KR20080042045A - Lipid construct for delivery of interferon to a mammal - Google Patents

Abstract

The instant invention is drawn to a hepatocyte targeted composition comprising interferon associated with a lipid construct comprising amphipathic lipid molecules and receptor binding molecule. The composition can comprise a mixture of free interferon and interferon associated with the complex. The composition can be modified to protect interferon and the complex from degradation. The invention also includes methods for the manufacture of the composition and loading interferon into the composition and recycling various components of the composition and methods of treating individuals infected with the hepatitis C and other hepatitis viruses.

Description

Lipid Construct for Delivery of Interferon to Mammal

Hepatitis C virus (HCV) infection is the most common chronic bloodstream infection in the United States. The Center for Disease Control (CDC) estimates that an average of 240,000 new infections occurred each year during the 1980s. Since the 1980s, the number of new infections has declined yearly, reaching approximately 30,000 in 2003. It is estimated that approximately 3.9 million Americans, about 1.8% of the US population, are infected with HCV. Approximately 2.7 million of these people are chronically infected and may not know they are infected because they are not clinically ill. Infected persons are a source of delivery to others and are at risk of chronic liver disease or other HCV-related chronic diseases for the first 20 years or more after the initial infection.

Current treatment protocols for hepatitis C are based on the use of various interferon-alpha preparations administered intramuscularly or subcutaneously. Interferon alpha is a natural glycoprotein secreted by cells in response to viral infections. Interferon-alpha has immunomodulatory, anti-proliferative and anti-viral properties and binds to membrane receptors to exert an effect. Interferon-alpha plays an important role in balancing the immune system and is generally at very low levels by the body when compared to conventional injectable interferon therapies, which require high doses to achieve the required concentration at the site of the disease. Is generated. If interferon alpha is administered directly into the bloodstream, very high dosages (millions of international units (IU)) are required to reach a sufficient amount in the diseased tissue. The released interferon-alpha is not delivered to the targeted body part but reaches a wide range of systems within the body. There is a need for a composition of interferon-alpha in which interferon-alpha is released at a relatively constant rate over an extended period of time and a portion of the contained interferon-alpha is targeted for delivery to the liver to further reduce or eliminate hepatitis C virus.

Interferon alfa-2a (ROFERON-A ^® , Hoffmann-La Roche), interferon alfa-2b (INTRON-A ^® , Schering-Plough )) And interferon alfacon-1 (INFERGEN ^® , Intermune) have been approved in the United States as the sole agent for the treatment of adults with chronic hepatitis C. In the treatment of chronic hepatitis C, the recommended dosages of interferon alpha-2b and alpha-2a are administered subcutaneously or intramuscularly using 3,000,000 units three times a week. The treatment period is administration for 6 months to 2 years. For Interferon alfacon-1, the recommended dose is 9 μg 3 times a week for the first treatment phase and 15 μg 3 times a week for an additional 6 months for patients with no response or relapse. Treatment with only interferon has a persistent response in less than 15% of the subjects. Ribavirin, a synthetic nucleoside that is active against a broad spectrum of viruses, is often administered in parallel with interferon-alpha in the treatment of chronic hepatitis C.

Recently, PEG-interferon-alpha, sometimes also called pegylated interferon, has been used for the treatment of chronic hepatitis C. Two formulations of the following PEG-interferon-alpha were studied in hepatitis C patients: PEG-interferon-alpha-2b (PEG-INTRON ^® , Schering-Flow) and PEG-interferon-alpha-2a (PEGASYS ^® , Hoffman-La Roche). PEG-interferon-alpha differs from unmodified interferon-alpha in that a polyethylene glycol molecule is attached to the interferon molecule. This structural modification allows for slower removal from the body, resulting in higher and more consistent blood levels of interferon-alpha even with less frequent administration. Unlike unmodified interferon-alpha, which must be injected three times a week for the treatment of chronic hepatitis C, PEG-interferon-alpha requires only one injection per week.

The main purpose of treating chronic hepatitis C is to remove detectable viral RNA from the blood. The absence of detectable hepatitis C virus RNA in the blood for six months after completion of therapy is known as a persistent response.

Thus, the need for compositions and methods for treating patients infected with hepatitis C virus has not been met in the art. The present invention meets this need by providing long acting compositions targeted for delivery to the liver.

Brief summary of the invention

In one aspect, the invention includes a lipid construct comprising at least one interferon, amphipathic lipids, and extended amphipathic lipids, wherein the extended amphipathic lipids include a proximal, midterm and distal portions, wherein the proximal portion Connects the extended amphipathic lipid to the structure, the distal portion targets the structure to a receptor presented by hepatocytes, and the medial portion connects the proximal and distal portions.

In another aspect, the interferon is interferon-alpha, interferon alpha-1a, PEGylated interferon alpha-1a, interferon-alpha-n1, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n3, interferon alphacon -1, interferon n-3, PEG-interferon alpha 2a, PEG-interferon alpha 2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, interferon gamma-1a, interferon gamma-1b, PEGylated interferon beta -1a, PEGylated interferon beta-1b, derivatives thereof, and combinations of any of the aforementioned interferons.

In another aspect, the lipid construct comprises one or more anti-viral agents, amphiphilic lipids, and extended amphipathic lipids that are not interferon or interferon derivatives, wherein the extended amphipathic lipids comprise a proximal, midterm, and distal portions. Where the proximal portion connects the extended amphipathic lipid to the structure, the distal portion targets the structure to a receptor presented by hepatocytes, and the middle portion connects the proximal and distal portions.

In another aspect, the active ingredient comprises one or more interferons and one or more anti-viral agents that are not interferon or interferon derivatives.

In another aspect, the lipid construct further comprises one or more active ingredients in insoluble form associated therewith.

In another aspect, the amphiphilic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetyl phosphate, 1,2-dipalmitoyl-sn-glycerol- [3- Phospho-rac- (1-glycero)], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-force At least one lipid selected from the group consisting of: ethanolamine-N- (succinyl), derivatives thereof, and mixtures of any of the foregoing compounds.

In another aspect, the proximal portion of the extended amphiphilic lipid comprises at least one but no more than two long chain acyl hydrocarbon chains attached to the backbone, wherein each hydrocarbon chain is comprised of a saturated hydrocarbon chain and an unsaturated hydrocarbon chain. Are independently selected.

In another aspect, the backbone comprises glycerol.

In another aspect, the distal portion of the extended amphipathic lipid is biotin, biotin derivative, iminobiotin, iminobiotin derivative, biocytin, biocitin derivative, iminobiocitin, iminobiocitin derivative, and At least one member selected from the group consisting of hepatocyte specific molecules that bind to receptors on hepatocytes.

In another aspect, the extended amphipathic lipids are N-hydroxysuccinimide (NHS) biotin, sulfo-NHS-biotin, N-hydroxysuccinimide long chain biotin, sulfo-N-hydroxysuccinimide long chain biotin , D-biotin, biocitin, sulfo-N-hydroxysuccinimide-SS-biotin, biotin-BMCC, biotin-HPDP, iodoacetyl-LC-biotin, biotin-hydrazide, biotin-LC-hydrazide , Biocytin hydrazide, biotin cardaberine, carboxybiotin, photobiotin, ρ-aminobenzoyl biocitin trifluoroacetate, ρ-diazobenzoyl biocitin, biotin DHPE, biotin-X-DHPE, 12- ((Biotinyl) amino) dodecanoic acid, 12-((biotinyl) amino) dodecanoic acid succinimidyl ester, S-biotinyl homocysteine, biocytin-X, biocytin x-hydrazide, biotinethylenediamine, Biotin-XL, Biotin-X- Ethylenediamine, Biotin-XX Hydrazide, Biotin-XX-SE, Biotin-XX, SSE, Biotin-X-Cadaberine, α- (t-BOC) Biocitin, N- (Biotinyl) -N '-( Iodoacetyl) ethylenediamine, DNP-X-biocitin-X-SE, biotin-X-hydrazide, norbiotinamine hydrochloride, 3- (N-maleimidylpropionyl) biocitin, ARP, biotin -1-sulfoxide, biotin methyl ester, biotin-maleimide, biotin-poly (ethyleneglycol) amine, (+) biotin 4-amidobenzoic acid sodium salt, biotin 2-N-acetylamino-2-deoxy-β -D-glucopyranoside, biotin-α-DN-acetylneuramid, biotin-α-L-fucoside, biotin lacto-N-bioside, biotin-Lewis-A trisaccharide, biotin-Lewis-Y Tetrasaccharide, biotin-α-D-mannopyranoside, biotin 6-O-phospho-α-D-mannopyranoside and polychromium-poly (bis)-[N -(2,6- (diisopropylphenyl) carbamoylmethyl) imino] diacetic acid.

In a further aspect, the median portion of the extended amphipathic lipid comprises a thio-acetyl triglycine polymer or derivative thereof, wherein the extended amphipathic lipid molecule extends out of the surface of the lipid construct.

