservative, a pharmaceutically acceptable carrier, and an optional pharmaceutically excipient. acceptable, in which the preservative demonstrates efficacy as a pharmaceutically acceptable antimicrobial preservative.

In a preferred embodiment, the β-cyclodextrin is 2-hydroxypropyl-β-cyclodextrin or sulfobutyl ether-β-cyclodextrin, preferably sulfobutyl ether-β-cyclodextrin. In another embodiment, the pharmaceutically acceptable preservative is selected from thimerosal, propylene glycol, phenol, or meta-cresol or a combination thereof. Preferably the preservative is meta-cresol. Preferably, the concentration of the preservative is between about 0.1 mg / ml and about 600 mg / ml. Preferably, the preservative is oxygen and is in a concentration of between about 0.1 mg / ml and about 20 mg / ml. In a preferred embodiment, the pharmaceutical composition has a pH in the range of between about 3 and about 6. In a preferred embodiment, the preservative has a cyclodextrin binding value that is less than an API binding value to the cyclodextrin .

Preferably, the binding value of the API to the cyclodextrin is between 500 M "1 and . 000 M "1. Preferably, the binding value of the API to the cyclodextrin is between 800 M-1 and 3,000 M "1. In another embodiment, the pharmaceutically active ingredient has a value greater than or equal to two times the binding constant with the cyclodextrin over that of the preservative In a preferred embodiment, the binding constant is greater than or equal to five times In a more preferred embodiment, the binding constant is greater than or equal to ten times In a preferred embodiment, from about 1 mg / ml to about 5 mg / ml of the preservative, preferably meta cresol, is un-sequestered in the cyclodextrin Preferably, approximately 2.5 mg / ml of the preservative, preferably meta-cresol, is uncapped in the cyclodextrin In a preferred embodiment, the pharmaceutical composition has an antimicrobial efficacy against the bacteria such that the concentration of bacteria decreases in a logarithmic reduction of 2 or greater after 6 hours, a logarithmic reduction of 3 or greater after 24 hours, and a recovery zero ion of bacteria after 28 days. Preferably, the bacteria are selected from Escherichia coli (bacteria, gram negative) (ATCC 8739), Pseudomonas aeruginosa (bacteria, gram negative) (ATCC 9027) or Staphylococcus aureus (batteries, gram positive) (ATCC 6538). In a preferred embodiment, the pharmaceutical composition has an antimicrobial efficacy against a fungus or mold so that the concentration of fungus or mold decreases in a logarithmic reduction of 2 or greater after 7 days, a logarithmic reduction of 1 after 14 days, and no increase in fungi or mold after 14 days to approximately 28 days. Preferably, the fungus is Candida albicans (fungus) (ATCC 10231) and the mold is Aspergillus niger (mold) (ATCC 16404). In a preferred embodiment, the pharmaceutical composition has an antimicrobial efficacy that satisfies criteria A and B of the European pharmacopoeia and the AET criterion of the USP. In another aspect, the invention relates to a pharmaceutical composition comprising a compound of formula I as an active pharmaceutical ingredient, or their pharmaceutically acceptable salts, wherein R2 is selected from the group consisting of methyl, ethyl, isopropyl, sec-butyl and tert-butyl, preferably tert-butyl, a pharmaceutically acceptable cyclodextrin, a pharmaceutically acceptable preservative, a pharmaceutically carrier acceptable and an optional pharmaceutically acceptable excipient. Preferably, the β-cyclodextrin is 2-hydroxypro-yl-β-cyclodextrin or sulfobutyl ether-β-cyclodextrin, preferably sulfobutyl ether-β-cyclodextrin.

Preferably, the pharmaceutically acceptable preservative is selected from thimerosal, propylene glycol, phenol, or meta-cresol, or a combination thereof. Preferably, the preservative is meta-cresol. Preferably, the pharmaceutical composition has a pH in a range of between about 4 and about 5. In a preferred embodiment, from about 1 mg / ml to about 5 mg / ml of the preservative, for example, meta-cresol, is not sequestered in the cyclodextrin. In a preferred embodiment, the compound of formula I, or a pharmaceutically acceptable salt thereof, is in an amount between about 0.1 mg / ml and about 100 mg / ml and cyclodextrin β is in an amount between about 20 mg / ml. ml and approximately 200 mg / ml and the preservative is meta-cresol. Preferably, the β-cyclodextrin is in an amount of between 55 mg / ml and 100 mg / ml and the meta-cresol is in an amount of between about 2.5 mg / ml and 3.5 mg / ml. In a preferred embodiment, the compound of formula I is the compound of formula la, or their pharmaceutically acceptable salts. Preferably the compound of formula la, a pharmaceutically acceptable salt, is in an amount of between about 0.1 mg / ml and about 100 mg / ml and the cyclodextrin β is in an amount of between about 20 mg / ml and about 200 mg / ml and the preservative is meta-cresol and is in an amount of between about 1 mg / ml and about 5 mg / ml. Preferably, the β-cyclodextrin is in an amount of between about 55 mg / ml and about 100 mg / ml and the preservative is meta-cresol and is in an amount of between about 2.5 mg / ml and about 3.5 mg / ml. Preferably, the β-cyclodextrin is sulfobutyl ether β-cyclodextrin. In a third aspect, the invention relates to a pharmaceutical composition comprising the compound of formula la, or their pharmaceutically acceptable salts, wherein the compound of formula Ia is 10 mgA / ml, sulfobutyl ether-cyclodextrin β is in an amount of about 63 mgA ml and meta-cresol is in an amount of about 3.3 mg / ml , a pharmaceutically acceptable carrier and an optional pharmaceutically acceptable excipient. Preferably, the pharmaceutically acceptable salt of the compound of formula la is citrate. In a fourth aspect, the invention relates to a method for treating emesis or improving the recovery of anesthesia in mammals comprising the parenteral injection in the mammal of an aqueous pharmaceutical composition comprising the pharmaceutical compositions described above of the compounds of formula I or la, the β-cyclodextrin being present in amounts which are sufficient for an improvement of injection tolerance at the injection site. Preferably, the pharmaceutically acceptable salt is citrate. Preferably, the composition is administered subcutaneously. In a fifth aspect, the invention relates to a method of improving the tolerance at the injection site during the treatment of emesis or the treatment for improving the recovery of anesthesia in a mammal comprising parenteral injection in the mammal of a pharmaceutically acceptable solution of the pharmaceutical compositions described above of the compounds of formula I or la. Preferably, the pharmaceutically acceptable salt is citrate. Preferably, the composition is administered subcutaneously. In a sixth aspect, the invention relates to a method for developing a conserved API composition comprising a therapeutically effective amount of an API, a β-cyclodextrin and a pharmaceutically acceptable preservative. In a preferred embodiment, the preservative has a cyclodextrin binding value that is lower than the binding value of the API to the cyclodextrin. Preferably, the preservative is selected from thimerosal, propylene glycol, phenol or meta-cresol or a combination thereof. In a preferred embodiment, the binding value of the API with the cyclodextrin is greater than 50 M "1. Preferably, the binding value of the API with the cyclodextrin is between 500 and 10,000 M" 1. Preferably, the binding value of the API with the cyclodextrin is between 800 and 3,000 M "1. In a preferred embodiment, the requirements of the antimicrobial efficacy test (AET) meet criteria A and B of the European Pharmacopoeia and the AET criterion. USP In a further aspect, the invention relates to a pharmaceutical composition, as defined herein, for use as a medicament especially in, when the composition comprises a compound of formula I or the treatment of a disease. for which a neurokinin receptor antagonist, such as an NK-1 receptor antagonist, is indicated.In a further aspect, the invention relates to the use of a pharmaceutical composition, as defined herein, comprising a compound of formula I or the, in the manufacture of a medicament for the treatment of a disease for which a neurokinin receptor antagonist, such as a NK-1 receptor antagonist is indicated. In a further aspect, the invention relates to a method for the treatment of a disease for which a neurokinin receptor antagonist, such. as an NK-1 receptor antagonist, it is indicated in mammals comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition as defined herein comprising a compound of formula I or la. Definitions The term (s) "compound (s) of formula I" and "compound of formula la" as used herein means a compound or compounds of formula I or the prodrugs thereof and pharmaceutically acceptable salts of the compounds or prodrugs. The compounds used in the present invention can be isolated and used per se or in the form of their pharmaceutically acceptable salt, solvate and / or hydrate. The term "pharmaceutically acceptable salt" refers to inorganic and organic salts of a compound of the present invention. These salts can be prepared in situ during the isolation and final purification of a compound, or by separately reacting the compound, or prodrug with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the salts hydrobromide, hydrochloride, hydroiodide, sulfate, bisulfate, nitrate, acetate, trifluoroacetate, oxalate, besylate, palmitate, pamoate, malonate, stearate, laurate, malate, maleate, borate, benzoate, lactate, phosphate, hexafluorophosphate, benzene sulfonate, tosylate, formate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and lauryl sulfonate, and the like. See, for example, Berge, et al., J. Pharm. Sci., 66, 1-19 (1977). Preferably, the pharmaceutically acceptable salt is citrate. The term "citrate salt" as used in this specification, refers to the monohydrated citrate salt of the compound of the formula la, which has a molecular weight of 660.82 and a theoretical power based on the active ingredient of 709 mg / g ,. The term "active pharmaceutical ingredient" or "API" as used in this specification, refers to a pharmaceutical drug substance having therapeutic properties and having the ability to bind or be "sequestered" in cyclodextrin. Preferably, the API has a cyclodextrin binding value greater than 50 M "1. More preferably, the API has a cyclodextrin binding value of between about 800 and about 3,000 M" 1. Even more preferably, the API has a cyclodextrin binding value of between about 500 and about 10,000 M "1. In addition, preferably, the API has a value of more than two times the cyclodextrin binding constant to the preservative. More preferably, the API has a value greater than 5 times the binding constant with the cyclodextrin Even more preferably, the API has a value greater than or equal to 10 times the binding constant with the cyclodextrin The term "active ingredient" or "mgA / ml", as used in this specification, refers to the free base of the compound of formula la, which has a molecular weight of 468.69 The term "cyclodextrin" refers to a compound that includes units of cyclic D-glucopyranose linked with a- (1? 4) bonds. Cyclodextrin a refers to a cyclodextrin with 6 cyclical D-glucopyranose units linked together, cyclodextrin? has 7 cyclic D-glucopyranose units joined together, and cyclodextrin and has 8 cyclic D-glucopyranose units linked together. These cyclically linked D-glucopyranose units define a hydrophobic cavity, and cyclodextrins are known to form inclusion compounds with other organic molecules, with salts, and with halogens either in the solid state or in aqueous solutions. Cyclodextrins vary in structure and properties. For example, the size (eg, diameter and depth) and functionality (eg, hydrophobicity, charge, reactivity and ability to form hydrogen bonds) of the hydrophobic cavity varies between the a, b and substituted and unsubstituted cyclodextrins. Typically, a cyclodextrin selected for a formulation has a size and functionality that binds the target component with the other components of the formulation. For the present formulations and procedures, it is believed that substituted cyclodextrins, such as hydroxyalkylcyclodextrins and sulfoalkylethercyclodextrins have a size and functionality that compliments the other components of the formulation. Preferred cyclodextrins include hydroxypropyl-β-cyclodextrin and sulfobutyl ether-β-cyclodextrin. More preferably, the cyclodextrin is sulfobutyl ether-β-cyclodextrin ("SBE-CD"). The phrase "therapeutically effective amount" means an amount of a compound of the present invention that (i) treats or prevents the particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of the disease, condition or particular disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition or disorder described in this specification. The term "mammals" or "animals", as used in this specification, refers to humans, companion animals such as, but not limited to, dogs, cats and horses, food source animals (e.g., cows, pigs and sheep), zoo animals and other similar animal species.

