WO2001003695A1 - Improvement of bioavailability with vitamin c fatty acid esters - Google Patents

Abstract

A method for increasing bioavailability of an orally administered pharmaceutical compound comprises orally coadministering the pharmaceutical compound to a mammal in need of treatment with the compound and a vitamin C fatty acid ester. Improved formulations of pharmaceutical compounds include a vitamin C fatty acid ester to enhance the bioavailability of the active ingredient of the pharmaceutical compound.

Description

IMPROVEMENT OF BIOAVAILABILITY WITH VITAMIN C FATTY ACID

ESTERS

INTRODUCTION Technical Field This invention is directed to the field of pharmacology and particularly to the formulation of oral pharmaceutical compositions for increased bioavailability and reduced inter- and intra-individual variability.

Background Pharmacokinetics is the study of the fate of pharmaceuticals from the time they are ingested until they are eliminated from the body. The sequence of events for an oral composition includes absorption through the various mucosal surfaces, distribution via the blood stream to various tissues, biotransformation in the liver and other tissues, action at the target site, and elimination of drug or metabolites in urine or bile. Bioavailability of a drug (pharmaceutical composition) following oral dosing is a critical pharmacokinetic determinant which can be approximated by the following formula:

where F_orai is the oral bioavailability fraction, which is the fraction of the oral dose that reaches the circulation in an active, unchanged form. F_orai is less than 100% of the active ingredient in the oral dose for four reasons: (1) drug is not absorbed out of the gut lumen into the cells of the intestine and is eliminated in the feces; (2) drug is absorbed into the cells of the intestine but back-transported into the gut lumen; (3) drug is biotransformed by the cells of the intestine (to an inactive metabolite); or (4) drug is eliminated by the cells of the liver, either by biotransformation and/or by transport into the bile. Thus, oral bioavailability is the product of the fraction of the oral dose that is absorbed (F_ABS), the fraction of the absorbed dose that successfully reaches the blood side of the gastrointestinal tract (F_G), and the fraction of the drug in the GI blood supply that reaches the heart side of the liver (F_H). The extent of gut wall absorption, back transport and metabolism, and liver elimination are all subject to wide inter- and intra-individual variability.

Previous investigations have resulted in new understandings of factors involved with bioavailability and in the invention described in U.S. Patent No. 5,567,592. The '592 patent describes general methods for increasing bioavailability of oral pharmaceutical compositions and methods for identifying compounds that increase bioavailability. However, although that invention made it possible to investigate a number of classes of compounds not previously thought to be useful in enhancing bioavailability, the actual process of identifying specific classes of compounds that are superior bioenhancers, among those bioenhancers which work to some degree, still remains a process of investigation and discovery. Within many classes of substances identified as showing general bioenhancing effects, there is surprising variance from class member to class member in the extent of each compound's bioenhancing effect, and some compounds that would at first thought appear to be enhancers of drug bioavailability because of their membership in a generally effective class of compounds, actually are found to be agents that interfere with the bioavailability of drugs, although the mechanism by which such interference takes place is not yet known. In some cases, a single compound or small group of compounds has been found to be particularly potent as a bioenhancer despite resembling in structure other compounds that have less activity or that even reduce bioavailability. Accordingly, it is important to identify and confirm the identity of individual compounds or classes of compounds that are particularly useful for enhancing bioavailability. For example, U.S. Patent No. 5,665,386 discloses the use of essential oils to enhance bioavailability.

SUMMARY OF THE INVENTION

An object of this invention is to identify compositions with superior ability to increase drug bioavailability, particularly by increasing net drug absorption and/or decreasing drug biotransformation in the gut wall by inhibiting cytochrome P450 drug metabolism. Another object of the invention is to provide compositions that strongly inhibit enzymes of the cytochrome P450 3 A class (CYP3A) in the gut in preference to in other locations, such as the liver, which was previously thought to be the primary site of drug metabolism.

One specific object of the present invention is to reduce inter-individual variability of the systemic concentrations of the active pharmaceutical compound, as well as intra-individual variability of the systemic concentrations of the pharmaceutical compound being administered.

The invention is carried out by coadministering a vitamin C fatty acid ester with an orally administered pharmaceutical compound (drug) or compounds to increase drug bioavailability. This may be done by simple coadministration or through a new formulation of the drug and vitamin C fatty acid ester. The compositions and methods of the invention can be used to increase drug efficacy in humans and in other mammals. Although veterinary use is specifically contemplated, the primary use will be in human treatment. Administration schemes include, but are not limited to, use of oral and topical formulations in humans and use of similar formulations for livestock. Ascorbyl palmitate is a preferred vitamin C fatty acid ester for use in the method of the invention.

BRIEF DESCRIPTION OF THE DRAWING The Figure is a graph showing mean cyclosporine concentration vs. time profile for cyclosporine (15 mg/kg) admimstered to male Sprague Dawley rats alone and with ascorbyl palmitate (30 mg/kg) or ketoconazole (30 mg/kg).

DESCRIPTION OF SPECIFIC EMBODIMENTS Vitamin C Fatty Acid Esters Increase Drug Bioavailability "Drug bioavailability" is defined here as the total amount of drug systemically available over time. The present invention increases drug bioavailability by inhibiting drug biotransformation in the gut. A class of compounds responsible for increased drug bioavailability is vitamin C fatty acid esters. A preferred member of this class is ascorbyl palmitate. It has been discovered that vitamin C fatty acid esters are capable of inhibiting the appropriate enzyme.

In general, the present invention provides a method for increasing the bioavailability of an orally admimstered pharmaceutical compound (particularly one which is hydrophobic) by orally coadministering the pharmaceutical compound to a mammal in need of treatment with a vitamin C fatty acid ester in sufficient amount to provide integrated systemic concentrations over time of the compound greater than the integrated systemic concentrations over time of the compound in the absence of the vitamin C fatty acid ester. Changes in the integrated systemic concentrations over time are indicated by "area under the curve" (AUC) measurements, an accepted pharmacological technique described in detail below.

Vitamin C Fatty Acid Esters

Vitamin C fatty acid esters are generally represented by the formula below:

The R group may be straight chain, branched chain, or substituted alkyl, alkenyl, or alkynyl substituents, as well as optionally substituted aryl, benzyl, phenyl, alicyclic and heterocyclic groups.

Specific alkenyl esters which may be attached to vitamin C and are useful in the practice of the invention include the following compounds, with common names given in parentheses: cis-9-hexadecenoate (pahnitoleate); trans-9-hexadecanoate (palmitelaidate); cis-9-octadecenoate (oleate); trans-9-octadecenoate (elaidyl); cis,cis-9, 12- octadecadienoate (linoleate); trans,trans-9, 12-octadecadienoate (linolelaidate); cis,cis,cis-9, 12, 15-octadecatrienyl (linolenate); trans,trans,trans-9, 12, 15- octadecatrienoate (linolenelaidate); cis,cis,cis-6, 9, 12-octadecatrienoate (gamma- linolenate).

