Use of gallic acid esters to increase bioavailability of orally administered pharmaceutical compounds

A method is disclosed for increasing bioavailability of an orally administered pharmaceutical compound comprising orally coadministering the pharmaceutical compound to a mammal in need of treatment with the compound and a gallic acid ester. Preferred gallic acid esters of the invention include octyl gallate, propyl gallate, lauryl gallate, and methyl gallate. Improved formulations of pharmaceutical compounds include the gallic acid ester to enhance the bioavailability of the active ingredient of the pharmaceutical compound.

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:
EQU F.sub.oral =F.sub.ABS.times.F.sub.G.times.F.sub.H
 where F.sub.oral is the oral bioavailability fraction, which is the
 fraction of the oral dose that reaches the circulation in an active,
 unchanged form. F.sub.oral 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.sub.ABS), the fraction of the absorbed dose that
 successfully reaches the blood side of the gastrointestinal tract
 (F.sub.G), and the fraction of the drug in the GI blood supply that
 reaches the heart side of the liver (F.sub.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 arising in the laboratory of one of the present
 inventors resulted in new understandings of factors involved with
 bioavailability and in the invention described in U.S. Pat. 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. For example, the use of essential
 oils to enhance bioavailability of an orally administered pharmaceutical
 composition is disclosed in U.S. Pat. No. 5,665,386.
 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 3A 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 co-administering a gallic acid ester with
 an oral pharmaceutical compound (drug) or compounds to increase drug
 bioavailability. Particularly preferred esters are octyl gallate, propyl
 gallate, lauryl gallate, and methyl gallate. 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.
 DESCRIPTION OF SPECIFIC EMBODIMENTS
 Gallic Acid Esters Increase Drug Bioavailability
 The present invention arises from continued research into the factors
 affecting drug bioavailability that were described in earlier applications
 arising from the laboratory of one of the present inventors. "Drug
 bioavailability" is defined here as the total amount of drug systemically
 available over time. The present invention provides a method for
 increasing drug bioavailability by inhibiting drug biotransformation in
 the gut using one or more of the compounds described herein. The
 compound(s) responsible for increased drug bioavailability is a gallic
 acid ester. The present inventors have discovered that gallic acid esters,
 in general, are capable of inhibiting the enzyme(s) responsible for drug
 biotransformation in the gut.
 In general, the present invention provides a method for increasing the
 bioavailability of an orally administered pharmaceutical compound
 (particularly one which is hydrophobic) by orally co-administering the
 pharmaceutical compound to a mammal in need of treatment with an amount of
 a gallic acid ester sufficient to provide integrated systemic
 concentrations over time of the pharmaceutical compound greater than the
 integrated systemic concentrations over time of the pharmaceutical
 compound in the absence of the gallic acid ester. At least one gallic acid
 ester is utilized in the method of the invention to increase
 bioavailability. However, two or more gallic acid esters may be used
 simultaneously in the practice of the invention, leading to further
 increased bioavailability of the pharmaceutical compound, depending on the
 particulars of the compound. 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.
 Gallic acid esters
 Gallic acid esters useful in the present invention have the general formula
 shown below:
 ##STR1##
 The R group may be an alkyl, alkenyl, alkynyl, aryl, benzyl, phenyl,
 alicyclic, or heterocyclic group, all of which groups may be substituted
 or unsubstituted. R is preferably a C.sub.1 -C.sub.22 alkyl group, a
 C.sub.2 -C.sub.22 alkenyl group or a C.sub.2 -C.sub.22 alkynyl group, all
 of which groups may be substituted or unsubstituted and may be straight
 chain or branched chain. R is more preferably a C.sub.1 -C.sub.12 alkyl
 group, particularly a methyl, propyl, octyl or dodecyl (lauryl) group, or
 a C.sub.2 -C.sub.19 alkenyl group, particularly a cis-9-hexadecenyl
 (palmitoleyl), cis-9-octadecenyl (oleyl), cis,cis-9,12-octadecadienyl
 (linoleyl), trans,trans-9,12-octadecadienyl (linolelaidyl),
 cis,cis,cis-9,12,15-octadecatrienyl (linolenyl),
 trans,transtrans-9,12,15-octadecatrienyl (linolenelaidyl),
 cis,cis,cis-6,9,12-octadecatrienyl (gamma-linolenyl), trans-9-octadecenyl
 (elaidyl) or trans-9-hexadecenyl (palmitelaidyl) group.