The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and / or toxicologically, with the other ingredients comprising a formulation, and / or the mammal being treated with them. The term "treating", "treating" or "treatment" includes both preventive treatment, that is, prophylactic and palliative treatment. The term "improved injection site tolerance" as used in this specification means a score of two or less, as defined in this specification in Table IV. The term "pharmaceutically acceptable preservative", as used in this specification, means a preservative. In particular, the preservative-containing formulation maintains efficacy according to the patterns set forth in Far. Eur. 4th edition, 2003 (5.1.3) for parenteral formulations and in USP26 NF21S2, < 51 > for the pharmaceutical products of category 1. Preferably, the preservative has a reduced cyclodextrin binding value compared to the API, so that sufficient preservative is "not sequestered" in the cyclodextrin, providing effective antimicrobial effectiveness.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the saturated solutions of meta-cresol of SBE-CD and the compound of formula la. Saturated meta-cresol solutions were prepared in varying amounts of SBE-CD and drug. The concentration of meta-cresol showed linear increase as SBE-CD grew. The drug concentration did not significantly alter the solubility of m-cresol in SBE-CD. In Figure 1, A: Solubility of m-cresol in SBECD with varying amounts of CJ-11, 972-10., B: m-Cresol [mM], C: SBE-CD [mM]. Figure 2 shows the concentration of the compound of formula la versus time at a concentration of 1, 0.5, and 0.25 mM of the compound of formula la, was adjusted to equation 11 using the Scientist software. The compound of formula la was charged to the donor side of the dialysis cell. The concentration of the drug in the donor and acceptor side is presented versus time. The drug reaches equilibrium in approximately 48 hours. In figure 2, A: donor cell, B: acceptor cell, C: total drug%, D: time (hours). Figure 3 shows the comparison between bacterial efficacy as a function of the total amount of meta-cresol and as a function of the kidnapped meta-cresol calculated for S. aureus at the 6-hour and 24-hour instants. The circles in black are the data corresponding to the formulations containing a total of 2.95 to 3.15 mg / ml meta-cresol. In figure 3, A: Bacteria: moment 6 hours, B: Bacteria: moment 6 hours, C: Bacteria: moment 24 hours, D: Bacteria: moment 24 hours, E: total metacresol in the formulation (mg / ml), F: Logarithmic reduction of S. Aureus, G: metacresol not sequestered calculated in the formulation (mg / ml), H: Logarithmic reduction of S. Aureus, I: total metacresol in the formulation (mg / ml), J: Logarithmic reduction of S. Aureus , K: non-sequestered metacresol calculated in the formulation (mg / ml), L: Logarithmic reduction of S.-Aureus, M: Logarithmic reduction of S. aureus presented as a function of total amount of meta-cresol in the formulation, N : Logarithmic reduction of S. aureus presented as a function of calculated non-sequestered amount of meta-cresol in the formulation. Figure 4 shows a formulation window that guarantees the efficacy of the preservative according to Farm criterion A. Eur., Painless in the injection, less than 3.5 mg / ml of meta-cresol, and less than 80 mg / ml of SBE-CD. In Figure 4, A: more than 2 mg / ml of non-sequestered compound, B: greater risk of pain in injection and drug precipitation, C: emission with robustness of preservative efficacy, D: more than 3.5 mg / ml of metacresol regulatory emission, E: Cyclodextrin concentration too high (more than 80 mg / ml), F: Compound not sequestered calculated (mg / ml), G: Metacresol not sequestered calculated (mg / ml), H: Criterion failed A of Farm. Eur., I: Past criterion B of Farm. Eur. DESCRIPTION OF THE INVENTION The development of parenteral formulations using cyclodextrin for solubilization, or for other purposes, requires an understanding of the interaction between the drug and the cyclodextrin. A pharmaceutical drug that is solubilized by the cyclodextrin is bound in a stoichiometric ratio relative to an inherent binding constant. This relationship varies based on various factors such as the structure of the drug, cyclodextrin, and the properties of the solution (e.g., pH, ionic strength, and co-dissolvency). Formulations that have multiple excipients further complicate the interaction. For example, in multiple-use parenteral formulations containing a preservative, the preservative may compete with the drug for cyclodextrin binding. It was previously reported that 2-hydroxypropyl-β-cyclodextrin interacts not only with drug molecules but can also form complexes with antimicrobial preservatives. Loftsson, T. et al., Drug Development and Industrial Pharmacy 1992, 18 (13), 1477-1484. However, the binding of preservative and cyclodextrin decreases the antimicrobial efficacy of the preservative, since the preservative needs to be unbound in the solution. A minimum requirement for conservation effectiveness for parenteral products is described in the European pharmacopoeia, criterion A being applicable, and in the United States pharmacopoeia. The antimicrobial preservatives for the proposed formulations were evaluated according to the antimicrobial efficacy test criterion ("AET"). A multiple dose formulation of the compound of the formula containing 10 mgA / ml of the compound of the formula la and 10% (w / v) cyclodextrin at pH 4.4 was used to identify an effective antimicrobial preservative that did not significantly interact with the cyclodextrin. From preliminary experiments, the solubility of the compound of the formula I in the presence of 2-hydroxypropyl-β-cyclodextrin was similar to the solubility in the presence of SBE-CD. In addition, both produced a formulation with tolerance at the acceptable injection site ("IST"). In addition to compatibility with cyclodextrin, for example, SBE-CD, there were additional criteria that limited acceptable antimicrobial preservatives for the formulation. These criteria were the physical and chemical compatibility with the compound of formula; the efficacy of the preservative against bacteria, fungi and yeasts at a pH of approximately 4.4 and tolerance at the acceptable injection site. As described more fully in the experimental section, a preliminary selection for an antimicrobial preservative for the multiple-dose compound of the formulation of the formula was carried out with chlorocresol, phenylethanol, benzyl alcohol, ethanol, bronopol, sucrose, gluconate of chlorhexidine, thimerosal, benzethonium chloride, benzalkonium chloride, chlorobutanol, benzoic acid, meta-cresol, phenol, and 25% propylene glycol. The initial results indicated that thimerosal, chlorobutanol / phenylethanol, ethanol and propylene glycol (50%) met the requirements of USP / Farm. Eur. (Table VII). When considering the tolerance issues at the injection site, chlorobutanol / phenylethanol, ethanol and propylene glycol showed little tolerance at the injection site (Table VIII). In contrast, thimerosal and meta-cresol provided good tolerance at the injection site.