In one aspect, the construct further comprises at least one active ingredient associated with a water insoluble target molecule complex, wherein the complex comprises a plurality of linked individual units, wherein the individual units are transition elements, internal A crosslinking component selected from the group consisting of a transition element, a neighboring element of the transition element, and a mixture of any of the above elements, and a complex forming component, but a chromium target molecule complex is formed when the transition element is chromium.

In another aspect, the construct further comprises one or more active ingredients not associated with the target molecule complex.

In another aspect, the crosslinking component is chromium.

In another aspect, the complex forming component comprises poly (bis)-[(N- (2,6-diisopropylphenyl) carbamoylmethyl) iminodiacetic acid].

In one aspect, the distal component of the extended amphiphilic lipid comprises a nonpolar derivatized benzene ring or heterobicyclic ring structure.

In another aspect, the structure includes a positive charge, a negative charge or a combination thereof.

In another aspect, the extended amphipathic lipid comprises one or more carbonyl moieties positioned at a distance of about 13.5 mm 3 or less from the distal end.

In a further aspect, the extended amphipathic lipid comprises one or more carbamoyl moieties comprising secondary amines.

In one aspect, the extended amphipathic lipid comprises chromium charged at the median point.

In another aspect, the lipid construct further comprises cellulose acetate hydrogen phthalate.

In one aspect, the invention is a lipid construct comprising at least one interferon, amphipathic lipids, and extended amphipathic lipids, wherein the extended amphipathic lipids comprise a proximal, a median, and a distal portion, wherein the proximal portion extends. The amphiphilic lipid to the construct, the distal portion to the receptor presented by the hepatocytes, and the medial portion to connect the proximal and distal portions, the method comprising the lipid in water This is accomplished by forming a suspension of the construct and loading the active ingredient into the lipid construct.

In another aspect, the step of loading the active ingredient into the lipid construct includes an equilibrium support and a non-equilibrium support.

In one aspect, the step of loading the active ingredient into the lipid construct comprises adding a free active ingredient containing solution to the mixture of lipid constructs in water and maintaining the active ingredient in contact with the mixture until equilibrium is reached.

In another aspect, the method of making an interferon binding lipid construct further comprises the step of finally supporting the active ingredient in the lipid construct after the mixture has reached equilibrium, and the free active ingredient containing solution is removed from the construct, The construct herein further contains one or more active ingredients associated therewith.

In another aspect, a method of making an interferon binding lipid construct comprises a rapid filtration procedure, centrifugation, filter centrifugation, and a streptavidin agarose affinity-resin gel having affinity for biotin, iminobiotin, or derivatives thereof. Or removing the free active ingredient containing solution from the lipid construct containing at least one active ingredient associated therewith via a process selected from the group consisting of chromatography with ion-exchange resins.

In another aspect, the method of making an interferon binding lipid construct further comprises adding a chromium complex comprising a plurality of linked individual units to the lipid construct.

In another aspect, the method of making an interferon binding lipid construct further comprises adding cellulose acetate hydrogen phthalate to the lipid construct.

In another aspect, the method of making an interferon binding lipid construct further comprises recovering from the process at least one substance selected from the group consisting of an active ingredient, an ion-exchange resin and a streptavidin agarose affinity gel.

In one aspect, a method of increasing the bioavailability of one or more active ingredients in a patient comprises combining the one or more active ingredients with a lipid construct comprising a plurality of non-covalent multi-dentate binding sites. And administering to the patient the construct containing the active ingredient.

In another aspect, the increase in bioavailability further comprises the step of isoelectric point adjustment of the one or more active ingredients.

In another aspect, the active ingredient is interferon-alpha, interferon alpha-1a, PEGylated interferon alpha-1a, interferon-alpha-n1, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n3, interferon Alphacon-1, interferon n-3, PEG-interferon alpha 2a, PEG-interferon alpha 2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, interferon gamma-1a, interferon gamma-1b, PEGylation Interferon beta-1a, PEGylated interferon beta-1b, derivatives thereof, and combinations of any of the aforementioned interferons.

In another aspect, the active ingredient is an anti-viral agent that is not an interferon or an interferon derivative.

In another aspect, the active ingredient comprises one or more interferons and one or more anti-viral agents that are not interferon or interferon derivatives.

In another aspect, the lipid construct is interferon, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetyl phosphate, 1,2-dipalmitoyl-sn-glycero- [ 3-phospho-rac- (1-glycerol)], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and 1,2-dipalmitoyl-sn-glycero-3- Phosphoethanolamine-N- (succinyl) or derivatives thereof and hepatocyte receptor binding molecules.

In one aspect, a method of forming a sustained release composition that increases the bioavailability of one or more active ingredients in a host comprises streptavidin agar having a pH of 9.5 or higher via a lipid comprising an iminobiotin or an iminobiotin derivative. Binding the los affinity gel to remove the structure from the bulk phase medium, separating the structure from the bulk phase medium, adjusting the pH of the affinity gel aqueous mixture to pH 4.5, Releasing from the affinity gel, wherein the released construct contains at least one insoluble active ingredient, wherein the construct is redissolved under physiological pH conditions in the host upon administration to a warm-blooded host.

In another aspect, a method of treating a hepatitis patient comprises administering to the hepatitis patient an effective amount of a lipid construct comprising one or more interferons, amphiphilic lipids and extended amphipathic lipids, wherein the extended amphipathic lipid is proximal , Proximal and distal, wherein the proximal portion connects the extended amphipathic lipids to the structure, the distal portion targets the structure to receptors presented by hepatocytes, and the medial portion connects the proximal and distal portions.

In another aspect, the patient is infected with one or more hepatitis selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F, and hepatitis G.

In one aspect, the active ingredient is interferon-alpha, interferon alpha-1a, PEGylated interferon alpha-1a, interferon-alpha-n1, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n3, interferon alphacon -1, interferon n-3, PEG-interferon alpha 2a, PEG-interferon alpha 2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, interferon gamma-1a, interferon gamma-1b, PEGylated interferon beta -1a, PEGylated interferon beta-1b, derivatives thereof, and combinations of any of the aforementioned interferons.

In another aspect, the active ingredient is an anti-viral agent that is not an interferon or an interferon derivative.

In another aspect, the active ingredient comprises one or more interferons and one or more anti-viral agents that are not interferon or interferon derivatives.

In another aspect, the lipid construct further comprises a target molecule complex, wherein the complex comprises a plurality of linked individual units, further wherein the linked individual units are transition elements, internal transition elements, adjacent to transition elements. A crosslinking component selected from the group consisting of an element and a mixture of any of the above elements, and a complex forming component, but when the transition element is chromium, a chromium target molecule complex is formed.

In another aspect, the lipid construct further comprises one or more active ingredients that are not associated with the target molecule complex.

In another aspect, the administration is oral or subcutaneous.

In one aspect, the invention comprises extended lipid molecules comprising interferon and hepatocyte receptor binding moieties to a hepatitis patient, and administering lipid structures that exist in various sizes, thereby increasing interferon delivery from liver to hepatocytes in hepatitis patients Wherein the hepatocyte receptor binds to an optimally sized construct to enhance endocytosis and induce the intended pharmacological action of the lipid construct.

In another aspect, the patient has at least one hepatitis virus or hepatitis A selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F, or hepatitis G. It is infected with a combination of viruses.

In another aspect, the active ingredient is interferon-alpha, interferon alpha-1a, PEGylated interferon alpha-1a, interferon-alpha-n1, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n3, interferon Alphacon-1, interferon n-3, PEG-interferon alpha 2a, PEG-interferon alpha 2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, interferon gamma-1a, interferon gamma-1b, PEGylation Interferon beta-1a, PEGylated interferon beta-1b, derivatives thereof, and combinations of any of the aforementioned interferons.

In another aspect, the active ingredient is an anti-viral agent that is not an interferon or an interferon derivative.

In another aspect, the active ingredient comprises one or more interferons and one or more anti-viral agents that are not interferon or interferon derivatives.

In another aspect, a method of treatment of a patient provides a three-dimensional array of lipid molecules that prevents the hydrolase from accessing the active ingredient in the lipid construct to protect the active ingredient in the lipid construct from hydrolysis. It further includes.

In another aspect, the method further comprises adding cellulose acetate hydrogen phthalate to the lipid construct and reacting with the individual lipid molecule.

In another aspect, the method further comprises producing the active ingredient in an insoluble dosage form in the lipid construct.

In one aspect, the invention includes a lipid construct, a physiological buffer, an applicator and instructions for use, wherein the lipid construct comprises at least one interferon, an amphipathic lipid and an extended amphipathic lipid, wherein the extension Amphiphilic lipids, including proximal, medial and distal portions, wherein the proximal portion connects the extended amphipathic lipids to the structure, the distal portion targets the structure to receptors presented by hepatocytes, and the proximal portion connects the proximal and distal portions. And a kit for treating a mammal infected with a virus.

In another aspect, the kit further comprises one or more active ingredients.

In another aspect, the kit treats a patient infected with a virus of one or more hepatitis selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F, and hepatitis G. It is to.

BRIEF DESCRIPTION OF THE DRAWINGS In order to illustrate the invention, there is shown a diagram of a particular embodiment of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

1 illustrates an interferon binding lipid construct comprising interferon, amphiphilic lipid molecules and extended amphipathic lipids.