The structure of a preferred vitamin C fatty acid ester for use in the methods and compositions of the invention, ascorbyl palmitate is shown below:

Ascorbyl palmitate is the ascorbyl ester of palmitic acid (hexadecanoic acid). Ascorbyl palmitate is also known as L-ascorbic acid-6-palmitate, vitamin C palmitate, and palmitoyl L-ascorbic acid, and is used routinely as an antioxidant to prevent the reaction of various food constituents with oxygen (Ensminger, A.H., et al., Foods and Nutrition Encyclopedia. 2^nd ed. CRC Press, Boca Raton, FL, 1:100, 119 (1994); Belitz, H.D. et al, Food Chemistry, Springer Verlag, Berlin, Heidelberg, 155-180 (1987); Stuckey, B.N., "Antioxidants as Food Stabilizers," CRC Handbook of Food Additives, 2^nd ed., ed. T.E. Furia, CRC Press, Boca Raton, FL, 1:185-196 (1980); Sims R.J., "Antioxidants as Stabilizers for Fats, Oils and Lipid-Containing Foods," CRC Handbook of Food Additives, 2^nd ed., ed. T.E. Furia, CRC Press, Boca Raton, FL 2:13-56 (1980)). Ascorbyl palmitate has the following identification and regulatory numbers: CAS 137-66-6 (Chemical Abstracts Registry); 21 CFR 182.3149 (U.S. Code of Federal Regulations, Title 21); EAFUS 1724 ASP (Everything Added to Food in the USA Database, full up- to-date toxicology information available).

In its current use, ascorbyl palmitate acts as a free radical and oxygen scavenger to prevent the deterioration of food and drug products (Belitz, H.D. et al., Food Chemistry, Springer Verlag, Berlin, Heidelberg, 155-180 (1987); Stuckey, B.N., "Antioxidants as Food Stabilizers," CRC Handbook of Food Additives, 2^nd ed., ed. T.E. Furia, CRC Press, Boca Raton, FL, 1 :185-196 (1980); Sims R.J., "Antioxidants as Stabilizers for Fats, Oils and Lipid-Containing Foods," CRC Handbook of Food Additives, 2^nd ed., ed. T.E. Furia, CRC Press, Boca Raton, FL, 2:13-56 (1980); Cort, W.M., "Antioxidant Activity of Tocopherols, Ascorbyl Palmitate, and Ascorbic Acid and Their Mode of Action," J. Am Oil Chem. Soc, 51:321-5 (1974)). This effect stabilizes the active ingredient in the food or drug product and serves to lengthen shelf life. Ascorbyl palmitate has been used as an antioxidant or preservative in foods, drugs, cosmetics and pesticide products since 1948. This compound is Generally Recognized As Safe (GRAS) by the FDA and is listed in the Everything Added to Food in the United States (EAFUS) database as well as the United States Pharmacopeia-National Formulary (USP-NF) and the Food Chemicals Codex. The Joint Food and Agricultural Organization/World Health Organization Expert Committee on Food Additives has established an acceptable daily intake of 0-1.25 mg/kg/day for each of ascorbyl palmitate and ascorbyl stearate. This value is 1/100 of the "no observed effect" level determined in a two year feeding study in rats ("Ascorbyl Palmitate and Stearate", WHO Food Additive Sengs, 5:146-147 (1974)).

Ascorbyl palmitate has now been found to significantly inhibit the metabolism of nifedipine and cyclosporine in human liver microsomes (see Example 1, below). Both nifedipine and cyclosporine are metabolized by CYP3 A in the system described (Gonzalez F.J., et al., "Human P450PCN1 : Sequence, Chromosome Localization, and Direct Evidence Through cDNA Expression That P450PCN1 is Nifedipine Oxidase," DNA, 7:79-86 (1988); Kronbach T., et. al, "Cyclosporine Metabolism in Human Liver: Identification of a Cytochrome P-450III Gene Family as the Major Cyclosporine- Metabolizing Enzyme Explains Interactions of Cyclosporine with Other Drugs," Clin. Pharmacol. Ther., 43:630-5 (1988)). The impact of ascorbyl palmitate on nifedipine and cyclosporine metabolism was not expected since the parent ascorbic acid was not a useful inhibitor of this metabolism and antioxidants as a class do not necessarily inhibit CYP3A. Moreover, previous studies demonstrated that ascorbyl palmitate induced metabolic enzymes in animal models (Sato P.H., et al., "Stimulation of Drug Metabolism by Ascorbic Acid in Weanling Guinea Pigs," Biochem. Pharmacol, 23:3121-3128 (1974)). Enzyme inhibition by vitamin or other esters of palmitic acid is similarly not predictive as retinyl, ethyl, and cholestryl palmitates are not effective inhibitors of nifedipine metabolism (as reported in Table 2).

The vitamin C fatty acid ester is preferably presented for coadministration in a vitamin C fatty acid ester to drug ratio in the range of 0.01 to 100 units of the vitamin C fatty acid ester to 1 unit of the drug. For example, a formulation having 1 mg of the vitamin C fatty acid ester per 100 mg drug represents the lower end of this range and a formulation having 500 mg of the vitamin C fatty acid ester per 5 mg drug represents the upper end of this range. A more preferred range of vitamin C fatty acid ester to drug in accordance with the present invention is 0.1 to 10 units vitamin C fatty acid ester to 1 unit of the drug. The most preferred range is 0.5 to 2 units vitamin C fatty acid ester per 1 unit of the drug. For instance, in Example 2 below, an approximately 2:1 vitamin C fatty acid ester to drug ratio was advantageously used. Because vitamin C fatty acid esters in very low concentrations, as have been used previously for the antioxidant purposes discussed above, are of low activity and thus not likely to be useful for the purposes described generally herein, only concentrations of the vitamin C fatty acid esters providing an inhibition activity are included in the invention. Preferred are those formulations having vitamin C fatty acid ester that show an inhibition of at least 20% at a 1 : 1 vitamin C fatty acid ester to drug ratio; even more preferred are formulations having vitamin C fatty acid ester that show an inhibition of at least 50% at a 1 : 1 vitamin C fatty acid ester to drug ratio.

Bioavailability Measurements

The increase in drug bioavailability attributable to administration of the vitamin C fatty acid ester can be determined by measuring total systemic drug concentrations over time after coadministration of a drug and the vitamin C fatty acid ester and after administration of only the drug. The increase in drug bioavailability is defined as an increase in the Area Under the Curve (AUC). AUC is the integrated measure of systemic drug concentrations over time in units of mass-time/volume. The AUC from time zero (the time of dosing) to time infinity (when no drug remains in the body) following the administration of a drug dose is a measure of the exposure of the patient to the drug. When efficacy of the vitamin C fatty acid ester is being measured, the amount and form of active drug administered should be the same in both the coadministration of drug and vitamin C fatty acid ester and the administration of the drug alone. For instance, administration of 10 mg of drug alone may result in total systemic drug delivered over time (as measured by AUC) of 500 μg-hr/ml. In coadministration (i.e., in the presence of the vitamin C fatty acid ester), the systemic drug AUC may increase to 700 μg-hr/ml. If significantly increased drug bioavailability in the presence of the vitamin C fatty acid ester is anticipated, drug doses may need to be reduced for safety. Systemic drug concentrations are measured using standard drug measurement techniques. "Systemic drug concentration" refers to a drug concentration in a mammal's bodily fluids, such as serum, plasma or blood; the term also includes drug concentrations in tissues bathed by the systemic fluids, including the skin. Systemic drug concentration does not refer to digestive fluids. The increase in total systemic drug concentrations is one way of defining an increase of drug bioavailability due to coadministration of the vitamin C fatty acid ester and the drug. For drugs excreted in part unmetabolized in the urine, an increased amount of unchanged drug in the urine will reflect the increase in systemic concentrations.

