EP1267898A2

EP1267898A2 - Bile acid containing prodrugs with enhanced bioavailability

Info

Publication number: EP1267898A2
Application number: EP01926709A
Authority: EP
Inventors: James E. Polli; Andrew Coop; Dean Y. Maeda; Kimberley A. Lentz
Original assignee: University of Maryland at Baltimore
Current assignee: University of Maryland at Baltimore
Priority date: 2000-04-07
Filing date: 2001-04-06
Publication date: 2003-01-02
Also published as: EP1267898A4; JP2003530329A; WO2001076531A3; CA2405151A1; WO2001076531A2; AU2001253226B2; AU5322601A; AU2006202213A1

Abstract

Many compounds have poor bioavailability or variable bioavailability because of poor absorption of the compound in the small intestine. Conjugation of the compound with bile acid to form a prodrug will increase the bioavailibility of the compound and/or reduce the bioavailability variability of the compound because of the active transport of the prodrug by the intestinal bile acid transporter and because of increased lipophilic nature of the prodrug. A linker group can be used between the bile acid and the compound. One example of a bile acid containing prodrug is acyclovir valylchenodeoxycholate, where valine is the linker group. Another example of this prodrug is atenolol cholic acid amide.

Description

BILE ACID CONTAINING PRODRUGS WITH

ENHANCED BIOAVAILABBLITY

Background of the Invention

Field of Invention

This invention relates to the method of increasing the bioavailability and

reducing the bioavailability variability of compounds by conjugating or linking a bile

acid to the compound. This invention also relates to the new composition of matter

obtained by attaching a bile acid to another compound to generate a prodrug. This

invention further relates to the usage of the bile acid transporter to actively move a

prodrug out of the lumen of the small intestine.

Description of the Related Art

Insufficient or variable intestinal permeability is a reason for inadequate oral

drug bioavailability. Many methods are available to increase bioavailability of various

drugs [1]. While some of these methods improve the bioavailability of some drugs,

not one method works for all compounds.

Some compounds that exhibit poor bioavailability, sub-optimal permeability,

or variable bioavailability may have some or all of the following characteristics [2]:

1. Less than complete oral absorption.

2. Permeability less than a suitable reference marker (e.g. metoprolol tartrate

[e.g. a permeability of 40 x IO^"6 cm/sec across Caco-2 monolayers]).

3. Molecular weight greater than 500 Daltons.

4. Hydrogen donors greater than five. 5. Hydrogen bond acceptors greater than 10.

6. Being substrates for P-glycoprotein efflux, Multi-drug Resistance-

associated Protein (MRP) efflux, or other efflux systems.

7. Not being substrates of or ligands for a carrier or transporter.

One potential method to increase the bioavailability of compounds with these

characteristics is to conjugate a bile acid to the drug or compound to create a prodrug.

A bile acid conjugated prodrug allows for an increase in bioavailability and/or a

reduction in the variability of the compound because of active transport of the

prodrug by the intestinal bile acid transporter (IB AT). The intestinal absorption of

bile acids is a sodium dependent process involving the human intestinal bile acid

transporter (hIBAT) and Na K⁺ ATP-ase [3].

The bile acid transporter is an ideal candidate for drug targeting because the

human IBAT has a high transport capacity of 10 grams per day [4, 5], and bile acids

are one of the largest molecules taken up by a carrier mediated system [6]. In light of

this biology, the bile acid transporter appears to be a promising mechanism to

improve oral drug absorption by incorporating a bile acid moiety with an active drug

in a prodrug fashion. The bile acid prodrug approach to targeting the bile acid

transporter in various tissues has been investigated with peptides [7, 8, 9], HMG-

CoA reductase inhibitors [8], and chlorambucil [10]. This work was directed either at

extremely small molecules or for targeting delivery of drugs to the liver. Other

attempts at drug therapy involve inhibiting the bile acid transporter to reduce

cholesterol synthesis in the liver [11]. One compound that may benefit from bile acid conjugation to improve uptake

in the small intestine is acyclovir. Acyclovir is an anti- viral compound, used to inhibit

herpes virus proliferation. Its target is not the liver but all tissues within the body.

Typical treatment requires 200 mg doses administered five times daily, with a

bioavailability after oral administration of 20% [12].

A different prodrug strategy has proved successful at improving the oral

bioavailability of acyclovir [13, 14]. Valacyclovir, the L-valine ester prodrug of

acyclovir, has an oral bioavailability of 54% [15]. This improved bioavailability for

valacyclovir allows for a more convenient dosing regimen of 1000 mg, twice daily,

with similar clinical efficacy as previously found for the acyclovir parent compound

[16]. Valacyclovir is a substrate for the human intestinal peptide transporter

(PepTl), with a Kj=4.08 mM in PepTl expressing Xenopus laevis oocytes [17] and

Kf=1.10 mM in stable lines of CHO/PepTl [18].

In comparison to PepTl, IB AT has the potential advantages of higher

capacity and micromolar affinity. Therefore, it is possible to further enhance the oral

bioavailability of acyclovir by synthesizing a cholic acid ester prodrug to target the

IB AT. It is also possible to increase the bioavailability of other compounds by

conjugating the compounds to a bile acid.

Furthermore, animals other than humans have an intestinal bile acid

transporter. One can use the animal intestinal bile acid transport and a bile acid

conjugated prodrug to increase the adsorption of the prodrug and bioavailability of the

compound in animals. Brief Description of the Invention

It is an object of this invention to increase the bioavailability of a compound

by conjugating a compound to a bile acid to create a prodrug. It is a further object of

this invention that the prodrug is administered orally to an animal or human. It is a

further object of this invention that the intestinal bile acid transporter binds to the

prodrug and moves the prodrug from the lumen of the small intestine into brush

border cells. It is a further object of this invention that the prodrug or compound

moves from inside the brush border cells into the blood stream.

It is an object of this invention to increase the bioavailability of a compound

by conjugating a compound to a bile acid to create a prodrug. It is another object of

this invention that a metabolically labile bond exist between the compound and the

bile acid. It is a further object of this invention that the prodrug is administered orally

to an animal or human. It is a further object of this invention that the intestinal bile

acid transporter binds to the prodrug and moves the prodrug from the lumen of the

small intestine into brush border cells. It is a further object of this invention that the

prodrug or compound moves from inside the brush border cells into the blood stream.

It is an object of this invention to increase the bioavailability of a compound

by linking a compound to a bile acid via a linker group to create a prodrug. It is a

further object of this invention that the prodrug is administered orally to an animal or

human. It is a further object of this invention that the intestinal bile acid transporter

binds to the prodrug and moves the prodrug from the lumen of the small intestine into

brush border cells. It is a further object of this invention that the prodrug or compound moves from inside the brush border cells into the blood stream. It is

another object of this invention that the linker group be any bifunctional chemical

moiety that achieves one or more of the following four functions: (1) facilitates the

synthesis of the prodrug, (2) aid or enhance the binding of the prodrug to the intestinal

bile acid transporter (IBAT), (3) make it easier for the compound to disassociate from

the bile acid after the prodrug has passed out of the lumen of the small intestine, (4)

enhance the solubility of the prodrug inside the body. It is another object of this

invention that the linker group be any size, but more preferably be less than 200

daltons. It is a further object of this invention that a metabolically labile bond exists

between the linker group and the compound, between the linker group and the bile

acid, or within the linker group itself.

It is an object of this invention to increase the bioavailability of a biologically

active compound by conjugating a biologically active compound to a bile acid to create

a prodrug. It is a further object of this invention that a metabolically labile bond exist

between the biologically active compound with the bile acid. It is a further object of

prodrug and moves the prodrug from the lumen of the small intestine into brush

moves from inside the brush border cells into the blood stream.

It is an object of this invention to conjugate a compound to a bile acid to form

a prodrug. It is an object of this invention to have a pharmaceutical compound containing

a compound and a bile acid.

It is another object of this invention to have a pharmaceutical compound

containing a compound, a metabolically labile bond, and a bile acid.

It is an object of this invention to have a pharmaceutical compound containing

an agent, a linker group, and a bile acid. It is another object of this invention to have a

pharmaceutical compound containing an agent, a linker group, a bile acid, and a

metabolically labile bond.

It is an object of this invention to have a pharmaceutical compound containing

an agent with biological activity and a bile acid.