Benzethonium chloride and benzoic acid were both ineffective in reducing microorganisms after 7 days. Propylene glycol (25%) was active against bacteria only in the presence of SBE-CD, but ineffective against fungi. On the other hand, the phenolic, phenol and meta-cresol compounds were effective in reducing the microorganisms, but their activity against bacteria was greatly diminished when the SBE-CD was present in the formulation. The inventors suspected and determined that the difficulties encountered in preserving the desired formulation were due to an interaction between the antimicrobial preservative (eg, meta-cresol) and the SBE-CD. In particular, the preservative, for example meta-cresol, was probably sequestered by the SBE-CD, making the meta-cresol inactive against bacteria and fungi. In order to demonstrate this theory, the binding constant of the compound of formula la to SBE-CD and meta-cresol to SBE-CD (Kp) was determined. These constants were used to calculate the concentration of meta-cresol not sequestered in the formulations tested to evaluate the antimicrobial efficacy. The average values used for the calculations are the binding constant for the drug ("KD" = 1000) and the binding constant for the preservative ("Kp" = 28). In cases where the preferential binding of a component is desired, it is desirable to quantify the binding part of each component at equilibrium. The binding of one component to another in solution can be measured using techniques such as spectroscopy, or calorimetry. Gadre, A., and Connors, K. A. "Binding of Substituted Acetic Acids to a-Cyclodextrin in Aqueous Solution" J. Pharm. Sci. 1997 86 (11): 1210-1214.). In order to differentiate the binding by inclusion of other possible effects of solubilization of a ternary formulation agent, such as stacking or hydrotropy, a method is required to determine the binding constant of a component bound to cyclodextrin in the presence of other competitive binders. . The ability to distinguish between union and other modes of interaction is meaningful to understand and design the optimal formulations. In the present invention, the method for determining binding constants utilizes equilibrium dialysis in the development of a multiple-use parenteral formulation containing SBE-CD and a preservative. In particular, the method was applied in the development of a parenteral formulation comprising the compound of formula la, a cyclodextrin (SBE-CD) and a preservative (meta-cresol). This approach is applicable to compounds other than the compound of formula la in the development of parenteral formulations and is within the scope of this invention. The development of the formulation using this approach resulted in the optimization of the cyclodextrin bound to the drug and not bound to the preservative. The significance of this procedure is its ability to measure the binding constant of multiple compounds that compete for binding with cyclodextrin. Experimental data from dialysis also provide an easily interpreted representation of binding in the formulation by visualizing the degree of interaction by the equilibrium established after dialysis. Equilibrium dialysis allows the calculation of the binding constants by modeling the resulting diffusion rate through a semipermeable membrane with a final equilibrium point. Equilibrium dialysis is performed by leaving the substrate in a solution containing bound substrate and ligating in a donor compartment of a dialysis apparatus by equilibrium (cell) that is balanced over time with an acceptor compartment. Ono, N., Hirayama, F., Arima, H., Uekama, K. "Determination of Stability Constant of ß-Cyclodextrin Complexes Using the Membrane Permeation Technique and the Permeation Behavior of Drug Competing Agent-ß-Cyclodextrin Ternary Systems" Eur J. Pharm. Sci. 1999 9: 133-139. The acceptor cell does not contain ligands. The membrane is semipermeable allowing substrates typically of low molecular weight to diffuse freely, while cyclodextrin (MW = 2163) remains in the donor compartment. Sampling of both compartments over time produces a concentration profile over time of the substrate in both the donor compartment and the acceptor of the dialysis cell. A mathematical model can be obtained that describes the speed of diffusion of a drug through the membrane for systems that contain two or more components in solution. The dialysis speed and binding constant for the substrates are obtained by solving the equation using a non-linear curve fitting software. Depending on the interactions between the components it is possible to describe the competitive union that occurs in the solution. The equilibrium binding constant is a measure of the relative concentration of meta-cresol bound to the SBE-CD according to the chemical equilibrium equation below: S = meta-cresol. L = SBE-CD. S: L indicates the complex formed between meta-cresol and SBE-CD.

K S + L S: L [S: J K = [S] [L] Solubility analysis. The citrate salt of the compound of formula la has a solubility of 2.7 mg / ml at a pH of 4.2 in 0.02 M phosphate buffered solution / 0.02 M acetate. The traditional solubility procedures were initially carried out for determining the constants of solubility and binding of the compound of formula la and of the preservative with SBE-CD. These studies allowed the determination of the binding stoichiometry between SBE-CD and a compound of formula la as observed by the linear slope in the ratio of molar solubility of the compound of formula la and SBE-CD (Fig. 1). The binding for meta-cresol was calculated using solubility analysis. The experiment was carried out at different concentrations of the compound of formula la to determine if the presence of drug in solution had an effect on the meta-cresol binding constant. The solubility of meta-cresol was measured in excess (saturated) meta-cresol and the equilibrium binding constant was calculated using the following equation: KnsQLt St - sQ l + ^ u ^ o Where St is the total solubility of meta-cresol, s0 is the inherent solubility of meta-cresol, Lt is the total concentration of SBE-CD (ligand) and Kp is the equilibrium constant of meta-cresol assuming a Binding stoichiometry from 1 to 1. Applying the solubility procedure, the average equilibrium constant of meta-cresol was 27.6 M "1 throughout all the studies There was a minimal effect on the binding from the presence of the compound of formula la as shown in table I. These data were used to compare the results to the equilibrium dialysis process that was currently investigated.The compound of formula had a binding constant of 1040 M "1 to pH 4.4. Table I Table I: Unit constants calculated from the experiments of saturated solubility of meta-cresol in SBE-CD and drug (compound of formula la) variables. The slope of the solubility against the concentration of SBE-CD was used to estimate the binding. The addition of the compound of formula la did not significantly alter the concentration of meta-cresol. Equilibrium dialysis procedure The initial experiments established equilibrium dialysis flow rates for the compound of formula la and meta-cresol through the dialysis membrane 500 MW CO. Three different concentrations of the compound of formula la were initially charged to the donor side of the dialysis well. The samples were removed at various time intervals and the concentration of the free component was measured using HPLC. The equilibrium was achieved for each condition tested after approximately 4 days. The smoothed line was an adjustment to the data using the model for a unitary system presented in the description. The equilibrium point for all these control experiments was reached after 50% of the total drug was distributed evenly across the donor and acceptor sides of the well. This asymptotic approach to equilibrium was modeled and the dialysis rates were calculated, table II.