Characteristics of Drugs Used With Vitamin C Fatty Acid Esters

The word "drug" as used herein is defined as a chemical capable of administration to an organism which modifies or alters the organism's physiology. More preferably the word "drug" as used herein is defined as any substance intended for use in the treatment or prevention of disease. Drug includes synthetic and naturally occurring toxins and bioaffecting substances as well as recognized pharmaceuticals, such as those listed in "The Physicians Desk Reference," 49th edition, 1995, pages 101-338; "Goodman and Gilman's The Pharmacological Basis of Therapeutics" 9th Edition (1996), pages 103-1645 and 1707-1792; and "The United States Pharmacopeia, The National Formulary", USP 23 NF 18 (1995), the compounds of these references being herein incorporated by reference. The term drug also includes compounds that have the indicated properties that are not yet discovered or available in the U.S. The term drug includes pro-active, activated and metabolized forms of drugs. The present invention can be used with drugs consisting of charged, uncharged, hydrophilic, zwitter-ionic, or hydrophobic species, as well as any combination of these physical characteristics. A hydrophobic drug is defined as a drug which in its non-ionized form is more soluble in lipid or fat than in water. A preferred class of hydrophobic drugs is those drugs more soluble in octanol than in water.

Compounds (or drugs) from a number of classes of compounds can be administered with the vitamin C fatty acid ester, for example, but not limited to, the following classes: acetamlides, anilides, aminoquinolines, benzhydryl compounds, benzodiazepines, benzofurans, cannabinoids, cyclic peptides, dibenzazepines, digitalis gylcosides, ergot alkaloids, flavonoids, imidazoles, quinolines, macrolides, naphthalenes, opiates (or morphinans), oxazines, oxazoles, phenylalkylamines, piperidines, polycyclic aromatic hydrocarbons, pyrrolidines, pyrrolidinones, stilbenes, sulfonylureas, sulfones, triazoles, tropanes, and vinca alkaloids.

Increased Drug Bioavailability by Inhibition of Cytochrome P450 Phase I Biotransformation

Inhibition of enterocyte cytochromes P450 participating in drug biotransformation is one objective of the present invention. The major enzymes involved in drug metabolism are present in the endoplasmic reticulum of many types of cells but are at the highest concentration in hepatocytes. Traditionally, enterocyte biotransformation was considered of minor importance in biotransformation compared to the liver. Many compounds inhibit cytochrome P450. These include, but are not limited to, ketoconazole, troleandomycin, gestodene, flavones such as quercetin and naringenin, erythromycin, ethynyl estradiol, and prednisolone. The primary goal of the invention is to use a vitamin C fatty acid ester to inhibit drug cytochrome P450 biotransformation in the gut to increase drug bioavailability.

Types Of Cytochromes And Tissue Location

The cytochromes P450 are members of a superfamily of hemoproteins. They represent the terminal oxidases of the mixed function oxidase system. The cytochrome P450 gene superfamily is composed of at least 207 genes that have been named based on their evolutionary relationships. For this nomenclature system, the sequences of all of the cytochrome P450 genes are compared, and those cytochromes P450 that share at least 40% identity are defined as a family (designated by CYP followed by a Roman or Arabic numeral, e.g. CYP3), further divided into subfamilies (designated by a capital letter, e.g. CYP3A), which are comprised of those forms that are at least 55% related by their deduced amino acid sequences. Finally, the gene for each individual form of cytochrome P450 is assigned an Arabic number (e.g. C7 3A4).

Three cytochrome P450 gene families (CYPl, CYP2 and CYP3) appear to be responsible for most drug metabolism. At least 15 cytochromes P450 have been characterized to varying degrees in the human liver. At concentrations of the substrates found under physiologic conditions, enzyme kinetics often favor a single form of cytochrome P450 as the primary catalyst of the metabolism of a particular drug or other enzyme substrate.

The CYP3 gene family encoding cytochromes P450 of type 3 is possibly the most important family in human drug metabolism. At least 5 forms of cytochrome P450 are found in the human 3 A subfamily, and these forms are responsible for the metabolism of a large number of structurally diverse drugs. In non-induced individuals, 3 A may constitute 20% of the P450 enzymes in the liver. In enterocytes, members of the 3 A subfamily constitute greater than 70% of the cytochrome-containing enzymes. The first two human 3 A subfamily members identified were 3A3 and 3A4. These two cytochromes P450 are so closely related that the majority of studies performed to date have not been able to distinguish their contributions, and thus they are often referred to as 3 A3/4. Erythromycin N-demethylation, cyclosporine oxidation, nifedipine oxidation, midazolam hydroxylation, testosterone 6β-hydroxylation, and cortisol 6β-hydroxylation are all in vitro probes of 3A3/4 catalytic activity. The levels of 3A3/4 vary by as much as 60-fold between human liver microsomal samples, with the levels of 3 A forms approaching 50% of the total cytochrome P450 present in human liver samples from individuals receiving inducers of 3A3/4. The recently studied CYP3A5 may also play a role as important as 3A3/4.

The liver contains many iso forms of cytochrome P450 and can biotransform a large variety of substances. The enterocytes lining the lumen of the intestine also have significant cytochrome P450 activity, and this activity is dominated by a single family of isozymes, 3A, the most important isoforms in drug metabolism.

Increased Drug Efficacy By Reducing CYP3A Drug Biotransformation

Vitamin C fatty acid esters, as used according to the invention, reduce drug biotransformation in the gut by inhibiting CYP3 A activity in gut epithelial cells which leads to a total increase in drug bioavailability in the serum. In the presence of the vitamin C fatty acid ester, fewer drug molecules will be metabolized by phase I enzymes in the gut and will not be available for phase II conjugation enzymes. This will lead to increased concentrations of untransformed drug passing from the gut into the blood and onto other tissues in the body.

Although the primary objective of the vitamin C fatty acid ester is to inhibit CYP3 A drug biotransformation in the gut, some biotransformation may be decreased in other tissues as well if the vitamin C fatty acid ester is absorbed into the blood stream. The decrease in biotransformation by other tissues will also increase drug bioavailability. The advantage of targeting the vitamin C fatty acid ester to the gut, however, is that it allows the use of lower systemic concentrations of the vitamin C fatty acid ester compared to inhibitors that target CYP3A in the liver. After oral administration of the vitamin C fatty acid ester, concentrations will be highest at the luminal surface of the gut epithelia, not having been diluted by systemic fluids and the tissues of the body. Luminal concentrations that are greater compared to blood concentrations will permit preferential inhibition of CYP3A in gut instead of the liver. The vitamin C fatty acid ester used according to the invention preferentially inhibits gut CYP3 A and so, will also be a particularly effective means of increasing drug bioavailability.

Coadministration of vitamin C fatty acid esters will also reduce variability of oral bioavailability. Reduction of drug biotransformation or increased drug absorption will decrease variability of oral bioavailability to some degree because the increase in bioavailability will begin to approach the theoretical maximum of 100% oral bioavailability. The increase in oral bioavailability will be generally larger in subjects with lower oral bioavailability. The result is a reduction in inter-individual and intra- individual variation. Addition of the vitamin C fatty acid ester will reduce inter- individual and intra-individual variation of systemic concentrations of a drug or compound.

A Net Increase in Drug Bioavailability Due to a Decrease in the Activity of CYP3A. The catalytic activities of CYP3A that are subject to inhibition include, but are not limited to, dealkyase, oxidase, and hydrolase activities. In addition to the different catalytic activities of CYP3 A, different forms of CYP3 A exist with a range in molecular weight (for example, from 51 kD to 54 kD, as shown in Komori et al., J. Biochem.. 104:912-16 (1988)). A vitamin C fatty acid ester reduces CYP3 A drug biotransformation by acting as an inhibitor of CYP3 A activity. Possible mechanisms include competitive, non- competitive, uncompetitive, mixed or irreversible inhibition of CYP3 A drug biotransformation.