2 depicts the pathway for making biocithin.

Figure 3 shows the route for making iminobiocitin.

Figure 4 shows the route to prepare benzoyl thioacetyl triglycine iminobiocitin (BTA-3-gly-iminobiocitin).

5 depicts the pathway for preparing benzoyl thioacetyl triglycine.

FIG. 6 shows the route to prepare benzoyl thioacetyl triglycine sulfo-N-hydroxysuccinimide (BTA-3-gly-sulfo-NHS).

FIG. 7 shows the route to prepare benzoyl thioacetyl triglycine iminobiocitin (BTA-3-gly-iminobiocitin).

8 depicts the pathways for preparing lipid anchors and hepatocyte receptor binding molecules (LA-HRBM).

9 shows the potential binding site between cellulose acetate hydrogen phthalate and interferon.

10 vs acidic conditions. The structural change of iminobiotin under basic conditions is shown.

11 schematically shows a method of making an interferon binding lipid construct comprising amphipathic lipid molecules and extended amphipathic lipids.

12 consists of two parts. 12A shows the relative expression levels in the liver and spleen of mice receiving interferon alpha. 12B shows relative expression levels in liver and spleen of mice receiving interferon alpha and HDV.

13 shows the effect of HDV targeting on hepatic PKR activation by interferon alpha in a mouse model.

The present invention includes hepatocyte targeting pharmaceutical compositions wherein the interferon is associated with a water-insoluble target molecule complex within the construct, the composition providing an effective means of targeting hepatocytes between patients to manage hepatitis C virus and other viruses.

The present invention includes lipid constructs comprising interferons, amphiphilic lipids and extended amphiphilic lipids (receptor binding molecules). Extended amphiphilic lipids include the proximal, middle and distal portions. The proximal connects the extended lipid to the construct, the distal connects the construct to the hepatocyte binding receptor in the liver, and the proximal connects the proximal and distal portions.

Lipid constructs are spherical lipid and phospholipid particles in which individual lipid molecules interact cooperatively to produce a bipolar lipid membrane that encapsulates and isolates the part in which it is formed. Lipid constructs can target the delivery of interferon to hepatocytes in the liver and can provide a sustained release of interferon to further reduce or eliminate hepatitis C virus or other viruses that damage the liver.

The composition of the present invention may be administered by various routes, including subcutaneous or oral, for the purpose of treating mammals infected with hepatitis C virus and other viruses.

In addition, the present invention provides a method of preparing a lipid construct comprising interferon, amphipathic lipids and extended amphipathic lipids. Extended amphiphilic lipids include the proximal, middle and distal portions. The proximal portion connects the extended lipid to the structure. The distal portion connects the structure to hepatocyte binding receptors, and the distal portion connects the proximal and distal portions.

The present invention also provides a method for preparing a composition comprising a free interferon in a construct and an interferon associated with a water insoluble target molecule complex and targeting delivery of the complex to hepatocytes. The target molecule complex consists of a plurality of linked individual units of the structure formed by the metal complex contained within the lipid structural matrix.

In addition, the present invention provides a method of treating an individual infected with hepatitis C virus and other viruses by administering an effective dose of a lipid construct that includes interferon, amphiphilic lipids, and extended amphiphilic lipids and is targeted for delivery to hepatocytes. To provide.

The invention also provides for individuals infected with hepatitis C virus and other viruses by administering an effective dose of a lipid construct targeted to delivery to hepatocytes, including interferon, amphiphilic lipids, extended amphiphilic lipids, and water insoluble target molecule complexes. Provides a way to treat it.

Justice

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the names and organic chemistry and protein chemistry experimental procedures used herein are those known in the art and commonly used.

As used herein, the articles “a” and “an” are used to refer to one or more than one (ie, one or more) grammatical references. For example, "element" means one element or more than one element.

The term "active ingredient" refers to interferon and other anti-viral compounds.

The term "lower" means a group containing 1 to 6 carbon atoms.

The term "alkyl" is used on its own or as part of another substituent, and unless stated otherwise straight chains having the indicated number of carbon atoms (eg C ₁ -C ₆ means 1 to 6 carbons), Means a branched or cyclic hydrocarbon, including straight chain, branched or cyclic groups. Examples are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, cyclohexyl and cyclopropylmethyl and the like. Most preferred are (C ₁ -C ₃ ) alkyl, in particular ethyl, methyl and isopropyl and the like.

The term "alkylene" is used on its own or as part of another substituent, and unless stated otherwise means a straight chain, branched or cyclic hydrocarbon having two substitution sites, for example methylene (-CH _2- ), Ethylene (-CH ₂ CH _2- ), isopropylene (-CH (CH ₃ ) = CH ₂ ), and the like.

The term "aryl", used alone or in combination with other terms, unless saturated or otherwise, contains one or more rings (typically one, two or three rings), wherein The ring refers to a cyclic carbon ring structure that may be attached together in a pendant manner, such as biphenyl, or may be fused, such as naphthalene. Examples are phenyl, anthracyl and naphthyl and the like. The structure may have one or more substitution sites to which functional groups such as alcohol, alkoxy, amide, amino, cyanide, halogen and nitro are bound.

The term "aryl lower alkyl" means a functional group in which an aryl group is attached to a lower alkylene group, for example -CH ₂ CH ₂ -phenyl.

The term "alkoxy" is used alone or in combination with other terms, and unless stated otherwise an alkyl group containing a substituent such as an alkyl group or hydroxyl group having the indicated number of carbon atoms and having a indicated number of carbon atoms represents an oxygen atom. Connected to the rest of the molecule through, for example, -OCHOH-, -OCH ₂ OH, methoxy (-OCH ₃ ), ethoxy (-OCH ₂ CH ₃ ), 1-propoxy (-OCH ₂ CH ₂ CH ₃ ), 2-propoxy (isopropoxy), butoxy (-OCH ₂ CH ₂ CH ₂ CH ₃ ), pentoxy (-OCH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ) and higher homologues and isomers Etc.

The term "acyl" means a functional group of the formula -C (= 0) R, wherein R is hydrogen, hydrocarbyl, amino or alkoxy. Examples include acetyl (-C (= 0) CH ₃ ), propionyl (-C (= 0) CH ₂ CH ₃ ), benzoyl (-C (= 0) C ₆ H ₅ ), phenylacetyl (-C (= O) CH ₂ C ₆ H ₅ ), carboethoxy (-CO ₂ CH ₂ CH ₃ ), dimethylcarbamoyl (-C (= 0) N (CH ₃ ) ₂ ), and the like.

The term "halo" or "halogen" is used on its own or as part of another substituent, meaning fluorine, chlorine, bromine or iodine atoms, unless stated otherwise.

The terms “heterocycle” or “heterocyclyl” or “heterocyclic” are used on their own or as part of another substituent, unless otherwise stated, are unsubstituted or substituted and stable, carbon atoms and N, O and A monocyclic or polycyclic heterocyclic ring system comprising at least one heteroatom selected from the group comprising S and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized and the nitrogen atom may be optionally quaternized do. Heterocyclic systems may be attached at any heteroatom or carbon atom that affects stable structure unless otherwise stated. Examples include pyrrole, imidazole, benzimidazole, phthalein, pyridenyl, pyranyl, furanyl, thiazole, thiophene, oxazole, pyrazole, 3-pyrroline, pyrrolidene, pyrimidine, purine, quinoline , Isoquinoline, carbazole and the like.

As used herein, an amino acid is represented by its full name or the corresponding three letter code, as indicated in the table below:

Full name 3 characters  code Full name 3 characters  code Alanine Ala Leucine Leu Arginine Arg Lee Sin Lys Asparagine Asn Methionine Met Aspartic acid Asp Phenylalanine Phe Cysteine Cys Proline Pro Cystine Cys-cys Serine Ser Glutamic acid Glu Threonine Thr Glutamine Gln Tryptophan Trp Glycine Gly Tyrosine Tyr Histidine His Valine Val Isoleucine Ile

The term "chromium target molecule complex" refers to a complex comprising a large number of individual units, wherein each unit up to 6 ligands, for example N- (2,6-diisopropylphenyl), due to the multivalent molecule Carbamoylmethyl) iminodiacetic acid includes chromium (Cr) atoms capable of accepting ligands from numerous molecules. Individual units are linked together to form complex polymeric structures connected in a three-dimensional array. Polymeric composites are insoluble in water but soluble in organic solvents.

The term “lipid structure” refers to spherical lipid and / or phospholipid particles in which individual lipid molecules interact cooperatively to produce a bipolar lipid membrane that seals and isolates the portion in which it is formed.

The term “amphiphilic lipid molecule” means a lipid molecule having polar and nonpolar ends.

The term “extended amphipathic lipid” means an amphipathic molecule having a structure in which a portion of the lipid construct extends from the lipid construct to the medium surrounding the construct and can bind or interact with the receptor.

"Compound formers" are compounds that form polymeric complexes with selected metal crosslinkers, such as chromium, zirconium, and the like salts, wherein the polymeric complexes exhibit polymeric properties that are substantially insoluble in water and soluble in organic solvents. Indicates.

