PERAMIVIR DERIVATIVE FOR ORAL ADMINISTRATION

BACKGROUND OF THE INVENTION

The present invention relates to novel orally active peramivir compositions, to a method for improving the oral bioavailability and bioactivity of peramivir, and to methods for preparing such orally active derivatives of peramivir. More specifically, the present invention relates to orally active peramivir prodrugs which comprise an adduct of peramivir with either an amino acid or a vicinal dicarbonyl compound.

Peramivir is a member of a class of antiviral agents that work by inhibiting viral neuraminidase, an enzyme essential for the influenza virus to replicate and infect its hosts. In addition to influenza A and B, avian influenza virus (H5N1) has been shown to be sensitive to peramivir. More specifically, depending on the influenza strain, peramivir has been determined to be 2 to 10 times more potent as a neuraminidase inhibitor than oseltamivir (Tamiflu ^® ) and zanimivir, based on experimentally determined concentrations of each required for 50% inhibition of neuraminidase activity in vitro. It has been reported that studies in rodents and primates have established the safety and efficacy of intramuscular injection of peramivir in a mouse influenza model. Further intravenous and intramuscular formulations of peramivir are reported to have been evaluated in pre-clinical animal models with success, and these preliminary studies indicate that the efficacy of a single IM injection of peramivir is comparable to five days of oral treatment with other agents.

However, human studies with oral forms of peramivir have demonstrated very poor oral bioavailability of peramivir. In particular it has been reported that in a phase III clinical trial of an oral formulation of peramivir, the antiviral activity against influenza A and B did not reach statistical significance. Accordingly there is a need for peramivir compositions which exhibit improved the bioavailability and efficacy of peramivir when administered orally for treatment or prevention of influenza.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a peramivir composition having improved oral activity. The composition includes an adduct selected from the group consisting of:

a) adducts of vicinal dicarbonyl compounds with peramivir or salts or esters thereof; b) adducts of amino acids with peramivir; and c) combinations of a) and b).

In another aspect, the invention provides a method for improving oral bioavailability and activity of peramivir that includes administering to a patient in need thereof a therapeutic amount of the above composition.

In yet another aspect, the invention provides a method of preparing a peramivir composition having improved oral activity and including a peramivir adduct. The method includes the steps of: contacting a vicinal dicarbonyl compound with peramivir or a Na, K, or NH ₄ salt or R ¹ OH ester thereof, wherein the vicinal dicarbonyl compound is according to the formula R ² C(O)C(O)R ³ , and wherein R ¹ , R ² and R ³ are each independently selected from the group consisting of hydrogen, phenyl, substituted phenyl, and alkyl, provided that R ¹ can not be H, and wherein R ² and R ³ may alternatively be linked together to form a C5-C8 cycloalkyl ring, thereby forming the adduct; and recovering the adduct.

In a further aspect, the invention provides a method of preparing a peramivir composition having improved oral activity and including a peramivir adduct. The method includes the steps of: in an aqueous medium, contacting peramivir with an amino acid to form a solution of peramivir dual amino(acid salt, then adding an organic solvent to the aqueous medium to precipitate the peramivir dual amino acid salt, or alternatively removing water from the aqueous medium, to provide the peramivir adduct as a solid dual salt of peramivir and the amino acid.

DETAILED DESCRIPTION OFTHE INVENTION

Peramivir has the chemical structure (I) shown below:

(D

Of particular significance is the presence of three functional groups: an alcohol - OH group, a carboxylic acid group, and a guanidino group. It is believed that poor oral absorption of peramivir and alkyl esters thereof may be due in large part to the highly polar nature of the guanidino group, particularly when in the protonated form such as is found in the zwitterϊonic form of peramivir. Yet the guanidino group is considered likely to be a major contributor to the improved activity of peramivir over other agents having neuraminidase activity, for example oseltamivir (Tamϊflu ^® ) and zanimivir. It has now been found that, if the guanidino group is masked to form an adduct that can be readily absorbed following oral administration and which can release free peramivir during or following absorption, a composition is provided which exhibits improved oral activity and efficacy. Without wishing to be bound by any particular theory or explanation, it is believed that the formation of such adducts reduces the polarity relative to the zwitterionϊc form of peramivir, i.e., the typical form of the molecule in aqueous solution. It is believed that this reduced polarity improves absorption of peramivir in vivo, thereby improving oral efficacy. The present invention will now be described with respect to two embodiments and variations thereof of adducts which are believed to provide peramivir compositions having improved oral efficacy.