Selection of Concentration of Vitamin C Fatty Acid Ester by Reduction of CYP3 A Drug Biotransformation

The ability of the vitamin C fatty acid ester to increase drug bioavailability of a particular drug can be estimated using in vitro and in vivo drug biotransformation measurements. In vivo measurements of drug bioavailability, such as measuring serum or blood drug concentrations over time, provide the closest measure of total drug systemic availability (bioavailability), as evidenced in Example 2, below. In vitro assays of CYP3A metabolism indirectly indicate drug bioavailability because CYP3A drug metabolism affects integrated systemic drug concentrations over time. Although even a minimally measured increase is all that is required for the vitamin C fatty acid ester to be useful, a preferred commercially desirable concentration of the vitamin C fatty acid ester acting as a CYP3 A modulator generally will increase drug bioavailability by at least 10%, preferably by at least 50%, and more preferably by at least 75% of the difference between bioavailability in its absence and complete oral bioavailability. For example, if the drug bioavailability is 40% without the fatty acid ester, then the addition of the vitamin C fatty acid ester may increase bioavailability to 85%, for a 75% increase. A sufficient amount of orally admimstered vitamin C fatty acid ester will provide integrated systemic drug concentrations over time greater than the integrated systemic drug concentrations over time in the absence of the vitamin C fatty acid ester. The actual amount or concentration of the vitamin C fatty acid ester to be included with a pharmaceutical compound for a particular composition or formulation will vary with the active ingredient of the compound. The amount of the vitamin C fatty acid ester to be used should be optimized using the AUC methods described herein, once the components for a particular pharmaceutical composition have been decided upon. As stated above, the recommended measure for the amount of the vitamin C fatty acid ester in a particular formulation is by direct comparison to the amount of drug, with a vitamin C fatty acid esteπdrug ratio in the range of (0.01-100):1 being preferred, (0.1-10):1 being more preferred, and (0.5-2): 1 being most preferred.

Inhibition of the P450 3 A class of enzymes by a vitamin C fatty acid ester can be studied by a variety of bioassays, several of which are set forth below.

In vitro CYP3 A Assays and Increased Drug Bioavailability

Cell Assays of CYP3A Function and Increased Drug Bioavailability

Cultured cells of either hepatocytes or enterocytes or freshly prepared cells from either liver or gut can be used to determine the activity of the vitamin C fatty acid ester as a CYP3A inhibitor. Various methods of gut epithelial cell isolation can be used such as the method of Watkins, et al., J. Clin. Invest. 80:1029-36 (1987). Cultured cells, as described in Schmiedlin-Ren, et al., Biochem. Pharmacol.. 46:905-918 (1993), can also be used. The production of CYP3 A metabolites in cells can be measured using high pressure liquid chromatograph (HPLC) methods as described in the following section for microsome assays of CYP3 A activity.

Microsome Assays of CYP3A Function and Increased Bioavailability

Microsomes from liver or intestine may be used for assays of CYP3A activity. Microsomes can be prepared from liver using conventional methods as discussed in Kronbach, et. al., Clin. Pharmacol. Ther.. 43:630-5 (1988). Alternatively, microsomes can be prepared from isolated enterocytes using the method of Watkins, et al., J. Clin. Invest.. 80:1029-1036 (1987). Microsomes from gut epithelial cells can also be prepared using calcium precipitation as described in Bonkovsky, et al., Gastroenterology. 88:458- 467 (1985). Microsomes can be incubated with drugs and the metabolites monitored as a function of time. In addition, the levels of these enzymes in tissue samples can be measured using radioimmunoassays or western blots. Additionally, the production of metabolites can be monitored using high pressure liquid chromatography systems (HPLC) and identified based on retention times. CYP3A activity can also be assayed colorimetrically measuring erythromycin demethylase activity as the production of formaldehyde as in Wrighton, et al, Mol. Pharmacol.. 28:312-321 (1985) and Nash, Biochem. I.. 55:416-421 (1953).

Characteristics of Vitamin C Fatty Acid Esters for Reducing CYP3A Drug Metabolism Vitamin C fatty acid esters bind CYP3A quickly and inhibit while the drug is passing through the enterocyte. After the vitamin C fatty acid ester reaches the heart and is distributed throughout the body the concentration of the vitamin C fatty acid ester will be diluted on future passes through the liver. Concentrations of the vitamin C fatty acid ester used in the gut lumen are preferably selected to be effective on gut CYP3 A metabolism but, due to dilution, to be less active in other tissues.

The amount of the vitamin C fatty acid ester used for oral administration can be selected to achieve small intestine luminal concentrations of at least 0.1 of the K, or apparent K, for CYP3A inhibition of drug metabolism or an amount sufficient to increase systemic drug concentration levels, whichever is less. Alternatively, the amount of an inhibitor of cytochrome P450 3 A enzyme that will be used in a formulation can be calculated by various assays that are described in detail below. For example, one such assay measures the conversion of nifedipine to its oxidation product in an assay system containing 50 μg human liver microsomes, 100 μM nifedipine, and 1mm NADPH in 500 μl of 0.1 M sodium phosphate buffer, pH 7.4. The initial inhibitor amount is selected to provide concentrations in the lumen of the small intestine equal or greater than concentrations that reduce the rate of conversion determined by this assay, preferably a rate reduction of at least 10%. While the actual dose of inhibitor in a clinical formulation might be optimized from this initial dosage depending on the results of a clinical trial, the assay as described is sufficient to establish a utilitarian dosage level.

More than one vitamin C fatty acid ester may be used in the method or formulation of the invention to achieve the necessary dosage. For example, ascorbyl palmitate may be combined with ascorbyl stearate and coadministered with the drug to provide increased bioavailability of the drug. In all of these cases, the goal of selecting a particular concentration is increased bioavailability of the pharmaceutical compound that is being administered. Thus, a desirable goal is to provide integrated systemic concentrations over time of the pharmaceutical compound in the presence of the inhibitor that is greater than the integrated systemic concentrations over time of the pharmaceutical compound in the absence of the inhibitor by at least 10% of the difference between bioavailability in its absence and complete oral bioavailability. Preferred is attainment of "complete bioavailability," which is 100% systemic bioavailability of the administered dosage. Screening Assay for Superior Vitamin C Fatty Acid Ester Formulations

In summary, the various techniques described above for screening vitamin C fatty acid ester concentrations for activity levels by assaying for inhibition in the gut of a mammal of activity of a cytochrome P450 enzyme are all generally useful as methods of creating useful formulations that are most useful for increasing bioavailability of the active ingredient of a given drug in a mammal. In all of these assays, the best amounts are those that best inhibit enzymatic destruction of a tested drug in the gut of the mammal (either by direct testing in vivo or by a test that predicts such activity). When testing for inhibition of activity of a cytochrome enzyme, assays that detect inhibition of members of a cytochrome P450 3A family (for a particular mammal, particularly human) are preferred. Although in vivo assays are preferred, because of the direct relationship between the measurement and gut activity, other assays, such as assays for inhibition of cytochrome P450 activity in isolated enterocytes or hepatocytes or microsomes obtained from either enterocytes or hepatocytes of the mammal in question or for inhibition of cytochrome P450 in a tissue or membrane from the gut of said mammal, are still useful as screening assays. It is possible to use enzymes from both the gut and liver interchangeably for these assays since it has been shown that CYP3 A enzymes are identical in the two locations (Kolars, J.C, et al., Identification of Rifampin-Inducible P450IIIA4 (CYP3A4) in Human Small Bowel Enterocytes, J. Clin. Investig.. 90:1871- 1878 (1992), Lown, K.S., et al., Sequences of Intestinal and Hepatic Cytochrome P450 3A4 cDNAs are Identical, Drug Metab. Dispos.. 26: 185-7 (1998)).