"Aqueous medium" means water or water containing a buffer or salt.

"Substantially soluble" means a material such as a polymeric chromium target molecule complex or other metal targeting complex that may be crystalline or amorphous, for example, in a composition formed from a complex former, and exhibits properties that are insoluble in water at room temperature. Such polymeric complexes or dissociated forms thereof, when associated with a lipid structural matrix, form a transporter that functions to transport and deliver interferon to hepatocytes in the liver of a warm-blooded host.

The use of the term "associated with" means that the mentioned material is incorporated into or on or within the surface of the lipid structural matrix.

The use of the term “glass” means that it is present in the mentioned solution of material and is not associated with the lipid construct or the target molecule complex.

The term “interferon” refers to interferon in natural or recombinant form, including alpha, beta, gamma and other forms of interferon, PEG-interferon and derivatives of the interferons mentioned above. Examples of interferons include interferon-alpha, interferon alfa-1a, PEGylated interferon alfa-1a, interferon-alpha-n1, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n3, interferon alfacon-1, Interferon n-3, PEG-interferon alpha 2a, PEG-interferon alpha 2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, interferon gamma-1a, interferon gamma-1b, PEGylated interferon beta-1a, PEGylated interferon beta-1b, derivatives thereof, and combinations of any of the aforementioned interferons, and the like.

The term "derivative" may be formed from a compound formed from a similar compound, a compound that can be expected to be derived from another compound if one atom is replaced by another atom or group of atoms, or at least in theory the named compound. Refers to a compound present.

The term “equilibrium” refers to a state where the rate at which the free active component associates with the lipid construct is approximately equal to the rate at which the active ingredient associated with the lipid construct dissociates from the lipid construct to become the free active ingredient.

The term “bioavailability” refers to a measure of the rate and extent at which an interferon or another anti-viral compound reaches the systemic circulation and becomes available at the site of action.

As used herein, the term "modulating" a biological or chemical process or state or the term "modulating" the state alters the normal progression of the biological or chemical process, or the state of the biological or chemical process It refers to a change to another new state. For example, isoelectric point modulation of a polypeptide may involve changes that increase the isoelectric point of the polypeptide. Alternatively, isoelectric point adjustment of the polypeptide may involve changes that reduce the isoelectric point of the polypeptide.

"HDV" or "Hepatocyte Delivery Vehicle" is a water-insoluble target molecule complex comprising a lipid structural matrix containing a plurality of linked individual units of the structure formed by the combination of metal crosslinkers and complex formers. "HDV" is described in WO 99/59545 (Targeted Liposomal Drug Delivery System).

"Statistical structure" means a structure formed from molecules that can migrate from one lipid structure to another, and the structure is present in a plurality of granularities that can be represented by a Gaussian distribution.

"Multi-toothed bond" is a chemical bonding process that utilizes multiple binding sites in a lipid construct, such as cellulose acetate hydrogen phthalate, phospholipids and interferons. These binding sites promote hydrogen bonds, ion-dipole and dipole-dipole interactions that form non-covalent bonds in which individual molecules operate in parallel to function to bind or connect two or more molecules.

As used herein, “treating” means reducing the frequency with which a patient has symptoms such as a disease, disorder or detrimental condition.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition that can be combined with the active ingredient and which can be used after administration to administer the active ingredient to the subject.

As used herein, the term “physiologically acceptable” means that the components are not harmful to the subject to which the composition will be administered.

Substrate of the Invention- Composition

An illustration of an interferon binding lipid construct comprising interferon, amphiphilic lipids and extended amphipathic lipids is shown in FIG. 1. Extended amphiphilic lipids, also known as receptor binding molecules, include the proximal, medial, and distal portions, where the proximal portion connects the extended lipids to the structure, the distal portion connects the structure to hepatocyte binding receptors in the liver, and the proximal portion is proximal. Connect the distal end with. Suitable amphiphilic lipids generally include polar head groups and nonpolar tail groups attached to each other via a glycerol-backbone.

Suitable amphiphilic lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-di Myristoyl-sn-glycero-3-phosphocholine, cholesterol, cholesterol oleate, dicetyl phosphate, 1,2-distearoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl -sn-glycero-3-phosphate, 1,2-dimyristoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (Cap biotinyl), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- ( Succinyl), 1,2-dipalmitoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (sodium salt), triethylammonium 2,3-diacetoxypropyl 2- ( 5-((3aS, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanamido) ethyl phosphate and mixtures of any of the foregoing lipids or Suitable derivatives of these lipids shown in Table 1, and the like are:

In one embodiment, the amphipathic lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetyl phosphate, 1,2-dipalmitoyl-sn-glycero-3 -Phosphoethanolamine-N- (Cap biotinyl), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3- Phosphoethanolamine-N- (succinyl), 1,2-dipalmitoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (sodium salt) triethylammonium 2,3- Diacetoxypropyl 2- (5-((3aS, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanamido) ethyl phosphate and any of the above Mixtures of lipids, and the like.

Extended amphiphilic lipids are also known as receptor binding molecules and include the proximal, mid and distal portions. The proximal connects the extended lipids to the construct and the distal connects the construct to hepatocyte binding receptors in the liver. The proximal and distal ends are connected through the midpoint. Compositions of various receptor binding molecules are described below. Within the lipid constructs one or more hepatocyte receptor binding molecules of the following groups may be present to bind the constructs to receptors in the hepatocytes.

Hepatocyte receptor binding molecules of the first group include terminal biotin or iminobiotin moieties and derivatives thereof. Structural formulas of biotin, iminobiotin, carboxybiotin and biocitin are shown in Table 2 below:

These molecules can be attached to phospholipid molecules using a variety of techniques to produce lipid anchoring molecules that can be intercalated into lipid constructs. These hepatocyte receptor binding molecules comprise a fixation site located proximal to the lipid construct. The anchoring site includes two lipophilic hydrocarbon chains that can associate and bind with other lipophilic hydrocarbon chains present on phospholipid molecules in the lipid construct.

In a preferred embodiment, the second group of hepatocyte receptor binding molecules comprises terminal biotin or iminobiotin moieties located at distal points from the lipid construct. Both biotin and iminobiotin contain a mild lipophilic bicyclic ring structure attached to the 5-carbon valeric acid chain at the 4-carbon point on the bicyclic ring. In one embodiment, the carboxyl group of valeric acid can be reacted covalently to the valeric acid C-terminal carboxyl functional group by reacting the N-terminal α-amino group or ε-amino group of L-lysine. This coupling reaction is carried out using the carbodiimide conjugation method, and an amide bond is formed between L-lysine and biotin as illustrated in FIG. 2.

Hepatocellular receptor binding molecules of the third group include iminobiotin, carboxybiotin and biocitin having valeric acid side chains attached via amide bonds to the α- or ε amino groups of amino acids L-lysine. A preferred embodiment uses iminobiotin in forming an iminobiocitin moiety as shown in FIG. 3. During the synthesis of hepatocyte receptor binding molecules, the α-amino group of iminobiocitin is activated ester benzoyl thioacetyl triglycine-sulfo-N-hydroxysuccinimide (BTA-3-gly-sulfo- as shown in FIG. 4). NHS) to form an active hepatocyte binding molecule (BTA-3-gly-iminobiocitin). BTA-3-gly-iminobiocitin eventually functions as a molecular spacer that represents an active nucleophilic sulfidral functional group that can be used in subsequent coupling reactions. The spacer is located at the median point in relation to the lipid construct and allows the terminal iminobiocitin moiety to extend approximately 30 mm 3 from the surface of the lipid construct, making it optimal and non-binding for iminobiocitin to bind to hepatocyte receptors. -Have a limited orientation. The medial spacer can include other derivatives that give the terminal biotin moiety the correct stereo-chemical orientation. The main function of the medial spacer is to connect the proximal and distal portions in a linear arrangement as appropriate and covalently coupled.

The BTA-3-gly-sulfo-NHS site of the hepatocyte receptor binding molecule can be synthesized by a number of means and can be linked to biocytin or iminobiocitin in a later step. The first step involves adding benzoyl chloride to thioacetic acid to form a protecting group for active thio functionality by nucleophilic addition. The product of the reaction is a benzoyl thioacetic acid complex and hydrochloric acid as shown in FIG. 5. A further step in this synthesis is sulfo-N using benzoyl thioacetic acid using dicyclohexylcarbodiimide or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide as coupling agent as shown in FIG. Reacting with hydroxysuccinimide to form benzoyl thioacetyl sulfo-N-hydroxysuccinimide (BTA-sulfo-NHS). The benzoyl thioacetyl sulfo-N-hydroxysuccinimide is then reacted with the amino acid polymer (glycine-glycine-glycine). As shown in FIG. 5, after nucleophilic attack by the α-amino group of triglycine, benzoyl thioacetyl triglycine (BTA-3-gly) is formed and the sulfo-N-hydroxysuccinimide leaving group is dissolved by an aqueous medium. do. As shown in FIG. 6, benzoyl thioacetyl triglycine was reacted with dicyclohexylcarbodiimide or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to react with sulfo-N-hydroxysuccinimide. To form ester bonds. Subsequently, sulfo-N-hydroxysuccinimide ester of activated benzoyl thioacetyl triglycine (BTA-3-gly-sulfo-NHS) was added to the α-amino group of the L-lysine functional group of biocithin or iminobiocitin. The reaction forms hepatocyte receptor binding moiety, which is an extended amphipathic lipid molecule of benzoyl thioacetyl triglycine-iminobiocitin (BTA-3-gly-iminobiocitin) illustrated in FIG. 7.