 Many of the gallic acid esters used in the practice of the present
 invention are commercially available compounds or may be readily
 synthesized by methods that are well known in the art, for example, by
 refluxing gallic acid and the appropriate alcohol (R-OH) in the presence
 of acid using standard conditions as described in Vogel, A., Vogel's
 Textbook of Organic Chemistry, 4.sup.th Edition, revised by Furniss, B. S.
 et al., Longman Inc., N.Y. (1978).
 In a recently published example, lauryl gallate was prepared in greater
 than 90% yield by refluxing gallic acid and lauryl alcohol in dioxane in
 the presence of p-toluenesulfonic acid and zeolite (Chen, L. and Wu, K.,
 New process of synthesis of lauryl gallate, Huaxue Shiji 19:382 (1997).
 The gallic acid ester is preferably presented for coadministration in a
 ratio of gallic acid ester to drug in the range of 0.01 to 100 units
 gallic acid ester to 1 unit of the drug. For example, a formulation having
 1 mg gallic acid ester per 100 mg drug represents the lower end of this
 range and a formulation having 500 mg gallic acid ester per 5 mg drug
 represents the upper end of this range. A more preferred range of gallic
 acid ester to drug in accordance with the present invention is 0.1 to 10
 units gallic acid ester to 1 unit of the drug. The most preferred range is
 0.5 to 2 units gallic acid ester 1 unit of the drug.
 Properties of Gallic Acid Esters
 The structure of propyl gallate (3,4,5-trihydroxybenzoic acid, n-propyl
 ester) is shown below:
 ##STR2##
 Propyl gallate 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 0-1.4 mg/kg/day for this compound. This value
 is 1/100 of the "no observed effect" level determined in a 90 day feeding
 study in rats ("Gallates: Propyl, Octyl, Dodecyl," WHO Food Additive
 Series, 32:3-23 (1993)).
 Octyl gallate and lauryl gallate have also been used as antioxidants in
 food; however, their current use is limited. The Joint Food and
 Agriculture Organization/World Health Organization Expert Committee on
 Food Additives has established temporary acceptable daily intake levels of
 0-0.1 mg/kg body weight for octyl gallate and 0-0.05 mg/kg body weight for
 lauryl gallate. These values are 1/200 of the "no observed effect level"
 determined in 90 day feeding studies in rats ("Gallates: Propyl, octyl,
 dodecyl." WHO Food Additive Series 32: 3-23 (1993); Forty-first Report of
 the Joint FAO/WHO Expert Committee on Food Additives. Evaluation of
 certain food additives and contaminants. WHO Technical Report Series
 837:6, 46 (1993)). Lauryl gallate and octyl gallate are listed in the
 Everything Added to Food in the US (EAFUS) database, but neither of these
 compounds is listed in the United States Pharmacopeia-National Formulary
 (USP-NF) or the Food Chemical Codex.
 Because these three alkyl gallate 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 a gallic acid ester
 providing an inhibition activity (resulting in increased bioavailability
 of a co-administered drug) are included in the invention. Preferred are
 those formulations of a gallic acid ester that show an inhibition of at
 least 20% at a 1:1 gallic acid ester:drug ratio; even more preferred are
 formulations of a gallic acid ester that show an inhibition of at least
 50% at the same gallic acid ester to drug ratio.
 Bioavailability Measurements
 The increase in drug bioavailability attributable to administration of the
 gallic acid ester can be determined by measuring total systemic drug
 concentrations over time after coadministration of a drug and the gallic
 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 gallic acid ester is being measured, the
 amount and form of active drug administered should be the same in both the
 coadministration of drug and gallic 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 .mu.g.cndot.hr/ml. In coadministration (i.e., in the presence of the
 gallic acid ester) the systemic drug AUC may increase to 700
 .mu.g.cndot.hr/ml. If significantly increased drug bioavailability in the
 presence of the gallic 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 gallic 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 Gallic 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.
 In the method of the present invention, compounds (or drugs) from a number
 of classes of compounds that can be administered with gallic acid esters
 include, for example, the following classes: acetanilides, 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 gallic 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 (Nelson et al.,
 P450 superfamily: update on new sequences, gene mapping accession numbers
 and nomenclature, Pharmacogenetics 6:1-42 (1996)). Finally, the gene for
 each individual form of cytochrome P450 is assigned an Arabic number (e.g.