Table II Table II: Union constants calculated from the equilibrium dialysis procedure. The asymptotic diffusion rates were adjusted to equation 11 using numerical linear adjustment software to generate the binding constants.

The primary procedure for analyzing the data was to perform the calculations from the equilibrium dialysis data, as described below. In particular, the velocity of diffusion through the membrane was calculated using the following equations: The diffusion rate of the donor phase is defined by the following relationship: Diffusion speed towards the acceptor phase: where k = constant of permeation speed, min "1 [D] o = concentration in donor or acceptor in time 0 [D] t = concentration in donor or acceptor in time t [D] eq = concentration in donor or acceptor in equilibrium t = time (min) The basis of the calculation in the presence of SBE-CD is to assume that complex formation occurs only in the donor phase according to the pattern complex formation reaction: K D + L < ? D: L The differential equation that governs the diffusion of the drug in the acceptor phase is given below: ~ ^ - = k [D] F -k [D] A (3) at The mass balance for the drug in the system is described below: [Dl? = [D] F + [DJA + [D: CyD] (4) wherein [D] F and [D] A are free drug in the donor well and free drug in the acceptor well, respectively. The mass balance for the cyclodextrin in the system, maintained within the donor phase, is given below: [CyD] tot = [CyD] F + [D: CyD] (5) Substituting the complexed drug from the mass balance (ec) in the equilibrium relation, we obtain: ([D] tol ~ [D] F - [D) A) K = [D] F [CyD} F (6) Solving the free drug and substituting it in equation 3 results in: Simplifying results in: Using the cyclodextrin mass balance and solving for free cyclodextrin in terms of known values, we obtain: CyDF = CyD? T - Dtat + DF + DA (9) Replacing the free drug, DF | by its relation in equilibrium it leads to: Solving the second degree equation for free cyclodextrin, CyDF provides: + K -DA - K-D0 + K'CyDm ± 4K-CyDlal +. { l- K-D? + K-D0 - K-CyDm) 2 CyDF = - (11) 2- K The value for CyDF can be substituted in equation 8. An implicit solution using equations 8 and 11 allows the determination of both the equilibrium binding constant K and the diffusion rate, k, in the acceptor phase using time , date of concentration, and initial conditions. Sampling removed the highest concentration of the drug (eg compound of formula la) from the donor side of the dialysis chamber, which resulted in raw data showing the concentrations reaching equilibrium with the midpoint biased below 50. %. This sampling bias was corrected, and the graphs were normalized to represent a midpoint of 50%. This normalization was applied before adjusting the curves to the model. The procedure used provided a measured binding constant for the drug and SBE-CD. The value obtained from the equilibrium dialysis procedure was 1092 M-1 (± 352 M-1, n = 3), compared to 1041 M-1 (n = 1) for the solubility procedure. The binding constant for the preservative and SBE-CD, using the solubility method was 28 M-1 (n = 1) compared to 83 M-1 (± 7 M-1) using equilibrium dialysis. The data show that, in binary systems, both the drug (for example, the compound of formula la) and the preservative bind to the cavity in SBE-CD, although in this case the drug binding constant was 13 times higher than the preservative. The data showed that in the ternary systems comprised of SBE-CD, drug (for example, compound of formula la), and preservative, at the ratios tested, the equilibrium profile indicated that the preservative did not bind to the cyclodextrin due to the competitive union with the drug. Based on the above calculations to obtain the amount of sequestered meta.-cresol and compound of formula la, proposed formulations were developed and evaluated for antimicrobial efficacy. Figure 3 shows no clear relationship between the total meta-cresol concentration contained in the formulation and the logarithmic reduction of the bacterial population, 6 or 24 hours after adding a known amount of Staphylococcus aureus (ie, formulations containing about 3 mg / ml meta-cresol seems to also have a logarithmic reduction as low as 0 or as high as greater than 4.6). However, when the same data set is plotted against the calculated concentration of meta-cresol not sequestered in the formulation (Figure 4), a relationship is visible. This data set was produced with a number of formulations containing between 9.0 and 11.0 mgA / ml of compound of formula, between 2,5 and 4,75 mg / ml of meta-cresol and between 60 and 100 mg / ml of SBE-CD. The appearance of a plateau at the highest concentrations is only due to the limitation in the procedure measuring bactericidal efficacy. Since the procedure consists of evaluating the population not eliminated by the preservative, when the total population is dead (that is, nothing is detected approximately 100%) the cited figure is of the form: a logarithmic reduction greater than a value usually between 3 and 5. Another factor was the concentration of non-sequestered compound of formula la, since higher concentrations were shown to cause pain in the injection. In addition, there was a risk of precipitation, if the concentration reached the limit of the aqueous solubility of the compound of formula la in the pH of the desired formulation of about 4.4. According to the above, the level of the non-sequestered compound of formula la was minimized in an attempt to maintain the concentration below 2 mg / ml. Two additional parameters were: (1) the total meta-cresol concentration level; and (2) the level of cyclodextrin (e.g., SBE-CD) should be kept as low as possible and, in particular, below 80 mg / ml to prevent binding and inactivation of meta-cresol. (See figure 4). According to the above, formulations containing between 9.0 and 11.0 mgA / ml of the compound of formula I, between 2.5 and 4.75 mg / ml of meta-cresol and between 60 and 100 mg / ml of SBE-CD were designed to contain a known amount of calculated non-sequestered compound of formula I and a known calculated amount of non-sequestered meta-cresol. The formulations were analyzed to evaluate the efficacy of the preservative. The results are indicated in Figure 4. From these results, a limit of confidence in the strong efficacy of the preservative was defined and outlined in Figure 4. Based on these results, the preferred formulation was selected containing calculated non-sequestered concentrations of meta-cresol (2.8 mg / ml) and the compound of formula I (1.4 mg / ml), corresponding to the black diamond in Figure 4 This formulation corresponded to the actual total concentrations of 10 mgA / ml of the compound of formula I, 63 mg / ml of SBE-CD and 3.3 mg / ml of meta-cresol at pH 4.4.