The second major coupling reaction for the synthesis of hepatocyte receptor binding molecules is covalently linked to N-para-maleimidophenylbutyrate phosphatidylethanolamine, where benzoyl thioacetyl triglycine iminobiocitin is a preferred phospholipid anchor molecule via thioether linkage. Illustrated as being attached. The reaction produces molecules that provide the correct molecular spacing between the terminal iminobiocitin ring and the lipid construct. The overall scheme for forming hepatocyte receptor binding molecules that function as extended amphipathic lipid molecules is shown in FIG. 8. Before the benzoyl thioacetyl triglycine iminobiocitin is reacted with N-para-maleimidophenylbutyrate phosphatidylethanolamine to form a thioether linkage, it is heated to remove the benzoyl protecting group to expose the free sulfidral functional group. The reaction must be carried out in an oxygen free environment to minimize the sulfidral oxidation to disulfide. Further oxidation can lead to the formation of sulfone, sulfoxide, sulfenic acid or sulfonic acid derivatives.

In one embodiment, the immobilized portion of the molecule contains a pair of acyl hydrocarbon chains that form the lipid moiety of the molecule. This site of the molecule is non-covalently bound within the lipid domain of the lipid construct. In one embodiment, the fixed moiety is produced from N-para-maleimidophenylbutyrate phosphatidylethanolamine. Other anchoring molecules can be used. In one embodiment, the fixing molecules include thio-cholesterol, cholesterol oleate, dicetyl phosphate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine-N- (succinyl), 1,2-dipalmitoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (sodium salt) And mixtures thereof. The overall molecular structure of the lipid anchor and hepatocyte receptor binding molecule, which is fully developed and named LA-HRBM, is shown in FIG. 8.

Hepatocellular receptor binding molecules of the fourth group include amphipathic organic molecules having both a water soluble moiety and a water insoluble moiety. The water insoluble moiety reacts with the medial or linker moiety in coordination and bioconjugation chemical reactions, and the water insoluble moiety binds to hepatocyte binding receptors in the liver. The molecule contains a distal component comprising a nonpolar derivatized benzene ring structure or a lipophilic heterobicyclic ring structure such as, for example, 2,6-diisopropylbenzene derivatives. The whole hepatocyte receptor binding molecule has a fixed charge or a transient charge, either positive or negative, or various combinations thereof. These molecules contain at least one carbonyl group and at least one carbamoyl moiety containing secondary amines and carbonyl groups located up to but not exceeding approximately 13.5 mm 3 from the distal end. The presence of the carbamoyl moiety (s) enhances the molecular stability of the organic molecule. There may be a plurality of secondary amines in the molecule. These secondary amines contain a pair of unshared electrons that allow ion-dipole and dipole-dipole bond interactions with other molecules in the structure. These amines enhance molecular stability and provide partially generated negative charges that interact with the distal to promote hepatocyte receptor binding and specificity. Examples of this group of receptor binding molecules include polychrome-poly (bis)-[N- (2,6- (diisopropylphenyl) carbamoylmethyl) imino diacetic acid]. In one embodiment, Chromium III is located at the medial point of the hepatocyte receptor binding molecule. The proximal portion of the hepatocyte specific binding molecule contains hydrophobic and / or nonpolar structures that allow the molecule to be inserted into and then bound therein. The medial and proximal also allow the hepatocyte receptor binding molecule distal to be properly stereochemically oriented.

The structure and properties of a lipid construct are governed by the structure of the lipid and the interactions between the lipids. The structure of the lipid is largely governed by covalent bonds. Covalent bonds are the molecular binding forces required to retain the structural integrity of a molecule comprising individual components of the lipid construct. Through non-covalent interactions between lipids, lipid structures are maintained in a three-dimensional shape.

Non-covalent bonds may be represented by the general terms ion-dipole or induced ion-dipole bonds, and hydrogen bonds associated with various polar groups of the lipid head. Hydrophobic bonds and van der Waals interactions can be generated through induced dipole bonds between lipid acyl chains. This binding mechanism is transient in nature and results in bond formation and bond breakdown processes that occur at time intervals that are less than femtoseconds. For example, van der Waals interactions are due to momentary changes in the dipole moments caused by orbital electrons moving briefly to one side of an atom or molecule, causing similar movement in adjacent atoms or molecules. Protons form dipoles because they are assumed to be δ ⁺ charge and single electron δ ⁻ charge. Dipole interactions occur very frequently between hydrocarbon acyl chains of amphiphilic lipid molecules. Once the individual dipoles are formed, they can momentarily induce new dipoles at neighboring atoms containing methylene-type (-CH _2- ) functional groups. A plurality of transiently induced dipole interactions are formed between the acyl lipid chains throughout the lipid construct. This induced dipole interaction lasts only for a fraction of femtoseconds (1 × 10 ⁻¹⁵ seconds), but working together is a powerful force. This interaction is constantly changing and has a force equal to approximately 1/20 of the covalent bond strength. Nevertheless, they are responsible for the transient binding between the three-dimensional statistical structure of the structure and stable covalent molecules that determine the stereo-specific molecular orientation of the molecules in the lipid structure.

As a result of this induced dipole interaction, the structure of the lipid construct is maintained by lipid component exchange between the constructs. While the composition of the individual components of the construct is fixed, the individual components of the lipid construct are subject to an exchange reaction between the constructs. This exchange is initially governed by zero order dynamics when the lipid component leaves the lipid structure. The lipid component may be recaptured by neighboring lipid structures after being released from the lipid construct. Recapture of the released component is controlled by secondary reaction kinetics, which is influenced by the concentration of the component and the concentration of the lipid construct that captures the released component in an aqueous medium around the structure that captured the released component. Receive.

Examples of extended amphipathic lipids and their respective numbers shown in Table 3 are as follows: N-hydroxysuccinimide (NHS) biotin [1], sulfo-NHS-biotin [2], N-hydroxysuccinate Imide long chain biotin [3], sulfo-N-hydroxysuccinimide long chain biotin [4], D-biotin [5], biocitin [6], sulfo-N-hydroxysuccinimide-SS-biotin [7], biotin-BMCC [8], biotin-HPDP [9], iodoacetyl-LC-biotin [10], biotin-hydrazide [11], biotin-LC-hydrazide [12], biocitin Hydrazide [13], biotin cardaberine [14], carboxybiotin [15], photobiotin [16], ρ-aminobenzoyl biocithin trifluoroacetate [17], ρ-diazobenzoyl biocitin [18] ], Biotin DHPE [19], biotin-X-DHPE [20], 12-((biotinyl) amino) dodecanoic acid [21], 12-((biotinyl) amino) dodecanoic acid succinimidyl ester [22] , S-by Orynyl homocysteine [23], biocitin-X [24], biocitin x-hydrazide [25], biotinethylenediamine [26], biotin-XL [27], biotin-X-ethylenediamine [28] , Biotin-XX hydrazide [29], biotin-XX-SE [30], biotin-XX, SSE [31], biotin-X-cardaberine [32], α- (t-BOC) biocitin [33] ], N- (biotinyl) -N '-(iodoacetyl) ethylenediamine [34], DNP-X-biocitin-X-SE [35], biotin-X-hydrazide [36], norbiotin Amine hydrochloride [37], 3- (N-maleimidylpropionyl) biocitin [38], ARP [39], biotin-1-sulfoxide [40], biotin methyl ester [41], biotin-maleic Mead [42], biotin-poly (ethyleneglycol) amine [43], (+) biotin 4-amidobenzoic acid sodium salt [44], biotin 2-N-acetylamino-2-deoxy-β-D-glu Copyranoside [45], biotin-α-DN-acetylneuramid [46], biotin-α-L-fucoside [47], biotin lacto-N-bioside [48], biotin-Lewis-A trisaccharide [49], biotin-Lewis-Y tetrasaccharide [50], biotin-α-D-mannopyranoside [ 51], biotin 6-O-phospho-α-D-mannopyranoside [52] and polychrome-poly (bis)-[N- (2,6- (diisopropylphenyl) carbamoylmethyl) Imino] diacetic acid [53].

In one embodiment, the cellulose acetate hydrogen phthalate polymer can be incorporated into the lipid construct to bind hydrophilic functional groups on the interferon molecule to protect the interferon from hydrolysis. Cellulose Acetate Hydrogen Phthalate

Two glucose molecules beta (1 → 4) linked in a polymer arrangement, wherein some of the hydrogen atoms present in the hydroxyl groups of the polymer are acetyl functional groups (methyl groups bonded to carbonyl carbons) or phthalate groups (of benzene rings And a benzene ring having two carboxyl groups in the first and second positions). The structural formula of the cellulose acetate hydrogen phthalate polymer is shown in FIG. 9. Only one carboxyl group in the phthalate ring structure is involved in covalent ester linkage with the cellulose acetate molecule. Other carboxyl groups contain carbonyl carbon and hydroxyl functional groups, which participate in hydrogen bonding with neighboring negative and positively charged dipoles present in the interferon and various lipid molecules.