 CYP3A4).
 Three cytochrome P450 gene families (CYP1, 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 3A subfamily, and these forms are
 responsible for the metabolism of a large number of structurally diverse
 drugs. In non-induced individuals, 3A may constitute 20% of the P450
 enzymes in the liver. In enterocytes, members of the 3A subfamily
 constitute greater than 70% of the cytochrome-containing enzymes. The
 present inventors have discovered that gallic acid esters preferentially
 inhibit the CYP3A form over the enzymes from the CYP1 and CYP2 families.
 The first two human 3A 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 3A3/4. Erythromycin
 N-demethylation, cyclosporine oxidation, nifedipine oxidation, midazolam
 hydroxylation, testosterone 6.beta.-hydroxylation, and cortisol
 6.beta.-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 3A 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 isoforms 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
 The gallic acid ester, as used according to the invention, reduces drug
 biotransformation in the gut by inhibiting CYP3A activity in gut
 epithelial cells which leads to a total increase in drug bioavailability
 in the serum. In the presence of the gallic 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 gallic acid ester is to inhibit CYP3A
 drug biotransformation in the gut, some biotransformation may be decreased
 in other tissues as well if the gallic 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 gallic acid
 ester to the gut, however, is that it allows the use of lower systemic
 concentrations of the gallic acid ester compared to inhibitors that target
 CYP3A in the liver. After oral administration of the gallic 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. Thus, as orally administered gallic acid esters
 preferentially inhibit CYP3A in the gut, they are a particularly effective
 means of increasing drug bioavailability of a co-administered drug.
 Coadministration of the gallic acid ester 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 gallic 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, dealkylase, oxidase, and hydrolase activities. In
 addition to the different catalytic activities of CYP3A, different forms
 of CYP3A 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)).
 The gallic acid esters reduce CYP3A drug biotransformation by acting as
 inhibitors of CYP3A activity. Possible mechanisms include competitive,
 non-competitive, uncompetitive, mixed or irreversible inhibition of CYP3A
 drug biotransformation.
 Selection of Gallic Acid Ester Concentration by Reduction of CYP3A Drug
 Biotransformation
 The ability of the gallic 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). 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 a gallic acid ester to be useful, a
 preferred commercially desirable concentration of a gallic acid ester
 acting as a CYP3A 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 gallic acid ester, then the addition of the gallic acid
 ester may increase bioavailability to 85%, for a 75% increase. A
 sufficient amount of orally administered gallic acid ester will provide
 integrated systemic drug concentrations over time greater than the
 integrated systemic drug concentrations over time in the absence of the
 gallic acid ester. The actual amount or concentration of the gallic 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 gallic 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 gallic acid ester in
 a particular formulation is by direct comparison to the amount of drug,
 with a gallic acid ester:drug 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 3A class of enzymes by gallic acid esters can be
 studied by a variety of bioassays, several of which are set forth below.
 In vitro CYP3A 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 gallic 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 CYP3A metabolites in cells can be measured using
 high pressure liquid chromatograph (HPLC) methods as described in the
 following section for microsome assays of CYP3A activity.
 Microsome Assays of CYP3A Function and Increased Bioavailability
 Microsomes from liver or intestine will 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
 calorimetrically measuring erythromycin demethylase activity as the
 production of formaldehyde as in Wrighton et al., Mol. Pharmacol.,
 28:312-321 (1985) and Nash, Biochem. J., 55:416-421 (1953).
 Characteristics of Gallic Acid Esters for Reducing CYP3A Drug Metabolism
 Gallic acid esters bind CYP3A quickly and inhibit while the drug is passing
 through the enterocyte. After the gallic acid ester reaches the heart and
 is distributed throughout the body the concentration of the gallic acid
 ester will be diluted on future passes through the liver. Concentrations
 of gallic acid ester used in the gut lumen are preferably selected to be
 effective on gut CYP3A metabolism but, due to dilution, to be less active
 in other tissues.
