WO2002087597A1

WO2002087597A1 - Oral delivery of macromolecules

Info

Publication number: WO2002087597A1
Application number: PCT/KR2001/001723
Authority: WO
Inventors: Young-Ro Byun; Yong-Kyu Lee
Original assignee: Mediplex Corp Korea
Current assignee: Mediplex Corp Korea
Priority date: 2001-04-30
Filing date: 2001-10-12
Publication date: 2002-11-07
Anticipated expiration: 2003-10-30
Also published as: US20020010153A1; EP1383518A4; EP1383518A1; US6656922B2; KR20020083905A; US20040220143A1; JP2004532851A; CN1518452A

Abstract

Polysaccharides, which are widely used as an anticoagulation drugs, especially heparin, are clinically administered only by intravenous or subcutaneous injection because of their strong hydrophilicity and high negative charge. Amphiphilic heparin derivatives were synthesized by conjugation to bile acids, sterols, and alkanoic acids, respectively. These heparin derivatives were slightly hydrophobic, exhibited good solubility in water, and have high anticoagulation activity. These slightly hydrophobic heparin derivatives are efficiently absorbed in the gastrointestinal tract and can be used in oral dosage forms. Methods of using these amphiphilic heparin derivatives and similarly modified macromolecules for oral administration are also disclosed.

Description

ORAL DELIVERY OF MACROMOLECULES

FIELD OF THE INVENTION

This invention relates to derivatives of

macromolecules, including polysaccharide derivatives,

having increased hydrophobicity as compared to the unmodified macromolecules or polysaccharides. More

particularly, the invention relates to oral delivery and absorption of hydrophobized macromolecules and amphiphilic

polysaccharide derivatives, such as amphiphilic heparin

derivatives, wherein the bioactivity of the macromolecule

or polysaccharide is preserved. In preferred embodiments of the invention, the hydrophobized macromolecules and

amphiphilic polysaccharide derivatives have a molecular weight of greater than 1000, yet are absorbed after oral

administration.

BACKGROUND OF THE INVENTION

Heparin is a polysaccharide composed of sulfated D-

glucosamine and D-glucuronic acid residues. Due to its

numerous ionizable sulfate groups, heparin possesses a

strong electronegative charge. It is also a relatively

strong acid that readily forms water-soluble salts, e.g. heparin sodium. It is found in mast cells and can be

extracted from many body organs, particularly those with

abundant mast cells. The liver and lungs are especially

rich in heparin. The circulating blood contains no heparin

except after profound disruption of mast cells. Heparin has many physiological roles, such as blood anticoagulation,

inhibition of smooth muscle cell proliferation, and others.

In particular, heparin is a potent anticoagulant agent that

interacts strongly with antithrombin III (A Till) to prevent

the formation of fibrin clots. Heparin is one of the most

potent anticoagulants used for treatment and prevention of

deep vein thrombosis and pulmonary embolism. In vivo,

however, applications of heparin are very limited. Because

of its hydrophilicity and high negative charge, heparin is

not absorbed efficiently from the Gl tract, nasal or buccal

mucosal layers, and the like. Therefore, the only routes of

administration used clinically are intravenous and

subcutaneous injections. Moreover, since heparin is soluble

in relatively few solvents, it is hard to use for coating

surfaces of medical devices or in delivery systems.

To improve the properties of heparin, R.J. Linhardt

et al . , 83 J. Pharm. Sci . , 1034-1039 (1994), coupled lauryl

(Cι₂) and stearyl (Cia) groups to single heparin chains, resulting in a derivatized heparin having increased hydrophobicity but with low anticoagulation activity. This

result demonstrated that coupling a small linear aliphatic

chain to heparin was ineffective in enhancing the

hydrophobicity of heparin while preserving activity. Thus,

known heparin derivatives have been ineffective in

preserving anticoagulation activity.

T.M. Rivera et al . , Oral Delivery of Heparin in

Combination with Sodium N-[8-(2-

Hydroxybenzolyl ) amino]caprylate: Pharmacological

Considerations, 14 Pharm. Res . , 1830-1834 (1997), disclosed

the possibility of oral delivery of heparin using heparin mixed with sodium N-[ 8- ( 2-hydroxybenzolyl) amino Jcaprylate .

M. Dryj ski et al . , Investigations on Plasma Activity of Low Molecular Weight Heparin after Intravenous and Oral

Administrations, 28 Br. J^". Clln . Pharma . , 188-192 (1989) described the possibility of oral absorption of low

molecular weight heparin using enhancers.

It is generally recognized that molecules having a

molecular weight greater than 1000 are poorly absorbed in

the gastrointestinal (Gl) tract after oral administration.

For example, J.G. Russell-Jones, Carrier-mediated Transport,

Oral Drug Delivery, in 1 Encyclopedia of Controlled Drug Delivery 173, 175 (Edith Mathiowitz ed. 1999), stated that

the work of W. Kramer et al . , 269 J. Biol . Chem. , 10621-

10627 (1994), suggested that the maximal size of a peptide

that could be transported via the bile acid transporter was

four amino acids, or about 600 Da. As another example, P. W.

Swaan et al . , Enhanced Transepithelial Transport of

Peptides by Conjugation to Cholic Acid, 8 Bioconjugate

Chemistry 520-525(1997), reported that bile acid conjugates with up to 6 amino acids (i.e., about 900 Da) showed

affinity for the intestinal bile acid transporter, but the only 6-amino-acid bile acid conjugate tested was not

transported by the bile acid carrier.

In view of the foregoing, it will be appreciated that

development of a method for obtaining absorption of

macromolecules having a molecular weight greater than 1000

after oral administration would be a significant advancement in the art. It will also be appreciated that

development of a method for obtaining absorption of

hydrophobized or amphiphilic heparin derivatives after oral

administration would be another significant advancement in

the art.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a

method for obtaining absorption of molecules having a

molecular weight greater than 1000 after oral

administration .