Peramivir Adducts With Dicarbonyl Compounds

In a first embodiment of the invention, the peramivir adduct comprises a reversible adduct of peramivir or a salt or ester thereof with a vicinal dicarbonyl compound, which may be a diketone, dialdehyde, or compound with both ketone and aldehyde functionality. The adduct is believed to have the structure shown below in formula (II), in which R ¹ , R ² , and R ³ are each independently selected from the group consisting of hydrogen, alkyl, phenyl, and substituted phenyl, and wherein R ¹ may also

be Na, K, or NH ₄ and R ² and R ³ may alternatively be linked together to form a C5-C8 cycloalkyl ring.

(II)

Suitable exemplary alkyl groups for R ¹ , R ² , and R ³ include methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, isobutyl, 1-pentyl, 2-pentyl, 3-pentyl, isopentyl, neopentyl, 3-methylbut-2-yl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methylpent-l-yl, isohexyl, 4-methyIpent-2-yl, 4-methylpent-3-yl, 1-heptyl, 2-methylhex-l-yl, 5-methylhex-2-yl, 2-nonbornyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, 4-octyl, 2-ethylhex-l-yl, 2-bornyl, adamantyl. In some embodiments, only H and lower aikyl (i.e., C1-C4) groups are used.

Reversible formation of the vicinal dicarbonyl adduct of the guanidino group of peramivir is exemplified with 2,3-butanedione in schema set forth below:

The reaction between the guanidino group of peramivir and 2,3-butanedione is a reversible reaction leading to formation of a 5-membered ring which masks the apparent polarity of the guanidino group. The forward reaction may be favored and stabilized by the presence of the optional borate ion, which is believed to form a borate ester with one or both of the OH moieties generated from the dicarbonyl compound, and the reverse reaction leading to the free guanidino group is favored in the absence of borate. Removal of free 2,3-butanedione by dilution, such as is expected to occur once the adduct has entered the bloodstream, shifts the equilibrium toward the free guanidino group by mass action, thus making the peramivir more biologically available.

In practical terms, to create a guanidino adduct of peramivir it is necessary to react together peramivir and the vicinal dicarbonyl compound in a suitable reaction medium, for example an aqueous medium. In one embodiment this may be achieved by first making an aqueous solution of peramivir, then adding the vicinal dicarbonyl compound to the resulting reaction mixture. However, the specific mode of addition is

not considered critical. Additionally, borate ion, suitably titrated to substantially neutral pH, may be added. Peramivir or a salt or ester thereof may suitably be used with an excess of the vicinal dicarbonyl compound. The reaction is suitably conducted in the presence of an equimolar amount or molar excess of borate ion, based on the amount of peramivir employed in the reaction. Thus the molar ratio of borate ion to peramivir is suitably at least 1 :1. Under these conditions the forward reaction toward adduct formation is favored. The resulting adduct can potentially be isolated by crystallization in the presence of borate ions for subsequent formulation of a dry tablet oral delivery system using conventional pharmaceutically acceptable excipients. The reaction is spontaneous and reversible at room temperature, and may be driven further toward desired product (adduct) by using excess di-carbonyl compound and optionally borate. Typical conditions include mixing peramivir with 50 mM NaHCO ₃ buffer, pH 8.0 with at least a 20% molar excess of di-carbonyl compound, and optionally with an equimolar or greater amount of borate, pH 8.0. The reaction mixture may merely be maintained at ambient temperature until equilibrium is established, which may take between about 1 and about 10 hours. Suitable reaction conditions for reaction of one dicarbonyl compound, methyl glyoxal with compounds bearing guanidino groups, may also be found in the Journal of Biological Chemistry, Vol. 269, No. 51, pages 32299-32305 (1994), Lo et al. Alternatively, in some cases it may be preferable to create a liquid capsule containing a therapeutically effective amount of soluble peramivir, an adequate excess of a vicinal dicarbonyl compound such as 2,3-butanedione, and optionally an adequate amount of borate ion wherein the adduct form is highly favored and stable. Such a dosage form might for example be targeted for release in the gastrointestinal tract, and the concentrations of 2,3-butanedione and borate ion may be based upon that needed to maintain a significant percentage of peramivir in the adduct form for absorption.