In one embodiment, the cellulose acetate hydrogen phthalate polymer interacts with lipids through ion-dipole bonds with 1,2-dstearoyl-sn-glycero-3-phosphocholine phosphate and dicetyl phosphate molecules. . Ion-dipole bonds occur between δ ⁺ hydrogen present in cellulose hydroxyl groups and negatively charged oxygen atoms present in the phosphate portion of the phospholipid molecule. The functional groups that play the greatest role in ion-dipole interaction are negatively charged oxygen atoms present in the phosphate group of the phospholipid molecule, hydrogen atoms present in the hydroxyl group, and hydrogen atoms present in the amide bond of the interferon molecule. The negatively charged functional groups undergo ion-dipole interactions and form sites that react with the δ ⁺ hydrogen atoms of the hydroxyl groups of the carboxyl functional groups present in the individual hydroxyl groups and cellulose acetate hydrogen phthalate. Ions can be formed between the carbonyl ^{oxygen-dipole} exists δ of the quaternary amine and cellulose acetate hydrogen phthalate, and interferon positively charged functional group of the phosphocholine. Sugar molecules comprising a branched hydrophilic structure in interferon can participate in hydrogen bonding and ion-dipole interactions.

The molecular arrangement and size of the polymer (molecular weight of about 15,000 or more) allows cellulose acetate hydrogen phthalate to coat individual phospholipid molecules of the lipid construct in the region of the hydrophilic head group. This coating protects interferon in the lipid construct from the acid environment of the stomach. There are several ways in which cellulose acetate hydrogen phthalate can be attached to the surface of molecules in lipid structures. A preferred means of linking cellulose acetate hydrogen phthalate to the surface of the lipid construct is to attach a polymeric cellulose species to the tail of the interferon molecule that provides sugars protruding from the surface of the lipid construct. This protects the proteinaceous tail of interferon from enzymatic hydrolysis.

Extended amphiphilic lipids include various multi-toothed binding sites for attachment to the receptor. Multi-toothed bonds as defined herein are the surface of the interferon and its accompanying sugar moieties, as well as on lipid structures that can interface with carbonyl, carboxyl and hydroxyl functional groups on the cellulose acetate hydrogen phthalate polymer. It requires a plurality of potential binding sites in the phase. This allows the cellulose acetate hydrogen phthalate polymer to bind the lipid construct as well as a plurality of hydrophilic regions on the molecule of interferon to establish the defense of hydrolysis protection against the lipid construct. In this way, both the interferon and lipid constructs are protected from the acidic environment of the stomach after oral administration of the interferon dosage form. Although cellulose acetate hydrogen phthalate passes through the stomach to cover or protect individual lipid molecules within and on the surface of the lipid structure, when the structure moves into the alkaline region of the small intestine, cellulose acetate hydrogen phthalate is degraded by hydrolysis. do. Cellulose acetate hydrogen phthalate is removed from the surface of the molecules of the lipid construct and then lipid anchoring-hepatocyte receptor binding molecules such as 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N -(Cap biotinyl) is exposed, which can then bind to the receptor. The use of cellulose acetate hydrogen phthalate coatings on interferon and lipid constructs is necessary to ensure that higher bioavailability of interferon is achieved.

In one embodiment, the lipid construct comprises a target molecule complex comprising a plurality of linked individual units obtained by forming a complex of a crosslinking component and a complex former. The crosslinking component is a water soluble salt of a metal capable of forming a water insoluble coordinated complex with a complex former. Suitable metals are selected from transition and internal transition metals or neighboring metals with transition metals. As transition and internal transition metals the metals are Sc (scandium), Y (yttrium), La (lanthanum), Ac (actinium), actinides, Ti (titanium), Zr (zirconium), Hf (hafnium), V (vanadium) , Nb (niobium), Ta (tantalum), Cr (chromium), Mo (molybdenum), W (tungsten), Mn (manganese), Tc (technetium), Re (renium), Fe (iron), Co (cobalt) , Ni (nickel), Ru (ruthenium), Rh (rhodium), Pd (palladium), Os (osmium), Ir (iridium) and Pt (platinum). Metals adjacent to transition metals are Cu (copper), Ag (silver), Au (gold), Zn (zinc), Cd (cadmium), Hg (mercury), Al (aluminum), Ga (gallium), In ( Indium), Tl (thallium), Ge (germanium), Sn (tin), Pb (lead), Sb (antimony) and Bi (bismuth), and Po (polonium). Examples of metal compounds useful as crosslinkers include chromium chloride (III) hexahydrate, chromium (III) fluoride tetrahydrate, chromium (III) bromide hexahydrate, zirconium (IV) citrate ammonium complex, zirconium (IV) chloride, zirconium ( IV) fluoride hydrate, zirconium (IV) iodide, molybdenum (III) bromide, molybdenum (III) chloride, molybdenum (IV) sulfide, iron (III) hydrate, iron (III) phosphate Tetrahydrate, iron (III) sulfate pentahydrate, and the like.

Complex formers are compounds capable of forming a water soluble coordinated complex with a crosslinking component. There are several families of suitable complex formers.

The complex former can be selected from the iminodiistic acid family of Formula 1, wherein R ₁ is lower alkyl, aryl, aryl lower alkyl, and heterocyclic substituents.

<Formula 1>

Suitable compounds of formula (I) include the following:

N- (2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid,

N- (2,6-diethylphenylcarbamoylmethyl) imino diacetic acid,

N- (2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid,

N- (4-isopropylphenylcarbamoylmethyl) iminodiacetic acid,

N- (4-butylphenylcarbamoylmethyl) iminodiacetic acid,

N- (2,3-dimethylphenylcarbamoylmethyl) iminodiacetic acid,

N- (2,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid,

N- (2,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid,

N- (3,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid,

N- (3,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid,

N- (3-butylphenylcarbamoylmethyl) iminodiacetic acid,

N- (2-butylphenylcarbamoylmethyl) imino diacetic acid,

N- (4-tert.butylphenylcarbamoylmethyl) iminodiacetic acid,

N- (3-butoxyphenylcarbamoylmethyl) iminodiacetic acid,

N- (2-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid,

N- (4-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid,

Aminopyrrole iminodiacetic acid,

N- (3-bromo-2,4,6-trimethylphenylcarbamoylmethyl) iminodiacetic acid,

Benzimidazole methyl iminodiacetic acid,

N- (3-cyano-4,5-dimethyl-2-pyrylcarbamoylmethyl) iminodiacetic acid,

N- (3-cyano-4-methyl-5-benzyl-2-pyrylcarbamoylmethyl) iminodiacetic acid, and

N- (3-cyano-4-methyl-2-pyrylcarbamoylmethyl) iminodiacetic acid and

Other derivatives of N- (3-cyano-4-methyl-2-pyrylcarbamoylmethyl) iminodiacetic acid of the formula

<Formula 2>

Wherein R ₂ and R ₃ are as follows:

Complex formers are a family of imino diacid derivatives of Formula 3 wherein R ₄ , R ₅ and R ₆ are independent of each other and may be hydrogen, lower alkyl, aryl, aryl lower alkyl, alkoxy lower alkyl and heterocyclic Is selected from.

<Formula 3>

Suitable compounds of formula 3 include N '-(2-acetylnaphthyl) iminodiacetic acid (NAIDA), N'-(2-naphthylmethyl) iminodiacetic acid (NMIDA), iminodicarboxymethyl-2-naphthyl Ketone phthalein complex, 3 (3: 7a: 12a: trihydroxy-24-norcol anyl-23-iminodiacetic acid, benzimidazole methyl iminodiacetic acid, and N- (5, pregnene-3-p- All-2-oil carbamoylmethyl) iminodiacetic acid.

Complex formers are selected from the family of amino acids of formula (4).

<Formula 4>

Wherein R ₇ is an amino acid side chain, R ₈ is lower alkyl, aryl, aryl lower alkyl, and R ₉ is pyridoxylidene.

Suitable amino acids of the formula (4) include aliphatic amino acids including glycine, alanine, valine, leucine, isoleucine, hydroxyamino acids including serine and threonine, aspartic acid, asparagine, glutamic acid, glutamine, dicarboxylic acid amino acids including glutamine and the like Amino acids having basic functional groups including amides, lysine, hydroxylysine, histidine, arginine, aromatic amino acids including phenylalanine, tyrosine, tryptophan, thyroxine, and sulfur-containing amino acids including cystine, methionine.