It is also an object of the invention to provide a

method for obtaining blood anticoagulation by oral

administration of amphiphilic heparin derivatives.

It is still another object of the invention to

provide heparin derivatives that can be absorbed from the

Gl tract, thereby facilitating oral delivery for preventing blood coagulation.

It is yet another object of the invention to provide

heparin derivatives comprising heparin coupled with a bile

acid, such as deoxycholic acid or glycocholic acid, or a

hydrophobic agent, such as cholesterol, or an alkanoic acid.

These and other objects can be addressed by providing

a method of treating a patient in need of anticoagulation

therapy comprising orally administering an effective amount

of a composition comprising heparin covalently bonded to a

hydrophobic agent selected from the group consisting of

bile acids, sterols, and alkanoic acids, and mixtures

thereof. The composition can also include a

pharmaceutically acceptable carrier. In one preferred embodiment of the invention the

hydrophobic agent is a bile acid selected from the group

consisting of cholic acid, deoxycholic acid,

chenodeoxycholic acid, lithocholic acid, ursocholic acid,

ursodeoxycholic acid, isoursodeoxycholic acid,

lagodeoxycholic acid, glycocholic acid, taurocholic acid,

glycodeoxycholic acid, glycochenodeoxycholic acid,

dehydrocholic acid, hyocholic acid, hyodeoxycholic acid, and mixtures thereof, and the like.

In another preferred embodiment of the invention, the

hydrophobic agent is a sterol selected from the group

consisting of cholestanol, coprostanol, cholesterol,

epicholesterol , ergosterol, ergocalciferol, and mixtures

thereof, and the like.

In still another preferred embodiment of the

invention, the hydrophobic agent is an alkanoic acid

comprising about 4 to 20 carbon atoms. Preferred alkanoic

acids include butyric acid, valeric acid, caproic acid,

caprylic acid, capric acid, lauric acid, myristic acid,

palmitic acid, stearic acid, and mixtures thereof and the like.

Preferably, the heparin comprises a molecular weight

of at least about 3000, and more preferably at least about 6000. In certain preferred embodiments, the heparin comprises a molecular weight less than about 12,000.

Another preferred embodiment of the invention

comprises a method for enhancing oral administration of a

macromolecular agent comprising:

(a) conjugating the macromolecular agent to a

hydrophobic agent selected from the group

consisting of bile acids, sterols, alkanoic acids, and mixtures thereof, and the like to

result in a hydrophobized macromolecular

agent; and,

(b) orally administering an effective amount of

the hydrophobized macromolecular agent to a

patient in need thereof.

Preferably, the macromolecular agent is a member

selected from the group consisting of heparin, heparan

sulfate, sulfonyl polysaccharide, heparinoids,

polysaccharide derivatives, and mixtures thereof, and the like. In another preferred embodiment of the invention, the

macromolecular agent is a peptide, such as insulin or

calcitonin.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows clotting time profiles as measured by

aPTT assay of heparin-DOCA conjugate after oral

administration in rats:

D - 100 g/kg raw heparin (control),

♦ - physical 20 mixture of heparin (200 mg/kg) and DOCA (200 mg/kg) ;

▼ - 50 mg/kg heparin-DOCA conjugate,

A - 80 mg/kg heparin-DOCA conjugate,

■ - 100 mg/kg heparin-DOCA conjugate, and

• - 200 mg/kg heparin-DOCA conjugate; data are plotted as mean ± SD, n=9.

FIG. 2 shows concentration profiles as measured by

Fxa assay of heparin-DOCA conjugate after oral

administration in rats:

D - 100 mg/kg raw heparin (control),

T - 50 mg/kg heparin-DOCA conjugate,

A - 80 mg/kg heparin-DOCA conjugate,

■ - 100 mg/kg heparin-DOCA conjugate, and

• - 200 mg/kg heparin-DOCA conjugate; data are plotted as mean ± SD, n=9.

FIG. 3 shows clotting time profiles in rats of

heparin-DOCA conjugates as a function of the mole ratio of DOCA to heparin: - raw heparin,

- 2.5 mole ratio,

- 5.0 mole ratio, and

- 10.0 mole ratio; data are plotted as mean ± SD, n=9

FIG. 4 shows clotting time profiles in rats of

heparin derivatives as a function of the hydrophobic agent

conjugate to heparin:

▼ - heparin-lauric acid conjugate,

A - heparin-palmitic acid conjugate,

I - heparin-cholesterol conjugate, and

Φ - heparin-DOCA conjugate; data are plotted as mean ± SD, n=9.

FIG. 5 shows micrographs of hematoxylin and eosin

stained gastrointestinal tissues that were isolated from rats after oral administration of 100 mg/kg of heparin-DOCA

conjugate: panels A, B, C and D show cross sections of the

stomach after 0, 1, 2 and 3 hours, respectively; panels E,

F, G and H show cross sections of the duodenum after 0, 1,

2 and 3 hours, respectively; panels I, J, K and L show

cross sections of the jejunum after 0, 1, 2 and 3 hours,

respectively; and panels M, N, 0 and P show cross sections

of the ileum after 0, 1, 2 and 3 hours, respectively; the original magnification was lOOx in all panels.

FIG. 6 shows electron micrographs of membrane or

microvilli in gastrointestinal tissues isolated from rats

after oral administration of 100 mg/kg of heparin-DOCA conjugate:

panels A, B, C and D show cross Sections of the

stomach after 0, 1, 2 and 3 hours, respectively;

panels F, F, G and H show cross sections of the

duodenum after 0, 1, 2 and 3 hours, respectively; panels I, J, K and L show cross sections of the

jejunum after 0, 1, 2 and 3 hours, respectively; and

panels M, N, 0 and P show cross sections of the ileum

after 0, 1, 2 and 3 hours, respectively; the original magnification was 25,OOOx in all panels.