While the foregoing embodiment has been illustrated with 2,3-butanedione, any suitable vicinal dicarbonyl compound may be employed. Either or both of the methyl groups shown above may be independently replaced with hydrogen, phenyl, substituted phenyl, or a straight or branched chain alkyl group, or they may be joined to form a cyclic alkyl group of 5 to 8 carbon atoms. Thus the reactant forming the cyclic adduct with the guanidino group may have the structure, R ² C(O)C(O)R ³ in which each of R ² and R ³ may be independently selected from hydrogen, straight or branched chain lower alkyl, or R ² and R ³ may be joined to form a cycloalkyl group of 5 to 8 carbon atoms. Thus suitable exemplary vicinal dicarbonyl compounds include glyoxal, methyl glyoxal, phenyl glyoxal and 2,3-butanedione.

It is thought that the adduct constitutes a peramivir prodrug that may be readily absorbed through the gastrointestinal tract following oral administration, and that once absorbed, dilution of borate and 2,3-butanedione favors formation of the free guanidino form over time, either in the cell or in the bloodstream. Thus the adduct form of peramivir is expected to provide substantially increased oral bioavailability.

Peramivir Adducts With Amino Acids

In a second embodiment of the invention, the peramivir adduct comprises a complex formed by the co-precipitation of peramivir and an amino acid in a 1:1 molar ratio as a dual salt. Any amino acid may be used, including D- and L- enantiomers and mixtures thereof, such as racemic mixtures. Suitable exemplary amino acids are according to the structure

in which the groups R ⁴ and R ⁵ , which may be separate or linked (to form a ring, e.g. proline), represent moieties such that the known naturally occurring amino acids (and their enantiomers), and derivatives thereof with modifications to the side chains (i.e., the substituent groups on the carbon atom between the carboxyl and amine groups), are encompassed by the structure. The amino acids are "inert," which for purposes of this invention means that the side chains or modifications thereto do not substantially interfere with dual salt formation or stability. Without wishing to be bound by any particular theory or explanation, it is believed that the structure of such adduct is as shown in formula (III)

(III)

In some embodiments, the amino acid is selected from the group consisting of GIy, Ala, Ser, Thr, VaI, He, Leu, GIu, GIn, Asp, Asn, Lys, Arg, Cys, Met, Orn, His, Pro, Tyr, Phe, and Trp. It will be appreciated by those skilled in the amino acid art that the foregoing abbreviations relate respectively to the naturally occurring or synthetic amino acids including glycine, alanine, serine, threonine, valine, isovaline, leucine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, arginine, cysteine, methionine, ornithine, hϊstϊdine, proline, tyrosine, phenylalanine, tryptophan. The amino acid may be a D- amino acid or an L- amino acid, or a mixture of these, provided that the amino and carboxyl groups are spatially oriented with respect to the peramivϊr molecule so as to allow dual salt formation. The presence of a dual salt may be demonstrated by the ability to repeatedly recrystallize the composition without substantial changes in the relative amounts of peramivir and amino acid.