It is another object of this invention to have a pharmaceutical compound

containing an agent with biological activity, a metabolically labile bond, and a bile acid.

It is an object of this invention to have a pharmaceutical compound containing

an agent with biological activity, a linker group, and a bile acid. It is another object of

this invention to have a pharmaceutical compound containing an agent with biological

activity, a linker group, a bile acid, and a metabolically labile bond.

It is an object of this invention to utilize the intestinal bile acid transporter to

actively uptake and remove from the lumen of the small intestine a bile acid containing

prodrug.

It is an object of this invention to have a method for increasing the

bioavailability of a compound by using the intestinal bile acid transporter to actively uptake and remove from the lumen of the small intestine a bile acid containing

prodrug.

It is another object of this invention to use a linker group to link a compound

to a bile acid to create a prodrug. It is further object of this invention that the linker

group be any bifunctional chemical moiety that achieves one or more of the following

four functions: (1) facilitates the synthesis of the prodrug, (2) aid or enhance the

binding of the prodrug to the intestinal bile acid transporter (IBAT), (3) make it easier

for the compound to disassociate from the bile acid after the prodrug has passed out

of the lumen of the small intestine, (4) enhance the solubility of the prodrug inside the

body. It is another object of this invention that the linker group be any size, more

preferably less than 200 daltons. It is also an object of this invention that the linker

group, when attached to the compound or the bile acid, result in a metabolically labile

bond being present.

It is an object of this invention to reduce the bioavailability variability of a

compound by conjugating a compound to a bile acid to create a prodrug. It is a further

object of this invention that the prodrug is administered orally to an animal or human.

It is a further object of this invention that the intestinal bile acid transporter binds to

the prodrug and moves the prodrug from the lumen of the small intestine into brush

moves from inside the brush border cells into the blood stream.

compound by conjugating a compound to a bile acid to create a prodrug. It is another object of this invention that a metabolically labile bond exist between the compound

and the bile acid. It is a further object of this invention that the prodrug is

administered orally to an animal or human. It is a further object of this invention that

the intestinal bile acid transporter binds to the prodrug and moves the prodrug from

the lumen of the small intestine into brush border cells. It is a further object of this

invention that the prodrug or compound moves from inside the brush border cells into

the blood stream.

compound by linking a compound to a bile acid via a linker group to create a prodrug.

It is a further object of this invention that the prodrug is administered orally to an

animal or human. It is a further object of this invention that the intestinal bile acid

transporter binds to the prodrug and moves the prodrug from the lumen of the small

intestine into brush border cells. It is a further object of this invention that the

It is another object of this invention that the linker group be a bifunctional chemical

moiety. It is another object of this invention that the linker group be any size, but

more preferably be less than 200 daltons. It is a further object of this invention that a

metabolically labile bond exists between the linker group and the compound, between

the linker group and the bile acid, or within the linker group itself.

biologically active compound by conjugating a biologically active compound to a bile

acid to create a prodrug. It is a further object of this invention that a metabolically labile bond exist between the biologically active compound with the bile acid. It is a

brush border cells. It is a further object of this invention that the prodrug or

compound moves from inside the brush border cells into the blood stream.

It is an object of this invention to reduce or prevent an adverse drug-drug

interaction by linking or conjugating a bile acid to at least one of the compounds which

is involved in the adverse drug-drug interaction to create a prodrug. It is a further

object of this invention to use the intestinal bile acid transporter to uptake the

prodrug, thereby avoiding a transporter that is involved in the adverse drug-drug

interaction.

It is an object of this invention to reduce or prevent an adverse drug-nutrient

interaction by linking or conjugating a bile acid to a compound which is involved in the

• adverse drug-nutrient interaction to create a prodrug. It is a further object of this

invention to use the intestinal bile acid transporter to uptake the prodrug, thereby

avoiding a transporter that is involved in the adverse drug-nutrient interaction.

It is an object of this invention to increase the lipophilicity of a compound by

linking or conjugating a bile acid to the compound. It is a further object of this

invention that the increased lipophilicity will increase the bioavailability and reduce

the variability of the bioavailability of the compound. It is an object of this invention that the compound component of the prodrug

will be cleaved from the prodrug after the prodrug binds to the intestinal bile acid

transporter.

It is an object of this invention that the prodrug be coated with a substance

that protects the prodrug from the acidic environment of the stomach and that does

not inhibit absorption of the prodrug in the intestine.

It is an object of this invention to have a pharmaceutical compound of

acyclovir valyldeoxycholate.

It is an object of this invention to have a pharmaceutical compound of

acyclovir valylchenodeoxycholate.

It is an object of this invention to have a pharmaceutical compound of atenolol

cholic acid amide.

Description of Several Views of the Drawings

Figure 1A illustrates the general structure of bile acids.

Figure IB illustrates the general structure of the prodrug.

Figure 1C generalizes the synthesis of acyclovir valylchenodexycholate.

Figure ID generalizes the synthesis of acyclovir valyldeoxycholate.

Figure IE generalizes the synthesis of atenolol cholic acid amide.

Figure 2 illustrates the competitive inhibition of uptake of ³H-taurocholate by

acyclovir valylchenodeoxycholate (acyclovir vCDC) (•), chenodeoxycholate (CDC)

(o), and valacyclovir (Δ). Figure 3 illustrates the competitive inhibition of uptake of ³H-taurocholate by

acyclovir valyldeoxycholate (acyclovir vDC) (•), deoxycholate (DC) (o), and

valacyclovir (Δ).

Figure 4 illustrates the competitive inhibition of uptake of ³H-taurocholate by atenolol

cholic acid amide (•), cholate (o), and atenolol (Δ).

Figure 5 shows the concentration dependence of ³H-taurocholate uptake in COS-

hlBAT (COS cells transfected with hIBAT) in HBSS (♦) and in MHBSS (no sodium)

(^■)-

Figure 6 illustrates the inhibition constant (Kj) of various bile acids and other agents

by inhibiting ³H-taurocholate uptake in COS cells transfected with hIBAT.

Detailed Description of the Invention

In the preferred embodiment, this invention is a prodrug containing a bile acid

attached to a compound. In an alternative embodiment, more than one bile acid can be

attached to a compound. Alternatively, more than one compound can be attached to

one bile acid. Alternatively, a complex of one or more bile acids can be attached to one

or more compounds.

When used wherein, the word "compound" includes, but is not limited to, a

pharmaceutical drug, a biologically active agent, a metabolic precursor to a

pharmaceutical drug, a metabolic precursor to a biologically active agent, or any other

agent which one would want to administer to an animal or human. A compound may

be therapeutic or diagnostic in nature. A compound can also be nutritionally beneficial

to an animal or human. It is preferable that a metabolically labile bond exist between a compound and

the bile acid to which the compound is attached or conjugated. By way of example

only, a metabolically labile bond can be an ester, amide, carbamate, carbonate, ether,

urea, anhydride, or sulfur containing derivatives, such as thioamides, thioesters,

thiocarbamates, and thioureas.

In an alternative embodiment, a linker group exists between a compound and

the bile acid to which the compound is attached. The linker group can be any

bifunctional chemical moiety but preferably has a molecular weight less than 200

daltons. Four reasons exist for using a linker group; any bifunctional chemical moiety

that achieves any one of these reasons is considered a linker group. First, the linker

group can facilitates the synthesis of the prodrug. Second, the linker group can aid or

enhance the binding of the prodrug to the intestinal bile acid transporter (IBAT).

Third, the linker group can make it easier for the compound to disassociate from the

bile acid after the prodrug has passed out of the lumen of the small intestine. Fourth,

the linker group can enhance the solubility of the prodrug inside the body.

Different types of linker groups can be used. Linker groups can be, for

example, natural and unnatural amino acids, di-acids, di-amines, di-alcohols, sulphate,

phosphate, sulfur containing moieties, amino alcohols, hydroxyacids, and polymers.