FIGS. 7A and 7B show clotting time profiles (FIG. 7A) and concentration profiles (FIG. 7B) of heparin-DOCA

conjugates after oral administration in rats:

A - LMWH(3K)-DOCA;

• - LMWH(6K)-DOCA;

■ - heparin-DOCA (also referred to herein as UFH- DOCA) .

FIGS. 8A and 8B show clotting time profiles (FIG. 8A)

and concentration profiles (FIG. 8B) of LMWH( 6K) -DOCA after after oral administration in rats:

♦ - 20 mg/kg of LMWH(6K) control;

▼ - 100 mg/kg of LMWH(6K) control; A - 20 mg/kg LMWH( 6K) -DOCA;

■ - 50mg/kg LMWH( 6K) -DOCA;

• - 100 mg/kg LMWH( 6K)-DOCA.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods for obtaining absorption

of orally delivered hydrophobized macromolecules and

amphiphilic polysaccharide compositions are disclosed and

described. It is to be understood that this invention is not limited to the particular configurations, process steps,

and materials disclosed herein as such configurations,

process steps, and materials may vary somewhat. It is also

to be understood that the terminology employed herein is

used for the purpose of describing particular embodiments

only and is not intended to be limiting since the scope of

the present invention will be limited only by the appended

claims and equivalents thereof.

It must be noted that, as used in this specification

and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a bile

acid" includes a mixture of two or more of such bile acids, reference to "an alkanoic acid" includes reference to one

or more of such alkanoic acids, and reference to "a sterol"

includes reference to a mixture of two or more sterols.

In describing and claiming the present invention, the

following terminology will be used in accordance with the definitions set out below.

As used herein, "bile acids" means natural and

synthetic derivatives of the steroid, cholanic acid,

including, without limitation, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholic

acid, ursodeoxycholic acid, isouirsodeoxycholic acid, lagodeoxycholic acid, glycocholic acid, taurocholic acid,

glycodeoxycholic acid, glycochenodeoxycholic acid,

dehydrocholic acid, hyocholic acid, hyodeoxycholic acid,

and mixtures thereof, and the like.

As used herein, "sterols" means alcohols structurally

related to the steroids including, without limitation,

eholestanol, coprostanol, cholesterol, epicholesterol ,

ergosterol, ergocalciferol, and mixtures thereof, and the like. As used herein, "alkanoic acids" means saturated

fatty acids of about 4 to 20 carbon atoms. Illustrative

alkanoic acids include, without limitation, butyric acid,

valeric acid, caproic acid, caprylic acid, eapric acid,

lauric acid, myristic acid, palmitic acid, stearic acid, and mixtures thereof, and the like.

As used herein, "hydrophobic heparin derivative" and

"amphiphilic heparin derivative" are used interchangeably.

Heparin is a very hydrophilic material. Increasing the

hydrophobicity of heparin by bonding a hydrophobic agent thereto results in what is termed herein an amphiphilic

heparin derivative or hydrophobic heparin derivative.

Either term is proper because the heparin derivative has

increased hydrophobicity as compared to native heparin and the heparin derivative has a hydrophilic portion and a

hydrophobic portion and is, thus, amphiphilic.

As used herein, "aPTT" means activated partial

thromboplastin time, and "FXa" means factor Xa.

As used herein, "DOCA" means deoxycholic acid, and

"heparin-DOCA" means a conjugate of heparin and deoxycholic acid.

As used herein, " acromolecule" means polypeptide, polysaccharide, and nucleic acid polymers with a molecular weight typically greater than 1000.

As used herein, "peptide' means peptides of any

length and includes proteins. The terms "polypeptide" and

"oligopeptide" are used herein without any particular

intended size limitation, unless a particular size is

otherwise stated. Typical of peptides that can be utilized are those selected from group consisting of oxytocin,

vasopressin, adrenocorticotrophic hormone, epidermal growth factor, prolactin, luliberin or luteinising hormone

releasing hormone, growth hormone, growth hormone releasing

factor, insulin, somatostatin, glucagon, interferon,

gastrin, tetragastrin, pentagastrin, urogastroine, secretin, calcitonin, enkephalins, endorphins, angiotensins , rennin,

bradykinin, bacitracins, polymixins, colistins, tyrocidin, gramicidines , and synthetic analogues, modifications and

pharmacologically active fragments thereof, monoclonal

antibodies and soluble vaccines. The only limitation to the

peptide or protein drug which may be utilized, is one of functionality .

As used herein, "effective amount" means an amount of

a pharmacologically active agent that is nontoxic but

sufficient to provide the desired local or systemic effect

and performance at a reasonable benefit/risk ratio attending any medical treatment. Thus, for example, an

effective amount of a heparin-DOCA conjugate is an amount

sufficient to provide a selected level of anticoagulation activity.

It is well known that heparin is used as an antithrombogenic agent to prevent blood coagulation.

Heparin is highly hydrophilic because of a high density of

negative charges such as are provided by sulfonic and

carboxylic groups. Due to this hydrophilicity, heparin is

usually administered by intravenous or subcutaneous

injection. Heparin derivatives with slightly hydrophobic

properties or amphiphilic properties and with high

bioactivity are described herein. Hydrophobic agents, such

as bile acids, e.g. deoxycholic acid (DOCA); sterols, e.g.

cholesterol; and alkanoic acids, e.g. lauric acid and

palmitic acid, were coupled to heparin. Both deoxycholic acid and cholesterol are non-toxic since they are naturally

occurring compounds found in the body. The amine groups of

heparin were coupled with carboxyl groups of the

hydrophobic agents. The end carboxylic groups in DOCA,

lauric acid, and palmitic acid were used directly for the

coupling reaction, while the hydroxy group of cholesterol

was activated by reaction with chloroacetic acid before coupling. It was determined that conjugating such

hydrophobic moieties to the amine groups of heparin had

little or no effect on heparin bioactivity. The coupling

between heparin and hydrophobic agents was confirmed by

detecting the resulting amide bond by FT-IR and ¹³C-NMR

analysis .

