CA2177175A1 - Method for increasing bioavailability of oral pharmaceutical compositions - Google Patents

Abstract

A method for increasing bioavailability of an orally administered hydrophobic pharmaceutical compound, which comprises orally administering the pharmaceutical compound to a mammal in need of treatment with the compound concurrently with a bioenhancer comprising an inhibitor of a cytochrome P450 3A enzyme or an inhibitor of P-glycoprotein-mediated membrane transport, the bioenhancer being present in sufficient amount to provide bioavailability of the compound in the presence of the bioenhancer greater than the bioavailability of the compound in the absence of the bioenhancer.

Description

2177~75 1.
METHOD FOR INCREASING BIOAVAILABILITY
OF ORAL PHARMACEUTICAL COMPOSITIONS

This invention was made with Government support under Grant No. GM
5 26691, awarded by the National ~n~titl~tes of Health. The Govcllllllenl has certain rights in this invention.

INTRODUCTION
Teçhnir~l Field This invention is directed to the field of l,h~.. ~rology and particularly to the form~ tion of ph~ e~ltif~l compositions for increased bioavailability.

Back~round Bioavailability pl.~ rokinPtirs is the study of the fate of ~h~ .açelltir~l~ from the time they are ingested until they are eli---i-~ from the body. The sequence of eventsfor an oral co,~o~ilion includes absol~,lion 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 elimin~tion of drug or metabolites 20 in urine or bile.
Bioavailability of a drug (ph~rm~re~tir~l composition) following oral dosing is a critical ph~ rokinetic det~....in~ which can be approxim~t~d by the following formula:
FOr"~ = FABS X FG X FH
Fora~ is oral bioavailability fraction, which is the fraction of the oral dose that reaches the circulation in an active, l~nrh~yed form. Fora~ is less than 100%
of the active h~lediellL in the oral dose for three reasons: drug is not absorbed through the GI tract and is elimin~ted in the feces; drug is biotransformed by the cells of the inttostin~ (to an inactive metabolite); or drug is elimin~ted 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 wossl2o98o 2 1 7 7 1 75 PCT/US~ 3q7 (FABS). the fraction of the absorbed dose that ~ucces!~rully reaches the blood side of the ga~Lloi~lesl;~-~l tract (FG), and the fraction of the drug in the GI blood supply that reaches the heart side of the liver (FH)- Previous drug formulationshave al~t~ d to increase drug efficacy by illcl.,a~ing drug absorption. For 5 example, methods have been used to illclease drug absorption using liposomes as carriers and designing more lipophilic drugs. These methods can increase drug absorption; however, they fail to address other ways of increasing drug bioavailability .
Liver Biotransformation and Biliary Secretion The liver affects drug bioavailability. All blood from the ga~lloil~
tract passes through the liver before going els~wh.,.e in the body in all m~mm~l.c, inrl~ ing hllm~nc. Due to its location, liver transformation of orally dosed drugs has a substantial "first-pass effect" on drug bioavailability that was thought to exceed effects in the gut, as liccl)ssed by Yun K. Tam in "Individual Variation in 15 First-Pass Metabolism," Clin. Pll~"-.~okinetics 25:300-328 (1993):
Enzyme activity in the small i-ll~li,-P is lower than in the liver. In humans, the liver to i..l~ i--P
~;ylochrollle P450 ratio has been reported as ~20, sugge~ g that the contribution of illlr~ l phase I
biollal~rollllation to the overall metabolism of a drug is unlikely to be illll)ol~ll~. (op. cit. 303) F.limin~tion of active drug by the liver occurs by one or both of two general pdtllways, namely biollallsr(~lllldlion of the drug and excretion of the drug into the bile. Biotransformation reactions have been cl~ified into two broadly defined phases. Phase I bio~ rolmation often utilizes reactions catalyzed by the cytochrome P450 enzymes, which are manifold and active in the liver and transform many ch~mir~lly diverse drugs. A second biotransformation phase can add a hydrophilic group, such as glutathione, glucuronic acid or sulfate, to increase water solubility and speed elimin~tion through the kidneys.
Hepatocytes have contact with many types of blood and other fluid-transport vessels, such as the portal vein (nutrient and drug-rich blood from the gut), the hepatic arteries (oxygenated blood direct from the heart), the hepatic veins (efflux), ~vo 95/20980 PCT/U~q~S~C317 _ Iymphatics (lipids and Iymphocytes), and bile ducts. The biliary ducts converge into the gall bladder and common bile duct that excretes bile into the upper ~ r~ P, aiding digestion. Bile also contains a variety of excretory products including hydrophobic drugs and drug metabolites.
Traditional solubility rate limhing approaches to increasing drug efficacy have focused on increasing solubility and membrane perrnP~bility. Where metabolism-based approaches have been considered, they have focused on biotransformation in liver. Although methods exist that affect biollallsrolll~ation in the liver, these mPth~lc are inadequate because they affect general liver 10 metabolism and can produce broad non-specific ~y~l~lllic effects.
Cytochromes Most biollallsrollllation is ~,clrolllled by el,,.yllles called "mixed function oxidases" co"l;.il,i,.~ cytochromes, molecules with iron-co"l; inil~g rings, that heip reduce oxygen to water. The ~;yloCh-c llle-cont~ining el,,yllRs that transform drugs 15 use radical oxygen. When oxygen is reduced to its reactive radical form, it reacts immPrli~tely with the drug at the oxygen reduction site.
Most l~,sealch on cytochromes involved in drug biollallsrollllalion focuses on inter-individual dirr~rel~ces in cytochrome activity beca~lse such dirrel~llces appear to be the dolllh~ll IllPch~ lll for dirr~lcl~ces in elimin~tion of 20 ph~ ir~l~ ~lween individuals. Large inter-individual dirr~,.e.l~es observed in the effects of drugs are at least in part ~elel-ll;llp~l by the variation of the eAl,lession and catalytic activity of the cytochl~,llles P450.
The sources of the inter-individual variation in the catalytic activity of the cytoch~ollles P450 can be divided into four general cdl~golies. The first is the25 inflmPn~e of genetics on the e~ression of the cytochromes P450. Si~llirlcalll inter-individual variability can occur for each of the cytochrollles P450. Genetic polymorphisms have been well characterized for the two cytochromes P450 responsible for debrisoquine/~alleille metabolism (CYP2D6; cytochrome families are defined below) and (S)-mephenytoin 4'-hydroxylation (possibly CYP2C19).
30 The second source of inter-individual differences is that several of the human cytochromes P450 are inducible. That is, the content as well as the catalytic wo gs/20980 2 1 7 7 1 7 5 PCTIUS95/00347 activity of these cytochrollles P450 is i~ cased by exposure of an individual toparticular classes of drugs, endogenous compounds, and environmental agents.
Thirdly, the activity of the cytochromes P450 can be inhibited or the cytochromes P450 can be inactivated by drugs and environmental compounds. This includes 5 competitive inhibition bclweell substrates of the same cytochrome P450, inhibition by agents that bind sites on the cytochrome P450 other than the active site, andsuicidal inactivation of the cytochrome P450 by reactive intP~.~nPdi~tP formed during the metabolism of an agent. Another source of inter-individual dirrelcnces is host factors. These factors include disease states, diet, and hormonal influences.
10 Inter-individual dirrercllces in the level of e~lcssion and catalytic activity of the various cylochlollRs P450 can result in an altered response to a drug (individuals can be hypo- or hyper-responsive), a toxic Ic~ol~.e to nnllsll~l levels of a drug or metabolite, and individual sensitivity to chemical carcinogens.
Multiple Drug Resistance Cancer cells that become resistant to one chemolllcl~eutic drug often become resistant to an entire group of chemothcla~culic drugs. This phenomenon is usually called multiple drug ~~ e (MDR).
Many patients on chemc ~l,.a~y initially have striking remissions but later relapse and die from cancer that exhibits recict~n~-e to a wide array of structurally 20 unrelated alllille~lastic agents. The MDR phPn-....l non in~ de cross-l~ re among the all~acyclines, the epipodophyllotoxins, the vinca aLkaloids, taxol, and other colll~oullds. A number of drugs are able to reverse MDR, including calciumchannel blockers, phello~ 7inPs~ qnini~linP~ ~ntim~l~rial agents, antiesllogel~ic and other steroids, and cyclosporine. Liposome therapy also lc~ Ses MDR, with or 25 without a drug on board.
In vitro studies in the past indicate that this form of resi~t~nre is associatedwith amplification or over-e~l~,;,sion of the mdr-l gene in tumors. The m~lr-l gene codes for the expression of a cell surface protein, P-glycoprvleill (P-gp), a tr~n~mPn hrane protein which acts as an energy-dependent efflux pump that 30 transports drugs associated with MDR out of the tumor cell before cytotoxic effects _ 5.
occur. ATP hydrolysis on the cytoplasmic face of P-gp is required for export of hydrophobic compounds from a tumor cell.
Normal mdr-l c~ ession occurs in secretory epithelial cells of the liver, ~anc,cas, small illlr,~ P, colon, and kidney; endothelial capillary cells of the brain 5 and testis; pl~r,çnt~l trophoblasts; and the adrenal gland. In the liver, P-gp is localized on the biliary domain of the hepatocyte membrane. In the small intestine and colon, P-gp is present on the luminal side of epithelial cells. P-gp transports dietary toxins back into the lumen and lh~lc~le helps prevent the toxins from being absorbed into the portal circulation.
Clinical studies have also previously shown that ph~rm~rel-tir~lc that are effective in eli---;--~ MDR of tumor cells in vitro (appdlclllly by inhibition of P-gp) restore chemotherapeutic cytotoxicity in vivo. Studies with small numbers of patients suggest that the addition of 'v~lap~llil, ~lilti~7Pm, quinine, trifluoperazine, or cyclosporine to chemotherapeutic regimPn.c has the potential to reverse MDR.
Absorption By The Gut Absorption across epithelia, in particular intPstin~l epithelia, also affects drug bioavailability. The illlr~ lumen plesclll~ a convoluted surface that h~lcases the surface area of the i.llr~l;nr to facilitate absorption of both nutrients and drugs. The membrane of the enterocyte contains many transport ~ Lcins that actively carry lluLIiclltS from the lumen of the gut into the interior of the enterocytes. Many molecules, inrlntling many drugs, passively diffuse or are actively lldlLspolLed through the melll~ldlle and into the cytoplasm. Most nutrients and drugs pass through the enterocyte and eventually diffuse into the capillary net on route to the portal circulation system and the liver.
The i~lc~l;nP can also pump drugs out of the int~stinP and back into the lumen. The ability of the i..lP~Iin~ to pump drugs out of the tissue has been thought to be important in protection against potentially ~l~m~ging hydrophobic cations and toxins and for protection against small intpstin~ cancer. No drugs or formulations have been designed to reduce pumping of drugs back into the intestine 30 to increase drug bioavailability prior to the present invention.