The principles described above for the development of a pharmaceutical formulation of the citrate salt of the compound of formula la are applicable in the development of other parenteral formulations comprising drugs, cyclodextrin and preservative. In particular the concentrations of drug, cyclodextrin and preservative should be adjusted to have a minimum concentration of non-sequestered preservative (2.1 mg / ml when meta-cresol is used). Formulation. In general, the formulations are prepared by dissolving a therapeutically effective amount of the compound of formula la in a pharmaceutically acceptable aqueous diluent. A pharmaceutically acceptable salt of the compound of formula I, such as the citrate or malate salts, can also be used. A cyclodextrin is added to the solution in a concentration range of between about 2% and about 40%. Preferably, the cyclodextrin comprises between about 5% and about 20% of the pharmaceutical composition and more preferably between about 5% and about 10%. Preferably, the cyclodextrin is a β-cyclodextrin: hydroxypropyl β-cyclodextrin, sulfobutyl ether β-cyclodextrin or other pharmaceutically acceptable substituted β-cyclodextrin. A preservative is added to the formulation based on weight. As used in this specification, a "therapeutically effective amount" for a dosage unit may typically be between about 0.5 mg and about 500 mg of active ingredient. However, the dose may vary, depending on the species, variety, etc., of the animal to be treated, the gravity and the body weight of the animal. According to the above, based on body weight, typical dose ranges of the active ingredient may be between about 0.01 and about 100 mg per kg of body weight of the animal. Preferably, the range is between about 0.10 mg and about 10 mg per kg of body weight, and more preferably, between about 0.2 and about 2 mg per kg of body weight. For example, 10 mgA / ml of the compound of the formulation of formula allows the preferred injection volume of 0.5 to 3.0 ml at a dose of 1 mg / kg to treat animals of 5 to 30 kg, which covers the most of the patients. The use of the product in larger mammals can be accommodated by the use of a larger syringe or multiple injections. The use of the product in small dogs and cats will require lower injection volumes. The veterinary professional, or those skilled in the art, will be able to determine the appropriate dosage for the particular individual patient, which may vary with the species, age, weight and response of the particular patient. The above dosages are exemplary of the middle case. According to the above, greater or lesser dosage intervals can be guaranteed, depending on the above factors, and are within the scope of this invention. The pharmaceutical compositions of the compound of formula la were developed such that a therapeutically effective amount of the compound of formula la could be administered to a patient with an acceptable injection site tolerance. Tolerance at the injection site was measured by inspecting the patient to assess the signs of reaction, including erythema (size), skin thickening (size), pain after palpation and edema. Table VI provides a detailed explanation of the scoring system: a score of 0 (no reaction) to 4 (severe reaction) was provided for each characteristic and each injection site daily. The formulation of the citrate salt of the compound of the formula is self-buffered by the counterion citrate (21.3 mM) at the native pH of about 4.4. However, if other pharmaceutically acceptable salts are used, a pharmaceutically acceptable buffer may be required. The preferred formulation is 10 mgA / ml of the formulation compound in the form of the citrate salt monohydrate, approximately 63 mg / ml of SBE-CD, and approximately 3.3 mg / ml of meta-cresol at pH 4.4.

GENERAL EXPERIMENTAL PROCEDURES A. Dialysis procedure by equilibrium to determine the binding constants Materials. Meta-cresol (MW = 108.14) was obtained from Aldrich, San Luis, MO. A 20-cell equilibrium dialyzer was used, equipped with 2 ml Teflon cells and 500 MWCO asymmetric cellulose ester membranes (Spectrum, Rancho Dominguez, CA). The compound of 1a (free base = 468.69) can be prepared as set forth in section B of the experimental procedures. Preparation of the formulations. Three different test formulations composed of any of the single component controls were prepared; binary systems containing either drug or m-cresol, and SBE-CD; or ternary systems containing drug, m-cresol, and SBE-CD. The formulations were prepared at room temperature at different ratios and concentrations 24 hours before the test to ensure equilibrium binding. The formulations were prepared by first dissolving the SBE-CD at the appropriate concentration and then adding drug or m-cresol and allowing it to dissolve in the cyclodextrin solution. Dialysis procedure. One ml of complexed or control formulation was loaded on the donor side of the membrane. The acceptor side was charged with 1 ml of sodium citrate (pH 4.4) to maintain ionic equilibrium through the chamber. At various times, 50 μl aliquots were removed from both the donor and acceptor sides of the dialysis chamber by equilibrium and analyzed using HPLC. The concentration versus time profile (mM) of ligand on each side was plotted for each ratio. HPLC procedure. Pure samples were loaded into an HP 1100 HPLC equipped with an Agilent Eclipse XDB-C8 column. The total processing time was 10 minutes. The mobile phase consisted of 25 mM 25% ammonium acetate and 75% methanol. Detection was performed using absorbance at 271 nm or fluorescence detection. The peaks were integrated using Turbochrome software [Perkin Elmer \ San José, CA]. Control experiments. The dialysis rates of the compound of the formula la and meta-cresol were measured alone through the 500 MWCO membrane. Different concentrations of meta-cresol and compound of formula la were placed on the donor side of the equilibrium dialyzer. The concentrations of the corresponding complex formation experiments were chosen to be equal to the concentration of drug or preservative in the single component systems. Binary systems These experiments were performed to quantify the binding of either drug or m-cresol with SBE-CD. Three separate mixtures were tested which consisted of: compound of formula la with SBE-CD, meta-cresol with SBE-CD, and drug with meta-cresol. The molar ratios of SBE-CD to drug or preservative were 1: 1, 2: 1, and 4: 1. Ternary systems. Several experiments were performed to test the effects of the three components of the formulation on the rate of dialysis of drug and preservative. In these, the concentration of SBE-CD was set while varying the amounts / ratios of the compound of formula la and meta-cresol. Data processing. The raw data was normalized to correct the concentration variation in the donor and acceptor well sides. The corrected percentages of the total were converted into theoretical mM concentrations. These data were then adjusted simultaneously to the equations presented in the discussion section using the Micromath Scientist software. B. Preparation of the compounds of formula I and II In general, the compounds of formula I and I can be prepared by methods including procedures known in the chemical art, particularly in light of the description contained in this specification and described in US 6,222,038 and US 6,255,320. The compounds of formula I and the can be prepared by various different synthetic routes. In particular, the compound of formula la can also be prepared as described in the provisional application of EE. UU in accordance with the present disclosure No. 60 / 541,323, of assignment and ownership of Pfizer, Inc. Certain procedures for the synthesis of the compound of the formula la, as more fully described in the above provisional interim application with the present, are illustrated in the following reaction scheme. The following reaction scheme illustrates a possible preparation of the monohydrated citrate salt of the compound of formula la, the compound of formula le.