Examples of the amino acids are valine, glycine, taurine, alanine, leucine, tyrosine,

aspartate, glutamate, lysine, arginine, asparagine, cysteine. Examples of the di-acids

are oxalic acid, fumaric acid, succinic acid, maleic acid, and tartaric acid. Examples of

the di-amines include ethylenediamine, propylenediamine, 1,3-diaminopropane, 1,4- diaminobutane, 1,5-diaminopentane, piperazine, homopiperazine, and 3-

aminopiperidine. Examples of the di-alcohols are ethyleneglycol, propyleneglycol,

1,4-butanediol, polyethyleneglycol, and 1,5-pentanediol. Examples of the sulfur

containing moieties are mercaptoacetic acid, mercaptopropanoic acid, mercaptobenzyl

alcohol, 2-mercaptoethanol, 3-mercaptopropanol, and 4-mercaptobutanol. Examples

of the amino alcohols are 2-aminoethanol, 3-aminopropanol, 4-aminobutanol, 4-

hydroxypiperidine, and 3-hydroxypiperidine. Examples of the hydroxyacids include

3-hydroxypropanoic acid, 4-hydroxybutanoic acid, and 5-hydroxypentanoic acid.

One example of polymers is polyethylene glycol.

It is preferably that the linker group has a metabolically labile bond for easy

cleavage of the prodrug into the compound and the bile acid. By way of example

thiocarbamates, and thioureas.

These bile acid containing prodrugs are administered orally and absorbed in the

small intestine. The intestinal bile acid transporter actively transports the prodrug

from inside the lumen of the small intestine into brush border cells. Once inside brush

border cells, the prodrug may pass passively through the cell membrane down a

concentration gradient into the blood stream or be actively transported out of the

brush border cells into the blood stream. This active transport of the prodrug out of

the lumen of the small intestine occurs because of the affinity of the intestinal bile acid

transporter for its ligand, bile acids. Intracellular or extracellular enzymes cleave one of the bonds between the bile

acid and the compound either (1) after the prodrug is inside the brush border cells, (2)

attached to the outside of the brush border cells, (3) located in proximity to the brush

border cells, (4) in the blood stream, or (5) at other location inside the body, except

cleavage should not occur in the stomach or lumen of the small intestine.

This active transport of a prodrug by the intestinal bile acid transport

produces enhanced bioavailability of the compound when compared to the

imconjugated compound. It also reduces bioavailability variability and provides for an

alternative mechanism for compound permeation, including the avoidance of efflux

pumps such as P-glycoprotein used by some drugs (e.g. , fexofenadine) and other drug

transporters and/or nutrient transporters. By avoiding other drug transporters and/or

nutrient transporters, drug-drug and/or drug-nutrient interactions can be reduced or

eliminated.

Any bile acid may be conjugated or linked to a compound. More than one bile

acid may be conjugated or linked to one compound. Also more than one compound

may be conjugated or linked to one bile acid. The bile acids are, by way of example,

cholate, glycocholate, taurocholate, deoxycholate, glycodeoxycholate,

taurodeoxycholate, chenodeoxycholate, ursodeoxycholate, glycochenodeoxycholate,

taurochenodeoxy cholate, and lithocholate. The general structures of the bile acids are

shown in Figure 1 A.

The following structural requirements generally promote recognition of a bile

acid by the intestinal bile acid transporter (IBAT): negatively charged side groups on the bile acid; at least one alpha oriented hydroxyl group at the steroid nucleus at

position 3, 7, or 12; and a cis configuration of rings A and B of the steroid nucleus [3].

By way of example, compounds that have improved bioavailability as a result

of conjugating or linking the compound to a bile acid are those compounds which

contain (Group 1) an alcohol function which can be chemically coupled to the bile acid

to give an ester; or (Group 2) a primary or secondary amine function which can be

chemically coupled to the bile acid to give an amide; or (Group 3) an acid function

which can be chemically coupled to the bile acid to give an anhydride.

Compounds which contain an alcohol function (Group 1) may include, but are

not limited to, anti-viral agents (e.g. 6-deoxyacyclovir, ganciclovir,

dihydroxybutylguanine, foscarnet, penciclovir, famciclovir, zidovudine, idoxuridine, 5-

trifluorothymidine, vidarabine, cytarabine, ribavirin). Compounds which contain a

primary or secondary amine function (Group 2) may include, but are not limited to,

H-2 antagonists (e.g. derivatives of cimetidine, ranitidine, nizatidine, famotidine,

roxatidine). Compounds which contain an acid function (Group 3) may include, but

are not limited to, bisphosphonates (e.g. alendronate, etidronate, pamidronate,

tiludronate, clonronate) and ACE inhibitors (e.g. enalapril, captopril, lisonopril). This

list is not exhaustive and is not meant to limit the scope of this invention, but is

provided by way of illustration only.

Any salts, solvates, isomeric compositions, and physical forms of these

compounds and/or their derivatives can be conjugated or linked to a bile acid to form a prodrug. One can also use known in the art field enteric coating to protect prodrugs

from the acidity of the stomach.

Figure 1 A illustrates the general structure of bile acids. Bile acids can vary in

their substituents at R_1; R₂, and R₃ (i.e. positions 12, 7, and 3, respectively). For

example, for chenodeoxycholate, Ri is H, R₂ is -OH, and R₃ is -OH. For

deoxycholate, R_! is α-OH, R₂ is H, and R₃ is α-OH. For cholate, R_l9 R₂, and R₃ are

all α-OH. Many natural and synthetic bile acids exist and are well-known in the art

field. Any of these natural or synthetic bile acids can be used to create a prodrug.

However, it may be preferable to have at least one alpha orientated hydroxyl group in

the bile acid and this alpha orientated hydroxyl group can be located at Rj, R₂, or R₃.

Figure IB illustrates the general structure of bile acid component of the

prodrug. Either directly or via a linker group, a compound can be attached to any

natural or synthetic bile acid at either R_l5 R₂, R₃, or R . While it may be preferable to

have only one compound conjugated or linked to one bile acid, more than one

compound can be conjugated or linked to the same bile acid. In addition, while it may

be preferable to have the compound linked or conjugated to a bile acid at ₄ (because

of potential structural requirements necessary for recognition of a bile acid by the

intestinal bile acid transporter), it is possible to link or conjugate compounds to a bile

acid at R_ls R₂, or R₃. When a compound is linked or conjugated to either R_l3 R₂, or R₃,

then it is preferable that R be any chemical moiety which can aid or enhance the

binding of the prodrug to the IBAT and/or can increase the solubility of the prodrug inside the body. Examples of such chemical moieties include hydroxyl, any amino

acid (such as glycyl, valyl, alanyl, tauryl, and leucyl), any di-amino acid (such as

glycyl-valyl, glycyl-glycyl, valyl-glycyl, alanyl-glycyl, alanyl-valyl, and leucyl-valyl),

any tri-amino acid (such as glycyl-glycyl-glycyl, glycyl-valyl-glycyl, glycyl-glycyl-

alanyl, valyl-glycyl-leucyl, alanyl-rglycyl-glycyl, alanyl-valyl-glycyl, and leucyl-valyl-

valyl). It is preferable that this chemical moiety have a molecular weight less than

1500 daltons.

Figures 1C, ID, and IE generalize the synthesis of three prodrugs, acyclovir

valylchenodeoxycholate, acyclovir valyldeoxycholate, and atenolol cholic acid amide,

which have increased bioavailability and reduced bioavailability variability as a result

of conjugating or linking the compounds, acyclovir and atenolol, to a bile acid. For

these three prodrugs, the compounds are attached to a bile acid (chenodeoxycholate,

deoxycholate, or cholic acid) at R For acyclovir valylchenodeoxycholate and

acyclovir valyldeoxycholate, valine is utilized as a linker group. Furthermore, both

acyclovir valylchenodeoxycholate and acyclovir valyldeoxycholate use an ester as a

metabolically labile bond that can be cleaved to release acyclovir. For atenolol cholic

acid amide, no linker group is used, but rather atenolol is conjugated directly to cholic

acid. Furthermore, atenolol cholic acid uses an amide as a metabolically labile bond

that can be cleaved to release atenolol.

Acyclovir Valylchenodeoxycholate Synthesis

As in Figure 1C, to synthesize acyclovir valylchenodeoxycholate, 1,

isobutylchloroformate (iBuOCOCl; 130 μL, 1 mmol) is added dropwise to a cooled (-15 °C) solution of chenodeoxycholate, 2, (1 mmol) and triethylamine (140 μL, 1

mmol) in N,N-dimethylformamide (DMF) (10 mL) under an nitrogen (N₂)

atmosphere. After 1.5 minutes, valacyclovir, 3, (0.42 g, 1.3 mmol) and triethylamine

(NEt₃; 280 μL, 2 mmol) are then added to the reaction mixture as a solution in DMF

(5 mL). The reaction is kept at -15 °C for 0.5 hours, then is warmed to room

temperature for 1 hour. The triethylammonium chloride formed during the reaction is

filtered off, and the filtrate is concentrated by rotary evaporation. The crude material

is then purified using silica gel flash chromatograpy with MeOH/CHCl₃ (1 :4, 250 mL)

as the eluent.