The yield of the coupling reaction was about 70 to

80% and was not significantly changed by changing the

hydrophobic agents or feed molar ratios. In the case of the

heparin-DOCA conjugate, as the feed ratio was increased,

the amount of DOCA in the conjugate was also increased. The

weight % of DOCA in heparin-DOCA was 24% when the feed

molar ratio of heparin to DOCA was 1:200. This molar ratio

was very high compared to the ratio of amine groups in heparin to DOCA. Therefore, this feed ratio is estimated as

an excess amount of DOCA.

The hydrophobic heparin derivatives according to the

present invention would have many medical applications. For

example, the hydrophobic heparin can be administered orally.

The oral administration of heparin can greatly extend the

usage of heparin as an oral anti-coagulant drug. The

heparin derivative is formulated with a pharmaceutically

acceptable carrier such as is well known in the art. By way of further example, hydrophobic heparin derivatives can be

used as a coating material for medical devices such as

catheters, cardiopulmonary bypass circuits, heart lung

oxygenators, kidney dialyzers, stent or balloon coating for

preventing restenosis, and the like. The hydrophobic heparin derivative is typically mixed with a carrier, and

then coated On the surface of the medical device by a film

casting technique such as is well known in the art.

After modification, heparin-hydrophobic agents were

also found to have a tendency in fast protein liquid

chromatography (FPLC®) to exhibit hydrophobic interactions

with hydrophobic media, as shown by chromatography on

Phenyl Sepharose® (eluting in ammonium sulfate buffer

rather than phosphate buffer). These heparin derivatives showed enhanced binding affinity when compared to

unmodified heparin. The increased interaction of modified

heparin derivatives with Phenyl Sepharose® is attributable

to its enhanced hydrophobicity, the result of the

hydrophobic functional groups present. These results

suggest hydrophobic heparin can be obtained by conjugating

a bile acid, sterol, or alkanoic acid to heparin. In

solubility tests, polar solvents or organic solvents were suitable to dissolve the heparin-hydrophobic agent conjugates. For example, the heparin-deoxycholic acid

conjugate showed good solubility in 65% acetone solution

(35% water). Finally, it was determined that bioactivity of

modified heparin derivatives was not appreciably influenced

by conjugation with hydrophobic agents. The role of a hydrophobic agent conjugated to heparin was studied with

respect to two biological activities of heparin as

determined by anticoagulation and factor Xa assays.

Although hydrophobicity is associated with a somewhat

reduced anticoagulant activity and antifactor Xa activity,

the decrease of bioactivity was not considered serious.

These results indicate that blocking the amine groups of heparin had little effect on its bioactivity. The

bioactivity of heparin in heparin hydrophobic agent

conjugates exhibited a progressive reduction, however, when the amount of hydrophobic agent in the conjugate exceeded

20 wt%. At less than 20 wt% of hydrophobic agent in the

conjugates, the bioactivity of the conjugates was greater

than 80% of the bioactivity of unmodified heparin. It is

suggested that 80% of bioactivity in hydrophobic heparin is

enough to support bioactivity in medical applications.

Example 1 Synthesis of Heparin-DOCA Conjugates

Five ml of N-hydroxylsuccinimide (HOSu, 92 mg/5 ml)

in dimethylformamide (DMF) was mixed with 5 ml of dicyclohexylcarbodiimi.de (DCC) (165 mg/5 ml) in DMF,

followed by adding 5 ml of DOCA (196 mg/5 ml) in DMF. The

mole ratio of DOCA, HOSu, and DCC was 1:1.6:1.6. The

concentrations of HOSu and DCC were slightly higher than

that of DOCA to activate DOCA completely. The resulting

solution was reacted for 5 hours at room temperature under

vacuum, and then the byproduct dicyclohexylurea (DCU),

which precipitated during the reaction, was removed. The

unreacted DCC was removed by adding a drop of distilled

water and filtering. The remaining HOSu was also removed by

adding 15 ml of distilled water. The activated DOCA was

precipitated and then lyophilized. The activated DOCA was then dissolved in DMF and reacted with heparin for 4 hours

at room temperature. The amounts of heparin used in such

reactions ranged from 40 to 400 mg. After reaction, there

were two types of products: a water-soluble product and a

water- insoluble product. These products were separated by

filtration through a 0.45 μm membrane filter, and the water-insoluble product (i.e., activated DOCA) was dried in

a vacuum oven. The water-soluble product (i.e., heparin- DOCA) was dialyzed for 1 day against water using a membrane

(MWCO 3,500), and then heparin-DOCA was freezing dried.

The synthesized heparin-DOCA was further purified by

reverse phase chromatography. A phenyl-Sepharose CL-4B

column (HR 16/30 I.D.) was washed with 100 ml of distilled

water, 40 ml of 50 mM phosphate buffer (pH 7.0), 40 ml of

50 mM phosphate buffer (pH 7.0) containing 1.7 M ammonium

sulfate, and 40 ml of 50 mM phosphate buffer, respectively.

Five milliliters of the heparin-DOCA solution (1 mg/ml) was

loaded in the column and the heparin-DOCA was fractionated by step elution with an ammonium sulfate solution. Elution

was carried out with phosphate buffer for 20 minutes,

followed by the ammonium sulfate solution (50 mM phosphate

buffer (pH 7.0) + 1.7 M ammonium sulfate) with the flow

rate of 1 ml/min. Heparin-DOCA was eluted in the ammonium

sulfate solution. The purified heparin-DOCA solution was dialyzed in distilled water and lyophilized.