SCHEME you In step A of scheme I, a mixture of the compound of formula Via in an alcohol solvent such as methanol, ethanol or n-propanol but preferably propan-2-ol, optionally also in the presence of water, is hydrogenated on a palladium catalyst on carbon at elevated temperature (typically 75-80 ° C) and pressure (typically 50 psig hydrogen (344.83 kPa gauge)). Those skilled in the art would appreciate that other catalysts may be suitable, such as palladium on carbon, palladium on carbon hydroxide, platinum on carbon, palladium on calcium carbonate, or palladium on alumina (AI2O3). Once the formation of the intermediate, compound VII, has been completed, typically 1 hour, the compound of formula VIII, typically in the form of a solution in the respective alcohol solvent, preferably in propan-2-ol (isopropanol, "IPA") is added to the reaction, without isolating the compound of formula VII, and the mixture is optionally stirred at elevated temperature (30-120 ° C) under a nitrogen atmosphere. Once a sufficient amount of the intermediate compound IXa has been formed, the nitrogen atmosphere is replaced with hydrogen. The reaction is then optionally stirred at elevated temperature (about 30-120 ° C) and at elevated pressure (typically 50 psig (344.83 kPa gauge)) until the formation of compound Ib is found to be complete (typically 18 hours) . Then the reaction mixture is cooled (approximately 20-25 ° C) and the hydrogen gas is removed. The palladium on carbon catalyst is removed by filtration, and the resulting solution of compound Ib is taken directly for stage B. In step B of reaction scheme I, the solution of compound Ib, typically in a mixture of propan-2 -ol and water, concentrated by distillation followed by the addition of toluene. The mixture is then concentrated again by distillation, adding additional toluene and water as needed during the distillation until sufficient isopropanol has been removed from the mixture and an appropriate volume of solution is obtained (typically, 2-4 volumes per kg of compound). Ib). Water and toluene are added as necessary (typically about 3.5 volumes of water and about 5 volumes of toluene). Those skilled in the art would appreciate that other solvents, other than toluene, such as methylene chloride, ethyl acetate, isopropyl acetate or methyl tert-butyl ether, can be used. The pH is adjusted to an appropriate point (approximately 11.5 to 13.5) by the addition of aqueous sodium hydroxide and if necessary aqueous hydrochloric acid with stirring. Once an appropriate pH has been obtained, the aqueous phase is removed by separation. Then the organic phase containing the product is concentrated by distillation. Then a mixture of propan-2-ol and water is added and the mixture is again concentrated by distillation. The addition of water and propan-2-ol and subsequent concentration by distillation is repeated as necessary until enough toluene (typically less than 3% w / w of toluene by GC analysis) has been removed from the mixture and obtained an appropriate volume of solution (approximately 4 volumes with respect to compound Ib), resulting in a solvent composition in the final granulation suspension typically greater than 80% w / w propan-2-ol, less than 20% p / p of water and less than 3% w / w of toluene. Once sufficient toluene has been removed, the mixture is cooled until crystallization occurs (typically 70-75 ° C). The resulting suspension is then further cooled (typically 20-25 ° C) and then granulated for a period of time before it is optionally further cooled to about 0-5 ° C and stirred for a period of time. The solid is then collected by filtration and the filter cake washed with propan-2-ol and dried under vacuum at elevated temperature (typically 45-55 ° C) to provide the compound of the formula, in the form of a crystalline solid. Those skilled in the art would appreciate that other solvents than propan-2-ol, such as methanol, ethanol, n-propanol, acetonitrile, isopropyl acetate, tere-amyl alcohol and 4-methyl-2-pentanone can be used. As indicated in optional step BX of the reaction scheme, which is not typically required, compound Ib can be further purified. Compound Ib is suspended in propan-2-ol and the mixture is heated to reflux to provide a solution. The mixture is then heated to an elevated temperature below the reflux temperature (about 70-75 ° C) for about 1 hour during which crystallization typically occurs. The resulting suspension is maintained at this temperature for a period of about 1 to 2 hours and then cooled (to about 20 -25 ° C). After stirring at room temperature for a period of time (typically 1-18 hours), the solid is collected by filtration. The filter cake is washed with propan-2-ol and then vacuum dried at elevated temperature (about 45-55 ° C) to provide a purified compound Ib, as a crystalline solid. Those skilled in the art would appreciate that other solvents, other than propan-2-ol, can be used, such as methanol, ethanol, n-propanol, acetonitrile, isopropyl acetate, tere-amyl alcohol and 4-methyl-2-pentanone. In step C of the reaction scheme, compound Ib (1 molar equivalent) and anhydrous citric acid (typically 1.1 molar equivalents) are combined in a mixture of acetone (typically about 8-10 volumes) and water (typically about 0). , 4 volumes), and the resulting solution is filtered. Then more acetone (typically about 2 volumes) is added to wash the transfer equipment. To the filtrate is added a filtered ether solvent such as methyl tert-butyl ether (tert-butyl methyl ether, "MTBE") or isopropyl ether ("IPE") (typically about 10 volumes), optionally at elevated temperature (30-45 °). C). Once crystallization occurs, which can optionally be initiated by the addition of some seed crystals, the mixture is granulated over a period of time (typically 18 hours), typically at 20-25 ° C but optionally at elevated temperature ( 30 - 45 ° C) during a part of this time. The solid is then collected by filtration. The filter cake is washed with the respective filtered ether solvent and then dried at a temperature of less than 60 ° C (room temperature, if isopropyl ether is used) under vacuum optionally without air or with nitrogen purge to provide the compound le, Citrate monohydrate, in the form of a crystalline solid. Optionally the product can then be milled or sifted. In the optional stage CX, the purity of the compound can be improved by dissolving it in a mixture of acetone (typically 7 volumes) and water (typically 0.3 volumes) at elevated temperature (approximately 35-50 ° C). The mixture is then cooled (to approximately 20-35 ° C) and optionally filtered. Then a filtered ether solvent, such as methyl tert-butyl ether or isopropyl ether, is added to the resulting mixture, optionally at elevated temperature (about 30-40 ° C). Once crystallization occurs, which may optionally be initiated by additions of some seed crystals, the mixture is granulated over a period of time (typically 18 hours), typically at 20-25 ° C but optionally at elevated temperature (30 ° C). - 45 ° C) during a part of this time. The solid is then collected by filtration. The filter cake is washed with the respective filtered ether solvent and then dried at a temperature below 60 ° C (room temperature, if isopropyl ether is used) under vacuum optionally without air or with nitrogen purge to provide the compound le, citrate monohydrate, in the form of a crystalline solid. The product can then optionally be milled or sieved. Other pharmaceutically acceptable salts, other than citrate, can be used. For example, the salts malate, maleate, mesylate, lactate, and hydrochloride or their in situ equivalents can be prepared by adding an equimolar amount of the appropriate acid to the compound, free base solutions.

C. Antimicrobial Preservatives Evaluated for Pharmaceutical Compositions Table III summarizes the antimicrobial preservatives evaluated for use in the formulation. Each antimicrobial preservative was tested at the highest concentration currently used in commercial products. Antimicrobial preservatives were purchased in general chemical sources. Table III: Selected antimicrobial preservatives Evaluation of phen ethane between, -, in ncrements e, Preparation of conserved formulations. Formulations were prepared, when the solubility allowed, at 5% and 10% (weight / volume) of SBE-CD. Antimicrobial preservatives with optimal activity at a pH outside the nominal formulation value (pH 4.4) were assessed at either 3.5 or 5.0 using 1 N HCl or 1 N NaOH. A 10% stock solution was prepared. or 5% (weight / volume) of SBE-CD containing 10 mgA / ml of the citrate compound of the formula la. Preservative was added to the respective formulation on a weight basis. Antimicrobial efficacy test. A hybrid antimicrobial efficacy test USP < 24 > / Farm. Eur. 2000 (AET), as follows: 20 ml of drug product was inoculated individually with 0.1 - 0.2 ml of bacterial or fungal culture, according to the compendial requirements of USP / Farm. Eur. The final concentration of the organisms in the test sample was between 1 x 105 and 1 x 106 cfu / ml. At initial time intervals of 6 hours, 24 hours, 7 days, 14 days, and 28 days, 1 ml of the inoculated product was transferred into 9 ml of a recovery diluent, which was validated to confirm the neutralization of the antimicrobial preservative. One ml of the diluted sample was then transferred to a sterile petri dish and combined with 15-20 ml of an agar broth to grow the organisms. The plates were then incubated for 3 to 5 days, after which the colonies were counted. The initial contamination of organisms was then calculated based on the dilution of the initial sample. The values are recorded as "logarithmic reduction". The organisms used in the AET assay are listed in Table IV. Table IV: Organisms tested in the hybrid antimicrobial efficacy assay (USP / Farm. the requirements of the Farm. Eur., Which typically have an immediate bactericidal activity requirement. The requirements of Farm. Eur. Shown in Table III have either a "Criterion A" or "Criterion B" specification depending on the rate of microorganism reduction, criterion A requiring an increased bactericidal rate. In order to find the combined hybrid assay, the initial inoculum count of microorganisms needs to be reduced by the amounts listed in table V. Table V. USP / Farm requirements. Eur for AET (aqueous parenteral) (24 USP and Farm, Eur. 2000 individual) Combined requirements of USP / Farm. Eur. Logarithmic reduction required in count of organisms Stability measurements. Potential guiding formulations were evaluated under various conditions of accelerated stability in order to determine the potency and purity of the compound of formula la, preservative content and SBE-CD content. For example, in an accelerated stability study, potential guide formulations were placed in stability furnaces to measure short-term thermal stability. Sample vials (20 ml) were placed in temperature chambers of 70 ° C, 50 ° C, 30 ° C, and 5 ° C and analyzed to evaluate the potency and purity of the compound of formula la, antimicrobial preservative and content of SBE-CD, at time intervals of 1, 3, 6, and 12 weeks. Purity and potency tests to measure the compound of formula la, as well as antimicrobial preservatives and SBE-CD content, were performed using validated HPLC methodology. The SBE-CD was tested using GTP 5984. D. Tolerance at the site of Invention The formulations of the compound of formula la were evaluated with respect to the tolerance at the site of injection (hereinafter "IST"). In general, formulations that did not contain SBE-CD were, in general, poorly tolerated. Formulations consisting of 10 mgA / ml of compound of formula la, 10% in excess of meta-cresol (0.33% w / v) and approximately 6.8% to 7.6% of SBE-CD were evaluated for IST . In particular, formulations containing 10 mgA / ml of compound of formula la, 61 to 72 mg / ml of SBE-CD and 3.2 to 4.2 mg / ml of meta-cresol were tested to assess tolerance in the injection site and all were well tolerated. The formulations were tested in groups of 4 dogs comprised by hounds and crossed. On each of the four consecutive days, the dogs received two subcutaneous injections of vehicle daily as a control in the left shoulder at 0.1 ml / kg and active formulation (10 mgA / ml of compound of the formula at 1 mg / kg). ) on the right shoulder. Dogs were observed daily for evidence of reaction at the site of injection and a score of 0-4 was given (see Table VI) for each of the following parameters: pain with injection, erythema, tissue thickening, pain after palpation and edema. The dogs were observed daily until day 5 (24 hours after the last dose).