 The amount of the gallic acid ester used for oral administration can be
 selected to achieve small intestine luminal concentrations of at least 0.1
 times the K.sub.i or apparent K.sub.i for CYP3A inhibition of drug
 metabolism or an amount sufficient to increase systemic drug concentration
 levels, whichever is less. Alternatively, the amount of a gallic acid
 ester inhibitor of cytochrome P450 3A 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 50-500
 .mu.g human liver microsomes, 10-100 .mu.M nifedipine, and 1 mm NADPH in
 500 .mu.l of 0.1M sodium phosphate buffer, pH 7.4. In the practice of the
 method of the present invention, the initial amount of gallic acid ester
 is selected to provide concentrations in the lumen of the small intestine
 equal to 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 gallic acid ester 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.
 In all of these cases, the goal in selecting a particular concentration of
 a gallic acid ester 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 gallic acid ester that is greater than the
 integrated systemic concentrations over time of the pharmaceutical
 compound in the absence of the gallic acid ester 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 Gallic Acid Ester Formulations
 In summary, the various techniques described above for screening gallic
 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 CYP3A 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., 26:185-187(1998)).
 Coadministration and Delivery of the Gallic Acid Ester
 Coadministration of the Gallic Acid Ester and a Drug
 The present invention will increase the bioavailability of a drug in
 systemic fluids or tissues by co-administering the gallic acid ester with
 a drug. "Co-administration" includes concurrent administration
 (administration of the gallic acid ester and drug at the same time) and
 time-varied administration (administration of the gallic acid ester at a
 time different from that of the drug), as long as both the gallic 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 gallic 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 gallic acid ester can be separately
 formulated with different coatings possessing different time constants for
 release of the gallic acid ester and the drug. The gallic acid ester can
 also be bound to the drug being protected, either by covalent bonding or
 by ionic or polar attractions.
 The gallic acid ester also increases 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 3A enzymes are present in the skin, and a gallic acid
 ester 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 3A enzymes by a gallic acid
 ester in epithelia other than the gut provides the same mechanism of
 action.
 Formulations Having a Gallic Acid Ester
 The invention is carried out in part by formulating an oral pharmaceutical
 composition to contain at least one gallic acid ester. This is
 accomplished in some embodiments by admixing a pharmaceutical compound,
 usually a pharmaceutical carrier, and the gallic acid ester, the gallic
 acid ester being present in an amount sufficient to provide integrated
 systemic concentrations over time of the pharmaceutical compound (as
 measured by AUCs greater than the integrated systemic concentrations over
 time of the pharmaceutical compound in the absence of the gallic acid
 ester) when the pharmaceutical composition is administered orally to an
 animal being treated. Additionally, more than one gallic acid ester may be
 used in the formulation. 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 the gallic acid ester, the gallic 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
 gallic 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 gallic 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 the gallic acid ester. Optimal
 gallic acid ester concentrations can be determined by varying the amount
 and timing of gallic acid ester administration and monitoring
 bioavailability. Once the optimal gallic acid ester concentration or
 gallic acid ester to drug ratio is established for a particular drug, the
 formulation (gallic 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 preferred to
 establish the optimal gallic acid ester concentration using such
 formulations from the start of the bioavailability experiments.
 Several gallic 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 antioxidants, gallic acid esters are 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 at least 1% by weight
 gallic 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%. For example, propyl gallate, when used as an
 antioxidant, is used in an amount that is less than 0.1% of the materials
 being protected or preserved. Other gallic acid esters, for example octyl
 gallate or lauryl gallate, are used as antioxidants at equivalent or lower
 levels. 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 gallic 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 example, which is
 offered for illustration only and is not to be considered limiting of the
 invention unless otherwise specified.

EXAMPLES
 Example 1
 Inhibition of Drug Degradation by Gallic Acid Esters
 The known CYP3A substrate nifedipine (Gonzalez, F. J., et al., Human
 P45OPCN1: sequence, chromosome localization, and direct evidence through
 cDNA expression that P45OPCN1 is nifedipine oxidase, DNA, 2:79-86 (1988))
 was used as a test substrate for evaluating the potential of various
 gallic acid esters to inhibit CYP3A metabolism in a human liver microsome
 study.
 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 1 mM EDTA and 20 mM BHT. 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.
 777-814 (1989)) and were stored at -80.degree. 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.