The heparin derivatives prepared according to this procedure were characterized by FT-IR and NMR according to

methods well known in the art to prove the successful coupling between heparin and the hydrophobic agent. Y. Lee,

H.T. Moon & Y. Byun, Preparation of Slightly Hydrophobic Heparin Derivatives Which Can Be Used for Solvent Casting in Polymeric Formulation , 92 Thromb . Res . , 149156 ( 1998 ) .

Example 2

Preparation of Heparin-Cholesterol Conjugates

The hydroxyi group of cholesterol was activated by

reaction with chloroacetic acid to result in a free

carboxyl group. This modified cholesterol was then reacted

with HOSu and DCC in 10 ml of DMF according to the procedure of Example 1. The mole ratio of cholesterol, HOSu,

and DCC was 1:1.6:1.6 and reaction was for 5 hours at room

temperature. To remove the unreacted DCC and HOSu, water

was added and the solution was filtered with a 0.45 μm membrane. Next, the activated cholesterol was reacted with

heparin solution for 4 hours. Two products, a water-soluble

product and a water-insoluble product, were obtained from

the reaction. These products were treated according to the procedure described above in Example 1.

Example 3

Synthesis of Heparin-Alkanoic Acid Conjugates

Laurie acid and palmitic acid were coupled to heparin

according to the procedure of Example 1. The carboxyl group

of the alkanoic acids was coupled with amine groups of heparin to form amide bonds. Coupling agents were also HOSu and DCC.

Example 4

Synthesis of Heparin-Cholic Acid Conjugates

In this example, the procedure of Example 1 is

followed except that cholic acid is substituted for DOCA.

Example 5

Synthesis of Heparin-Chenodeoxycholic Acid Conjugates

In this example, the procedure of Example 1 is

followed except that chenodeoxycholic acid is substituted for DOCK.

Example 6

Synthesis of Heparin-Ergosterol Conjugates

In this example, the procedure of Example 2 is

followed except that ergosterol is substituted for cholesterol .

Example 7

Synthesis of Insulin-DOCA Conjugates

In this example, the procedure of Example 1 is followed except that insulin is substituted for heparin.

The amine groups of insulin, i.e., GlyAl , PheBl, and LysB29,

are coupled with carboxyl groups of DOCA.

Example 8

Synthesis of Calcitonin-DOCA Conjugates

In this example, the procedure of Example 1 is

followed except that calcitonin is substituted for heparin.

An amine group of calcitonin is coupled to a carboxyl group

of DOCA.

Example 9

Measurement of Bioactivity of Heparin Derivatives

For heparin-DOCA, heparin cholesterol, and heparin-

alkanoic acid prepared according to the procedures of

Examples 1-3, the production yield, molecular weight, and binding mole ratios between heparin and hydrophobic agents

varied according to the mole ratio of reactants. The yield

of heparin-DOCA conjugates was in the range of 71 to 77%.

The amount of hydrophobic agent in modified heparin

derivatives was calculated by subtracting the molecular

weight of heparin (i.e., 12,386 daltons as determined by light scattering) from the measured molecular weight of each heparin derivative. As the feed mole ratio of

deoxycholic acid to heparin was increased from 1:6 to 1:200,

the amount of DOCA in heparin-DOCA conjugates was increased from 7 to 24%. For the heparin-cholesterol conjugates, the

yield also was in the range from 73 to 78%. The amount of

cholesterol in such hydrophobic heparin conjugates, however,

was slightly lower than the amount of DOCA in heparin-DOCA

conjugates. In heparin-lauric acid and heparin-palmitic acid conjugates, similar amounts of alkanoic acid were

coupled to heparin.

Anticoagulant activities of heparin derivatives were

determined by aPTT assay and FXa chromogenic assay. The

activities of heparin derivatives in the prevention of

fibrin clot formation were measured by aPYF assay. Each of the platelet-poor-plasma containing heparin standards (0.1

to 0.7 U/ml, 0.1 ml) and plasma samples containing heparin derivatives (0.1 ml) was incubated with 0.1 ml of aPTT

reagent for 2 min at 37 ^°C . After the incubation, 0.1 ml of 0.02 M calcium chloride was added, and the time was

recorded from this point until the fibrin clot was formed.

The bioactivity of the heparin derivative was calculated by

comparing the clotting time with the heparin standard curve.

The clotting time was linearly proportional to the activity of heparin in the plasma.

The activity and the concentration of heparin

derivatives were also determined by FXa chromogenic assay.

Each of the heparin standards and plasma samples containing

heparin derivatives (25 μl was mixed with 200 μl of AT III solution (0.1 IU/ml), where the AT III concentration was in

excess of the heparin concentration. This solution was

incubated at 37 ^°C for 2 min, and 200 μl of FXa (4 nkcat/ml) was added. The resulting solution was then incubated for an

additional 1 min. The concentration of FXa was also in

excess of the heparin concentration. FXa substrate (200 μl, 0.8 μmol/ml) was then added and incubated at 37 ^°C for 5 min.

The reaction was terminated by adding 200 μl of acetic acid (50% solution). The bioactivity and the concentration of

heparin in the plasma sample were calculated from the

absorbance at 405 nm. These data are summarized in Table I.

Table I

a Mole ratio of hydrophobic agent to heparin

Example 10

Oral Administration of Heparin-DOCA

Sprague-Dawley rats (male, 250-260 g) were fasted for 12 hours before dosing. The rats were anesthetized with

diethyl ether and then were administered a single dose of heparin derivative through an oral gavage that was

carefully passed down the esophagus into the stomach. The

gavage was made of stainless steel with a blunt end to

avoid causing lesions on the tissue surface. The solution

containing the heparin derivative was prepared in a sodium bicarbonate buffer (pH 7.4). The total administered volume

of heparin-derivative-containing solution was 0.3 ml. The

dose amount was varied at 50, 80, 100, and 200 mg/kg, respectively. There were 9 rats in each group. Blood (450 μ 1) was collected serially by capillary from the retro-

orbital plexus at each time point and directly mixed with

50 μl of sodium citrate (3.8% solution). The blood samples

were immediately centrifuged at 2500 x g and 4 ^°C for 5 minutes. The clotting time and the concentration of heparin derivative in the plasma were measured by aPTT assay and

FXa assay, respectively.