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SUMMARY OF THE INVENTION
The invention is col-r-~ d with ~I;...i,Alion of drug bioavailability. The 25 invention m~Li...i~Ps drug bioavailability by hlcreasillg net drug absorption or decl,,asillg drug biullal~rollllalion in the gut by using either cytochrome P450 drug metabolism inhibitors or P-glycoprotehl (P-pg) drug llal~lJll inhibitors, both of which are called "bioenh~nrrrs" for the purposes of this invention.
An important object of the invention is inhibiting el~yllRs of the 30 cytochrome P450 3A class (CYP3A) in the gut in pl~r~l~,nce to other locations, such as the liver, which was previously thought to be the prima~y site of drug metabolism. Another object of the invention is to inhibit P-gp-controlled back transport to increase the net transport of drugs through the enterocyte layer, causing an hlcl~,ase in the bioavailability of the drug, since the protein P-gp pumps 35 drugs that have been transported into the cytoplasm of the enterocytes back into the lumen of the gut.
The invention is carried out by coa~lminictration of one or more bioenhAnrers with a drug or drugs to increase drug bioavailability. The WO95/20980 2 ~ 15 PCTIUS95/00347 -11.
compositions and methods of the present invention can be used to increase drug efficacy in h-lm~n~ and in other ~ al~, Although v~lelin~ly use is specifically culllelllplated the ~ al~ use will be in human tre~tmP~t ~tlmini~tration s~h~mPs include but are not limited to use of oral and topical formulations in 5 hllm~n~ and use of similar formulations for livestock.
One specific object of the present invention is to reduce inter-individual variability of the systemic cc,ncelllldlions of the colllpuund as well as intra-individual variability of the systemic concellll~lions of the pharm~eeutic~l compound being ^~ in;~ d.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a diagram of ellt. r~;yte cytochrome P450 3A drug metabolism and P-glycol,lo~eh~ drug transport ..-~ch~ that lead to decreased drug bioavailability .
Figure 2 is a graph showing the increase in cyclosporine (CYA) bioavailability caused by co-a~ tion of ketoconazole (KC) a bioenh~nrer.
Pre-IV i~ . s ~ .alion of CYA prior to IV ~ alion of KC. Post-IV
intlie~t~s a~lmini~tration of CYA after IV ~lmini~tration of KC. Pre-PO int~ tes oral ~-lmini.~tration of CYA prior to oral ~ lion of KC. Post-PO imlie~tes 20 oral a~lmini~tration of CYA after oral ~ l.a~ion of KC. The data illustrate an increase in ill~lated Sy~liC drug concellLIalions over tirne due to the addition of a bioenh~nrer as inrlic~ted by the increase in the area under the curve from pre-PO to post-PO which is m~rke-lly greater than the enh~nrçm~nt seen for the increase in the area under the curve (AUC) from pre-IV to post-IV.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Bioenhancers Increase Dru~ Bioavailability The present invention is based on a new discovery of various factors 30 affecting drug bioavailability. "Drug bioavailability" is defined as the total amount of drug systçmir~lly available over time. The present invention increases drug woss/20980 2 1 7 7 1 75 PCT/US95/00347 bioavailability by inhibiting drug biollar~rollllation in the gut and/or by inhibiting active llal~OlL systems in the gut which decrease the net transport of drugs across gut epithelia. ln either case the composition responsible for increased drug bioavailability is called a bioellhancel in this specification. It has been discovered S that, in contrast to previous t.,achil~ aboue the primacy of liver metabolism, the gut is the plimaly location of drug ll~lsrollllation for many drugs, if not the majority of drugs dosed orally. Thus, bioellhallce.~ specifically targeted to the gut provide a llulllber of advantages, as described in detail below.
In general, the present invention provides a method for increasing the 10 bioavailability of an orally aArnini~tçred ph~rmarelltir~l compound (particularly one which is hydro~hobic), which co~ lises orally af~ n;x~ g the ph~rm~reutic~l colllp~,ulld to a ...~..n~.~l in need of treatment collcullclllly with a bioenh~nrer colll~lising an inhibitor of a lllelllber of the cytochrome P450 3A enzyme family or an inhi~ilor of P-glycoproleill-mPAi~ted membrane ll~r~oll (or both), the 15 bioenh~nrer being present in sufficient amount to provide hlteglal~d systemicconce.lllalions over time of the compound greater than the integrated systemic collce.lt,alions over time of the compound in the ahsenre of the composition.
Changes in the integrated systemic collcellllalions over time are inAir~ted by the area under the curve (AUC) defined below. In pl~:fe.l~d emboA;..-~nl~ side effects are reduced by providing a bioPnh~nrer that is active only (or primarily) in the gut, either because of its sllu~;lul~ or because of deliberately selected concel~llàtion effects.

Bioavailability Measul~melll~
The increase in drug bioavailability attributable to a~ .ation of the bioenh~nrer can be de~llllilled by measuring total systemic drug conctllllalionsover time after coa~h--ini!il.alion of a drug and a bioenhancer and after ~Amini~tration 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 collcellllations 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 21771;~5 ~vo 95/20980 PCT/US95/00347 -13.
body) following the s~ l, alion of a drug dose is a measure of the exposure of the patient to the drug. When efficacy of the bioenh~nrer is being measured, theamount and form of active drug ~rlmini~tered should be the same in both the co~l.";";~l~ation of drug and bioenhancer and the ~r~ .alion of the drug alone.
5 For in~t~nre, ~rlmini.~tration of 10 mg of drug alone may result in total systemic drug delivered over time (as measured by AUC) of 500 ,~g-hr/ml. In co. ~l.";~ l,àlion (i.e., in the pl~sence of the bioel-h~-rer) the systemic drug AUC
will increase to 700 ~g-hr/ml. However, if sigll;rlr~ ly increased drug bioavailability in the presence of the bioenhancel is anticipated, drug doses may 10 need to be reduced for safety. Systemic drug col1cellL-alions are measured using standard in vitro or in vivo drug IlRasulellltlll techniques. "Systemic drug collcellllalion" refers to a drug concentration in a "I~"""~l'S bodily fluids, such as serum, plasma or blood; the term also includes drug concellllalions in tissues bathed by the systemic fluids, including the skin. Systemic drug concentration 15 does not refer to digestive fluids. The illcl~,ase in total systemic drug collcelll~alions is one way of defining an increase of drug bioavailability due to co~lmini~tration of bioç~h~n~el and drug. For drugs excreted lmm~t~holized in the urine, an i~ ased amount of u~lrh~l-ged drug in the urine will reflect the increase in systemic concentrations.
Chara~ lics of Dru~s Used With Bioe~h~nrers The word "drug" as used herein is defined as a chrmir~l capable of a~l",;"i~l-ation to an organism which modifies or alters the (Jl~al)lSIII'S physiology.
More preferably the word "drug" as used herein is defined as any substance 25 intPnrle~l for use in the ll~ or prevention of disease. Drug includes synthetic and naturally OC~;ullil g toxins and bioaffecting ~ -r-es as well as recognized ph~rm~e~ltir~ , such as those listed in "The Physicial s Desk Reference," 47th edition, 1993, pages 101-321; "Goodman and Gilman's The Ph~ rological Basis of Therapeutics" 8th Edition (1990), pages 84-1614 and 1655-1715; and "The 30 United States Ph~ ropeia, The National Formulary", USP XXII NF XVII
(1990), the compounds of these references being herein incorporated by reference.

woss/20980 2 1 7 7 1 75 PcT/us35~r~17 14.
The term drug also includes co~ ,unds that have the inrli~ted plo~lLies that arenot 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 withdrugs co~ l;"g of charged, unchalged, hydrophilic, zwitter-ionic, or hydrophobicspecies, 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. Preferably, a hydrophobic drug is defined as a drug more soluble in octanol than in water.

Incleased Dru~ Bioavailability by Inhibition of Cytochrome P450 Phase I Biotransformation Reduction of enterocyte cytochromes P450 pal li~ a~ion in drug bi-~L,al~rollllation is one objective of the present invention. The major el~yllles involved in drug metabolism are present in the endoplasmic retic~ m of many types of cells but are at the highest concell~ ion in hepatocytes. Traditionally, el~t~,lu(;yle bio~ rolllldlion was considered of minor importance in biol,a,~rollllalion coll~al~,d to the liver. Many colll~ ul~ds inhibit cytochrome P450. These include, but are not limited to, ketoconazole, troleandomycin, gestoclenP, flavones such as quercetin and na,i,lgelf"l",l~Ll~olllycill, ethynylestradiol, and prednisolone. The first goal of the invention is to use cytochrome P450 inhibitors to inhibit drug cytochrome P450 biu~ rollllalion in the gut to increase drug bioavailability.
Types Of C~l~hro~l~es And Tissue Location The cytochrollles P450 are a su~clr~llily of hemoproLeil~s. They lc~l~sel,L
the terminal oxidases of the mixed function oxidase system. The cytochrome P450 gene ~u~clralllily is composed of at least 207 genes that have been named based on the evolutionary relationships of the cytochromes P450. For this nomPn~l~h-resystem, 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 numeMl, e.g. CYP3), further 2177~75 "'O 95/20980 PCTIUS9S/00~17 divided into subf~mili~s (de~ign~t~d by a capital letter, e.g. CYP3A), which arecolllylised of those forms that are at least 55 % related by their de~l~lred amino acid sequences. Finally, the gene for each individual form of cytochrome P450 is acsign~d an Arabic number (e.g. CYP3A4).
Three cytochrome P450 gene families (CYPl, CYP2 and CYP3) appear to be les~onsible for most drug metabolism. At least 15 cytochromes P450 have been characterized to varying degrees in the human liver. At cGllc~llllalions of the substrates found under physiologic conditions, enzyme kin~til~c often favor a single form of cytochrome P450 as the primary catalyst of the metabolism of a particular drug or other enzyme ~.lbsllate.
The CYP3 gene family encoding cylochrollles P450 of type 3 is possibly the most important family in human drug metabolism. At least S 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-in~ ced individuals 3A may co~ u~ 15 % of the P450 el~ymes in the liver. In elllero.;yles, members of the 3A ~ubfalllily con~lilule greater than 70% of the cytochrome-co~ g enzymes. The first two human 3A ~ubf~lily members identifi~-d 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 distinguishtheir coll1libulions~ and thus they are often le~ll.,d to as 3A3/4. ~lyllllclllycill N-delllt~lylation, cyclo~pulille oxidation, nifedipille oxidation, midazolam hydroxylation, le~losle,olle 6,~-hydroxylation, and cortisol 6,B-hydroxylation are all in vilro probes of 3A3/4 catalytic activity. The levels of 3A3/4 vary by as muchas 60-fold belwæn human liver miclosolllal samples with the leve~s of 3A forms approacl-ing 50% of the total cytochrome P450 present in human liver samples from individuals receiving inducers of 3A3/4. The l._celllly 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 signifir~nt cytochrome P450 activity, and this activity is do~ cl by a single family of isozymes, 3A, the most hllpol~nl isoforms in drug metabolism.