Table VI: tolerance score at the injection site EXPERIMENTAL Experimental 1: Selection of antimicrobial preservatives for an injectable compound of the formula in Study A (selection of a large antimicrobial preservative) The efficacy of several different antimicrobial preservatives in combination with a compound of the formula la and SBE-CD was investigated. The bibliography indicated that the antimicrobial preservatives met both the USP requirements and the A or B criteria of the Farm. Eur. Were ethanol, propylene glycol, benzoic acid, thimerosal, meta-cresol, (Lucchini, JJ; Corre, J .; and Cremieux, A. "Antibacterial activity of phenolic compounds and aromatic alcohols" Res. Microbiol. 141, 499-510 , (1990)) and the combination of chlorobutanol / phenylethanol. Table VII shows the results of the selection of various antimicrobial preservatives or combinations thereof.

TABLE VII. ANTIMICROBIAL EFFECTIVENESS ESSAY: SELECTION FOR ANTIMICROBIAL PRESERVATIVE SYSTEM and designates USP and / or Farm criteria. Satisfied Eur. The formulations containing these antimicrobial preservatives were further evaluated for physical and chemical stability and tolerance at the injection site, (see Table VIII). The approaches of antimicrobial preservatives co-solvents, ethanol and propylene glycol, failed to satisfy IST acceptable. In addition, the benzoic acid formulations also provided poor IST results. TABLAVIH. RESULTS OF THE STUDY A All formulations contain a compound mg a a mg m and Designate USP and / or Farm criteria. Eur. Satisfied. Study B (Selection of antimicrobial preservatives that meet criterion B of Farm.

All antimicrobial preservatives that meet Farm B criteria. Eur. Were also selected to assess injection site tolerance and stability. The guides identified in table VII and table IX that met criterion B were thimerosal, meta-cresol, and benzoic acid. These formulations were evaluated to detect stability and IST (Table VII). The results of the studies indicated that the thimerosal stability was commercially undesirable for the formulation. Only 30% of thimerosal remained in the formulation after 1 week at 70 ° C storage and complete loss was observed after 6 weeks (Tan, M., Parkin, LE "Route of decompositlon of thimerosal" Int. J. Pharm 195 N ° 1 - 2, 23-34, 2000). Benzoic acid showed no detectable loss at 12 weeks at 70 ° C storage, which was stable enough for the formulation.

Although the stability of benzoic acid was acceptable, moderate to severe pain after the injection eliminated it from further consideration. On the other hand, the formulations containing meta-cresol showed excellent stability and tolerance at the injection site. In accordance with the above, meta-cresol was identified as the preferable antimicrobial preservative due to the excellent tolerability at the site of injection, as well as by a strong adherence to the A criterion of the Farm. Eur. For the effectiveness of the preservative. Due to these favorable performance characteristics, the formulation was optimized with respect to the concentration of SBE-CD, resulting in a formulation with a high margin of solubility, strong efficacy of the antimicrobial preservative, and acceptable tolerance at the injection site. The stability of meta-cresol and compound of formula la in formulations containing 3 mg / ml of meta-cresol, 100 mg / ml of SBE-CD and 10 mgA / ml of compound of formula la is shown in table IX. The strong stability was demonstrated for both the compound of the formula la and for meta-cresol. The compound of formula experienced a 3% loss (compared to week 1 at 5 ° C) after 12 weeks at 70 ° C, while the potency of meta-cresol decreased by 2%. Table IX: stability of meta-cresol v compound of formula Preferred Embodiments A. A pharmaceutical composition comprising a therapeutically effective amount of an active pharmaceutical ingredient, a β-cyclodextrin, a pharmaceutically acceptable preservative, a pharmaceutically acceptable carrier, and an optional pharmaceutically acceptable excipient, wherein the preservative demonstrates efficacy as an antimicrobial preservative pharmaceutically acceptable.

B. The pharmaceutical composition according to the preferred embodiment A wherein the β-cyclodextrin is 2-hydroxypropyl-β-cyclodextrin or sulfobutyl ether-β-cyclodextrin.

C. The pharmaceutical composition according to preferred embodiment B wherein the preservative is selected from thimerosal, propylene glycol, phenol, or meta-cresol or a combination thereof. D. The pharmaceutical composition according to preferred embodiments B or C wherein the preservative has a cyclodextrin binding value that is less than a binding value of the active pharmaceutical ingredient to cyclodextrin. E. The pharmaceutical composition according to the preferred embodiment D, wherein the concentration of preservative is between about 0.1 mg / ml and about 600 mg / ml. F. The pharmaceutical composition according to the preferred embodiment E, wherein the preservative is meta-cresol and the concentration of the preservative is between about 0.1 mg / ml and about 20 mg / ml. G. The pharmaceutical composition according to the preferred embodiment F wherein from about 1 mg / ml to about 5 mg / ml of the meta-cresol is not sequestered in the cyclodextrin. H. The pharmaceutical composition according to the preferred embodiment G wherein approximately 2.5 mg / ml of the preservative is not sequestered in the cyclodextrin.

I. The pharmaceutical composition according to preferred embodiment D wherein the binding value of the active pharmaceutical ingredient to cyclodextrin is between 500 M "1 and . 000 M "1 J. The pharmaceutical composition according to the preferred embodiment I wherein the binding value of the active pharmaceutical ingredient to cyclodextrin is between 800 M" 1 and 3. 000 M "1 K. The pharmaceutical composition according to the preferred embodiment D wherein the active pharmaceutical ingredient has a value greater than or equal to twice the cyclodextrin binding constant to that of the preservative L. The pharmaceutical composition according to preferred embodiment K wherein the binding constant is greater than or equal to five times M. The pharmaceutical composition according to the preferred embodiment L wherein the binding constant is greater than or equal to ten times.