Suitable amino acids also include amino acids, such as the foregoing, which have been modified in minor ways. For example in the cases of Asp and GIu, the carboxyl-bearing side chains may be converted to acid chlorides, esters, amides, aldehydes, and Schiff bases derived from the aldehydes. Similarly, for Lys and Orn, the side chain amino groups may be converted to Schiff bases by reaction with suitable aldehydes or ketones, or may be alkylated to form secondary or tertiary amines, or may be converted to short amides of C2-C12 straight chain or branched carboxylic acids. Similarly, the amino acid may be either GIu amidated on the side chain carboxylic acid group with a C2-C12 straight chain or branched aliphatic amine, or Asp so amidated. These and other modifications may be made to the amino acid side chains according to the invention, provided that the resulting amino acid derivative forms a dual salt of peramivir in a 1 :1 molar ratio with the amino acid. Although formula (III) is shown in a way that may suggest a particular orientation of the amino acid with respect to the peramivir, no such implication is intended. Thus, although the ammonium group on an amino acid may be adjacent the carboxylate anion on a peramivir molecule, with the guanidinium and carboxylate ions respectively on those same molecules also being adjacent, some other arrangement may also (or instead) be present. For example, an alternating sequence of peramivir and amino acid groups may be present, or yet some other arrangement, and all such configurations are contemplated according to the invention.

Methods of making the peramivir adduct (III) include merely contacting peramivir with an amino acid in an aqueous medium. Generally this reaction may be conducted at ambient or elevated temperature in the range of about 20°C up to about 50 ⁰ C. While the order of addition is not considered critical, it is contemplated that best

results are to be achieved if the molar ratio of amino acid to peramivir acid is at least 1:1, and it may be preferable to employ excess amino acid to assure maximization of dual salt formation. The reaction mixture is suitably stirred for a desired period of time to assure complete contact of the peramivir and amino acid, for example from about 0.25 hr to about 5 hours or more depending on the amino acid employed. The resulting product is a solution or suspension of the desired peramivir dual amino acid salt.

Thereafter the salt may be precipitated and recovered as a solid by adding a sufficient amount of an organic solvent to the aqueous reaction mixture to precipitate the peramivir dual amino acid salt, or alternatively by removing water from the aqueous solution, for example by lyophilization, to provide the peramivir dual amino acid salt as a solid. The resulting solid may then be dried, further purified as needed, and then incorporated into a biologically effective pharmaceutical composition.

Pharmaceutical Compositions Comprising Peramivir Adducts Peramivir adducts of this invention, including the adducts of formula (II) or (III), may be combined with one or more pharmaceutically acceptable excipients for delivery to the patient by any means known in the medical art. Methods of treatment contemplated according to the invention may include, for example, administration by intramuscular or intravenous injection of the adducts. However, it is expected that particular improvement in bioavailability and activity is to be evidenced when compositions comprising the adduct or adducts of this invention are administered orally as indicated above.

The invention also provides a method for improving the bioavailability of peramivir, comprising orally administering to a patient in need thereof a therapeutic amount of a peramivir adduct of this invention, including an adduct of formula (II) or (III), or a pharmaceutical formulation or dosage form thereof which is suitable for oral administration. Examples of suitable forms include, for example, capsules, tablets, caplets, various sustained or controlled release dosage forms, and the like. In one embodiment, the method comprises orally administering a composition comprising a therapeutic amount of the peramivir adduct and from about 5% to about 95% by weight of an excipient or mixture thereof.

Suitable excipients are well known to those skilled in the formulation art, and any excipient or combination of excipients known in the pharmaceutical art may be used. Examples may include flow aids, stabilizers, surface active agents, binders, dispersing agents, flavorings, taste masking agents, coatings, release control agents

and/or other excipients typically employed for formulation of oral dosage forms. In some embodiments, the excipient may comprise one or more materials selected from the group consisting of microcrystalline cellulose, di-calcium phosphate, lactose, pre- gelatinized starch, carnuba wax, candelilla wax, silica, and magnesium stearate. Particularly for peramivir adducts of formula (II), the excipient may comprise a stabilizing amount of borate ion, for example in a peramivir: borate molar ratio in the range of about 4:1 to about 1:4. Typically, the peramivir: borate molar ratio will be no greater than 1.

Although the invention is illustrated and described herein with reference to specific embodiments, the invention is not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims without departing from the invention.