Complex formers include, but are not limited to, (3-alanine-y-amino) butyric acid, O-diazoacetylserine (azaserine), homoserine, ornithine, citrulline, penicillamine, And pyridoxylidene glutamate, pyridoxylidene isoleucine, pyridoxylidene phenylalanine, pyridoxylidene tryptophan, pyridoxylidene-5-methyl tryptophan, pyridoxylidene-5-hydroxytrytamine, and Selected from the members of the pyridoxylidene compound group, including but not limited to pyridoxylidene-5-butyltrytamine. The complex former is selected from the family of diamines of the formula

<Formula 6>

Wherein R ₁₀ is hydrogen, lower alkyl, or aryl, R ₁₁ is lower alkylene or aryl lower alkyl, and R ₁₂ and R ₁₃ are independently hydrogen, lower alkyl, alkyl, aryl, aryl lower alkyl, acylhetero Cyclic, toluene, sulfonyl or tosylate.

Some suitable diamines of formula 6 include ethylenediamine-N, N diacetic acid, ethylenediamine-N, N-bis (-2-hydroxy-5-bromophenyl) acetate, N'-acetylethylenediamine-N, N Diacetic acid, N'-benzoyl ethylenediamine-N, N Diacetic acid, N '-(p-toluenesulfonyl) ethylenediamine-N, N Diacetic acid, N'-(pt-butylbenzoyl) ethylenediamine-N, N Diacetic acid, N '-(benzenesulfonyl) ethylenediamine-N, N diacetic acid, N'-(p-chlorobenzenesulfonyl) ethylenediamine-N, N diacetic acid, N '-(p-ethylbenzenesulfonyl Ethylenediamine-N, N diacetic acid, N'-acyl and N'-sulfonyl ethylenediamine-N, N diacetic acid, N '-(pn-propylbenzenesulfonyl) ethylenediamine-N, N diacetic acid, N' -(Naphthalene-2-sulfonyl) ethylenediamine-N, N diacetic acid, and N '-(2,5-dimethylbenzenesulfonyl) ethylenediamine-N, N diacetic acid, and the like.

Other suitable complex forming compounds or formers include penicillamine, p-mercaptoisobutyric acid, dihydrothioctic acid, 6-mercaptopurine, ketoxal-bis (thiosemicabazone), hepatobiliary amine complex , 1-hydrazinophthalazine (hydralazine), sulfonyl urea, hepatobiliary amino acid Schiff base complex, pyridoxylidene glutamate, pyridoxylidene isoleucine, pyridoxylidene phenylalanine, pyridoxy Silyden tryptophan, pyridoxylidene 5-methyl tryptophan, pyridoxylidene-5-hydroxytrytamine, pyridoxylidene-5-butyl trytamine, tetracycline, 7-carboxy-p-hydroxyquinoline, phenolphthalein , Eosin I blueish, eosin I yellowish, verographene, 3-hydroxyl-4-formyl-pyriden glutamic acid, azo substituted iminodiacetic acid, hepatobiliary dye complexes such as rose bengal (rose bengal), congo red , Bromosulfophthalein, bromophenol blue, toluidine blue, and indocyanine green, hepatobiliary contrast agents such as iodipamide, and ioglycamic acid, blue salts such as bilirubin, colgisiliodohistamine And thyroxine, hepatobiliary thio complexes such as penicillamine, p-mercaptoisobutyric acid, dihydrothiocitic acid, 6-mercaptopurine, and ketoxal-bis (thiosemicabazone), hepatobiliary Amine complexes such as 1-hydrazinophthalazine (hydralazine), and hepatobiliary, including sulfonyl urea, pyridoxylidene-5-hydroxytrytamine and pyridoxylidene-5-butyltrytamine Amino acid Schiff base complexes, hepatobiliary protein complexes such as protamine, ferritin, and asialo-orosomucoids, and asialo complexes such as lactosamined albumin, immunoglobulin G, IgG, hemoglobin, etc. But not limited to this Neunda.

Three-dimensional target molecule complexes prepared by complexing crosslinkers and complex formers are described in WO 99/59545, which is incorporated herein by reference. In one embodiment, the crosslinking agent is a chromium capable of forming a complex coordinated with a metal salt, such as a complex former, such as N- (2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid. Metal salts such as chloride hexahydrates. The crosslinker and the complex former are complexed to form a complex consisting of multiple linked units in a three-dimensional array. In a preferred embodiment, the complex consists of multiple units of chromium (bis) [N- (2,6- (diisopropylphenyl) carbamoylmethyl) imino diacetic acid] linked together. In one embodiment, the chromium target molecule complex material is soluble in a mixture of lipids containing 1,2-dstearoyl-sn-glycero-3-phosphocholine, dicetyl phosphate and cholesterol. The complex is incorporated into a lipid construct formed from the group of lipids described above.

In one embodiment, the interferon is effective in antiviral agents such as ribivirine, acyclovir, double stranded DNA, oligonucleotides, protease inhibitors, reverse transcriptase inhibitors, and when delivered in HDV without effect on their own. Mix in appropriate proportions with other possible anti-viral agents.

Description of the method of the present invention for preparing a lipid construct

11 shows a schematic of a process for preparing a lipid construct comprising amphipathic lipids, extended amphipathic lipids, and interferon.

Preparation of the composition comprises three steps: preparing a mixture of amphiphilic lipids and extended amphipathic lipids, preparing a lipid construct from a mixture of amphiphilic lipids and extended amphiphilic lipids, and combining interferon into the lipid construct. Includes overall steps.

Lipids are prepared and loaded by the methods disclosed herein and by the methods described in US Pat. Nos. 4,946,787, 4,603,044, and 5,104,661 and the references cited therein. Typically, the aqueous lipid construct formulations of the invention optionally contain 0.1% to 4% lipids and 0.1% to 10% by weight active agent (ie 1 to 10 mg of drug per ml) in an aqueous solution containing salts and buffers. In amounts for producing 100% by volume. Preferred are formulations comprising 0.1% to 5% active agent. Most preferred is a formulation comprising from 0.01% to 5% by weight of the active agent and up to 2% by weight of the lipid component in an amount of a (titrate) aqueous solution sufficient to produce 100% by volume.

In one embodiment, the lipid construct is prepared by the following procedure. The individual lipid components are mixed together in an organic solvent system in which the solvent is dried over molecular sieves for approximately 2 hours to remove any residual water that may accompany the solvent. In one embodiment, the solvent system comprises a mixture of chloroform and methanol in a volume ratio of 2: 1. Other organic solvents may also be used that can be easily removed from the mixture of dried lipids. By using the lipid components in a one-step addition in the initial mixing procedure, the structure of the lipid construct is unnecessarily complicated and eliminates the need to introduce any additional coupling reactions requiring additional separation procedures. The lipid component and hepatocyte receptor binding molecule are dissolved in a solvent and then the solvent is removed under high vacuum until a dried mixture of lipids is formed. In one embodiment, the solvent is removed by rotating slowly at approximately 60 ° C. for about 2 hours using a rotary evaporator or other methods known in the art. Such lipid mixtures can be stored or used directly for further use.

The lipid construct is prepared from a dried mixture of amphiphilic lipids and extended amphiphilic lipids. The dried mixture of lipids is added to an appropriate amount of aqueous buffered medium, followed by swirling the mixture to form a homogeneous suspension. The lipid mixture is then heated with mixing at approximately 80 ° C. for about 30 minutes under a dry nitrogen atmosphere. The heated homogeneous suspension is immediately delivered to a microfluidizer preheated to about 70 ° C. Pass the suspension through the microfluidizer. It may further be necessary to pass the suspension through the microfluidizer to obtain a homogenous lipid microsuspension. In one embodiment, a Model # M-110 EHI microfluidizer was used wherein the pressure at the first pass was approximately 9,000 psig. To produce a product that exhibits the properties of a homogeneous lipid microsuspension, it is necessary to pass the lipid suspension through the microfluidizer a second time. This product is defined structurally and morphologically as a three-dimensional lipid construct containing hepatocyte receptor binding molecules.

Interferon is loaded into the lipid construct using one of two methods, equilibrium loading and non-equilibrium loading. Equilibrium loading of interferon begins when interferon is added to the suspension of the lipid construct. Over time, interferon molecules migrate into and out of the lipid construct. Migration is controlled by first dispensing the equilibrium by introducing interferon into the suspension and then moving the interferon to the lipid construct.

Non-equilibrium loading of interferon into the lipid construct localizes the interferon into the lipid construct. Equilibrium loading of the free interferon to the lipid construct removes the bulk phase medium containing the free interferon. The non-equilibrium loading procedure is a vector-induced process that begins when removing enamel on the outer bulk. When the aqueous phase containing interferon is removed, the concentration gradient potential that interferon migrates out of the lipid construct is removed. Since the movement of interferon from within the construct is eliminated, there is a higher concentration of interferon inside the final lipid construct by the whole process. The equilibrium loading of interferon is a time-dependent phenomenon, while the non-equilibrium loading procedure is virtually instantaneous. Non-equilibrium loading can be initiated by various processes that separate the material in solution from the lipid construct. Examples of such processes include, but are not limited to, filtration, centricon filtration, centrifugation, batch style affinity chromatography, streptavidin agarose affinity gel chromatography or batch style ion-exchange chromatography. Any means of removing the concentration gradient potential for interferon diffusion and exposure and allowing the interferon to be retained by the lipid construct can be used.