Coadministration and Delivery of the Vitamin C Fatty Acid Ester Coadministration of Vitamin C Fatty Acid Ester and a Drug

The present invention will increase the bioavailability of a drug in systemic fluids or tissues by coadministering the vitamin C fatty acid ester with a drug.

"Coadministration" includes concurrent administration (administration of the vitamin C fatty acid ester and drug at the same time) and time-varied administration (administration of the vitamin C fatty acid ester at a time different from that of the drug), as long as both the vitamin C fatty acid ester and the drug are present in the gut lumen and/or membranes during at least partially overlapping times. "Systemic fluids or tissues" refers to blood, plasma, or serum and to other body fluids or tissues in which drug measurements can be obtained. Delivery Vehicles and Methods

Coadministration can occur with the same delivery vehicle or with different delivery vehicles. The vitamin C fatty acid ester and the drug can be administered using, as examples, but not limited to, time release matrices, time release coatings, companion ions, and successive oral administrations. Alternatively, the drug and the vitamin C fatty acid ester can be separately formulated with different coatings possessing different time constants for release of the vitamin C fatty acid ester and drug. The vitamin C fatty acid ester can also be bound to the drug being protected, either by covalent bonding or by ionic or polar attractions.

Vitamin C fatty acid esters also increase bioavailability when used with epithelia tissues other than the gut. The discussion above of the invention as used in the gut is appropriate for other types of epithelia. For example, CYP 3 A enzymes are present in the skin, and vitamin C fatty acid esters can be used in transdermal formulations to increase drug bioavailability to systemic fluids and tissues. Such applications are part of the invention, since inhibition of CYP 3 A enzymes by vitamin C fatty acid esters in epithelia other than the gut provides the same mechanism of action.

Formulations Having a Vitamin C Fatty Acid Ester

The invention is carried out in part by formulating an oral pharmaceutical composition to contain at least one vitamin C fatty acid ester. This is accomplished in some embodiments by admixing a pharmaceutical compound, usually a pharmaceutical carrier, and the vitamin C fatty acid ester, the vitamin C fatty acid ester being present in an amount sufficient to provide integrated systemic concentrations over time of the compound (as measured by AUC's) greater than the integrated systemic concentrations over time of the compound in the absence of the vitamin C fatty ester when the pharmaceutical composition is administered orally to an animal being treated. A pharmaceutical carrier is generally an inert bulk agent added to make the active ingredients easier to handle and can be solid or liquid in the usual manner as is well understood in the art. Pharmaceutical compositions produced by the process described herein are also part of the present invention.

The present invention can also be used to increase the bioavailability of the active compound of an existing oral pharmaceutical composition. When practiced in this manner, the invention is carried out by reformulating the existing composition to provide a reformulated composition by admixing the active compound with at least one vitamin C fatty acid ester, the vitamin C fatty acid ester being present in an amount sufficient to provide integrated systemic concentrations over time of the compound when administered in the reformulated composition greater than the integrated systemic concentrations over time of the compound when administered in the existing pharmaceutical composition. All of the criteria described for new formulations also apply to reformulation of old compositions. In preferred aspects of reformulations, the reformulated composition comprises all components present in the existing pharmaceutical composition plus the vitamin C fatty acid ester, thus simplifying practice of the invention, although it is also possible to eliminate existing components of formulations because of the increase in bioavailability. Thus, the invention also covers reformulated compositions that contain less than all components present in the existing pharmaceutical composition plus the vitamin C fatty acid ester. However, this invention does not cover already existing compositions that contain a component which increases bioavailability by mechanisms described in this specification (without knowledge of the mechanisms), should such compositions exist.

Traditional formulations can be used with vitamin C fatty acid esters. Optimal vitamin C fatty acid ester concentrations can be determined by varying the amount and timing of the vitamin C fatty acid ester administration and monitoring bioavailability. Once the optimal vitamin C fatty acid ester concentration or vitamin C fatty acid ester to drug ratio is established for a particular drug, the formulation (vitamin C fatty acid ester, drug, and other formulation components, if any) is tested clinically to verify the increased bioavailability. In the case of time- or sustained- release formulations, it will be preferced to establish the optimal vitamin C fatty acid ester concentration using such formulations from the start of the bioavailability experiments.

Vitamin C fatty acid esters have been used as antioxidants under many different circumstances, including as part of a pharmaceutical composition or formulation. Their use has been limited to preventing decomposition of the materials in the formulation, rather than for a physiological effect. As an antioxidant, a vitamin C fatty acid ester, and particularly ascorbyl palmitate, is used in small quantities, and such materials are not likely to approach even the outer limits of the present invention as defined by the specification and claims. In particular, preferred formulations of the invention contain greater than 0.5%, preferably at least 1%, by weight of the vitamin C fatty acid ester relative to the total weight of the formulation (including the capsule, if present), more preferably at least 2%, even more preferably at least 5%. In most cases the vitamin C fatty acid ester used as an antioxidant is used at less than 0.1% of the materials they are being used to protect or preserve. In considering these percentages, it should be recalled that these are percentages of the formulation in which the active ingredient is being presented, not percentages by weight or volume as concentrations in the medium in which the pharmaceutical composition will become dissolved or suspended after ingestion of the formulation. Furthermore, the vitamin C fatty acid ester may be used in capsules (either hard or soft standard pharmaceutical gel capsules, for example).

The invention now being generally described, the same will be better understood by reference to the following detailed examples, which are offered for illustration only and are not to be considered limiting of the invention unless otherwise specified.

EXAMPLE 1 Inhibition of Drug Degradation by a Vitamin C Fatty Acid Ester

The ability of a vitamin C fatty acid ester at various concentrations to inhibit metabolism for two representative drugs was tested. Human liver microsomes were prepared and each of three drugs were incubated with the microsomes in the presence of a vitamin C fatty acid ester or a known inhibitor of CYP3A metabolism. Metabolism in the presence of the vitamin C fatty acid ester or known CYP3A inhibitor was compared to a control treated only with the vehicle in which the inhibitor was dissolved.

Human liver pieces from a male donor were obtained from the International Institute for the Advancement of Medicine (HAM, Exton, PA). To prepare the microsomes, human liver pieces were perfused with 1.15% potassium chloride, then homogenized in 0.1 mM Tris-acetate, pH 7.4, containing ImM EDTA and 20 mM BHT (butylated hydroxytoluene). Microsomal pellets were prepared from the homogenate using standard differential centrifugation procedures (Guengerich, Analysis and characterization of enzymes in Principles and Methods of Toxicology. A.W. Hayes (ed.), Raven Press, New York. pp. 774-814 (1989)) and were stored at -80°C in Tris-acetate buffer, pH 7.4, containing 20% w/v glycerol. Microsomes were diluted in 100 mM potassium phosphate buffer, pH 7.4, for use in metabolic incubations. Microsomal protein and CYP content of the human liver microsomes were determined using methods of Bradford (Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72:248-254 (1976)) and Omura and Sato (Omura, T. et. al. The carbon monoxide-binding pigment of liver microsomes II. Solubilization, purification and properties. J. Biol. Chem. 239:2370-2378 (1964)), respectively.