W095/20980 2 1 7 7 1 7 5 PCT/US551~317 16.
Il,c~ d Drug Efficacy By ~J~ CYP3A Drug Biotransformation P~r~ ,d bioenh~nrers of the invention reduce drug biotransformation in the gut by inhibiting CYP3A activity in gut epithelial cells. Inhibition of CYP3A
by bioenh~nrers in gut epithelia will lead to a total increase in drug bioavailability 5 in the serum. Fewer drug molecules will be metabolized by phase I enzymes in the gut and will not be available for p_ase II conjugation enzymes. This will lead to increased concellLlalions of ullLlal~folllled drug passing from gut into the blood and onto other tissues in the body.
Another object of the invention is to reduce variability of oral 10 bioavailability. Reduction of drug bio~ldl~rolmalion or increased drug absorption will decrease variability of oral bioavailability to some degree because the increase in bioavailability will begin to approach the theoretical m~ximllm 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-15 individual and intra-individual variation. Addition of bioenh~nrer will reduce inter-individual and intra-individual variation of systemic concellLIdLions of a drug or compound.
Although the ~ llaly objective of the bioenh~nrer is to inhibit CYP3A drug bioLlal~fo~ on in the gut, some biollal~r,lllldlion may be decl~ased in other 20 tissues as well if the bioenh~-re- is absorbed into the blood stream. The decrease in biuLlal~follllalion by other tissues will also increase drug bioavailability. The advantage of ~l~elillg a bioe~-h~l-r-el to the gut, however, is that it allows the use of lower ~y~l~lllic coluellLlalions of bioenh~nrer colllpaled to inhibitors that target CYP3A in the liver. After oral ~A~ ation of a bioel-h~nr~-, concellllalions 25 will be highest at the luminal surface of the gut epithelia, not having been diluted by systemic fluids and the tissues of the body. T llmin~l concellllalions that are greater compared to blood col1cellLlalions will permit plefelenlial inhibition of CYP3A in gut instead of the liver. Bioenh~nrers that p,~felelllially inhibit gutCYP3A will also be a particularly effective means of increasing drug 30 bioavailability while ..,illi",i~.i"g the effects of greater concellllations of bioenhancers in tissues other than the gut.

~vo 95/20980 PCTtUS9StO0347 ._ , 17.
A Net I.,.,ea~e in Drug Bioavailability Due to a Dec,ea~e in the Activity of CYP3A.
The activity of CYP3A is defined as CYP3A catalyzed production of reaction product from CYP3A substrates. Substrates for CYP3A can be naturally 5 occurring substrates or other components such as those listed in Table 1. In addition, some of the CYP3A inhibitors listed in Table 1 have been identified as~ul,~L,ates, as decignqted in the table. Most likely many if not all of these inhibitors will be shown to be 3A ~ul,~lldtes through further research, althoughallosteric effects are also possible. The catalytic activities of CYP3A, subject to 10 inhibition, include, but are not limited to, dealkyase, oxidase, and hydrolase activities. In addition to the dirr~ catalytic activities of CYP3A, dirrt;~ 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. 1988, 104:912-16).

18.

P450 3A _ '' P450 3A i ' ' a 5 A ,h~; A
A ~ ~ C'' Lidocaine Te"
Quinidine P ' . ~
Mi~ ' *
F ' ~- Calcium channel blocker 7 '- Diltiazem A ' , ~ re l~
I '. ' Nical.i,: -Tianeptine 15 P- ' . - Verapamil C!
Diazepam Clc,t- ' Triazolam Er~ lh- u.. y~illt ~,, .. .. r Dapsone I~
Josamycin El.. h. ' toxina Kl -1.6-dinitropyrene 1~,' 1 1: U,U~
6 u~h. ~ Na~ t Aflatoxin B 1 F~
Benzo(a)pyrene Tl ia~ei~' ' J ,~- ' *
MOCA' V ' ' -phlp2 V *
30 1 , Vindesine*
C~cluOl~ul Fl~
FK-506 ll~,.. Lùllà~ -J ~.ill 1~-- r I
Narcotic N, Alfentanil Quercetin Cocaine Steroid hormone Codeine Cortisol*
r ~ ~ F Jl~ollal;ùl~
Steroid h~ C
40 17 oc ~ ~ .. ~ I ' 1~, ~ ' Estradiol N~ oh~,l Flutamide T~ une Fu ~ -r ~ t 1; ~ . I T~ ' ~
Al r Tl ~ -E . r ,~
D~ ' Bl~ - ir Digitoxin DDEP
Lovastatin D ~ ll o~
NoHA3 E. ~. -Retinoic acid Selegiline Terfenadine Drugs nlarked ' have also been identified as P450 3A substrates MOCA: 4,4'-Methylene-bis(2-~'1 ' . ' I
2 PhlP:2-ar.^ino-1 hyl 6, ' ,: ;~1,5-b~pyridine 3 NOHA: I`J: ~ h, ' ~ -L-arginine 4 DDEP: 3,5 ~ -2,6 :' ' ,1 ~ 1,~1 1,~ ' ', ',~ ", ' 21~7~
~VO 95/20980 PCT/US95/00347 19.
Some bioenh~l-r~ ~ reduce CYP3A drug bioLl~ ro~llld~ion by acting either as an inhibitor of CYP3A activity or as a substrate of CYP3A activity. The bioenhancer acting either as the inhibitor or the ~ub~ Lc of CYP3A can act as a co~ cLiLi~/e, non-colllpcLili~e, uncollll~etitive, mixed or irreversible inhibitor of CYP3A drug 5 bioLl~l~rollllation. Additionally, bioenhqnrer can have propellies of being a ligand for P-gp or cylochrollle P450 or a ligand for either p~oteills. Bioenhq-nrers can also include combinations of compounds of dirrclelll plupcllies. For example, a firstcompound can act as a P-gp inhibitor while a second compound acts as a CYP3A
inhibitor. Bioenhancer can also be bound to the drug being ploteclcd, either by 10 covalent bonding or by ionic or polar attractions. Colllpounds (or drugs) from a number of classes of colll~ounds can be ~h~ lcd with a bioenhq-nrer or can act as a bioe~-h~nrer, for example, but not limited to, the following classes: q-retqnili~lPs, anili~l~s, aminoquinolines, benzhydryl compounds, benzodiazephles, benzofurans, cannabinoids, cyclic peptides, diben2aze~ es, digit~lis gylcosides, ergot alkaloids, 15 flavonoids, imidq.7Oles, quinolines, macrolides, llaphLllalenes, opiates (or morphinans), oxazines, oxazoles, phenylalkylamines, piperidines, polycyclic aromatic hydroc~llL ons, pyrrolidines, pyrrolidinones, stilbenes, sulro-l~lulcas, sulfones, triazoles, llopalles, and vinca alkaloids.
Selection of Compounds for use as Bi~^~h~rlcers by R~ln-^tion of CYP3A Drug 20 Biotransformation The relative ability of colllpuullds to act as bioei^hqnrers and to increase drug bioavailability can be e~ c~l using in vitro and in vivo drug biotransformation measurements. In vivo measulclllellls of drug bioavailability, such as measuringserum or blood drug conccllll~lions over time, provide the closest measure of total 25 drug systemic availability (bioavailability). In vitro assays of CYP3A metabolism and P-pg-transport, as rli~cl~s.~ed below, indirectly in-licqt~ drug bioavailability because CYP3A drug metabolism and P-pg drug transport affect integrated systemic drug concentrations over time. Generally, the ability of a compound being tested to act as a bioenhq-nrer is demonstrated when the addition of the compound to a dru~
30 biotransformation assay decreases CYP3A drug biotransformation. Although evena minimqlly measured increase is all that is required for a compound to be a wo 9~120980 2 1 7 7 1 7~ PCT/US95/00347 20.
bio~l-h~.-rel, a collllllel.;ially desirable bioenh~llrer 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 the ~resellce of the bioe~-h~l-rer and total availability of the ingested dosage in the absence of the bioenhal-re~. A sllfflri~ont amount of orally ~l."i";~ d bioenhancer will provide integrated systemic drug conce~ ions over time greater than the illle~laled systemic drug conce~ lions over time in the absence of bioenh~nrçr.
Compounds that can inhibit el~yllles of the P450 3A class can be identified by a variety of bioassays, several of which are set out below.
In vitro CYP3A Assays and Increased Dru~ Bioavailability Cell Assays of CYP3A Function and I~ d Drug Bioavailability Cultured cells of either hepatocytes or enterocytes or fresh~y prepared cells from either liver or gut can be used to determine the ability of a compound to act 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. 1985; 80:1029-36. Cultured cells, as described in SçhmiP~llin-Ren, P. et al., Biochem. Pharmacol. 1993; 46:905-918, can also be used. The production of CYP3A metabolites in cells can be measured using high pl~ u~e liquid chroll,alograph (HPLC) m~tholc as described in the following section for microsome assays of CYP3A activity.

Mi~,oso",c Assays of CYP3A Function and In~ d Bioavailability Mi~;losollles from h~.p~tocytes or enterocytes will be used for CYP3A assays.
Micr~sollles can be pl~pal~d from liver using conve~tion~l methods as ~ c~sed inKronbach et al., Clin. Pharmacol. Ther 1988; 43:630-5. Alt~rn~tively, microsomescan be ~lep~,d from isolated enterocytes using the method of Watkins et al., J. Clin.
Invest. 1987; 80:1029-1037. Microsomes from gut epithelial cells can also be prepared using calcium pl~,ci~i~lion as described in Bonkovsly, H.L. et al., Gastroenterology 1985; 88:458-467. Microsomes can be incubated with drugs and the metabolites monitored as a function of time. In addition the levels of these .