 In the experiments, 100 .mu.M nifedipine and either 5 .mu.l of a solution
 of one of the gallic acid esters (at a concentration indicated in Table 1)
 or 5 .mu.l of solvent alone (control) were preincubated along with 0.1
 mg/ml of the human liver microsomal proteins and 1 mM
 diethylenetriarninepentaacetic acid (DETA) in 100 mM phosphate buffer,
 pH 7.4, for 5 minutes at 37.degree.0 C. Metabolic reactions were started
 by addition of reduced .beta.-nicotinamide adenine dinucleotide phosphate
 (NADPH) to give a final concentration of 1 mM and a final volume of 0.5
 ml. Metabolic reactions were stopped after 3 minutes by vortex mixing with
 0.2 ml of an extraction solvent of (94:6) acetonitrile:glacial acetic
 acid. Protein was precipitated by centrifugation (3000 rpm.times.10
 minutes) and supernatants were analyzed for nifedipine and its oxidation
 product 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid
 dimethyl ester by High Performance Liquid Chromatography (HPLC).
 All experiments were conducted in triplicate and compared to incubations
 carried out without NADPH or without substrate. The data are the
 mean.+-.standard deviation of three measurements.
 The results, shown in Table 1, indicate that nifedipine oxidation rates in
 the presence of all gallic acid esters tested at the indicated
 concentrations were significantly different from the control (P&lt;0.05)
 using ANOVA with Dunnett's post hoc comparison.
 TABLE 1
 Inhibition of CYP3A-Mediated Metabolism in
 Human Liver Microsomes by Gallic Acid Esters
 Relative
 Nifedipine
 Conc. Oxidation
 Inhibitor.sup.a (.mu.M) Rate (.+-. S.D.) Comment
 Control -- 100 (3)
 Methyl gallate 100 53 (2)
 500 16.0 (0.1)
 Propyl gallate 50 59 (2) Non-competitive inhibitor of
 100 34 (1) nifedipine metabolism K.sub.i =
 500 6.1 (0.1) 64 .+-. 2 .mu.M (mean .+-. S.E. of
 estimate; r.sup.2 = 0.999).sup.b
 Octyl gallate 10 44 (2) Non-competitive inhibitor of
 25 17.5 (0.5) nifedipine metabolism K.sub.i =
 50 4.9 (0.6) 5.2 .+-. 0.2 .mu.M (mean .+-. S.E. of
 100 1.1 (0.3) estimate; r.sup.2 = 0.996).sup.b
 Lauryl gallate 10 61 (1)
 (Dodecyl gallate) 25 31 (3)
 50 12.6 (0.1)
 100 6.4 (0.5)
 .sup.a Substrate and inhibitor were dissolved in acetonitrile except for
 lauryl gallate which required methanol as vehicle.
 .sup.b Inhibition constant K.sub.i determinations utilized 10, 20, 50 and
 100 .mu.M nifedipine substrate concentrations and experiments were run in
 duplicate. K.sub.i values were determined by regression analysis of rate
 data using SigmaPlot V4.OS software (SPSS Inc., San Rafael, California).
 The various gallic acid esters, at all tested concentrations, served as
 effective inhibitors of CYP3A-mediated metabolism. Octyl gallate proved to
 be an especially good inhibitor of the metabolism at relatively low
 concentrations. The usefulness of gallic acid esters to increase
 bioavailability of pharmaceutical compounds given to patients by
 coadministration of the gallic acid ester with the pharmaceutical compound
 is thus self-evident.
 Example 2
 Inhibition of Drug Degradation by Propyl Gallate
 The ability of propyl gallate at various concentrations to inhibit
 metabolism for three representative drugs through inhibition of the
 cytochrome P450 mechanism was tested. Human liver microsomes were prepared
 and each of three drugs, amiodarone, buspirone, or nifedipine, were
 incubated with the microsomes in the presence of propyl gallate or a known
 inhibitor of CYP3A metabolism. Metabolism in the presence of propyl
 gallate or known CYP3A inhibitor was compared to a control treated only
 with the solvent in which the inhibitor was dissolved.
 Inhibition of metabolism of the known CYP3A substrates amiodarone (Fabre,
 G., et al., Evidence for CYP3A-mediated N-deethylation of amiodarone in
 human liver microsomal fractions, Drug Metab. Dispos., 21:978-985 (1993),
 Triver, J. M., et al., Amiodarone N-deethylation in human liver
 microsomes: involvement of cytochrome P450 3A enzymes (first report), Life
 Sci., 52:PL91-96 (1993)), nifedipine (Gonzalez, F. J., et al., Human
 P45OPCN1: sequence, chromosome localization, and direct evidence through
 cDNA expression that P45OPCN1 is nifedipine oxidase, DNA, 7:79-86 (1988)),
 and buspirone (Kivisto K. T. et al. Plasma buspirone concentrations are
 greatly increased by erythromycin and itraconazole. Clin. Pharmacol. Ther.