Cyclosporine Reactions Cyclosporine (50 μM) and inhibitor or inhibitor vehicle (5 μl) were preincubated with human liver microsomes (1 mg/ml) and diethylenetriaminepentaacetic acid (DETAPAC; 1 mM) in 100 mM phosphate buffer pH 7.4 for 5 min at 37°C. Metabolic reactions were started by addition of reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) to give a final concentration of 1 mM and a final volume of 0.5 ml. Reactions were stopped after 40 minutes by addition of 1 ml extraction solvent (50:30:20 methanol-acetonitrile- water saturated with zinc sulfate). Ten μl of internal standard solution (5 mM lauryl gallate in acetonitrile) was then added and the precipitated materials were separated by centrifugation (3000 rpm x 10 min). Supernatants were then subjected to solid-phase extraction. Solid-phase extraction utilized Bond-Elut® C-18 sample preparation columns (10 ml/lOOmg) fitted to a Vac-Elut SPS 24 vacuum manifold (Varian Sample Preparation Products, Harbor City, CA). Prior to applying extraction supernatants, the Bond-Elut® columns were primed by washing with 1 volume reagent alcohol followed by 2 volumes water. Supernatants from extractions were applied to the column by aspiration, then the columns were washed with 1 ml 80:20 water (pH 3)-acetomtrile. Cyclosporine and metabolites were eluted with 400 μl 95% ethanol. The eluent was diluted with 200 μl water, then washed by vortex mixing for 60 seconds with 500 μl hexane. Phases were separated by centrifugation (3000 rpm x 5 minutes), then the hexane phase was removed by aspiration through a Pasteur pipette and the remaining ethanol eluent was injected directly for HPLC.

Cyclosporine and metabolites were separated using a Rainin Microsorb C-18 column (4.6 x 250 mm; 5 μm), maintained at 70 °C with a Hot Pocket heating jacket (Keystone Scientific Inc.). Detection was at 214 nm. Cyclosporine was eluted from the column using a binary solvent gradient system (Table 1) where solvent A was water which had been adjusted to pH 3 with phosphoric acid, and solvent B was 80:20 acetonitrile-methanol. The solvent flow rate was 0.75 ml/min.

TABLE 1

HPLC gradient used for cyclosporine analysis

Time (nun) Initial %B Final %B Time (mm) %B per nun

0 65 85 25 0.8

25 85 65 0.2 Step

32 (end) 65

Nifedipine reactions

Nifedipine (100 μM) and inhibitor or inhibitor vehicle (5 μl) were preincubated with human liver microsomes (0.1 mg/ml) and DETAPAC (1 mM) in 100 mM phosphate buffer pH 7.4 for 5 min at 37°C. Metabolic reactions were started by addition of reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) to give a final concentration of 1 mM and a final volume of 0.5 ml. Reactions were stopped after 3 min at 37°C by addition of 200 μl stop solution (94:6 acetonitrile:glacial acetic acid). Protein was precipitated by centrifugation (3000 rpm x 10 min). Supernatants were analyzed for nifedipine and its oxidation product 2,6-dimethyl-4-(2-mtrophenyl)-3,5- pyridinedicarboxylic acid, dimethyl ester by HPLC with UV detection (Gonzalez F.J. et al. Human P450PCN1: sequence, chromosome localization and direct evidence through cDNA expression that P450PCN1 is nifedipine oxidase. DNA 2: 79-86 (1988)). All experiments were conducted in triplicate and compared to reactions with inhibitor and substrate but without NADPH or reactions with NADPH and inhibitor in the absence of substrate. Inhibitor efficacy was confirmed in repeated experiments. HPLC analysis utilized a Beckman model 126 binary solvent module with detection by a Beckman model 166 UV detector. Samples were injected using a Beckman model 507e autosampler fitted with a Rheodyne model 7010 sample injection valve (100 μl sample loop volume). Data were collected and analyzed using Beckman System Gold Nouveau™ chromatography software. Possible interfering peaks in the HPLC traces were identified by analysis of metabolic incubations with and without

NADPH in the absence of substrate. Nifedipine and its oxidation product were separated on a Rainin Microsorb-MV™ C-18 analytical column (5 μm; 4.6 mm x 150 mm). Compounds were eluted using one of two systems depending on any interfering peaks in the HPLC trace. System 1 : solvent A = 70% pH 3 water 30% acetonitrile; solvent B = methanol; mobile phase flow rate = 1.0 ml/min; detector wavelength = 280 nm; column temperature = 50°C. The initial mobile phase was 70% A and 30% B. Immediately upon sample injection the concentration of B was increased linearly over 6 min to a final concentration of 86% B which was maintained for 2 min at which time the system was returned to the initial conditions.

System 2: solvent A = water brought to pH 8.5 using ammonium hydroxide; solvent B = 50:50 methanol-acetonitrile; mobile phase flow rate = 1.5 ml/min; detector wavelength = 237 nm. The initial mobile phase was 65% A and 35% B. Immediately upon sample injection the concentration of B was increased linearly over 16 min to a final concentration of 75% B at which time the system was returned to the initial conditions.

The results of the cyclosporine and nifedipine experiments are presented in Table

TABLE 2 Effect of inhibitors on the metabolism of CYP3 A substrates by human liver microsomes (HLM). Metabolism in the presence of the inhibitor is expressed as percentage (± standard deviation; n = 3) of the uninhibited reaction (control)³

Inhibitor μM Nifedipine oxidation Cyclosporine hydroxylation AMI AM9

Control — 100 (3) 100 (1) 100 (1)

Ketoconazole (3A)^C 1 10.3 (0.4)* 64 (3)* 57 (3)*

Ascorbyl palmitate^d 20 not tested 70 (2)* 69 (2)*

25 74.6 (0.4)* not tested not tested

50 41 (2)* 49 (2)* 46 (2)*

100 6 (1)* 36 (1)* 34 (2)*

Ascorbic acid 100 97 (3) 90 (1)* 92 (3)*

Retinyl palmitate 100 103 (1) 102 (3) 101 (3)

Ethyl palmitate 100 81 (2)* not tested not tested

Cholesteryl palmitate 100 84 (1)* not tested not tested ^a A smaller number indicates greater inhibition of metabolism. Metabolites were identified by comparison with authentic standards. ^b AMI and AM9 represent different cyclospoπne hydroxylation products (Kronbach T., et. al., "Cyclospoπne Metabolism in Human Liver: Identification of a Cytochrome P-450III Gene Family as the Major Cyclosponne-Metabohzing Enzyme Explains Interactions of Cyclospoπne with Other Drugs," Clin Pharmacol. Ther., 43:630-5 (1988)) ^cCYP3A-selectιve inhibitor (Bourne M. et al. Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J Pharmacol. Exp. Ther. 277: 321-32 (1996). Newton D.J. et al. Cytochrome P450 inhibitors. Evaluation of specificities m the in vitro metabolism of therapeutic agents by human liver microsomes. Drug Metab. Dispos. 23 : 154-8 ( 1995)) ^d Competitive inhibitor of nifedipine metabolism K, = 5.4 ± 0.4 μM (mean ± standard error of estimate, r = 0.996). Inhibinon constant (K,) determinations utilized 10, 20, 50 and 100 μM nifedipine substrate concentrations and expenments were run m duplicate. K, values were determined by regression analysis of rate data using SigmaPlot V4.0S software (SPSS Inc., San Rafael, California) * Statistically significant difference compared to control (P<0 05) as determined by ANOVA with Dunnett's post-hoc compaπson