217~175 -vo 95/20980 PCTIUS95/00347 el~yllRs in tissue samples can be measured using radio;--...-..,-o~cs~ys or we~lblots.
Isolated microsomes will be used to deltllllille bioenh~nrer inhibition of CYP3A drug biollal~.rolll-ation. Generally, the drug will be a substrate of CYP3A.
The addition of the bioel-h~nrer will decrease the ability of CYP3A to catalyze drug metabolism. Bioenh~nrers iderltifi~d in this assay will be inhibitors of CYP3A
function and dimini~h substrate catalysis. The production of metabolites can be monitored using high L~ iUl~: liquid chromatography systems (HPLC) and identified based on retention times. CYP3A activity can also be assayed by colorimetricallymeasuring c.~llllolnycill delll~lylase activity as the production of fonn~ldellyde as in Wrighton, et al., Mol. Pharmacol. 1985; 28:312-321 and Nash, T., Biochem. J.
1953; 55:416-421.
Characteristics of BiD(~nh~ncPrs that R~dnce CYP3A Drug Metabolism Bio~--h~nrel~ that reduce CYP3A drug metabolism will generally be hydrophobic colllpvullds that can pass across cell melllblalles and reduce CYP3 drug metabolism in the manner previously i~ fe~l Preferably the bioenh~nrer(s) will bind CY;P3A quickly and inhibit while the drug is passing t_rough the enterocyte. After the bioenh~ er diffuses out of theenterocyte, normal CYP3A function will return. Reversible and i~ ible inhibitorswill both have marked effects on gut drug metabolism following oral dosing. After the bioenh~nrers reach the heart and are distributed throughout the body the concellllalions of the bioenhancers will be diluted on future passes through the liver.
CollcellLlaLions of bioe-~h~l-rel in the gut lumen are preferably select~d to be effective on gut CYP3A metabolism but, due to dilution, to be less active in other tissues.
The amount of bioenh~nrer used for oral ~tlmini~tration can be selected tO
achieve small hll~s~ e luminal cuncell~lalions of at least 1/10 of the Kj for CYP3A
inhibition of drug metabolism or an amount sufficient to increase systemic drug concellLlalion levels, whichever is less. Alternatively, the amount of an 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 cyclosporine to hydroxylated products in an assay system wo 95/20980 2 1 7 7 1 7 5 PCTtUS95/00347 cont~ining 100 ~4g human liver microsomes, 25 ,uM cyclosporine, and an NADPH
regeneldlillg system in 100 ~4l of 0.1 M sodium phosphate buffer, pH 7.4. The initial inhibitor amount is selectrcl to provide collcellll~lions in the lumen of the small i"~ equal or greater than collcell~ldlions that reduce the rate of conversion 5 d~t~...;.-r~l by this assay, preferably a rate reduction of at least 10%. While the actual dose of inhibitor in a clinical formulation might be op~hlli~ed from this initial dosage depending on the results of a clinical trial, the assay as described is sufficient to establish a utilitarian dosage level.

10 Increased Dru~ Bioavailability by Inhibition of P-glycoprolein (P-p~) d Drug Absorption By 1~ , P-gp Drug Transport One embodiment of the present invention further increases bioavailability by inc~asillg net drug absorption in the gut. Traditionally, drug absorption by the gut was considered to be the result of a passive diffusion process. Drugs were thought 15 to diffuse into the gut based on the concell~dlion gradient across the gut epithelial cells. Net drug transport across the gut, however, is the net result of drug influx and back flux, some of which is active drug ~la~ l. Drug influx is the flux from lumen to blood. Drug back flux is from blood or epithelium cytoplasm into the lumen. The invention reduces P-gp active drug lldl~7~ across the luminal 20 mt;l~ldlle to prevent return of drugs absorbed into the cytoplasm of the enterocytes back to the lumen of the gut.
Generally, the invention will reduce P-gp active drug transport in order to h~l l.,ase the net transport of drugs across the gut ephh~ lm An epithelium exists in a number of dirr.,lell~ tissue types inrl~ ing, but not limited to, the epithelia of the 25 skin, liver, kidneys, adrenals, i..~ lr, and colon. Such epithelia would be affected by systemic a~lminictration of P-gp inhibitors. However, the major effects of the invention will be limited to the gut because of concentration effects resulting from oral delivery.
In embodiments of the invention where the bioenhancer comprises an inhibitor 30 of P-glycul)loteill-m~di~trd membrane transport the structure of the bioenhancer can vary widely as long as P-gp-m~di~t~d transport is reduced. A number of different ~vo 95/20980 PCT/US95/00347 molecules are known to inhibit this llalLs~olL system, and a number of examples are given below. However, whether a given compound acts as an inhibitor is best d~ inPd by activity assays, such as those described below, rather than by reliance on the structure of the molecule.
Because of the many dirr~lelll structures that can act as inhibitors, the oral dosage of inhibitor to be present in the formulation (or elsewise as described below) is best ~e~ d empirically, as the dosage will depend on the affinity of the inhibitor for P-gp relative to the drug's affinity for P-gp. There are a number of assays available that allow the desired dosage to be readily detcllllh~ed without requiring clinical trials. While the actual dosage of inhibitor in a clinical formulation might be ol,lillli~ed from this initial dosage depelldil~g on results of a clinical trial, the assay as described is sufficient to establish a utilit. rian dosage level.
Selection of Compounds for Use as Bi~Pnl-~ ~c~-~ by R~d~ tion of P-gp Drug Transport/Activity The relative ability of colllp~,ullds to act as bioenhancers and to increase drug bioavailability can be çsl;.--~l~d using in vitro and in vivo drug transport llleasul~"llell~. Colll~.~ullds acting as a bioenhqn~er will cause a net increase in drug diffusion resnlting from a decrease in active P-gp drug transport activity. The activity of P-gp is defined either as ATP depen~lPnt membrane llal~ ll of a drug or as drug-~l~pen-l~nt ATP hydrolysis. P-gp activity or drug flux can be measured using in vitro or in vivo mea~ur~lllell~ such as, but not limited to, voltage sel~ilive electrodes or dyes, or chlomirql sel~ilive electrodes or dyes, Sub~LIal~ or product analysis, electron microsco~y or coupled assays. The form of P-gp used in the assay can vary in molecular weight depending on the species, isoform, amount of glycosylation, andmolecular weight assay method. Typically, the molecular weight of the P-gp will be approximately 170 kilodaltons.
The bioçllh~ el, acting as either the inhibitor or the substrate of P-gp, acts as a colll~elilive, uncollll,~lilive, non-conll,elilive, mixed or irreversible inhibitor of P-gp drug transport. The bioenh~m~er. as an inhibitor or substrate of P-gp, can be either a transportable or non-transportable ligand of P-gp. The bioenhancer can bind to the P-gp on its lumen ~ccessible surface, cytoplasrnic accessible surface or WO 95/20980 2 1 ~ 7 1 7 5 PCT/US95/00347 24.
llleln~lalle ~ ,-i..g region. The bioenh~nrer can be a ligand of P-gp, a ligand of cytochrome P450, or a ligand of both, or any combination of the three types of ligands. For example a bioenh~.-r-t;l can comprise a ligand of P-pg plus a ligand of cytochrome P450 or a ligand of P-gp plus a ligand that binds to both P-gp and 5 cytochrome P450.

Characteristics of bioe~-h~.-r~-~ that reduce P-~p drug transport Some of the structural f~alùles that have been found for inhibitors of P-glycoploleill-mP~liqtPcl me~ ,aile transport include hydrophobic character of the 10 molecule, especially those colll~lisillg two co-planar aromatic rings, a positively chalged nitrogen group, or a C&IIJOIIYI group. However, these ch~lacleli~lics are not essenti~l. The bio~h~r~r can be a(lmini~tered with coll,~-,uilds from classes, such as, but not limited to, aminoarridines~ aminoquinolines, qnilir~P,s, anthracycline antibiotics, antiestrogens, bel~orulails, benzhydryl compounds, bel-7~zel~ines, 15 cannabioids, cephalos~o,illes, colchicine, cyclic peptides, dibenzàzepil es, epipodophyllotoxins, flavonoids, flavones, im~ 7ole~ isoquinolines, macrolides, opioids, phenylalkylamines, phel~lh;~.i,-Ps, pi~l.azii~es, pire-ri~1inps~ polyethylene glycols, pyridines, pyridones, pyrimi(linPs, pyrrolidines, ql~inq7olines~ quinolines, ~uhlolles~ rauwolfia alkaloids, retinoids, salicylates, sOlbilails, steroids, taxol, 20 triazoles, ....~ f~ fatty acids, and vinca alkaloids. The bioe~-h~-rer can also be made of a compound listed above.
The ~Ihl~ al common chala.;l~lislic of these cc,llll~oullds is that they act as inhibitors of P-gp drug LlallSpOll. When the bioe~ -el~ are used in sufficient amounts, the activity of P-gp will be reduced; in particular P-gp drug transport back 25 into the i.~ l lumen will be reduced. Sufficient amounts would include amounts nPcess~y to increase h~ laled systemic concentrations over time of the drug usedin conjul~;lion with the bioenh-q-nter. The concentration of bioenhq-nrer required to produce a sufficient amount of bioenh~nrer for inhibition of P-gp drug transportvaries with the delivery vehicle used for the bioenhancer and the drug. The luminal 30 concentration of the bioenh~nrer should be related to the drug's and bioenhancer's relative afflnitiPs for P-gp and the drug concentration used. As the affinity of drug -vo 95/20980 PCT/US95/00347 for P-gp increases, the required concellLlalion of the applol,liale bioenh~nrer will increase. Most bioçnh~nrers of collllll.,rcial application will decrease P-gp drug L~ JOll by at least 10%, more preferably by at least 50%, and even more preferably by at least 75 % .
Several compounds that are themselves normally thought of as drugs can be used as bioe.-h~l-re,~, inrl~ ing calcium channel blockers, phenolhi~7i~-~s, quini-linr, ~ntim~l~rial agents, antiestrogenic and other steroids, and cyclosporine and other compounds listed in Table 2.

1 7 7 1 7 ~ PCT/Ug5S~CA317 wo 55/20980 L

26.