The acyclovir valylchenodeoxycholate, 1, prodrug synthesis is monitored

using thin layer chromotography (TLC) plates coated with silica gel GHLF-0.25 mm

plates (60 F₂₅ ) manufactured by Analtech, Inc. (Newark, DE). Fast-atom

bombardment mass spectrometry (FAB-MS) and high resolution mass spectrometry

(HRMS) spectra are obtained on a Jeol SX 102 mass spectrometer in the positive ion

mode. Proton nuclear magnetic resonance (NMR) spectrometry is performed in de-

dimethyl sulfoxide (DMSO) on a 300 MHz General Electric Aquerius model

spectrometer controlled by a Macintosh Power Mac 7100 using MacNMR v. 5.0

software. The purity of the bile acid conjugate is determined by analysis on a

Beckman System Gold high pressure liquid chromotography (HPLC) system

consisting of a model 126 solvent module, model 168 detector, and model 507

autosampler. The HPLC column used is a Vydac analytical column (C₁₈, 300 A, 5μm, 4.6 x 250 mm) equipped with a guard cartridge. Solvent A is aqueous 0.1%

trifluoroacetic acid (TFA) and solvent B is acetonitrile containing 0.1% TFA. The

conjugate is eluted using a linear gradient of 5 to 75% B over 50 minutes at a flow rate

of 1.0 mL/min and detected at 214 nm.

The amount of acyclovir valylchenodeoxycholate, 1, purified is 0.62 g (89%).

Additionally, TLC R_f(MeOH/CHCl₃, 1:4) = 0.46; HPLC R_t = 32.1 min (99.3 %

purity); and ESI-MS [M+H] = 700.4. HRMS (calculated for C₃₇H₅₉O₇N₆): 699.4445.

found 699.4454. The NMR spectra contained peaks consistent with both

chenodeoxychloate and valacyclovir portions. Coupling through the amino acid amine

(and not the aniline) was confirmed through the presence of the NH₂ signal at 5.3

ppm.

, Acyclovir Valyldeoxycholate Synthesis

As in Figure ID, to synthesize acyclovir valyldeoxycholate, 4,

isobutylchloroformate (iBuOCOCl; 130 μL, 1 mmol) is added dropwise to a cooled

(-15 °C) solution of deoxy cholate, 5, (1 mmol) and triethylamine (140 μL, 1 mmol) in

N,N-dimethylformamide (DMF) (10 mL) under an nitrogen (N₂) atmosphere. After

1.5 minutes, valacyclovir, 3, (0.42 g, 1.3 mmol) and triethylamine (NEt₃; 280 μL, 2

mmol) are then added to the reaction mixture as a solution in DMF (5 mL). The

reaction is kept at -15 °C for 0.5 hours, then is warmed to room temperature for 1

hour. The triethylammonium chloride formed during the reaction is filtered off, and the filtrate is concentrated by rotary evaporation. The crude material is then purified

using silica gel flash chromatograpy with MeOH/CHCl₃ (1 :4, 250 mL) as the eluent.

The acyclovir valyldeoxycholate, 4, prodrug synthesis is monitored using thin

layer chromotography (TLC) plates coated with silica gel GHLF-0.25 mm plates (60

F ₅ ) manufactured by Analtech, Inc. (Newark, DE). Fast-atom bombardment mass

spectrometry (FAB-MS) and high resolution mass spectrometry (HRMS) spectra are

obtained on a Jeol SX 102 mass spectrometer in the positive ion mode. Proton

nuclear magnetic resonance (NMR) spectrometry is performed in d₆-dimethyl

sulfoxide (DMSO) on a 300 MHz General Electric Aquerius model spectrometer

controlled by a Macintosh Power Mac 7100 using MacNMR v. 5.0 software. The

purity of the bile acid conjugate is determined by analysis on a Beckman System Gold

high pressure liquid chromotography (HPLC) system consisting of a model 126

solvent module, model 168 detector, and model 507 autosampler. The HPLC column

used is a Vydac analytical column (C₁₈, 300 A, 5μm, 4.6 x 250 mm) equipped with a

guard cartridge. Solvent A is aqueous 0.1%> trifluoroacetic acid (TFA) and solvent B is

acetonitrile containing 0.1% TFA. The conjugate is eluted using a linear gradient of 5

to 75% B over 50 minutes at a flow rate of 1.0 mL/min and detected at 214 nm.

The amount of acyclovir valyldeoxycholate, 4, purified is 0.59 g (85%).

Additionally, TLC R_f(MeOH/CHCl₃, 1:4) = 0.47; HPLC R_t = 33.0 min (98.1 %

purity); FAB-MS [M+H]⁺ = 699.54; HRMS (calculated for C₃₇H₅₉O₆N₇): 699.4445,

found: 699.4448. The NMR spectrum contained peaks consistent with both deoxycholic acid and valacyclovir portions. Coupling through the amino acid amine

(and not the aniline) was confirmed through the presence of the NH₂ signal at 5.3ppm.

Atenolol Cholic Acid Amide Synthesis

As in Figure IE, to synthesize atenolol cholic acid amide, 6,

isobutylchloroformate (iBuOCOCl; 130 μL, 1 mmol) is added dropwise to a cooled

(-15 °C) solution of cholic acid, 7, (0.5 mmol) and triethylamine (140 μL, 1 mmol) in

N,N-dimethylformamide (DMF) (10 mL) under an nitrogen (N₂) atmosphere. After

1.5 minutes, atenolol, 8, (0.42 g, 1.3 mmol) and triethylamine (NEt₃; 280 μL, 2 mmol)

are then added to the reaction mixture as a solution in DMF (5 mL). The reaction is

kept at -15 °C for 0.5 hours, then is warmed to room temperature for 1 hour. The

triethylammonium chloride formed during the reaction is filtered off, and the filtrate is

concentrated by rotary evaporation. The crude material is then purified using silica gel

flash chromatograpy with MeOH/CHCl₃ (1:4, 250 mL) as the eluent.

The atenolol cholic acid amide, 6, prodrug synthesis is monitored using thin

F₂₅ ) manufactured by Analtech, Inc. (Newark, DE). Fast-atom bombardment mass

spectrometry (FAB-MS) and high resolution mass spectrometry (HRMS) spectra are

obtained on a Jeol SX 102 mass spectrometer in the positive ion mode. Proton

nuclear magnetic resonance (NMR) spectrometry is performed in d₆-dimethyl

sulfoxide (DMSO) on a 300 MHz General Electric Aquerius model spectrometer

controlled by a Macintosh Power Mac 7100 using MacNMR v. 5.0 software. The purity of the bile acid conjugate is determined by analysis on a Beckman System Gold

high pressure liquid chromotography (HPLC) system consisting of a model 126

solvent module, model 168 detector, and model 507 autosampler. The HPLC column

guard cartridge. Solvent A is aqueous 0.1 % trifluoroacetic acid (TFA) and solvent B is

acetonitrile containing 0.1 % TFA. The conjugate is eluted using a linear gradient of 5

to 75% B over 50 minutes at a flow rate of 1.0 mL/min and detected at 214 nm.

The amount of atenolol cholic acid amide, 6, purified is 0.19 g (58%).

Additionally, TLC R_f(AcOH/MeOH/CHCl₃, 1:1:4) = 0.47; HPLC R_t = 34.9 min (98.3

% purity); FAB-MS [M+H]⁺ = 657.6; HRMS (calculated for C₃₈H₆₁N₂O₇):

657.4479, found: 657.4470; IR (CHC1₃) 3394, 2932, 2870, 1671, and 1610 cm^"1.