As demonstrated in this experiment, ascorbyl palmitate significantly inhibits the metabolism of nifedipine and cyclosporine in human liver microsomes. Both nifedipine and cyclosporine are metabolized by CYP3A in the system described (Gonzalez F.J., et al., "Human P450PCN1 : Sequence, Chromosome Localization, and Direct Evidence Through cDNA Expression That P450PCN1 is Nifedipine Oxidase," DNA, 7:79-86 (1988); Kronbach T., et. al., "Cyclosporine Metabolism in Human Liver: Identification of a Cytochrome P-450III Gene Family as the Major Cyclosporine-Metabolizing Enzyme Explains Interactions of Cyclosporine with Other Drugs," Clin. Pharmacol. Ther., 43:630-5 (1988). As noted above, the impact of ascorbyl palmitate on nifedipine and cyclosporine metabolism was not expected since the parent compound, ascorbic acid, is not a useful inhibitor of this metabolism and antioxidants as a class do not necessarily inhibit CYP3A. Moreover, previous studies demonstrated that ascorbyl palmitate induced metabolic enzymes in animal models (Sato P.H., et al., "Stimulation of Drug Metabolism by Ascorbic Acid in Weanling Guinea Pigs," Biochem. Pharmacol,

23:3121-3128 (1974)). Enzyme inhibition by palmitic esters of vitamins is similarly not predictive as retinyl palmitate is not an effective CYP3A inhibitor (see Table 2).

As evidenced by this experiment, at all tested concentrations, ascorbyl palmitate acted as an effective inhibitor of CYP3A-mediated metabolism. Greater inhibition of the metabolism occurred with increasing concentrations of the ascorbyl palmitate. Ascorbyl palmitate also compared favorably with known CYP3A inhibitors. This demonstrates the utility of a vitamin C fatty acid ester to increase bioavailability of a pharmaceutical compound by coadministration of the vitamin C fatty acid ester with the pharmaceutical compound. EXAMPLE 2 Improvement of Bioavailability in an In Vivo Model by a Vitamin C Fatty Acid Ester

Ascorbyl palmitate and the established CYP3 A-inhibitor ketoconazole were evaluated for their potential to improve the bioavailability of a cyclosporine oral solution administered by gavage to male Sprague-Dawley rats.

Formulations

The control formulation was prepared by addition of 0.5 ml ethanol to 0.5 ml cyclosporine oral solution (100 mg/ml cyclosporine (SangCyA™)) followed by dilution with 4 ml propylene glycol. Ascorbyl palmitate (100 mg) was mixed with 400 μl ethanol then dissolved in 0.5 ml of the cyclosporine oral solution and diluted with 4 ml propylene glycol. Ketoconazole (100 mg) did not completely dissolve in ethanol or the cyclosporine oral solution and formed a white suspension which was diluted with 4 ml propylene glycol. Formulations were evaluated by HPLC for cyclosporine content.

Dosing and Sampling

Formulations were administered by gavage to 3 separate groups of 6 male Sprague-Dawley rats. Inhibitor and cyclosporine doses were 30 mg/kg and 15 mg/kg respectively. Clearly, the 2:1 ratio of inhibitor to drug is within the most preferred range. Serial blood samples (0.5 ml) were drawn prior to the dose and at 0.5, 1, 2, 3, 4, and 6 hours post-dose (encompassing the cyclosporine absorption phase) through a cannula inserted into the jugular vein. Blood samples were stored at 4°C in micro-centrifuge tubes containing EDTA as anticoagulant. No hemolysis or coagulation of blood samples was observed over the course of the study. No adverse effects were reported for any of the doses.

Analysis

Whole blood samples (0.4 ml) were analyzed for cyclosporine using a validated HPLC method with UV detection. The cyclosporine concentration at 1 hr (Ci_hr), the maximum cyclosporine concentration (C_max) and the time to achieve this concentration (T_max) were measured by observation of the concentration vs. time curve. The area under the concentration vs. time curve from 0 to 6 hr (AUC_0-6) was calculated using the linear trapezoidal method. Statistical analysis of normally-distributed data utilized one-way ANOVA with the Dunnett and Tukey post-hoc comparisons. Tm_ax data were compared by ANOVA based on ranks.

The results from the bioavailability experiments in rats is presented in Table 3 and in the Figure.

TABLE 3 Pharmacokinetic parameters for cyclosporine (15 mg/kg) administered by gavage to male Sprague-Dawley rats alone and with ascorbyl palmitate (AscP, 30 mg/kg) or ketoconazole (KC, 30 mg/kg). Data are mean (Standard Deviation; Coefficient of Variation %) except for T_ma which are median (range).

Dose (number of rats)

Parameter cyclosporine alone (6) cyclosporine with cyclosporine with ascorbyl palmitate (6) ketoconazole (5)

Cihr (ng/ml) 904 (190; 21) 1551 (298; 19)* 1291 (253; 20)*

% of control 100 172 143

C_max (ng/ml) 1123 (158; 14) 1811 (364; 20)* 1707 (174; 10)*

% of control 100 161 152

AUC_0-6 5457 (669; 12) 8521 (1609; 19)* 8300 (1093; 13)*

(ng-hr/ml)

% of control 100 156 152 max (hr) 3 (2-6) 2 (1-6) 3 (2-4)

Statistically different from cyclosporine alone (P<0.05). The pharmacokinetic data are presented in Table 3. The Figure presents the mean cyclosporine concentration vs. time profile for cyclosporine (15 mg/kg) administered to male Sprague Dawley rats alone and with ascorbyl palmitate (30 mg/kg) or ketoconazole (30 mg/kg). Ascorbyl palmitate and the known CYP3A-inhibitor ketoconazole both caused statistically significant increases in C_ln,-, C_max and AUC_0-6 however T_max was unaffected.

As evidenced by this experiment, ascorbyl palmitate, an exemplary vitamin C fatty acid ester, increased the bioavailability of cyclosporine in an in vivo model. Thus, this experiment demonstrates that a vitamin C fatty acid ester can be used to increased the bioavailability of a pharmaceutical compound.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

WE CLAIM:

1. A method for increasing bioavailability of an orally administered pharmaceutical compound, the method comprising: orally coadministering (1) the pharmaceutical compound to a mammal in need of treatment with the compound and (2) a vitamin C fatty acid ester in an amount sufficient to provide bioavailability of the compound in the presence of the vitamin C fatty acid ester greater than bioavailability of the compound in the absence of the vitamin C fatty acid ester.

2. The method of claim 1, wherein the vitamin C fatty acid ester has the formula

wherein R is a straight chain or branched chain, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, benzyl, phenyl, alicyclic or heterocyclic group.

3. The method of claim 2, wherein R is selected from a cis-9-hexadecenoate group, a trans-9-hexadecanoate group, a cis-9-octadecenoate group, a trans-9- octadecenoate group, a cis,cis-9,12-octadecadienoate group, a trans,trans-9, 12- octadecadienoate group, a cis,cis,cis-9, 12, 15-octadecatrienyl group, a trans,trans,trans-9, 12, 15-octadecatrienoate group, a cis,cis,cis-6, or a 9, 12- octadecatrienoate group.

4. The method of claim 1 , wherein the vitamin C fatty acid ester is ascorbyl palmitate.

5. The method of claim 1, wherein the vitamin C fatty acid ester is administered in a range of 0.01 to 100 units vitamin C fatty acid ester per 1 unit of the pharmaceutical compound.

6. The method of claim 5, wherein the vitamin C fatty acid ester is administered in a range of 0.1 to 10 units vitamin C fatty acid ester per 1 unit of the pharmaceutical compound.