Classes of P-Glyco~rote;.l Substrates or Inhibitors with Specific Examples A .h~i; E ".~acai. e NSAlDs Lldocame Quinidine Aspirin ,~ ' & ~ ~ ' Ph~
10 C Cr;mophor EL
Triton X-100 r~ Tween 80 .~ ~' ' & ~ '.i, Tricyclic ' .
15 Em t'n D
E~. u~ - r Quinacrine D
Quinine Reserpine Calcium Charmel Blockers C~.,los~,u.i.. c Bepridil FK-506 Diltiazem re~ r~
Ni' li,: Quercetin 25 Ni ~' 'i;; - SDZ 280-446 Tiapamll Tumor Necrosis Factor Cancer c! ' . Vltamin A
~ regimens A~ ~. D
T _ ~
DUAU~ .
c 35 Taxol T C.~a:~C
Vinblastine Vincristine 1' 40 Aldo;,~. ~ -Cl '. ' Cortisol D~
45 ~uc u.,~
Tamoxifen 217~175 ''VO 95/20980 PCT/US95/00347 27.
Any bioassay that d~ ...;..PS whether a given co~ uulld has the inhibition chal~ ics required of a bioe~-hA~-rel can be used to identify compounds that canbe used in the practice of the present invention. A number of such assays are set out below.
s n vitro P-p~ Assays for Bioavailability Everted Gut Assays Everted i..l~sl;i~p can be plel)a~ed by methods known in the art Hsing et al. Gastrobentsrology 1992; 102:879-85). In these studies rat small i-.lP~ PS10 turned "inside out" (i.e. the mllcosql (or luminal) surface turned outside and the serosal surface inside) are bathed in a drug contq-ining solution with and without the addition of the bioel hAnrer. The serosal surface of the small i..l~sl;.-P is bathed in a solution that is periodically monitored or changed for the purpose of drug or bioenhAI-~el mea~ul-,lllc;lll. For illclAIlre the everted rat small illlr~ PS can be bathed in a physiological saline solution loaded with Rho~lqminP 123 (Rhl23) and the flux of Rh 123 monitored into the serosal solution. The addition of a bioenhq-nrer in this set-up will increase Rh 123 Ill~oll into the serosal solution. An increase in drug or Rh 123 bioavailability will be d~l~- .--i.~Pd as follows:
X(100) Y
where Y is the initial rate of Rh 123 L~ ,u,l, and X is the initial rate of rhodqminP
transport in the ~ sellce of a bioP~-h~.-rel. The initial rates will be del~l".hRd as a linear relationship btlween time and Rh 123 conce~Ll_Lion in the luminal solution.
l!~lLf~--AIiVely~ the serosal side of rat small ;--I~I;--PS is bathed with the drug or bioPnh~nrer of interest and the mllros~l solution is monitored, as described in Hsing et al. (1992).
Selection of a P-gp Inhibitor Based on Cell Growth Assays This assay will be used to select cAn~ AtP bioenhancers. Cells cultured with cytotoxic agents that are known P-gp transport substrates will be grown as controls in the absence of either drug or bioenh~nrer. The appK; (apparent inhibition constant) for cell growth by drugs will be de~llllined by varying the drug WO 95/20980 2 1 7 7 1 7 ~ PCT/US95/00347 28.
collcelllla~iol1 in the culture m~dillm. The appKj will be expressed as the concentration of drug required to produce 50% inhibition of cell growth. Cells will also be grown in the presence of drug and bioel~hallcel. The bioenhal~cer will act to shift the appKj to lower drug concellLI~-lions nrcess~ry for inhibition of cell growth.
5 Cells with MDR can be used in this assay as described in Hait, W. N., et al., Bioc~-rnir~l Ph~rm~rology 1993, 45:401406. The method sections of Hait, W.N., et al. (1993) are herein incorporated by lcfelc;nce. P~f~lled bioenh~nr-ers willdecrease the appK; for a drug by at least 2 times, more preferably by at least 3 times, and even more preferably by at least 6 times.
Rhodamine (Rh 123) Cellular Assay of P-gp Drug Transport and Drug Bioavailabilib Rh 123 can be used in a cellular assay to monitor the bioavailability of drugs.
Rh 123 LlallS~Oll~;d by P-gp in this system acts as a drug, where P-gp pumps the Rh 123 out of the cell. Single cells or a population of cells can be monitored for the Rh 15 123 fluolescel~ce which is indicative of P-gp transport. The cell types used will contain a P-gp ~ ,ollel from a MDR strain such as those listed in Nielsen and Skov~ga~l, Biochimica et Biophysica Acta 1993; 1139:169 183 and herein incorporated by lefel.,llce. Cells are loaded with Rh 123 in the ~l~sence of 15 nanograms per ml to 500 l~lOglalllS per ml of Rh 123 in a physiologically colllpalible 20 buffer such as 3-N-morpholinoprop~ lfonic acid (MOPS) with the suitable collc~ dlions of sodium, p~ --, and calcium chloride and an energy source.
The cells are loaded with Rh 123 for 30 - 60 ~ s de~~-Ai~g on the telll~el~lulc (37 or room Iclllp~ lule ). The loaded cells are then washed and resuspended inbuffer free of Rh 123. The efflux of Rh 123 can be delellllilled u~ing a fluorimeter.
25 In the absence of any bio~nh~-rer Rh 123 will be pumped out of the cell due to the action of P-gp, leading to a reduced amount of Rh 123 fluoresc--~re from the cell.
Addition of a P-gp substrate or inhibitor either by preincubation after the cells have been washed with Rh 123 free buffer or during the efflux of Rh 123 from thecell will cause retention of Rh 123 within the cell. Retention of Rh 123 in the cell 30 will be caused by the addition of a bioenhancer. Increased drug bioavailability is '`'O95/20980 2 1 7 i 1 75 PCT/I~S95/00347 29.
defined as the increase in Rh 123 retention within the cell. Compounds that increase Rh 123 retention are bioenhA~-rc~
Rh 123 retention in the absence of a bioenhAnr-er will be determined by total Rh 123 cell fluorescence minus background Rh 123 cell fluo,~scellce. An increase5 in drug bioavailability due to the addition of the bioel-h inrer will be the percentage increase in Rh 123 fluorescellce retention as described by:
X(100) y where X equals Rh 123 fluorescence in the presence of the bioenhancer minus the background Rh 123 fluorescellce and Y equals the Rh 123 flu~ scence in the absence of the bioenhAnrer minus the background Rh 123 fluorescç--re.
The background Rh 123 fluolescellce can be measured in a variety of ways inrll~rling, but not lirnited to the residual amount of Rh 123 fluorescenre at the end of the ~ c~ Rnt, the residual amount of Rh 123 fluorescence rçm~ining based on an extrapolation of first order rate kinPtics describing the efflux of Rh 123 from the cell, the residual amount of Rh 123 fluorescellce in the p~sel ce of a sufficient amount of membrane d~lc,~e"l~ such as triton or ligitonin or the amount of Rh 123 fluo,,sce-~-re in the p,~scnce of a po~ ;.-."-valillo",ycill clamp.
The addition of both a second drug and a bio~l-h~l-rer to the Rh 123 assay will not I~Pcçc~-lily cause an i".;,._ased amount of Rh 123 retention colll~)al~d to the ~,~sellce of either the bioe~h~nrer alone or the second drug alone. This is because Rh 123 retention can already be very high due to the second drug or bioenhancer conce"l,~lion. Extra retention due to the addition of either the second drug or the bioel~hAI~rer can be ~liffirlllt to measure above the signal for Rh 123 in the presence of the second drug or bioenh~nrer alone. However, once it has been det~rminPd that the drug (or second drug alone) increases Rh 123 fluorescence, i.e. decreases Rh 123 efflux it can be ~csllmPd that the drug (or second drug alone) is transported by the P-gp transport system.
Vesicle Assays of P-pg Activity and Drug Bioavailability A particularly p,efe"cd assay uses brush border membranes. Brush border membrane vesicles are prepared from the small intestine by methods known in the W O95t20980 2 1 7 7 l 7 ~ PCTrUS95/00347 30.
art, suchas, Hsing, S. etal., Gastroenterology 1992;102:879-885. Thevesicleswillbe assayed for the ylcsellce of P-gp by using monoclonal antibodies directed to P-gp either using SDS page gel elecllopholesis and wc~ ll blotting t~clmi-luec or using immllm)ch.on i~try and elecllulllicluscopy. Vesicles cont~ining P-gp will be used for S drug transport assays.
Drug transport assays consist of m.o~Cllring the llallsyol~ of drugs into the vesicles in an ~c~eno,cin~ triphosphate (ATP) dependent fashion. Uptake of the drug in the ylcsellce of ATP will be monitored using fluolcscel1ce or absorbance t~e~lni~lues, for ;...~ e using Rh 123 as the fluolcscen~ drug transported into the 10 interior of the vesicle. I~ ioactively labeled drugs can also be used to monitor drug transport into the interior of the vesicle using a filter wash system. The addition of ATP will induce the transport of the drug into the vesicle and will increase drug transport cOlllyalcd to passive diffusion of the drug into the vesicle interior. Addition of non-hydrolyzable analogs of ATP such as ATP gamma S or adenosine 15 monophosphate para-nitrophenol (AMP-PNP) will not produce an ATP dependent influx of drug into the vesicle. Thus, the introduction of a non-hydrolyzable nucleotide can be used as a control to monitor whelll~,l drug ~ yull has actually occurred due to ATP hydrolysis from the P-gp LlallSyOll system.
The addition of a bioei-h~ er to this assay system using a fluolcscclll drug or 20 a ra~iio;lctive drug and monilolillg its uptake, will reduce the uptake of the drug into the interior of the vesicle with the addition of ATP. This reduction in drug llal~yOll lep~s~ an ill~ase of the bioavailability of the drug. The vesicles transporting drugs in an ATP dependent fashion are oriented with the cystolic face of the P-gp acceccihle to the ATP. It is these vesicles that hydrolyze the ATP and llallSyOll the 25 drug into the interior of the vesicle. The interior of the vesicle in turn collc~yollds to the luminal surface or the apical membrane of the brush border cells. Thus, ~ yOll into the lumen of the vesicle or interior of the vesicle corresponds tollal~yull into the lumen of the gut. A decrease in the transport of the lumen of the vesicle is the equivalent of increasing net drug absorption and increasing the drug bioavailability.