Atenolol, 8, contains both a secondary alcohol and a secondary amine, and

coupling to cholic acid, 7, through both of these nucleophiles could occur to give an

ester or an amide, respectively. Only the amide is formed during the above reaction of

cholic acid and atenolol which is confirmed through the presence of characteristic

amide C=O stretching bands at 1671 cm^"1 and 1610 cm^"1 in the IR spectrum. Also,

NMR spectrum is consistent with that expected for a conjugate between cholic acid

and atenolol {i.e., the absence of the secondary amine proton signal at 4.92 ppm in the

NMR). Coupling through the amine of atenolol (and not the alcohol) is also confirmed

by a negative chloranil test, a test for secondary and primary amines. Atenolol is used

as the positive control for the chloranil test. Bioavailability Assays

To test the increase of bioavailability of acyclovir valylchenodeoxycholate, 1,

acyclovir valyldeoxycholate, 4, and atenolol cholic acid amide, 6, the human intestinal

bile acid transporter (hIBAT) cDNA (specifically, pCMV5-hIBAT expression

plasmid) is transformed into competent DH5α cells according to the Life

Technologies protocol (Grand Island, NY), using LipofectAMINE 2000 transfection

reagent (Life Technologies, Grand Island, NY). Cell dilutions are streaked on nutrient

agar plates containing 50 μg/ml ampiciUin, and incubated at 37°C overnight. Isolated

colonies are selected aseptically, and are used to inoculate a 200 ml nutrient broth

culture containing 50 μg/ml ampiciUin. The culture is incubated for 24 hours at 225

RPM's and 37°C. cDNA is isolated from the broth cultures using a Quigan maxi-prep

kit (DNA plasmid maxi kit #12162; Valencia, CA). After an isopropanol

precipitation and ethanol wash, the DNA is reconstituted in 600 μl of sterile

deionized water. DNA concentration is 1.18 μg/μl, determined by spectrophotometry

at 260 nm. The 260/280 absorbance ratio is 1.50, indicating the DNA is free from

RNA contamination. pCMV5-hIBAT is also digested with the restriction

endonucleases, BAMH1 and NDE, and results in bands of appropriate size (4238 bp

and 1689 bp) when electrophoresed on a 1% agarose gel.

COS-7 cells are grown in T-75 flasks at 37°C, 5% CO₂ and 95% RH using

Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS. Cells

are passaged at 80-90%) confmency using a 0.25% trypsin/0.20% EDTA solution, and are plated at a density of 8x10⁴ cells per well (1.88 cm²). Cells are transfected at 24

hours post seeding. For each well transfected, 0.8 μg hIBAT cDNA is combined with

LipofectAMINE 2000 reagent and incubated at room temperature for 20 minutes to

allow complexes to form. A volume of 100 μl of hIBAT-lipid complex is added to

each well of cells. Transfected cells are incubated at 37°C, 5% CO₂ and 95% RH for

24 hours, until ready for uptake assay.

Uptake studies are performed^" on COS-hlBAT cells at 24 hours post-

transfection. Uptake buffer consists of either a Hank's Balanced Salts Solution

(HBSS) containing 137 mM NaCl or a Modified Hank's Balanced Salts Solution

(MHBSS) that replaced the sodium chloride with 137 mM tetraethylammonium

chloride [19]. Because bile acid transport is sodium dependent, this MHBSS

approach allows for the simple modification of the uptake buffer to exclude all sources

of sodium, and thus enables for a control uptake assay under sodium-free conditions.

For the K_m and V_max studies of taurocholate in the COS-hlBAT cells, cell

culture medium is removed from each well and is replaced with 0.5 ml of uptake

solution. 0.5 μM ³H-taurocholate is investigated in HBSS. Cells are incubated at

37°C and 100 RPM's for 10 minutes. Uptake solution is removed, and the cells are

washed three times with ice cold HBSS. Cells are lysed, neutralized, and counted for

associated radioactivity. Each well is analyzed for protein content using the Lowry

method [20]. This uptake assay is performed in triplicate, under both sodium and

sodium-free conditions. The saturable uptake of ³H-taurocholate is determined using the following

equation (Formula 1):

— = ^Vm* ^{' S} + k„S (Formula 1 ) dt K_m + S "

where V_max and K_m represent the Michaelis-Menten constants, k_p is the passive

uptake rate constant, S is the concentration of taurocholate, and dM/dt is the uptake

rate of taurocholate into the COS-hlBAT cells. This approach includes the

contribution of passive uptake to the saturable kinetics of taurocholate uptake into the

cells.

In the competitive inhibition studies (see Figures 2, 3, and 4), the

concentration of acyclovir valylchenodeoxycholate, 1, varies between 10 μM and 400

μM; the concentration of acyclovir valyldeoxycholate, 4, varies between 10 μM and

600 μM; and the concentration of atenolol cholic acid amide, 6, varies between 10 μM

and 400 μM. Chenodeoxycholate, 2, deoxycholate, 5, and cholate inhibition studies

are conduced as positive controls. Valacyclovir and atenolol inhibition studies are

performed as negative controls.

Figure 2 illustrates the inhibition study of valacyclovir, 3, (Δ),

chenodeoxycholate (CDC), 2, (o), and acyclovir valylchenodeoxycholate (acyclovir

vCDC), 1, (•). There is no inhibition of the bile acid transporter in COS-hlBAT cells

after application of up to 600 μM valacyclovir, indicating that valacyclovir is not a

substrate for hIBAT. A chenodeoxycholate inhibition study is performed as a positive control for this experiment, and results in Kj =5.4 (±0.4) μM, which indicates

that the COS-hlBAT model is suitable for the inhibition study. The calculated Kj for

acyclovir valylchenodeoxycholate is K; =35.6 (±4.4) μM, indicating a very strong

interaction of acyclovir valylchenodeoxycholate for the hIBAT transporter.

Similiarly for acyclovir valyldeoxycholate (Figure 3), inhibition study of

valacyclovir, 3, (Δ), deoxycholate (DC), 5, (o), and acyclovir valyldeoxycholate

(acyclovir vDC), 4, (•) are performed. Acyclovir valyldeoxycholate strongly interacts

with hIBAT with a Kj=401 (±50) μM.

Likewise, in Figure 4, atenolol cholic acid amide, 6, (•) strongly interacts with

hIBAT with a Kf= 160 (±21) μM, while atenolol, 8, (Δ) itself does not interact. The

natural bile acid cholate, 7, (o) also strongly interacts with hIBAT (± 2.4)

μM].

Figure 5 shows both the sodium dependence and saturation of ³H-taurocholate

uptake. The uptake rate is measured at ³H-taurocholate concentrations from 0.1 to

125 μM in HBSS with 137 mM NaCl and in MHBSS which contains no sodium. As

expected, carrier mediated ³H-taurocholate uptake is not present in the absence of

sodium ions, thus establishing a baseline permeability due to the passive permeability

of taurocholate into the cells. A passive uptake rate constant (k_p) of 0.12 (pmoles/min

mg proteinVμM is estimated using linear regression. Control uptake studies are also

performed in untransfected COS-7 cells, and in COS-7 cells transfected with the

antibiotic resistant vector, pcDNA3. The passive uptake of ³H-taurocholate in these experiments is the same as that obtained under sodium-free conditions (data not

shown). The kinetic parameters for carrier mediated uptake are estimated using

WinNonlin (version 1.0), and yields a K_m=12.0(±2.2) μM and a V_max=126.0(±5.9)

pmoles/min/mg protein. These Michaelis-Menten parameters are in close agreement

to those obtained previously [21].

Competitive inhibition studies are performed in HBSS containing 0.25 μM ³H-

taurocholate, and varying the concentration of unlabeled bile acid from 1 to 100 μM.

Glycine, taurine, acyclovir, and atenolol inhibition studies are performed as negative

controls. Incubation conditions and analysis are performed as described for the K_m

and V_max studies. The following equation (Formula 2) is used to estimate the Kj for a

series of naturally occurring bile acids:

(Formula 2) where V_max and K_m are the Michaelis-Menten parameters for taurocholate uptake, S is

0.25 μM ³H-taurocholate, dM/dt is the uptake rate of taurocholate, and I is the

concentration of inhibitor applied to the cells.

The effect of various bile acids (cholate, glycocholate, taurocholate,

deoxycholate, taurodeoxycholate, glycodeoxycholate, chenodeoxycholate,

glycochenodeoxycholate, taurochenodeoxycholate, ursodeoxycholate, and lithocholate)

on the uptake of ³H-taurocholate into COS-hlBAT cells is determined (see Figure 6).

Inhibition studies using glycine, taurine, valacyclovir, and atenolol are also performed as negative controls. Figure 6 shows the K; value (± SEM) for each compound tested.

All bile acids inhibit the uptake of ³H-taurocholate into COS-hlBAT cells. However,

the uptake of ³H-taurocholate is not reduced in the presence of glycine or taurine.