7. The method of claim 6, wherein the vitamin C fatty acid ester is administered in a range of 0.5 to 2 units vitamin C fatty acid ester per 1 unit of the pharmaceutical compound.

8. The method of claim 1, wherein the pharmaceutical compound is hydrophobic.

9. The method of claim 1, wherein the amount is sufficient to produce a concentration of the vitamin C fatty acid ester in the lumen of the gut of the mammal of at least 0.1 times a K, or apparent K, of CYP3A inhibition of the compound.

10. The method of claim 1, wherein bioavailability of the compound in the presence of the vitamin C fatty acid ester is greater than bioavailability of the compound in the absence of the vitamin C fatty acid ester by at least 10% of the difference between bioavailability in the absence of the vitamin C fatty acid ester and complete oral bioavailability.

11. The method of claim 10, wherein bioavailability of the compound in the presence of the vitamin C fatty acid ester is greater than bioavailability of the compound in the absence of the vitamin C fatty acid ester by at least 50% of the difference between bioavailability in the absence of the vitamin C fatty acid ester and complete oral bioavailability.

12. The method of claim 11 , wherein bioavailability of the compound in the presence of the vitamin C fatty acid ester is greater than bioavailability of the compound in the absence of the vitamin C fatty acid ester by at least 75% of the difference between bioavailability in the absence of the vitamin C fatty acid ester and complete oral bioavailability.

13. The method of claim 1, wherein the vitamin C fatty acid ester shows an inhibition of at least 20% when the vitamin C fatty acid ester and the compound are present in a 1 :1 vitamin C fatty acid esterxompound ratio.

14. The method of claim 1, wherein the pharmaceutical compound comprises an acetanilide, aminoacridine, aminoquinoline, anilide, anthracycline antibiotic, antiestrogen, benzazepine, benzhydryl compound, benzodiazapine, benzofuran, cannabinoid, cephalosporine, colchicine, cyclic peptide, dibenzazepine, digitalis glycoside, dihydropyridine, epiphodophyllotoxin, ergeline, ergot alkaloid, imidazole, isoquinoline, macrolide, naphthalene, nitrogen mustard, opioid, oxazine, oxazole, phenothiazine, phenylalkylamine, phenylpiperidine, piperazine, piperidine, polycyclic aromatic hydrocarbon, pyridine, pyridone, pyrimidine, pyrrolidine, pyrrolidinone, quinazoline, quinoline, quinone, rauwolfia alkaloid, retinoid, salicylate, steroid, stilbene, sulfone, sulfonylurea, taxol, triazole, tropane, or vinca alkaloid.

15. The method of claim 1, wherein the vitamin C fatty acid ester is present as a counter ion of the pharmaceutical compound.

16. The method of claim 1 wherein the vitamin C fatty acid ester is covalently bound to the pharmaceutical compound.

17. A method of formulating an oral pharmaceutical composition, which comprises: admixing a pharmaceutical compound, a pharmaceutical carrier, and vitamin C fatty acid ester, the vitamin C fatty acid ester being present in sufficient amount to provide bioavailability of the pharmaceutical compound in the presence of the vitamin C fatty acid ester greater than the bioavailability of the pharmaceutical compound in the absence of the vitamin C fatty acid ester when the pharmaceutical composition is administered orally to an mammal.

18. The method of claim 17, wherein the vitamin C fatty acid ester has the formula:

wherein R is a straight chain or branched chain, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, benzyl, phenyl, alicyclic or heterocyclic group.

19. The method of claim 18, wherein R is selected from a cis-9- hexadecenoate group, a trans-9-hexadecanoate group, a cis-9-octadecenoate group, a trans-9-octadecenoate group, a cis,cis-9,12-octadecadienoate group, a trans,trans-9, 12-octadecadienoate group, a cis,cis,cis-9, 12, 15-octadecatrienyl group, a trans,trans,trans-9, 12, 15-octadecatrienoate group, a cis,cis,cis-6, or a 9, 12-octadecatrienoate group.

20. The method of claim 17, wherein the vitamin C fatty acid ester is ascorbyl palmitate.

21. The method of claim 17, wherein the vitamin C fatty acid ester is present in an amount sufficient to produce a concentration of the vitamin C fatty acid ester in the lumen of the gut of the mammal of at least 0.1 times a Kj or apparent Kj of CYP3A inhibition of the compound.

22. The method of claim 17, wherein the vitamin C fatty acid ester is present in an amount sufficient to provide greater than 0.5% by weight vitamin C fatty acid ester relative to the total weight of the pharmaceutical composition.

23. The method of claim 17, wherein the vitamin C fatty acid ester is present as a counter ion of the pharmaceutical compound.

24. The method of claim 17, wherein the vitamin C fatty acid ester is covalently bound to the pharmaceutical compound.

25. The method of claim 17, wherein the pharmaceutical compound comprises an acetanilide, aminoacridine, aminoquinoline, anilide, anthracycline antibiotic, antiestrogen, benzazepine, benzhydryl compound, benzodiazapine, benzofuran, cannabinoid, cephalosporine, colchicine, cyclic peptide, dibenzazepine, digitalis glycoside, dihydropyridine, epiphodophyllotoxin, ergeline, ergot alkaloid, imidazole, isoquinoline, macrolide, naphthalene, nitrogen mustard, opioid, oxazine, oxazole, phenothiazine, phenylalkylamine, phenylpiperidine, piperazine, piperidine, polycyclic aromatic hydrocarbon, pyridine, pyridone, pyrimidine, pyrrolidine, pyrrolidinone, quinazoline, quinoline, quinone, rauwolfia alkaloid, retinoid, salicylate, steroid, stilbene, sulfone, sulfonylurea, taxol, triazole, tropane, or vinca alkaloid.

26. A pharmaceutical composition produced by the process of claim 17.

27. The composition of claim 26, wherein the vitamin C fatty acid ester is present in an amount sufficient to provide greater than 0.5% by weight vitamin C fatty acid ester relative to the total weight of the pharmaceutical composition.

28. A method of increasing bioavailability of the active compound of an existing oral pharmaceutical composition, which comprises: reformulating the existing composition to provide a reformulated composition by admixing the active compound with vitamin C fatty acid ester, the vitamin C fatty acid ester being present in sufficient amount to provide bioavailability of the active compound when administered in the reformulated composition greater than said bioavailability of the active compound when administered in the existing pharmaceutical composition.

29. The method of claim 28, wherein the vitamin C fatty acid ester has the formula:

wherein R is a straight chain or branched chain, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, benzyl, phenyl, alicyclic or heterocyclic group.

30. The method of claim 29, wherein R is selected from a cis-9-hexadecenoate group, a trans-9-hexadecanoate group, a cis-9-octadecenoate group, a trans-9- octadecenoate group, a cis,cis-9,12-octadecadienoate group, a trans,trans-9, 12- octadecadienoate group, a cis,cis,cis-9, 12, 15-octadecatrienyl group, a trans,trans,trans-9, 12, 15-octadecatrienoate group, a cis,cis,cis-6, or a 9, 12- octadecatrienoate group.

31. The method of claim 28, wherein the vitamin C fatty acid ester is ascorbyl palmitate.

32. The method of claim 28, wherein the reformulated composition comprises all components present in the existing pharmaceutical composition plus the vitamin C fatty acid ester.

33. The method of claim 28, wherein the reformulated composition contains less than all components present in the existing pharmaceutical composition plus the vitamin C fatty acid ester.

34. A reformulated pharmaceutical composition produced by the process of claim 28.