'~0 95/20980 PCT/US95/00347 P-gp ATPase Assays of P-gp Activity and Drug Bioavailability P-gp molecules can be isolated in vesicles suitable for measuring ATP'ase activity. P-gp ATP'ase activity will be measured in the presence of other types of ATP'ase inhibitors, such as, but not limited to, sodium potassium ATPase inhibitors 5 (ouabain and vanadate), mitocholld.ial ATP'ase inhibitors such as oligomycin, and ~lk~line phosphatase inhibitors. The ATP'ase assays will also be conducted in the absence of sodium and pol;~si~ to elimin~te background sodium and potassium ATP'ase activity. ATP'ase activity will be measured as ATP'ase activity dependent on the presence of a drug such as d"~ llycin. ATP'ase activity will be measured 10 using ATP or hydrolyzable ATP analogs such para-nill~ophel~olphosphate. The production of product will be .l,o~ ol-,d using phosphate assay procedures of those of Yoda, A. and Hokin, L., Biochem. Biophys. Res. Comm. 1970; 40:880-886 or by moniloling phosphatase activity as recognized in the lile.~lulc.
An increase in P-gp ATPase activity due to the addition of a drug is 15 recognized as an increase in drug bioavailability. P-gp molecules located in the brush border membrane vesicles are oriented so the cytosolic portion of the molecule finds and hydrolyzes ATP. It is these P-gp molecules that will give rise to the drug depen(lent ATPase activity. Bioenh~-rer that is able to stim~ te the ATPase activity will be able to comrete with the drug for the P-gp transport system. Such 20 bioe~-h-.~r~ls will decrease P-gp drug llal~Oll due to their increased ability to stim~ te P-gp activity. Bioe.-h ~n~e. ~ can also inhibit drug dependent P-gp ATP'ase activity wi~wul stim~ ting P-gp ATP'ase activity thus, inhibiting drug transport.
Another Illalll~r of ~ -i-.g the amount of bioenh~.~rel applopliate for an oral formulation is based on the Kj of the specific inhi~ilor (for whichever binding is 25 being measured). An applo~liale amount of inhil,ilur is one that is sufficient to produce a concellll~lion of the bioenh~neer in the lumen of the gut of the animal of at least 0.1 times the K; of the bioenhancel.
In all of these cases, the goal of selecting a particular concentration is increased bioavailability of the ph~ relltir~l compound that is being ~-lmini~tered.
30 Thus, a desirable goal is to provide integrated systemic concel~ ions over time of the ph~ re~ltir~l compound in the presence of the inhibitor that is greater than the wo gs/20980 2 1 7 7 1 7 ~ PCTrUS95/00347 integrated systemic collcc~ ons over time of the ph~nn~r,e~ltir~l compound in the absence of the inhibitor by at least 10% of the dirrelence between bioavailability in its absence and complete oral bioavailability. Plcrellcd is ~tt~ining of "complete bioavailability," which is 100% systemic bioavailability of the a~lmini.ct~red dosage.
Scrcel~ Assay for Bioel-hAI-rels In ~ z~y, the various techniql~es described above for scl.,.,l ing c~nr~ t~
bioenh~nrer cOlll~n)ullds for activity by assaying for inhibition in the gut of a ".~."..~l of activity of a cytochrome P450 enzyme or of transport by P glycol~n~Lein are all generally useful as methods of identifying compounds that are useful for increasing 10 bioavailability of a drug in a ~ l. In all of these assays, the best bio~nh~nrers are those colllpoul~ds selected from the c~n~ te colllpoul~ds being tested that best inhibit either lla~ or el~ylllalic destruction (preferably the latter) of a tested drug in the gut of the -.~ l (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 llle.llb.,... of a cytochrome P450 3A family (for a particular ",~ l, particularly human) are IJlcfcllcd. Although in vivo assays are plefc~l~d, because of the direct l, lalionship bclwccn the llleasulelllclll and gut activity, other assays, such as assays for inhibition of cytochrome P450 activity in isolated elllero.;ytes or microsomes obtained from cl~locyles of the --~------~l in question or for inhibition of cyloc~llle P450 in a tissue or mtm~ from the gut of said ... ~.. ~l, are still useful as sclc;~,nil~g assays. The same ordering of plerell~,d sclccl~illg assays (i.e., in vivo being pler~,lled over in vitro) is also ~,leÇellcd for sclcenillg of inhibition of P-gp llal~llOll. Sclccnillg by assaying for both inhibitions is plefcll~d, with inhibition of cyloc~llle P450 activity generally being more important than that of P-gp-mt~ tP~ llanspolL.

Co~lmini~tration and Delivery of Bioenhallccl~
as~ in Drug Bioavailability with Coadmi,-i~lr.~tion of a ~2iopnh~nc~pr and a Drug The present invention will increase the bioavailability of the drug in the systemic fluids or tissues by co-a~lmini~tering the bioenh~n~er with a drug. "Co-~VO 95/20980 2 1 7 7 1 7 5 PCT/US5S~I~C317 `_ a~ ation" includes coll~;ull~ a~ aLion (a~lministration of the bioenh~nrer and drug at the same time) and time varied a~l.--il-i~l.dLion (a~lministration of the bioellhallcer at a time dirrc~llL from that of the drug), as long as both the bioe~hqnrer and the drug are present in the gut lumen and/or membranes during at least partially 5 ovellap~ing times. Systemic fluids or tissues refer to drug concentration measured in blood, plasma or serum, and other body fluids or tissues in which drug l,leasu,~;.llcllt~ can be obtained.
D~ Vehicles ~v dF For Coa~ alion Co~-l...i..;~l.àtion can vary in the type of delivery vehicle. The bioenhqnrer 10 and the drug can use dirÇelelll delivery vehicles such as, but not limited to, time release lllaLlices, time release co.,l ;-.g~, companion ions, and successive oral ~q~ alions. Alternatively, the drug and the bioenhq-ncer can be form~ trd with dirr~ coali,lgs posses~;ug dirr.,rel,l time Col~Ldll~ of bioenhqnrer and drug release.
The use of bic~nhqnrers also applies to epithelia tissues other than the gut. Aspects 15 of the invention used in the gut are a~ro~liàtely used in other types of epithelia. For example, CYP 3A enzymes and P-glycoprotein have also been demor~sllated in the skin and bioenh~l-rel~ used in ~~ d~ l formnlqtions would increase drug bioavailability to ~y~l~llliC fluids and tissues. Such applications are included as part of the invention herein because of inhibition by bioenhq-nrers of CYP 3A enzymes and 20 P-glyc~rot~,m in epithelia other than the gut.

Formlllqtion~ of Bioenl-~nr-el~
In some embo~im~ntc, the bioe--h~nrer colll~ises a cytochrome-P450-3A-i~i~illg col.lpuu,ld and a sepalate P-glycoploleill-inhibiting compound. In other 25 cases, the bioenh~l-rel comprises a single compound that inhibits both CYP3A and P- glycop,olein, or just one of the two p,ocesses. The bioenhq-nrer is preferably present as a counter ion of the ph~....~reutirql compound in order to ensure that the bioenhq-nrer is present at mqximnm concentration in the presence of the drug that it is protecting.
The cytochrome P450 3A family of enzymes and the P-gp transporting protein both have a wide range of substrates, and thus potential inhibitors, as exemplified by wo gs/20980 2 1 7 7 1 7 5 PCT/US95/00347 34.
the variety of structures present in co~ oullds that can act as inhibitors as set forth above.
The invention is carried out in part by formlllAting an oral phA, ...lreutirAl composition to contain a bioe~-hAnrel . This is accomplished in some 5 embodi,nelll~ by ~Irlmixin~ a ph,.- ",ArelltirAl compound, a ph~rmAre~ltirAl carrier, and a bioenhAn~er colllll,isillg an inhibitor of P-~lycoproleil~ d membrane transport or an illhibilol of a cytochrome P450 3A enzyme, the bioe~h~nrer being present in s~fflri~nt amount to provide illte~5laled systemic concentrations over time of the colllpuulld as measured by AUC's greater than the illlt;~lated systemic concentrations 10 over time of the compound in the absence of the composition when the phAIlll~e~lti~Al composition is AA..-i--;~t~,;cd orally to an animal being treated with the ph~ e~ltirAl composition. A ph~....~cc~ Al carrier increases drug solubility or plolecl~ drug structure or aids in drug delivery or any combinalioll thereof.
Pl.~. ",~ irAl compositions produced by the process described herein are also 15 part of the present invention.
In addition to use with new formulations, the present invention can also be used to ill~,.,ase the bioavailability of the active compound of an exictin~ oral ~h~- ".~ A1 composition. When p,acliced in this manner, the invention is carriedout by ~cfc~ ...lAtin~ the e~i.cting cûlllposiLion to provide a ,~rO- ....-lAted composition 20 by ,~lmixing the active cûulpuul~d with a bioenh~l-rel co,lll"ising an inhibitor of a cytochrome P450 3A enzyme or an inhibitor of P-glycoploLcill-m~diAt~d membrane transport, the bioenhAnr-er being present in sllffiri~nt amount to provide integrated systemic col,ce"L,aLions over time of the colll~uulld when ~.h"in;~ ,d in the l.,fc,-~ lAted co",po~ilion greater than the integrated ~y~lel~ic collce"Llations over 25 time of the co"l~,oulld when ~ "i~ L~ red in the existing ph~ e~ltirAl composition.
All of the criteria described for new formulations also apply to lcfollllulation of old compositions. In plcrell~d aspects of lcrollllulations, the reformlllAted composition c~,lll~lises all components present in the existing phArmAre~ltirAl composition plus the - bioenhancer, thus simplifying practice of the invention, although it is also possible to 30 eliminAte existing components of formulations because of the increase in bioavailability. Thus, the invention also covers lerol..---l~t~cl compositions that -v095/20980 2 1 77 1 75 PCrtUS95/00347 contain less than all components present in the e~i~ting lJha~ c~ ir~l composition plus the bioenh~nrer. However, this invention does not cover already existing con~osilions that contain a colllpollelll which increases bioavailability by mechani~m~
described in this specification (without knowledge of the mech~ni~m~), should such 5 conlposiLions exist.
Traditional formulations can be used with bioenhdllce~s. Optimal bioenh~nrer doses can be d~lellllh ed by varying the coa~h.~ alion of bioenhA,-rel and drug in time and amount dependent fashion and monitoring bioavailability. Once the optimal biot~-hanr-e~ dose is established for a drug the formulation (bioenhanrer, drug and 10 formulation composition(s)) is tested to verify the illcleased bioavailability. In the case of time or sllst~inrd release formulations it will be pl~Ç~ d to establish the optimal bioe nhal-cel dose using such formulations from the start of the bioavailability EXAMPLE - QUANTITATION OF BIOENHANCER IN VIVO
Cyclosporin (CYA) Bioavailability in the Absence and ~,se.~ce of Kc;loconazole (KC) As A Bior ~h~nrel.

1. GENERAL DESIGN OF METHODOLOGY:
Six rnale/female healthy volunteers served as ~ubjec~ for the procedure. Pre-,locedule laboldl~l,y tests, physical ~ tiQns, and consent was obtained at leastfive days prior to the procedure date.
The procedure was ~lÇollllld in two phases. During the initial phase (I), baseline oral and intravenous ph,.,...~r,okinetic parameters were established. Phase 25 II consisted of ketoconazole ~h"i~ l,dLion and oral/intravenous ph~rmar,okinetic procedures. Each phase followed identir~l procedures: after an overnight fast, subjects were ~rlmittrd for initial CYA ph,.. ~rokinPtirc plocedul~s. The order of CYA route of aflmini~tration was randomized during phase I, and the same order was n~int~inPd in phase II. After the insertion of an indwelling catheter, each subject 30 received an oral or intravenous dose of CYA with 5mL blood draws obtained at 0, 15, 30, 45, and 60 ..,i.,..les, then 2, 3, 4, 5, 6, 8, 10, 12, 14, and 24 hours (total .