The absorption of heparin-DOCA in the Gl tract was

determined according to the dose amount in the range of 50 to 200 mg/kg. In this experiment, the mole ratio of coupled

DOCA to heparin in the heparin-DOCA conjugate was 10. When raw heparin was administered orally to rats, the clotting

time, measured by aPTT assay, was about 18 seconds and this

value did not change over time. The average value of the

baseline was 18 seconds, indicating that the raw heparin was not absorbed in the Gl tract. When the physical mixture

or admixture of heparin and DOCA was administered orally,

the aPTT value was about 20 seconds, and this value did not

change over time. On the other hand, when heparin-DOCA conjugate was orally administered, the clotting time

increased as shown in FIG. 1. Since the blood sampling was

carried out at one-hour intervals and the maximum clotting time was shown at the one-hour time point, the real maximum

clotting time could not be determined. However, the

clotting time at one hour was linearly increased with the

increase of dosage. When heparin-DOCA conjugate was given

at 50, 80, 100, and 200 mg/kg, the clotting times at one

hour were 25.8±2.6, 43.1+4.0, 51.2+9.3, and 136±33 seconds,

respectively. When heparin-DOCA conjugate was administered

at 200 mg/kg, the clotting time at one hour increased greatly, above 7-times the baseline. Since the therapeutic

window of heparin is 1.5 to 2.5 times the baseline, the

therapeutic effect can be seen at an 80-100 mg/kg dose.

Therefore, the heparin-DOCA conjugate greatly enhanced the absorption of heparin in the Gl tract, in contrast to DOCA

mixed with heparin in a physical mixture, which did not

enhance heparin absorption.

The concentration of heparin-DOCA conjugate in the plasma was determined by FXa assay, as shown in FIG. 2. The

concentration profiles of heparin-DOCA conjugate over time

were similar to the results of the aPTT assay shown in FIG.

1.. The concentration of absorbed heparin-DOCA increased with the increase of the dosage. The therapeutic target

range was 0.1 to 0.2 IU/ml. For a 200 mg/kg does of

heparin-DOCA conjugate, the mean concentration peak at one hour was about 9-10 times the baseline and the

concentration at that time was about 1.0 IU/ml. The plasma

concentration of heparin-DOCA conjugate returned to the baseline after 3 hours. Therefore, the absorption of

heparin-DOCA in the Gl tract was confirmed.

Example 11

Heparin-DOCA Conjugate Absorption in the Gl Tract of Rats

To determine the absorption of heparin-DOCA conjugate in the Gl tract as a function of the ratio of DOCA to

heparin, heparin-DOCA conjugates were synthesized with DOCA: heparin mole ratios of 2.5, 5.0, and 10.0, as

described in Example 1. As shown in Table 1, the

bioactivity of heparin-DOCA conjugates decreased slightly

as the mole ratio of DOCA to heparin increased. However, since the molecular weight of heparin-DOCA increased as the

mole ratio of DOCA to heparin increased, the bioactivity of heparin-DOCA conjugates as a function of mole ratio

decreased only about 5%. That is, the bioactivities of

heparin and heparin-DOCA conjugate (10:1 mole ratio) were 1,734 and 1,632±7 IU/mol, respectively.

FIG. 3 shows the change in the clotting time according to the coupled mole ratio of DOCA to heparin. In this experiment, the dosage of heparin-DOCA conjugate was

100 mg/kg. When the mole ratio of the coupled DOCA to

heparin increased, the bioactivity of heparin DOCA

conjugate slightly decreased, as shown in Table 1, whereas

the maximum clotting time increased. This result indicates

that the heparin-DOCA conjugate facilitated absorption of

heparin in the Gl tract of rats.

Example 12

Effect of Hydrophobic Agent Coupled to Heparin on Gl

Absorption in Rats

To show the effect of a hydrophobic agent coupled to

heparin on Gl absorption, heparin-DOCA, heparin-cholesterol,

heparin-palmitic acid, and heparin-lauric acid prepared

according to Examples 1-3 were tested. As shown in Table 1, the mole ratio of hydrophobic agent to heparin was

controlled in the range of 4 to 4.5. The bioactivities of these heparin derivatives were similar to each other, i.e.,

in the range of 113 to 123 IU/mg. FIG. 4 shows the

absorption values obtained after oral administration of 100

mg/kg of these heparin derivatives. Maximum clotting times at one hour after administration, as measured by the aPTT assay, were 32+6.1 seconds for heparin-cholesterol, 29+8.3

seconds for heparin-palmitic acid, and 25.9+6.6 seconds for

heparin-lauric acid. The carbon numbers of cholesterol, palmitic acid, and lauric acid were 24, 16, and 12,

respectively, and the hydrophobicity of the hydrophobic

agent is proportion to the number of carbon atoms. Thus,

the maximum clotting time increase with the hydrophobicity

of the coupled hydrophobic agent. This result indicated that the hydrophobicity of the heparin derivative was an

important property for increasing the absorption of heparin

in the Gl tract. Even though cholesterol is more

hydrophobic than DOCA, however, heparin-DOCA conjugate

exhibited a higher clotting time than heparin-cholesterol

conjugate. Possible explanations for this observation

include (1) the amphiphilic properties of heparin-DOCA

conjugate, which may improve the permeability of the

heparin derivative in the Gl wall, and (2) the interaction

between the DOCA moiety of the heparin-DOCA conjugate and

the DOCA receptors in the Gl wall, especially in the ileum,

which might increase the adhesion of heparin-DOCA conjugate

to the Gl wall, thereby increasing the probability of absorption. Example 13

Histological Evaluation of Gl Tract

In this example heparin-DOCA conjugate was

administered to rats by oral gavage according to the

procedure of Example 10. The mole ratio of coupled DOCA to

heparin in the heparin-DOCA conjugate was 10. That is, ten

molecules of DOCA were coupled to one molecule of heparin.