 62: 348-54 (1997); Lilja J. J. Grapefruitjuice substantially increases
 plasma concentrations of buspirone. Clin. Pharmacol. Ther. 64: 655-60
 (1998)) by human liver microsomes was tested. The microsomes were prepared
 as in Example 1.
 The amiodarone was present in a concentration of 100 .mu.M, the buspirone
 was in a concentration of 25 .mu.M, and the nifedipine was present in a
 concentration of 25 .mu.M. The propyl gallate was tested with each of
 these drugs at concentrations of 25, 50, and 100 .mu.M. Other inhibitors
 of CYP3A metabolism were utilized at known inhibition concentrations, i.e.
 ketoconazole at 1 .mu.M, cyclosporine at 25 .mu.M, and diltiazem,
 erythromycin, and verapamil at 100 .mu.M.
 The drug and optionally the inhibitor were preincubated with the microsomes
 at 1 nmol CYP/ml and 1 mM DETA in 100 mM phosphate buffer, pH 7.4 for 5
 minutes at 37.degree. C. After the preincubation, metabolic reactions were
 started by the addition of 1 mM NADPH. Samples were taken at 1, 2, and 3
 minutes after the start of the reaction and analyzed by HPLC.
 Disappearance of substrate and/or formation of metabolite were quantitated
 by comparison to standard curves.
 The results are presented in Table 2. The metabolism rates (nmol/ml/min)
 are the mean.+-.standard deviation of three measurements. Also shown in
 Table 2 are the metabolism rates expressed as a percentage of the control
 for each drug. These numbers are presented in parentheses.
 TABLE 2
 Inhibition of CYP3A-Mediated Metabolism in
 Human Liver Microsomes by Propyl Gallate
 Mean .+-. SD Metabolism rate (% control)
 Inhibitor .mu.M Amiodarone.sup.a Buspirone.sup.b Nifedipine.sup.c
 Control 1.92 .+-. 0.08 (100) 5.37 .+-. 0.56 (100) 4.36 .+-.
 0.17 (100)
 Propyl Gallate 25 0.94 .+-. 0.02 (49) 3.49 .+-. 0.49 (65) 3.57 .+-.
 0.29 (82)
 50 0.55 .+-. 0.02 (28) 2.25 .+-. 0.25 (42) 2.35 .+-.
 0.10 (54)
 100 0.32 .+-. 0.03 (17) 1.55 .+-. 0.23 (29) 1.43 .+-.
 0.04 (33)
 Ketoconazole 1 0.79 .+-. 0.004 (4) 1.47 .+-. 0.39 (28) 0.48 .+-. 0.06
 (11)
 Cyclosporine 25 0.32 .+-. 0.03 (17) 2.21 .+-. 0.38 (41) 1.05 .+-.
 0.02 (24)
 Diltiazem 100 1.06 .+-. 0.02 (55) 2.80 .+-. 0.18 (52) 3.74 .+-.
 0.16 (86)
 Erythromycin 100 0.84 .+-. 0.07 (44) 3.59 .+-. 0.46 (67) 2.67 .+-.
 0.11 (61)
 Verapamil 100 0.81 .+-. 0.04 (42) 2.34 .+-. 0.46 (44) 3.00 .+-.
 0.05 (69)
 .sup.a Formation rate of N-desethylamiodarone metabolite (nmol/ml/min)
 .sup.b Buspirone disappearance (nmol/ml/min)
 .sup.c Formation of nifedipine oxidation product
 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid, dimethyl
 ester (nmol/ml/min)
 As evidenced above, propyl gallate at all tested concentrations and against
 each drug, served as an effective inhibitor of CYP3A-mediated metabolism.
 Greater inhibition of the metabolism occurred with increasing
 concentrations of propyl gallate. Propyl gallate also compared favorably
 with the known CYP3A inhibitors tested. Specifically, propyl gallate was
 found to be better at inhibiting drug metabolism than the established
 CYP3A inhibitors diltiazem, erythromycin, and verapamil. This demonstrates
 the utility of propyl gallate to increase bioavailability of compounds by
 coadministration of propyl gallate with 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.