WO 9S/20980 2 1 7 7 t ~ 5 PCT/I~S9S/00347 36.
volume of blood: 300mL). During the intravenous infusion, contralateral catheters were inserted. Intravenous CYA was a~lmini~tered over 2.5 hours by AVI infusion pump. After completing the i.v. infusion, the infusion catheter was removed.
Subjects either returned to the testing location in the morning for each 24 hour blood 5 draw or stayed overnight in the testing location.
During phase I, ~uLje~;~ received oral or i.v. CYA (8mg/kg and 2.5mg/kg, ~ ively) and followed the above plocedules (Day 1). After a three day washout period (Day 5), subjects received CYA i.v. or p.o. depçn~ling on initial randomi_ation. Blood samples were drawn as described above. When subjects 10 returned to the testing location for the 24 hour blood draw (Day 6), they were given KC 200mg and instructed to take one tablet daily at lOPM for eight days. The last dose of ketocona_ole was taken the night of procedure day 14, just before the last ph~....~rokinetic procedure day.
In study sections designPd to evaluate Ketoconazole's effects (Phase II) on 15 both intravenous and oral CYA metabolism, dosing of the bioenh~l.rel and the drug (CYA) were sepalated by approximately 10 hours.
During phase II (Day 11), subjects were a~mhted to the testing location after an overnight fast for further CYA ph~rm~rokinetic procedures. Subjects received either a single oral dose of CYA at a reduced dose of 2mg/kg or intravenous CYA
20 (0.6mg/kg) dep~ .u1i~g on the previous ran~lomi7~tion sclled~ . Blood samples were drawn as descrihed above. After a three day washout period, on Day 15, subjects received the oral (2mg/kg) or i.v. (0.6mg/kg) dose, again dependillg on the initial randomization sel~ lllP. Blood samples were drawn as described above.
In phase II, in healthy volunteers, dosing of CYA in the presence of the 25 enh~nrer was reduced from that ~ lmini~tered when no bioenh~l-re- was present for safety considerations.
The total volume of blood drawn for this procedure was 380mL (pre/post labs and ph~rm~eokinetic procedures.

2tt71~

-37.
2. SAMPLE AND DATA ANALYSIS:
Whole blood samples were assayed for CYA and metabolites (AM1, AM4N, AM9, AMlc9) by HPLC. Ph~rm~rokinetic parameters, including bioavailability, were obtained from data and cclllpalcd for dirr~,le.1ces between baseline (-KC) and 5 inhibition ( +KC) .

3. SUBJECr SELECrlON CRrrERIA:
a. Healthy adult volunteers were used to ~ interpatient variation in response to drug a~mini~tration, which can occur with hepatic or renal dy~ru,1clion, 10 and to ...il.i.-.i,~ risks associated with drug a~ alion.
b. Eight healthy male or female subjects were recruited for this procedure to assure that at least six subjects completed both phases.
c. INCLUSION CRITERIA:

>than 18 years of age * Weight not more than 10% above or below the ideal body weight for age, height and weight. (Metropolitan Life Il~u~ ce Co. tables) * Good health on the basis of history and physical exam.
* No history of cardiovascular, renal, hepatic, ga~Lloi--~
atOl y, hematologic disease, or other ~ ces which could affect the distribution, absorption, metabolism, or excretion of either plvcedulc drugs.
* Have laboratory tests within normal limits.
* Ability to provide written and illÇollllcd consent.
d. EXCLUSION CRITERIA:
* Use of any drugs, including both p,escli~Lion and chronic over-the-counter m~lirAtions within one week of the procedure.
Specifically, use of antacids, H2-antagonists, or other agents known to decrease KC absorption or interactions with CYA.
* Participation in experimental drug procedure within one month preceding the procedure.

WO 95/20980 2 1 7 ~ 1 7 5 PCT/US95/00347 38.
* History of hy~ ensilivily of KC, azole-alllirungal agents, or CYA.
* History of my~P~ema hypcllllyloidism, hepatic disease, hPpatiti~, alcohol, or recreational drug use, cardiac arrhythmias, sei ules, tobacco, or any other condition which can alter drug metabolism, absorption, or distribution.

4. SUBJECT RECRUITMENT:
Subjects were recruited from the population in and around a ullivel~i~y campus. Potential ~ubje~ were screened for their fitness to participate in the procedure within S days prior to ~ ,ce-lule date. The scl~el~illg procedure included the following: m.o~ l and drug hi~lolics, physical exam and baseline laboratory procedures (hemoglobin, hPmatorrit, RBC, WBC, dirr~ ial, platelets, potac~illm, chloride, bicarbonate, serum cl~ in~, BUN, glucose, albumin, total bilirubin, alkalin.o phosphatase, AST, ALT, cholesterol, HOL, LOL, and a urine pregnancy test when female volunteers were used).

5. SPECIFIC PROOEDURES:
a. At least five days prior to the procedure date, a 40mL blood sample was drawn from each subject for baseline laboratory procedures as stated above. At this time a history and physical exam was ~lrolllRd.
b. The procedul~ was divided into two phases. During phase I, baseline CYA ph~--..~rokin~ti~ after intravenous and oral dosing were established.
25 During phase II, subjects were given eight days of KC therapy, and post-KC
hlL,~venous and oral CYA pl---.--~rokinetic procedures were ~lrolllled. Subjectswere randomized to receive an oral or i.v. dose as the initial route of a~lmini~tration and followed this ran~1omi7~tion throughout the procedure.
Day 1: Subjects were a.(lmitted to the testing location at 07:00 after an 30 overnight fast from 22:00 the previous evening. An indwelling polyethylene catheter with a teflon obturator was inserted aseptically into the for~dllll vein for blood withdraw. Tmm~iat~ly prior to drug a-1mini~tration, a SmL blood sample was collected. CYA was then aflmini~tered orally (8mg/kg) or intravenously (2.5mg/kg .
.

~vo 95/20980 PCT/U~ C317 _ 39.
over 2.5 hours) based on body weight. Five millilher blood samples were drawn atthe following times post during ~-l."i~ l.alion: 15, 30, 45, 60 minutes and 2, 3, 4, 5, 6, 8, 10, 12, 14, and 24 hours. Subjects returned to the testing location thefollowing day (Day 2) for the 24 hour blood draw (peripheral venipuncture). Whole 5 blood was analyzed for parent drug and metabolites. Some samples were frozen prior to extraction. Bre~kf~t, lunch, a light snack, and dinner were supplied during all days spent in the testing location. All subjects were given oral CYA with chocolate milk to ensure a~eqll~te absorption and to .-i~-i..-i,~ variability in absorption.
Subjects were allowed to drink water freely.
Day 5: After a three day wash-out period, subjects were again ~Amitted to the testing location, and i~1enti~l procedures were followed as described above in Day 1 except that ~ubjec~ received CYA by the alternate route of lalion not used previously on Day 1. Subjects receiving CYA by the i.v.
route had the i.v. catheter removed after the infusion was completed. Blood samples were drawn as described above.
Day 6: When l~lulllhlg for the 24 hour blood draw, subjects received a lOOmg dose of kelocol~ole (half-dose) and were observed for one hour, the other lOOmg being taken at lOpm that evening. Each subject was instructed to take one 200mg tablet each evening at lOpm with food. Subjects were instructed to take KCfor a total of eight days. The last dose of KC was taken on Day 14.
Day 11: The proce-lulc went fOl~al.l as described for Day 1 with the exception of CYA dosing. Subjects received an oral dose of 2mg/kg of CYA or an i.v. dose of 0.6mg/kg over 2.5 hours. Blood samples were drawn as previously described. An i~ ntir~l diet WâS provided to --i--i--.i~e ch~l~ges in CYA absorption.
Day 15: After the three day wash-out period, subjects were ~-lmitted to the testing location and received the final CYA i.v. or oral dose. Blood samples were drawn as previously described. The 24 hour blood draw included an additional 40mL to pelrollll post-procedure laboratory tests. Prior to discharge, each subject - underwent a physical exam and history to detect any adverse reactions due to drugs or catheter insertion.

40.
Since CYA absorption can be highly variable, especially with diet content, food outside of the procedure diet was not pe. ~ led. All meals (breakfast, lunch, snack, dinner) were supplied on all procedure days.

5 F. RESULTS
KC inhi~iled the metabolism of cyclosporine (CYA),res~llting in elevated CYA levels. CYA as a single dose was ~ r~cd orally and intravenously, pre-and post-KC l1~,A~ to 6 normal healthy volullt~ . The mean pre-KC
bioavailability (F) was 22.2% colllpa~d to 62.0% post-KC (P<0.003). Utilizing 10 Fheptjc=l-ER~ where ER=CLIv/hepatic blood flow (1.28Llhr/kg), F can be brokendown into its collll~oll~nls: FhepatjcxFabs xFg",. FabsxFg"~ increased ~ig~.irir~lly post-KC
(68.5%) cOlll~alcd to pre-KC (26.2%) (P<0.006), whereas FH changed minim~lly, 90%PSt-KCvs86%Pre~KC(P=O.lO). CYAis well absorbed (>62%) by the gut under these conditions.
The hlte.a.;lion b~,lwcen CYA and kelocona~ole is also e~ ssed graphically in Figure 1. Subject CYA levels were measured after IV ~dlllil~;~llalion of CYA pre-KC ~ .l (pre-IV) and post-KC ll~ (post-IV). Subject CYA levels were also llleasulcd after oral ~ l.alion of CYA pre-KC tre~tm~nt (pre-PO) and post-KC llc~ l (post-PO). The ill;lease in CyAconccllllalion was ~l-,al~l post-KC
20 l,.,~ -.l of orally ~ e,~,dCYA.
This cA~plc ~P-nn~ l."t~s the effect of ill1r~ Al P450 enzymes on the bioavailability of CYA. It shows that ;--l~s~ l P450 enzymes are illlpOl~ll~
d~ of the bioavailability of CYA and other agents which undergo ~ignifir~nt metabolism by P450 enzymes.
All publications and patent applications mentioned in this specification are herein incorporated by refe,~nce to the same extent as if each individual publication or patent application was specifically and individually intlir~t~d to be incorporated by reference.