The dose amount was 200 mg/kg. At 1, 2, and 3 hours after

dosing, rats were anesthetized with diethyl ether and were

sacrificed by cutting the diaphragm. Gastric, duodenal, jejunal, and ileal tissues were removed from the rats and

fixed in neutral buffered formalin for processing. Gl tissues sampled before administration of heparin-DOCA

conjugate were prepared as control samples. The tissue

specimens were washed with alcohol to remove any water.

Specimens were perfused with colored silicone and embedded

in paraffin. The embedded specimens were cut into 5 μm sections using a microtome at -20 ^°C , and picked up on a glass slide. The tissue sections were then washed with

xylene and absolute alcohol, respectively, to remove the

paraffin. The prepared 5 μm sections were then stained with hematoxylin and eosin (H&E) according to procedures well

known in the art. At least 4 rats were used for each treatment .

For evaluation by transmission electron microscopy

(TEM) , the gastric, duodenal, jejunal, and ileal tissues were fixed with 1% osmium tetroxide in PBS (0.1 M, pH 7.4),

and then hydrated by changing the alcohol concentration

gradually from 50 to 100%. The hydrated tissues were

infiltrated with propylene oxide and embedded with an epon

mixture. The embedded tissues were sectioned as about 50-60

nm thickness slides. These slides were stained very lightly

with uranyl acetate and lead citrate for 1 minute, and were

observed by TEM (Hitachi 7100, Tokyo, Japan).

FIG. 5 shows that there was no evidence of damage to

the Gl wall, such as occasional epithelial cell shedding,

villi fusion, congestion of mucosal capillary with blood, or focal trauma, in any parts of the stomach, duodenum,

jejunum, or ileu . These results confirm that increased

absorption of heparin derivatives was not caused by the

disruption of the gastrointestinal epithelium.

FIG. 6 shows the electron-microscopic morphology of

microvilli after exposure to heparin derivatives. The control samples showed healthy tight junctions, microvilli,

and mitochondria. After 1, 2, and 3 hours, the cell

appearance in all sections showed on signs of damage, such as microvilli fusion, dissolution, disoriented cell layer

with porosity, or cytotoxic effect. Microvilli exposed to

heparin derivatives were also found to be as healthy as the

control. The absence of tissue damage indicates that the

enhancing effect of the coupled DOCA on heparin absorption in the Gl tract was not caused by changing the tissue

structure.

Example 14

Conjugation of Lower Molecular Weight Heparin to DOCA

Conjugates of heparin to DOCA were synthesized

according to the procedure of Example 1 except that

unfractionated heparin ("UFH"), i.e., the compound referred

to simply as "heparin" in previous examples, 6000 molecular

weight heparin ( "LMWH( 6K) " ) , and 3000 molecular weight

heparin ("LMWH(3K)") were used. The resulting conjugates,

UFH-DOCA, LMWH(6K)-DOCA, and LMWH( 3K) -DOCA, were then

characterized, as shown in Table 2. Table 2

a Measured by light scattering

The maximum ratio of DOCA to heparin obtained in UFH-

DOCA was 10 when the feed ratio of UFH to DOCA was 1:200.

Under these conditions, the ratios obtained with lower

molecular weight heparins were 1.3 for LMWH( 3K) -DOCA and

3.6 for LMWH(6K)-DOCA.

The mole ratio of DOCA to heparin decreased with the

decrease in molecular weight of heparin because of the

fewer number of amine groups available for bonding to DOCA. Bioactivities of the heparin-DOCA conjugates also decreased

with the decrease of molecular weight of heparin, although

all heparin-DOCA conjugates demonstrated similar

bioactivities in the range of 116.9±1.6 to 134.3+0.8 by FXa

assay. After conjugation with DOCA, all of the heparin-DOCA conjugates showed above 70% relative bioactivity compared

to the unmodified heparin.

Example 15

Oral Absorption of Lower Molecular Weight Heparin-

DOCA Conjugates

The lower molecular weight heparin-DOCA conjugates

prepared in Example 14 were tested for absorption in the Gl

tract of rats after oral administration according to the

procedure of Example 10. FIG. 7 shows the effect of

molecular weight of heparin on the absorption of heparin-

DOCA conjugates in the Gl tract. LMWH( 3K) -DOCA, LMWH(6K)-

DOCA, and UFH-DOCA (i.e., heparin-DOCA) were each

administered by oral gavage at 100 mg/kg dosage. The

clotting times of LMWH( 3K) -DOCA and UFH-DOCA were lower than that of LMWH( 6K) -DOCA; the mean aPTT times at 1 hour were 31.0+6.01 and 51.0+8.7, respectively (p<0.005). These

data suggest that the clotting time of LMWH( 6K) -DOCA was

1.5- and 3-fold greater than those of LMWH( 3K) -DOCA and

UFH-DOCA, respectively. The concentration profiles of heparin-DOCA conjugates with time were similar to the

results of the aPTT assay. When UFH-DOCA was administered

at 100 mg/kg dosage, the peak concentrations of plasma was 4.10±1.3 μg/ml, which was very low compared to the concentration of LMWH( 6K) -DOCA at the same dosage level.