Uptake of ³H-taurocholate decreases in the presence of lithocholate, but never reaches

50% maximal velocity at a lithocholate concentration of 100 μM. Lithocholate

concentrations above 100 μM were not investigated. All bile acids inhibit the uptake

of ³H-taurocholate in COS-hlBAT cells. Valacyclovir and atenolol do not inhibit the

uptake of ³H-taurocholate. In these inhibition studies, ³H-taurocholate concentration

is held constant at 0.25 μM and inhibitor concentration is varied between 1 and 100

μM; except for glycine and taurine, which range in concentration from 50 to 200 μM,

valacyclovir which range from 10 to 600 μM, and atenolol which range from 10 to 200

μM. Inhibition studies are performed in triplicate. N/A denotes not applicable,

because there was no evidence of inhibition.

Compounds can be conjugated or linked via a linker group to bile acids to

improve the bioavailability and reduce the bioavailability variability of the

compounds. It is preferable that a metabolically labile bond exist between the bile acid

and the compound (with or without a linker group) for easy cleavage of the compound

from the bile acid. While it is preferable that the compound or compound and linker

group be attached to the R₄ of the bile acid, one can also attach the compound or

compound and linker group at R_l5 R₂, or R₃. Also, one can link or conjugate different or the same compounds to multiple positions on the bile acid. Furthermore, one may

attach more than one bile acid to a compound.

The prodrugs can be coated with various coating compounds known in the art

field to protect the prodrug from the acidic environment in the stomach. These

coating compounds dissolve in the basic environment in the small intestine, thereby

permitting the prodrug to be available for uptake by the IBAT.

The prodrugs can also be converted into a pharmaceutically acceptable salt or

pharmaceutically acceptable solvate or other physical forms {e.g., polymorphs by

way of example only and not limitation) via known in the art field methods.

Pharmaceutically acceptable carriers can be used along with the prodrugs. In making

the compositions of the present invention, the prodrug can be mixed with an excipient,

diluted by an excipient or enclosed within such a carrier which can be in the form of a

capsule, sachet, paper or other container. When the excipient serves as a diluent, it

can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier, or medium

for the prodrug. Thus, the compositions can be in the form of tablets, pills, powers,

lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, soft and

hard gelatin capsules, and other orally ingestible formulations.

Some examples of suitable excipients include lactose, dextrose, sucrose,

sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth,

gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose,

water, syrup, and methyl cellulose. The formulations can additionally include

lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propyl-

hydroxybenzoates, sweetening agents; and flavoring agents. The compositions of the

present invention can also be formulated so as to provide quick, sustained or delayed

release of the prodrug after administration to the patient by employing procedures

known in the art.

The compositions are preferably formulated in a unit dosage form, each dosage

containing from about 0.005 mg to 100 mg, more usually about 1.0 mg to about 300

mg, of the prodrug. Unit dosage form generally refers to physically discrete units

suitable as unitary dosages for human and animal patients, each unit containing a

predetermined quantity of the prodrug calculated to produce the desired therapeutic

effect, in association with a suitable pharmaceutical excipient.

A prodrug is used for treatment of human or animal patients which are in need

of treatment with the compound that is contained in the prodrug. The specific

purpose of the treatment, and the dose range to be administered, depends on the

identity of the compound and the condition for which the patient is to be treated.

While a list of compounds for which this prodrug approach will work is too

large to be contained here, below is a short list of compounds and their "drug" class:

foscarnet, valacyclovir, acyclovir, ganciclovir, penciclovir, famciclovir (anti-

virals); alendronate, etidronate disodium, pamidronate, risedronate, tiludronate,

clodronic acid (bisphosphonates); cimetidine, ranitidine (H-2 antagonists);

enalaprilate, captopril, lisonopril (ACE inhibitors); losartan, E-3171

(angiotensin II antagonists); levofloxacin, norfloxacin (quinalone antibiotic which have decreased absorption with antacids); formycin B; acetbutalol,

pindolol, alprenolol, atenolol, nadolol (beta adrenergic blockers); bretylium

tosylate (antiarrhythmic agents); cefuroxime sodium (cephalosporins);

chlorothiazide, hydrochlorothiazide, furosemide (diuretic agents); gabapentin,

lamotrigine (anticonvulsant); didanosine (nucleoside reverse transcriptase

inhibitors); neviriapine (non-nucleoside reverse transcriptase inhibitors);

ritinavir, saquinavir, amprinavir (HIV protease inhibitors); tacrolimus,

cyclosporin (immunosuppresants); zafirlukast (leukotriene receptor

antagonists); leuprorelin actetate (LHRH analogues); dDAVP (l-deamino-8-D-

arginine-vasopressin; desmopressin), calcitonin, thyrotropin releasing hormone

(polypeptide hormones); loratidine, cetirizine (non-sedating antihistamines);

penicillin V, amoxicillin, cefacor, cefixime, cefuroxime axetil, cefuroxime

sodium, ampiciUin (antibiotics); terbutaline hemisulfate (adrenergic agonist

agents); metformin (anti-diabetics); celecoxib, refecoxib (COX-2 inhibitors);

sumatriptan, naratriptan, araztriptan, zolmitriptan (anti-migraines); 6-

mercaptopurine; ziprasidone; RGD mimetic (alpha lib beta 3-antagonists); leu-

enkephalin analogues; alpha-methyldopa; 5-fluorouracil (fluoropoyrimidines);

tacrine (acetylcholinesterase inhibitors); DZ-2640 (the ester-type oral

carbapenem prodrug of an active parent compound, DU-6681, and other

carbapenems); vitamin B12 (nutrients and minerals); 7-chlorokynurenic acid;

oseltamivir or its active moiety; RGD (Arg-Gly-Asp) analogs (glycoprotein

(GP) Ilb/IIIa agonists and antagonists; platelet aggregation inhibitors); sibrafiban (oral platelet aggregation inhibitors); nelarabine, 9-beta-D-

arabinofuranosyl guanine (ara-G), and ara-G; mycophenolate mofetil (MMF)

and its active immunosuppressant mycophenolic acid (MPA); nabumetone

and its active metabolite 6-methoxy-2-naphthylacetic acid (anti-osteoarthritis

agents); adefovir (9-[2-phosphonylmethoxyethyl]-adenine [PMEA]) and

adefovir dipivoxil [bis-(POM)-PMEA], and cidofovir (antiviral nucleotides);

cromoglicate lisetil and cromoglycic acid (anti-arthritis agents); oseltamivir or

its active moiety; its parent Ro 64-0802 (inhibitors of influenza virus

neuraminidase); peptidomimetics; nucleic acids;

This is for illustrative purposes only and is not meant to be exhaustive. Other

compounds will have increased bioavailability and/or reduced bioavailability

variability with bile acid conjugation.

All references cited herein are incorporated by reference in their entirety.

References:

[1] Remington: The Science and Practice of Pharmacy (A.R. Gennaro, editor).

Mack Publishing Compant, Easton, PA. 1995.

[2] Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Adv. Drug Del.

Rev. 1997, 25, 3-25.

[3] L. Lack, Properties and biological significance of the ileal bile salt transport

system, Environ Health Perspect 33 (1979) 79-90. [4] W. Kramer, G. Wess, A. Enhsen, E. Falk, A. Hoffmann, G. Neckermann, G.

Schubert and M. Urmann, Modified bile acids as carriers for peptides and

drugs, J Cont Rel 46 (1997) 17-30.

[5] S. Matern and W. Gerok, Pathophysiology of the enterohepatic circulation of

bile acids, Rev Physiol Biochem Pharmacol 85 (1979) 126-204.

[6] G. Burckhardt, W. Kramer, G. Kurz and F.A. Wilson, Inhibition of bile salt

transport in brush border membrane vesicles from rat small intestine by

photoaffinity labeling, J Biol Chem 258 (1983) 3618-3622.

[7] M. Kagedahl, P.W. Swaan, C.T. Redemann, M. Tang, C.S. Craik, F.C. Szoka,

Jr. and S. Oie, Use of the intestinal bile acid transporter for the uptake of

cholic acid conjugates with HIV-1 protease inhibitory activity, Pharm Res 14

(1997) 176-80.

[8] W. Kramer, G. Wess, A. Enhsen, K. Bock, E. Falk, A. Hoffmann, G.