N. The pharmaceutical composition according to the preferred embodiment D having antimicrobial efficacy against bacteria so that the concentration of bacteria decreases to a logarithmic reduction of 2 or greater after 6 hours, a logarithmic reduction of 3 or greater after 24 hours, and zero recovery of bacteria after 28 days. O. The pharmaceutical composition according to the preferred embodiment N wherein the bacteria are Escherichia coli (bacteria, gram negative) (ATCC 8739), Pseudomonas aeruginosa (bacteria, gram negative) (ATCC 9027) and Staphylococcus aureus (bacteria, gram positive) (ATCC 6538) P. The pharmaceutical composition according to the preferred embodiment O having antimicrobial efficacy against a fungus or mold such that the concentration of the fungus or mold decreases in a logarithmic reduction of 2 or greater after 7 days, a logarithmic reduction of 1 after 14 days, and no increase in fungi or molds after 14 days to approximately 28 days. Q. The pharmaceutical composition according to the preferred embodiment P wherein the fungus is Candida albicans (fungus) (ATCC 10231). R. The pharmaceutical composition according to the preferred embodiment P wherein the mold is Aspergillus niger (mold) (ATCC 16404). S. A pharmaceutical composition according to the preferred embodiment D wherein the antimicrobial efficacy meets the A and B criteria of Farm. Eur. And the USP AET criteria. T. A pharmaceutical composition comprising a compound of formula I, wherein R2 is selected from the group consisting of methyl, ethyl, isopropyl, sec-butyl, and tert-butyl, a pharmaceutically acceptable cyclodextrin, a pharmaceutically acceptable preservative, a pharmaceutically acceptable carrier and an optional pharmaceutically acceptable excipient. U. The pharmaceutical composition according to the preferred embodiment T wherein the β-cyclodextrin is 2-hydroxypropyl-β-cyclodextrin or sulfobutyl-ether-β-cyclodextrin. V. The pharmaceutical composition according to the preferred embodiment U wherein the pharmaceutically acceptable preservative is selected from thimerosal, propylene glycol, phenol or meta-cresol, or a combination thereof. W. The pharmaceutical composition according to the preferred embodiment V wherein the preservative is meta-cresol. X. The pharmaceutical composition according to the preferred embodiment W having a pH in the range between about 4 and about 5. Y. The pharmaceutical composition according to preferred embodiments W or X wherein from about 1 mg / ml to about 5 mg / ml of the preservative is not sequestered in the cyclodextrin. z. The pharmaceutical composition according to the preferred embodiment Y wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is in an amount between about 0.1 mg / ml and about 100 mg / ml and cyclodextrin β is in a amount between about 20 mg / ml and about 200 mg / ml and the preservative is meta-cresol.

A1. A pharmaceutical composition according to the preferred embodiment Z wherein the β-cyclodextrin is in the amount of between 55 mg / ml and 100 mg / ml and the meta-cresol is in an amount between about 2.5 mg / ml and 3.5 mg / ml.

B1 A pharmaceutical composition according to preferred embodiments T, U, W or X wherein the compound of formula I is the compound of formula la, or their pharmaceutically acceptable salts. C1. A pharmaceutical composition according to preferred embodiment B1 wherein the compound of formula la, or a pharmaceutically acceptable salt thereof, is in an amount of between about 0.1 mg / ml and about 100 mg / ml and cyclodextrin β is in an amount between about 20 mg / ml and about 200 mg / ml and the preservative is meta-cresol and is in an amount of between about 1 mg / ml and 5 mg / ml.

D1. The pharmaceutical composition according to the preferred embodiment C1 in which the β-cyclodextrin is in an amount between about 55 mg / ml and about 100 mg / ml and the preservative is meta-cresol and is in an amount between about 2.5 mg / ml and 3.5 mg / ml. E1. The pharmaceutical composition according to the preferred embodiment D1 in which the β-cyclodextrin is sulfobutyl ether-β-cyclodextrin. F1. A pharmaceutical composition comprising the compound of formula la, or their pharmaceutically acceptable salts, wherein the compound of formula la is mgA / ml, sulfobutyl ether-β-cyclodextrin is in an amount of about 63 mg / ml and meta-cresol is in an amount of about 3.3 mg / ml, a pharmaceutically acceptable carrier and an optional pharmaceutically acceptable excipient. G1 The pharmaceutical composition of the preferred embodiment F1 wherein the pharmaceutically acceptable salt of the compound of formula la is citrate. H1 A method for the treatment of emesis or improvement of the recovery of anesthesia in mammals comprising the parenteral injection in the mammal of an aqueous pharmaceutical composition comprising the pharmaceutical composition of the preferred embodiments T, U, V, W, X , F1 or G1, the β-cyclodextrin being present in amounts that are sufficient for an improved injection tolerance at the injection site. 11. A method for the treatment of emesis or improvement of the recovery of anesthesia in mammals comprising the parenteral injection in the mammal of an aqueous pharmaceutical composition comprising the pharmaceutical composition of the preferred embodiment F1. J1 The method according to the preferred embodiment 11 wherein the pharmaceutically acceptable salt is citrate. K1 The method according to preferred embodiments 11 or J1 wherein the administration is subcutaneous. L1. A method of improving tolerance at the site of injection during the treatment of emesis or treatment for improvement in the recovery of anesthesia in a mammal comprising the parenteral injection in the mammal of a pharmaceutically acceptable solution of the pharmaceutical composition according to preferred embodiments T, U, V, W, X, F1 or G1. M1 A method of improving tolerance at the site of injection during the treatment of emesis or treatment for improvement in the recovery of anesthesia in a mammal comprising the parenteral injection in the mammal of a pharmaceutically acceptable solution of the pharmaceutical composition according to preferred embodiment F1. N1 The process of the preferred embodiment M1 wherein the pharmaceutically acceptable salt is citrate. 01. A method for developing preserved API compositions comprising a therapeutically effective amount of an API, a β-cyclodextrin and a pharmaceutically acceptable preservative. P1 The process according to the preferred embodiment 01 wherein the preservative has a cyclodextrin binding value that is less than an API binding value to the cyclodextrin. Q1 The process according to the preferred embodiment P1 wherein the preservative is selected from thimerosal, glycol, phenol or meta-cresol or a combination thereof. R1 The process of the preferred embodiments P1 or Q1 wherein the binding value of the API with the cyclodextrin is greater than 50 M "1 S1 The method of the preferred embodiment R1 wherein the binding value of the API with the cyclodextrin it is between 500 and 10,000 M ". T1. The process of the preferred embodiment S1 wherein the binding value of the API with the cyclodextrin is between 800 and 3,000 M "1. U1 The method of the preferred embodiment T1 in which the requirements of the antimicrobial efficacy test (AET) they meet criteria A and B of the European Pharmacopoeia and the USP AET criteria.

Claims (10)

1. CLAIMS 1. A pharmaceutical composition comprising a therapeutically effective amount of an active pharmaceutical ingredient, a β-cyclodextrin, a pharmaceutically acceptable preservative, a pharmaceutically acceptable carrier, and an optional pharmaceutically acceptable excipient, wherein the preservative demonstrates efficacy as a pharmaceutically antimicrobial preservative. acceptable.
2. 2. A pharmaceutical composition according to claim 1 wherein the active pharmaceutical ingredient is a compound of formula I, or their pharmaceutically acceptable salts, wherein R 2 is selected from the group consisting of methyl, ethyl, isopropyl, sec-butyl and tert-butyl.
3. 3. The pharmaceutical composition according to claims 1 or 2 wherein the β-cyclodextrin is 2-hydroxypropyl-β-cyclodextrin or sulfobutyl ether-β-cyclodextrin.
4. 4. The pharmaceutical composition according to any preceding claim wherein the preservative is selected from thimerosal, propylene glycol, phenol, or meta-cresol or a combination thereof.
5. 5. The pharmaceutical composition according to any preceding claim wherein the preservative has a cyclodextrin binding value that is less than a binding value of the active pharmaceutical ingredient to the cyclodextrin.
6. 6. The pharmaceutical composition according to any preceding claim wherein from about 1 mg / ml to about 5 mg / ml of the preservative is not sequestered in the cyclodextrin.
7. 7. The pharmaceutical composition according to any preceding claim wherein the binding value of the active pharmaceutical ingredient to the cyclodextrin is between 500 M'1 and 10,000 M "1.
8. 8. The pharmaceutical composition according to any preceding claim for use as a medicament.
9. 9. The use of a composition according to any of claims 2 to 7 in the manufacture of a medicament for the treatment of a disease for which a neurokinin receptor antagonist is indicated.
10. 10. A method for the treatment of a disease for which a neurokinin receptor antagonist is indicated in mammals comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition of any of claims 2 to 7.