The absorption of LMWH( 6K) -DOCA in the Gl tract was

determined according to the dose amount in the range of 20

to 100 mg/kg, as shown in FIG. 8. When 100 mg/kg of

LMWH(6K) was administered orally to rats, the clotting time as measured by aPTT assay was about 30 seconds at 1 hour

after dosing. This curve fell to baseline at 2 hours after

dosing. On the other hand, oral delivery of LMWH( 6K)-DOCA

resulted in the increased heparin absorption in rats as

shown by the highly elevated aPTT values. When LMWH(6K)-

DOCA was dosed at 100 mg/kg, the peak plasma aPTT value was

about 87.8±11.1 seconds (the baseline aPTT values averaged

20 seconds). Heparin derivatives dosed at 20 mg/kg and 50

mg/kg gave mean peak aPTT responses of 52.5±4.7 and 68.4±7.2 seconds, respectively (p<0.005). The therapeutic

range of heparin, which is about 1.5-2.5 times baseline in

aPTT, is matched with a dose of 20 mg/kg, as shown in FIG.

8A.

Concentrations of heparin derivatives in the plasma

could be determined using the anti FXa assay. When 100

mg/kg of LMWH( 6K) -DOCA was administered orally, the

concentration of LMWH(6K) was 1.34±0.28 μg/ml. The low facilitate anticoagulation activity. However, the maximum

peak of LMWH( 6K) -DOCA was 8.21+1.6 μg/ml at a dose of 100 mg/kg, as shown in FIG. 8B. The therapeutic target range

was 0.1 to 0.2 IU/ml. The mean concentration peaks were about 9-10 times the baseline. These results suggest that heparin derivatives can perform as an oral anticoagulant

drug for patients at risk for deep vein thrombosis and

pulmonary embolism.

Example 16

Histological Evaluation of the Gl Tract after Oral Administration of Lower Molecular Weight Heparin-DOCA

Conjugates

Gl tract tissues from rats given a single dose of 100

mg/kg of lower molecular weight heparin-DOCA conjugates prepared according to the procedure of Example 14 were

examined histologically according to the procedures of Example 13. The results were substantially similar to those

of Example 13. That is, no evidence of damage to any of the

tissues of the Gl wall was detected.

Claims

The subject matter claimed is:

1. A method of treating a patient in need of

anticoagulation therapy comprising orally administering an effective amount of a

composition comprising heparin covalently bonded

to a hydrophobic agent selected from the group

consisting of bile acids, sterols, and alkanoic

acids, and mixtures thereof.

2. The method of claim 1 wherein said hydrophobic agent is a bile acid selected from the group

consisting of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid,

ursocholic acid, ursodeoxyeholic acid,

isoursodeoxycholie acid, lagodeoxycholic acid,

glycocholic acid, taurocholic acid,

glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid,

hyodeoxycholic acid, and mixtures thereof.

3. The method of claim 2 wherein said bile acid is

deoxycholic acid.

. The method of claim 1 wherein said hydrophobic

agent is a sterol selected from the group

consisting of cholestanol, coprostanol,

cholesterol, epicholesterol, ergosterol,

ergocalciferol, and mixtures thereof.

5. The method of claim 1 wherein said hydrophobic

agent is an alkanoic acid comprising about 4 to

20 carbon atoms.

6. The method of claim 5 wherein said alkanoic acid is a member selected from the group consisting

of butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid,

myristic acid, palmitic acid, stearic acid, and

mixtures thereof.

7. The method of claim 1 wherein said composition

further comprises a pharmaceutically acceptable

carrier.

8. The method of claim 1 wherein said heparin

comprises a molecular weight of at least about

3000.

9. The method of claim 8 wherein said heparin

comprises a molecular weight of at least about

6000.

10. The method of claim 1 wherein said heparin

comprises a molecular weight less than about 12,000.

11. A method for enhancing oral administration of a

macromolecular agent comprising:

(a) conjugating said macromolecular agent

to a hydrophobic agent selected from

the group consisting of bile acids,

sterols, alkanoic acids, and mixtures

thereof to result in a hydrophobized

macromolecular agent; and

(b) orally administering an effective

amount of said hydrophobized

macromolecular agent to a patient in need thereof.

2. The method of claim 11 wherein said hydrophobic

agent is a bile acid selected from the group

consisting of cholic acid, deoxycholic acid,

chenodeoxycholic acid, lithocholic acid,

ursocholic acid, ursodeoxyeholic acid,

isoursodeoxycholie acid, lagodeoxycholic acid,

glycocholic acid, taurocholic acid,

glycodeoxycholic acid, glycochenodeoxycholic

acid, dehydrocholic acid, hyocholic acid,

hyodeoxycholic acid, and mixtures thereof.

13. The method of claim 12 wherein said bile acid is

deoxycholic acid.

14. The method of claim 11 wherein said hydrophobic

agent is a sterol selected from the group

consisting of cholestanol, coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciferol , and mixtures thereof.

15. The method of claim 11 wherein said hydrophobic

agent is an alkanoic acid comprising about 4 to 20 carbon atoms.

16. The method of claim 15 wherein said alkanoic

acid is a member selected from the group

consisting of butyric acid, valeric acid,

caproic acid, caprylic acid, capric acid, lauric

acid, myristic acid, palmitic acid, stearic acid,

and mixtures thereof .

17. The method of claim 11 wherein said

macromolecular agent is a member selected from

the group consisting of heparin, heparan sulfate,

sulfonyl polysaccharide, polysaccharide

derivatives, and mixtures thereof.

18. The method of claim 17 wherein said

macromolecular agent is heparin.

19. The method of claim 11 wherein said

macromolecular agent is a peptide.

20. The method of claim 19 wherein said

macromolecular agent is insulin.

21. The method of claim 19 wherein said

macromolecular agent is calcitonin.

22. A method of treating a patient in need of

composition comprising a member selected from

the group consisting of heparin, heparan sulfate,

sulfonyl polysaccharide, heparinoids, and

mixtures thereof covalently bonded to a

hydrophobic agent selected from the group

consisting of bile acids, sterols, and alkanoic

acids, and mixtures thereof.