Neckermann, D. Gantz, S. Schulz and L. Nickau, Bile Acid derived HMG-

CoA reductase inhibitors., Biochim Biophys Acta 1227 (1994) 137-154.

[9] D.C. Kim, A.W. Harrison, M.J. Ruwart, K.F. Wilkinson, J.F. Fisher, I.J.

Hidalgo and R.T. Borchardt, Evaluation of the bile acid transporter in

enhancing intestinal permeability to renin-inhibitory peptides., J Drug Target 1

(1993) 347-359.

[10] W. Kramer, G. Wess, G. Schubert, M. Bickel, F. Girbig, U. Gutjahr, S.

Kowalewski, K.H. Baringhaus, A. Enhsen and H.G. H, Liver-specific drug

targeting by coupling to bile acids., J Biol Chem 267 (1992) 18598-18604. [11] M.C. Lewis, L.E. Brieaddy and C. Root, Effects of 2164U90 on ileal bile acid

absorption and serum cholesterol in rats and mice, J Lipid Res 36 (1995) 1098-

1105.

[12] P. deMiranda and M.R. Blum, Pharmacokinetics of acyclovir after intravenous

and oral administration, J Antimicrob Chemother 12 (1983) 29-37.

[13] M.A. Jacobson, Valaciclovir (BW256U87): the L-valyl ester of acyclovir., J

Med Virol Supplement 1 (1993) 150-153.

[14] H. Gao, A.K. Mitra. Synthesis of acyclovir, gancyclovir, and their prodrugs:

A review. Synthesis (2000): 329-359.

[15] J. Soul-Lawton, E. Seaber, N. On, R. Wootton, P. Rolan and J. Posner,

Absolute bioavailability and metabolic disposition of valaciclovir, the L-valyl

ester of acyclovir, following oral administration to humans, Antimicrob Agents

Chemother 39 (1995) 2759-2764.

[16] K.H. Fife, R.A. Barbarash, T. Rudolph, B. Degregorio and R. Roth,

Valaciclovir versus acyclovir in the treatment of first-episode genital herpes

infection. Results of an international, multicenter, double-blind, randomized

clinical trial, Sex Transm Dis 24 (1997) 481-486.

[17] P.V. Balimane, I. Tamai, A. Guo, T. Nakanishi, H. Kitada, F.H. Leibach, A.

Tsuji and P.J. Sinko, Direct evidence for peptide transporter (PepTl)-

mediated uptake of a nonpeptide prodrug, valacyclovir, Biochem Biophys Res

Comm 250 (1998) 246-251. [18] H. Han, R.L. deVrueh, J.K. Rhie, K.M. Covitz, P.L. Smith, C.P. Lee, D.M.

Oh, W. Sadee and G.L. Amidon, 5'-Amino acid esters of antiviral nucleosides,

acyclovir, and AZT are absorbed by the intestina; PEPT1 peptide transporter,

Pharm Res 15 (1998) 1154-1159.

[19] C.E. Chandler, L.M. Zaccaro and J.B. Moberly, Transepithelial transport of

cholyltaurine by Caco-2 cell monolayers is sodium dependent, Am J Physiol

264 (1993) Gi l 18-25.

[20] O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein

measurement with the folin phenol reagent, J Biol Chem 193 (1951) 265-275.

[21] M.H. Wong, P. Oelkers and P.A. Dawson, Identification of a mutation in the

ileal sodium-dependent bile acid transporter gene that abolishes transport

activity, J Biol Chem 270 (1995) 27228-27234.

[22] F. Gaspari and M. Bonati, Correlation between n-octanol/water partition

coefficient and liquid chromatographic retention for caffeine and its

metabolites, and some structure-pharmacokinetic considerations, J Pharm

Pharmacol 39 (1987) 252-60.

[23] K.A. Lentz, J. Hayashi, L.J. Lucisano and J.E. Polli, Development of a more

rapid, reduced serum culture system for Caco-2 monolayers and application to

the Biopharmaceu ics Classification System, Int J Pharm 200 (2000) 41-51.

[24] G.A. Sawada, C.L. Barsuhn, B.S. Lutzke, M.E. Hooughton, G.E. Padbury,

N.F.H. Ho and TJ. Raub, Increased lipophilicity and subsequent cell partitioning decrease passive transcellular diffusion of novel, highly lipophilic

antioxidants, J Pharm Exp Ther 288 (1999) 1317-1326.

[25] G.A. Sawada, L.R. Williams, B.S. Lutzke and TJ. Raub, Novel, highly

lipophilic antioxidants readily diffuse across the blood-brain barrier and access

intracellular sites, J Pharm Exp Ther 288 (1999) 1327-1333.

While the invention has been described in detail, and with reference to specific

embodiments thereof, it will be apparent to one of ordinary skill in the art that various

changes and modifications can be made therein without departing from the spirit and

scope thereof. The artisan will further acknowledge that the Examples recited herein

are demonstrative only and are not meant to be limiting.

Claims

Claims:We, the inventors, claim

1. A method of increasing the bioavailability of a compound, said method comprising:

conjugating a bile acid to said compound to form a prodrug, and

orally administering said prodrug to an animal or human.

2. The method of Claim 1 further comprising the step of:

coating said prodrug with a coating agent prior to orally administering said

prodrug.

3. A method of reducing the bioavailability variability of a compound, said method

comprising:

conjugating a bile acid to said compound to form a prodrug, and

orally administering said prodrug to an animal or human.

4. The method of Claim 3 further comprising the step of:

coating said prodrug with a coating agent prior to orally administering said

prodrug.

5. A method of increasing the bioavailability of a compound, said method comprising:

linking a bile acid to a compound to form a prodrug, and

orally administering said prodrug to an animal or human.

6. The method of Claim 5 further comprising the step of:

coating said prodrug with a coating agent prior to orally administering said

prodrug.

7. A method of reducing the bioavailability variability of a compound, said method

comprising:

linking a bile acid to a compound to form a prodrug, and

orally administering said prodrug to an animal or human.

8. The method of Claim 7 further comprising the step of:

coating said prodrug with a coating agent prior to orally administering said

prodrug.

9. A method of eliminating adverse interactions between two compounds wherein

said adverse interactions result from intestinal absorption of at least one of said

compounds comprising:

linking a bile acid to at least one of said compounds to form a prodrug, and

orally administering said prodrug to an animal or human.

10. A method of eliminating adverse interactions between a compound and a nutrient

wherein said adverse interactions result from intestinal absorption of said compound

comprising:

linking a bile acid to said compound to form a prodrug, and

orally administering said prodrug to an animal or human.

11. A method of eliminating adverse interactions between two compounds wherein

compounds comprising:

conjugating a bile acid to at least one of said compounds to form a prodrug, and

orally administering said prodrug to an animal or human.

12. A method of eliminating adverse interactions between a compound and a nutrient

comprising:

conjugating a bile acid to said compound to form a prodrug, and

orally administering said prodrug to an animal or human.

13. The method of Claims 9, 10, 11, and 12, further comprising:

coating said prodrug with a coating agent prior to orally administering said

prodrug.

14. A pharmaceutical compound comprising:

acyclovir valyldeoxycholate.

15. A pharmaceutical compound comprising:

acyclovir valylchendeoxycholate.

16. A pharmaceutical compound comprising:

atenolol cholic acid amide.

17. A pharmaceutical compound comprising:

a compound;

a linker group; and

a bile acid.

18. The pharmaceutical compound of Claim 17 further comprising:

wherein said pharmaceutical compound contains a metabolically labile bond.

19. The pharmaceutical compound of Claim 18 further comprising: wherein said metabolically labile bond is selected from a group comprising an

amide, ester, carbamate, carbonate, ether, thio, urea, anhydride, thioamide, thioester,

thiocarbamate, and thiourea.

20. The pharmaceutical compound of Claim 17 further comprising:

wherein said linker group has a molecular weight of less than 200 daltons.

21. A pharmaceutical compound comprising:

a compound; and

a bile acid.

22. The pharmaceutical compound of Claim 21 further comprising: s

wherein said pharmaceutical compound contains a metabolically labile bond.

23. The pharmaceutical compound of Claim 22 further comprising:

wherein said metabolically labile bond is selected from a group comprising an

thiocarbamate, and thiourea.