2~77175 'YO 95/20980 PCTtUS95tO0347 -41 .
The invention now being fully described, it will be ap~alcll~ to one of oldillal~ 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 (45)

42.
WE CLAIM:

1. A method for increasing bioavailability of an orally administered hydrophobicpharmaceutical compound, which comprises:
orally administering said pharmaceutical compound to a mammal in need of treatment with said compound concurrently with a bioenhancer comprising an inhibitor of a cytochrome P450 3A enzyme or an inhibitor of P-glycoprotein-mediated membrane transport, said bioenhancer being present in sufficient amount to provide bioavailability of said compound in the presence of said bioenhancer greater than bioavailability of said compound in the absence of said bioenhancer.

2. The method of Claim 1, wherein said bioenhancer comprises an inhibitor of a cytochrome P450 3A enzyme and said inhibitor is a hydrophobic molecule.

3. The method of Claim 1, wherein said bioenhancer comprises an inhibitor of a cytochrome P450 3A enzyme and said inhibitor is present at a concentration in the gut of said mammal equal to or greater than a concentration of said bioenhancer that reduces conversion of cyclosporine to hydroxylated products by 10%, compared to controls, in an assay system containing 100 µg human liver or enterocyte rnicrosomes, 25 µM cyclosporine, and an NADPH regenerating system in 100 µl of 0.1 M sodium phosphate buffer, pH 7.4.

4. The method of Claim 1, wherein said amount is sufficient to produce a concentration of said bioenhancer in the lumen of the gut of said mammal of at least 0.1 times said Ki or apparent Ki of the bioenhancer.

5. The method of Claim 1, wherein bioavailability in the presence of said inhibitor is greater than bioavailability of said compound in its absence by at least 10% of the difference between bioavailability in its absence and complete oral bioavailability.

43.

6. The method of Claim 1, wherein said bioenhancer comprises an inhibitor of P-glycoprotein-mediated membrane transport and said inhibitor is a hydrophobic molecule comprising two co-planar aromatic rings, a positively charged nitrogen group, or a carbonyl group.

7. The method of Claim 1, wherein said bioenhancer comprises an inhibitor of P-glycoprotein-mediated membrane transport and said inhibitor is present at concentrations equal to or greater than concentrations that reduce transport of Rh 123 by-glycoprotein in brush border membrane vesicles or P-gp containing cells by 10%.

8. The method of Claim 1, wherein said bioenhancer comprises a cytochrome-P450-3A-inhibiting compound and a separate P-glycoprotein-inhibiting compound.

9. The method of Claim 1, wherein said bioenhancer comprises a single compound that inhibits both cytochrome P450 3A and P glycoprotein.

10. The method of Claim 1, wherein said bioenhancer is present as a counter ion of said pharmaceutical compound.

11. The method of Claim 1, wherein said bioenhancer is covalently bound to said pharmaceutical compound.

44.

12. The method of Claim 1, wherein said 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.

13. The method of Claim 1, wherein said bioenhancer comprises an acetanilide, aminoacridine, aminoquinoline, anilide, anthracycline antibiotic, antiestrogen, benzazepine, benzhydryl compound, benzodiazapine, benzofuran, cannabinoid, cephalosporine, colchicine, cyclic peptide, dibenzepine, digitalis glycoside, dihydropylidine, epiphodophyllotoxin, ergeline, ergot alkaloid, flavone, flavonoid, imidazole, isoquinoline, macrolide, naphthalene, nitrogen mustard, opioid, oxazine, oxazole, phenothiazine, phenylalkylamine, phenylpiperidine, piperazine, piperidine, polycyclic aromatic hydrocarbon, polyethylene glycol, pyridine, pyridone, pyrimidine, pyrrolidine, pyrrolidinone, quinazoline, quinoline, quinone, rauwolfia alkaloid,retinoid, salicylate, sorbitan, steroid, stilbene, sulfone, sulfonylurea, taxol, triazole, tropane, unsaturated fatty acid, or vinca alkaloid.

14. The method of Claim 1, wherein said inhibitor is selected from said group consisting of the compounds listed in Tables 1 and 2.

15. The method of Claim 1, wherein said bioenhancer reduces inter-individual variability of said bioavailability of said pharmaceutical compound.

16. The method of Claim 1, wherein said bioenhancer reduces intra-individual variability of said bioavailability of said pharmaceutical compound.

17. A method of formulating an oral pharmaceutical composition, which comprises:admixing a pharmaceutical compound, a pharmaceutical carrier, and a bioenhancer comprising an inhibitor of P-glycoprotein-mediated membrane transport or an inhibitor of a cytochrome P450 3A enzyme, said bioenhancer being present in sufficient amount to provide bioavailability of said pharmaceutical compound in the presence of said bioenhancer greater than the bioavailability of said pharmaceutical compound in the absence of said bioenhancer when said pharmaceutical compositionis administered orally to an mammal.

18. The method of Claim 17, wherein said bioenhancer comprises an inhibitor of acytochrome P450 3A enzyme and said inhibitor is a hydrophobic molecule.

19. The method of Claim 17, wherein said bioenhancer comprises an inhibitor of acytochrome P450 3A enzyme and said inhibitor is present in an amount sufficient to provide a lumen concentration equal to or greater than a concentration of said bioenhancer that reduces conversion of cyclosporine to hydroxylated products by 10%
in an assay system containing 100 µg human liver or enterocyte microsomes, 25 µM
cyclosporine, and an NADPH regenerating system in 100 µl of 0.1 M sodium phosphate buffer, pH 7.4.

20. The method of Claim 17, wherein said bioenhancer comprises an inhibitor of P-glycoprotein-mediated membrane transport and said inhibitor is a hydrophobic molecule comprising two co-planar aromatic rings, a positively charged nitrogen group, or a carbonyl group.

21. The method of Claim 17, wherein said bioenhancer comprises an inhibitor of P-glycoprotein-mediated membrane transport and said inhibitor is present in the gut of said mammal at a concentration equal to or greater than a concentration that reduces transport of Rh 123 by P-glycoprotein in brush border membrane vesicles or P-gp-containing cells by 10%.

46.

22. The method of Claim 17, wherein said amount is sufficient to produce a concentration of said bioenhancer in the lumen of the gut of said mammal of at least 0.1 times said Ki or apparent Kiof said bioenhancer.

23. The method of Claim 17, wherein said inhibitor comprises a cytochrome-P450-3A-inhibiting compound and a separate P-glycoprotein-inhibiting compound.

24. The method of Claim 17, wherein said inhibitor comprises a single compound that inhibits both cytochrome P450 3A and P glycoprotein.

25. The method of Claim 17, wherein said bioenhancer is present as a counter ionof said pharmaceutical compound.

26. The method of Claim 17, wherein said bioenhancer is covalently bound to saidpharmaceutical compound.

27. The method of Claim 17, wherein said pharmaceutical compound comprises an acetanilide, aminoacridine, aminoquinoline, anilide, anthracycline antibiotic, antiestrogen, benzazapine, 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.

47.

28. The method of Claim 17, wherein said bioenhancer comprises an acetanilide, aminoacridine, aminoquinoline, anilide, anthracycline antibiotic, antiestrogen, benzazepine, benzhydryl compound, benzodiazapine, benzofuran, cannabinoid, cephalosporine, colchicine, cyclic peptide, dibenzazepine, digitalis glycoside, dihydropylidine, epiphodophyllotoxin, ergeline, ergot alkaloid, flavone, flavonoid, imidazole, isoquinoline, macrolide, naphthalene, nitrogen mustard, opioid, oxazine, oxazole, phenothiazine, phenylalkylamine, phenylpiperidine, piperazine, piperidine, polycyclic aromatic hydrocarbon, polyethylene glycol, pyridine, pyridone, pyrimidine, pyrrolidine, pyrrolidinone, quinazoline, quinoline, quinone, rauwolfia alkaloid,retinoid, salicylate, sorbitan, steroid, stilbene, sulfone, sulfonylurea, taxol, triazole, tropane, unsaturated fatty acid, or vinca alkaloid.

29. The method of Claim 17, whelcin said inhibitor is selected from said group consisting of the compounds listed in Tables 1 and 2.

30. A pharmaceutical composition produced by the process of Claim 17.

31. A method of increasing bioavailability of the active compound of an existing oral pharmaceutical composition, which comprises:
reformulating said existing composition to provide a reformulated composition by admixing said active compound with a bioenhancer comprising an inhibitor of cytochrome P450 3A enzyme or an inhibitor of P-glycoprotein-mediated membrane transport, said bioenhancer being present in sufficient amount to provide bioavailability of said pharmaceutical compound when administered in said reformulated composition greater than said bioavailability of said pharmaceutical compound when administered in said existing pharmaceutical composition.

32. The method of Claim 31, wherein said reformulated composition comprises all components present in said existing pharmaceutical composition plus said bioenhancer.

48.

33. The method of Claim 31, wherein said reformulated composition contains less than all components present in said existing pharmaceutical composition plus said bioenhancer.

34. A reformulated pharmaceutical composition produced by the process of Claim 31.

35. A method of identifying a compound useful for increasing bioavailability of a drug in a mammal, which comprises:
screening candidate compound by assaying for inhibition of a cytochrome P450 enzyme or of transport by P glycoprotein by said candidate compounds in the gut of said mammal, and selecting from said candidate compounds a compound or compounds that inhibit either said enzymes or said transport in said gut.

36. The method of Claim 35, wherein said assaying is for inhibition of said cytochrome P450 enzyme in said gut.

37. The method of Claim 35, wherein said cytochrome P450 enzyme is a member of a cytochrome P450 3A family.

38. The method of Claim 35, wherein said assaying measures inhibition of cytochrome P450 activity in isolated enterocytes or microsomes obtained from enterocytes or microsomes of said mammal.

39. The method of Claim 35, wherein said assaying measures inhibition of cytochrome P450 in a tissue or membrane from the gut of said mammal.

40. The method of Claim 35, wherein said assaying measures inhibition of cytochrome P450 in vivo in the gut of said mammal.

49.

41. The method of Claim 35, wherein said screening comprises assaying for both inhibitions.

42. The method of Claim 35, wherein said assaying measures inhibition of P
glycoprotein transport in isolated cells from the gut of said mammal.

43. The method of Claim 35, wherein said assaying measures inhibition of P
glycoprotein transport in a tissue or membrane from the gut of said mammal.

44. The method of Claim 35, wherein said assaying measures inhibition of P
glycoprotein transport in vivo in the gut of said mammal.

45. A method for increasing bioavailability of a topically administered hydrophobic pharmaceutical compound, which comprises:
topically administering said pharmaceutical compound to a mammal in need of treatment with said compound concurrently with a bioenhancer comprising an inhibitor of a cytochrome P450 3A enzyme or an inhabitor of P-glycoprotein-mediated membrane transport, said bioenhancer being present in sufficient amount to provide bioavailability of said compound in the presence of said bioenhancer greater than bioavailability of said compound in the absence of said bioenhancer.