24. The pharmaceutical compound of Claims 17, 18, 19, 20, 21, 22, and 23 further

comprising:

wherein said bile acid is selected from the group comprising cholate,

glycocholate, taurocholate, deoxycholate, glycodeoxycholate, taurodeoxycholate,

chenodeoxycholate, glycochenodeoxycholate, taurochenodeoxycholate,

ursodeoxycholate and lithocholate.

25. A method of increasing the bioavailability of a compound, said method

comprising: attaching a linker group to said compound;

attaching a bile acid to said linker group to form a prodrug, and

orally administering said prodrug to an animal or human.

26. The method of Claim 25 further comprising:

wherein said linker group has a molecular weight of less than 200 daltons.

27. The method of Claim 25 further comprising:

wherein said prodrug contains a metabolically labile bond.

28. The method of Claim 27 further comprising:

wherein said metabolically labile bond is selected from a group comprising an

thiocarbamate, and thiourea.

29. A method of reducing the bioavailability variability of a compound, said method

comprising:

attaching a linker group to said compound;

attaching a bile acid to said linker group to form a prodrug, and

orally administering said prodrug to an animal or human.

30. The method of Claim 29 further comprising:

wherein said linker group has a molecular weight of less than 200 daltons.

31. The method of Claim 29 further comprising:

wherein said prodrug contains a metabolically labile bond.

32. The method of Claim 31 further comprising: wherein said metabolically labile bond is selected from a group comprising an

thiocarbamate, and

thiourea.

33. A method ofincreasing the bioavailability of a compound, said method

comprising:

attaching a linker group to a bile acid;

attaching said compound to said linker group to form a prodrug; and

orally administering said prodrug to an animal or human.

34. The method of Claim 33 further comprising:

wherein said linker group has a molecular weight of less than 200 daltons.

35. The method of Claim 33 further comprising:

wherein said prodrug contains a metabolically labile bond.

36. The method of Claim 35 further comprising:

wherein said metabolically labile bond is selected from a group comprising an

thiocarbamate, and thiourea.

37. A method of reducing the bioavailability variability of a compound, said method

comprising:

attaching a linker group to a bile acid;

attaching said compound to said linker group to form a prodrug; and

orally administering said prodrug to an animal or human.

38. The method of Claim 37 further comprising:

wherein said linker group has a molecular weight of less than 200 daltons.

39. The method of Claim 37 further comprising:

wherein said prodrug contains a metabolically labile bond.

40. The method of Claim 39 further comprising:

wherein said metabolically labile bond is selected from a group comprising an

thiocarbamate, and

thiourea.

41. The method of Claims 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 39, and

40 further comprising the step of:

coating said prodrug with a coating agent.

42. The method of Claims 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 39, 40,

and 41 further comprising:

wherein said bile acid is selected from the group comprising cholate,

glycocholate, taurocholate, deoxycholate, glycodeoxycholate, taurodeoxycholate,

chenodeoxycholate, glycochenodeoxycholate, taurochenodeoxycholate,

ursodeoxycholate, and lithocholate.

43. A method for increasing the bioavailability of a compound comprising:

administering a prodrug containing said compound and a bile acid to an animal

or human; and using the intestinal bile acid transporter to actively uptake and remove from

the lumen of an intestine said prodrug.

44. A method for reducing the bioavailability variability of a compound comprising:

administering a prodrug containing said compound and a bile acid to an animal

or human; and

using the intestinal bile acid transporter to actively uptake and remove from

the lumen of an intestine said prodrug.

45. A compound of the formula:

wherein R_l5 R₂, and R₃ are independently selected from the group consisting of

hydrogen, alpha-hydroxyl, and beta-hydroxyl;

R is selected from the group consisting of an agent having biological activity and a

metabolic precursor of an agent having biological activity;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

46. A compound of the formula:

wherein R₂ and R₃ are independently selected from the group consisting of hydrogen,

alpha-hydroxyl, and beta-hydroxyl;

R_.i is selected from the group an agent having biological activity and a metabolic

precursor of an agent having biological activity;

R is any chemical moiety that enhances binding of the compound to the intestinal bile

acid transporter;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

47. A compound of the formula:

wherein Ri and R₃ are independently selected from the group consisting of hydrogen,

alpha-hydroxyl, and beta-hydroxyl; R₂ is selected from the group an agent having biological activity and a metabolic

precursor of an agent having biological activity;

R₄ is any chemical moiety that enhances binding of the compound to the intestinal bile

acid transporter;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

48. A compound of the formula:

wherein R] and R₂ are independently selected from the group consisting of hydrogen,

alpha-hydroxyl, and beta-hydroxyl;

R₃ is selected from the group an agent having biological activity and a metabolic

precursor of an agent having biological activity;

acid transporter;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

49. A compound of the formula:

alpha-hydroxyl, and beta-hydroxyl;

K_\ is selected from the group an agent having biological activity and a metabolic

precursor of an agent having biological activity;

R is any chemical moiety that increases solubility of the compound;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

50. A compound of the formula:

wherein R₂ and R are independently selected from the group consisting of hydrogen,

alpha-hydroxyl, and beta-hydroxyl;

R₂ is selected from the group an agent having biological activity and a metabolic

precursor of an agent having biological activity; R is any chemical moiety that increases solubility of the compound;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

51. A compound of the formula:

wherein R_ι and R₂ are independently selected from the group consisting of hydrogen,

alpha-hydroxyl, and beta-hydroxyl;

precursor of an agent having biological activity;

₄ is any chemical moiety that increases solubility of the compound;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

52. A compound of the formula:

wherein R_l3 R₂, and R₃ are independently selected from the group consisting of

hydrogen, alpha-hydroxyl, and beta-hydroxyl;

X is any chemical moiety resulting in a metabolically labile bond; and

R₄ is selected from the group consisting of an agent having biological activity or a

metabolic precursor of an agent having biological activity;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

53. The compound of Claim 52 wherein said metabolically labile bond is selected

from the group consisting of amide, ester, carbamate, carbonate, ether, thio, urea,

anhydride, thioamide, thioester, thiocarbamate, and thiourea.

54. A compound of the formula:

wherein R and R₃ are independently selected from the group consisting of hydrogen,

alpha-hydroxyl, and beta-hydroxyl;

R-i is any chemical moiety that enhances binding of the compound to the intestinal bile

acid transporter;

X is any chemical moiety resulting in a metabolically labile bond; and

R,, is selected from the group consisting of an agent having biological activity or a

metabolic precursor of an agent having biological activity;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

55. A compound of the formula:

alpha-hydroxyl, and beta-hydroxyl;

acid transporter;

X is any chemical moiety resulting in a metabolically labile bond; and

R₂ is selected from the group consisting of an agent having biological activity or a

metabolic precursor of an agent having biological activity;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

56. A compound of the formula:

wherein Rι and R₂ are independently selected from the group consisting of hydrogen,

alpha-hydroxyl, and beta-hydroxyl; R₄ is any chemical moiety that enhances binding of the compound to the intestinal bile

acid transporter;

X is any chemical moiety resulting in a metabolically labile bond; and

R₃ is selected from the group consisting of an agent having biological activity or a

metabolic precursor of an agent having biological activity;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

57. A compound of the formula:

alpha-hydroxyl, and beta-hydroxyl;

R₄ is any chemical moiety that increases solubility of the compound;

X is any chemical moiety resulting in a metabolically labile bond; and

Rι_ is selected from the group consisting of an agent having biological activity or a

metabolic precursor of an agent having biological activity;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

58. A compound of the formula:

wherein Ri. and R₃ are independently selected from the group consisting of hydrogen,

alpha-hydroxyl, and beta-hydroxyl;

R-_f is any chemical moiety that increases solubility of the compound;

X is any chemical moiety resulting in a metabolically labile bond; and

metabolic precursor of an agent having biological activity;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

59. A compound of the formula:

wherein Rj and R₂ are independently selected from the group consisting of hydrogen,

alpha-hydroxyl, and beta-hydroxyl;

is any chemical moiety that increases solubility of the compound;

X is any chemical moiety resulting in a metabolically labile bond; and R₃ is selected from the group consisting of an agent having biological activity or a

metabolic precursor of an agent having biological activity;

or a pharmaceutically acceptable salt, solvent, or polymorph thereof.

60. The compound of Claim 54, 55, 56, 57, 58 and 59 wherein said metabolically

labile bond is selected from the group consisting of amide, ester, carbamate, carbonate,

ether, thio, urea, anhydride, thioamide, thioester, thiocarbamate, and thiourea.