When using batch-style chromatography, an affinity or ion-exchange gel is mixed quickly with a mixture of interferon and the construct. Binding to the chromatography medium occurs rapidly and the chromatography medium is removed from the aqueous medium by decanting the aqueous phase or by using typical filtration techniques such as filter paper and Buchner funnels.

The lipid construct contains discrete amounts of loaded interferon located on the interior and surface of the lipid construct as well as inside the lipid construct. The resulting lipid construct is a new and novel composition of matter and is a composition for delivering an effective amount of interferon as a result of non-equilibrium loading. Subsequent loading of the interferon into the lipid construct followed by removal of the bulk phase interferon represents a high concentration of interferon in the lipid construct by shortening the length of time required for removal of the external phase medium. It is difficult to load the interferon into the construct at this level using time-dependent procedures such as ion-exchange or gel-filtration chromatography, because the procedure requires constant injection of buffers containing high concentrations of interferon. Because. For example, loading interferon into the construct using small scale column chromatography requires approximately 20 minutes to remove the external bulk phase medium containing the interferon from the construct containing the interferon. During this period, the equilibrium is reestablished by the movement of interferon from the construct. Maintaining high concentrations of interferon within and on lipid structures is one of the positive benefits of non-equilibrium loading.

As an extension of the non-equilibrium loading process, cellulose acetate hydrogen phthalate is added to the lipid construct during the step of loading the interferon into the lipid construct after the interferon performs equilibrium loading but before the non-equilibrium loading process is initiated. Depending on the nature and structure of the interferon molecule, the interferon molecule can be inserted into the lipid construct when the interferon is dispersed throughout the lipid construct. Hydrophilic sites of interferon, and branched complex sugars and additional functional groups extend from the surface of the lipid construct into the bulk phase medium. This extended hydrophilic site of interferon is subjected to hydrogen bond, dipole-dipole and ion-dipole interactions at the surface of the lipid construct with hydroxyl, carboxyl and carbonyl functional groups of cellulose acetate hydrogen phthalate as illustrated in FIG. 10. Can participate Cellulose acetate hydrogen phthalate provides excellent protection for shielding the contents of the lipid construct from the gastric digestive environment by conferring unique means of binding to molecules of the lipid construct. The digestion process in the stomach results from hydrolytic cleavage of proteinaceous substrates by the enzyme pepsin and cleavage by acid hydrolysis. The acidic environment of the stomach degrades free interferon and can hydrolyze ester bonds that retain the acyl hydrocarbon chain as the glycerol backbone in the phospholipid molecule. Hydrolytic cleavage can also occur on each side of the phosphate functional group in the phosphocholine group. The digestive system change from the acidic region of the stomach to the alkaline region of the small intestine was the enzymatic action of trypsin and chymotrypsin. Amino acid lytic enzymes such as alpha amino peptidase can distribute proteins such as interferon from the N-terminal end. The presence of cellulose acetate hydrogen phthalate in the lipid construct protects interferon from hydrolysis. As the alkaline environment of the small intestine degrades cellulose acetate hydrogen phthalate protection of lipid constructs by hydrolysis, hepatocyte receptor binding molecules can be used to directly bind the construct to hepatocyte binding receptors. While not wishing to be bound by any particular theory, there is a synergy of hydrolytic protection upon the addition of cellulose acetate hydrogen phthalate at the end point of the non-equilibrium loading. The protection is distributed not only to the interferon and individual lipid molecules, but also to the entire lipid construct. This synergy protects not only individual molecules but whole molecules from enzymes and acid hydrolysis.

In one embodiment, cellulose acetate hydrogen phthalate is covalently bound to interferon or lipid constructs using a variety of methods. For example, one method uses a Mannich reaction to hydroxyl groups on cellulose acetate hydrogen phthalate to the ε-amino groups of the 10 L-lysines or 1,2-diacyl-sn-glycer in the interferon molecule. Coupling to an amine functional group on the 3-phosphoethanolamine.

In one embodiment, cellulose acetate hydrogen phthalate is loaded into the lipid construct during equilibrium loading of the interferon into the construct. The hydroxyl and carbonyl functional groups of cellulose acetate hydrogen phthalate hydrogen bond with lipid molecules in the lipid construct. Hydrogen bonds between cellulose acetate hydrogen phthalate and the structure are formed simultaneously with the interferon being loaded into the lipid construct under equilibrium to create a protective film around the interferon and the construct.

HDV interferon is recovered from the aqueous medium and recycled by binding the HDV interferon to streptavidin-agarose iminobiotin. Streptavidin covalently bound to cyanogen bromide activated agarose provides a means to separate iminobiotin-based lipid constructs from interferons in an aqueous medium at the end of non-equilibrium loading of interferon into constructs. In one embodiment, the iminobiotin derivative forms the hepatocyte receptor binding site of the phospholipid moiety in the lipid construct. The water-soluble site of the lipid anchoring molecule extends approximately 30 mm 3 from the lipid surface to promote the binding of the hepatocyte receptor binding site of the phospholipid portion to the hepatocyte receptor to aid in attaching the lipid construct to streptavidin.

Streptavidin reversibly binds iminobiotin at a pH value of 9.5 or higher, wherein the uncharged guanidino function of iminobiotin is one of four binding sites on streptavidin located approximately 9 Hz below the surface of the protein. Strongly bound to one. Lipid constructs containing iminobiotin are removed from the buffered medium by raising the pH of the aqueous mixture of constructs to pH 9.5 by addition of 20 mM sodium carbonate-sodium bicarbonate buffer. At this pH, the bulk phase medium contains free interferon regenerated and separated from the lipid constructs using a variety of procedures including, but not limited to, filtration, centrifugation, or chromatography.

The mixture at pH 9.5 is then mixed with streptavidin-agarose cross-linked beads, wherein the construct is absorbed onto streptavidin. Beads of approximately 120 μm in diameter are separated from the solution by filtration. The lipid construct is released from the streptavidin-agarose affinity gel by adding 20 mM sodium acetate-acetic acid buffer of pH 4.5 to reduce the pH from pH 9.5 to pH 4.5. At pH 4.5, the guanidino groups of iminobiotin are protonated and positively charged as shown in FIG. Separation is achieved by releasing the lipid construct from streptavidin-agarose beads by filtration. Streptavidin-agarose beads are regenerated for further use. Thus, both free interferon and streptavidin agarose are preserved and can be reused.

In one embodiment, streptavidin-agarose beads are used to prepare a composition that provides prolonged release of interferon when loading iminobiotin or iminobiocitin lipid constructs with interferon alpha. When the pH of the above-mentioned construct is adjusted from pH 9.5 to pH 4.5, interferon-alpha will precipitate in the lipid construct at approximately pH 5.9. The isoelectric point of interferon-alpha is pH 5.9, indicating the pH at which interferon-alpha has its lowest water solubility. In the pH range of pH 5.9 to pH 6.7, interferon-alpha remains essentially insoluble and typically exhibits properties due to particulate matter. Insoluble interferon-alpha in lipid constructs result in novel interferon-alpha preparations that provide sustained release interferon-alpha molecules when administered by subcutaneous injection or via oral administration. Solubilization of interferon-alpha is initiated as the pH of the lipid construct approaches pH 7.4.

The lipid construct is freeze-dried or maintained in a non-aqueous environment prior to administration. In the interferon-alpha of the aqueous dosage form, the pH of the interferon-alpha solution is maintained at approximately pH 6.5 to keep the interferon-alpha in insoluble form. When interferon-alpha is exposed to an external pH gradient, in vivo interferon-alpha dissolves and migrates out of the lipid construct, thereby interferon-alpha is supplied to other virus-harboring tissue. Interferon remaining with the lipid constructs retains the ability to be designated as hepatocyte binding receptors on hepatocytes in the liver. Thus, two forms of interferon-alpha are produced from this particular lipid construct. In an in vivo environment, free and lipid associated interferon-alpha are produced in a time-dependent manner. It is expected that solubilization of lipid-associated interferon-alpha as described above can be made to release interferon over a designated sustained release period. This results in a less frequent dosing schedule for patients infected with the virus.

In a preferred embodiment, the interferon molecule migrates to the lipid construct and sequesters inside the lipid domain of the loaded lipid construct. When the chemical equilibrium is broken, the interferon molecules are moved in one direction using a vector-derived process during the last step of the interferon loading procedure. During the last step of the interferon loading, the buffer or aqueous medium is quickly removed to remove the external medium to which the interferon molecules associated with the lipid constructs migrate. Removal of the external medium effectively quenchs the equilibrium between the interferon associated with the lipid construct and the solubilized interferon in the external medium. This process is referred to as non-equilibrium support as described herein.

In one embodiment, the lipid construct is loaded with interferon using an equilibrium method. The loading procedure is initiated by selecting an interferon concentration of 273,000 units of interferon per μg of protein. Equilibrium loading continues until the lipid construct is saturated with interferon.

Termination of non-equilibrium loading of interferon into lipid constructs requires the use of a procedure to separate solid lipid constructs from buffered media containing free interferon. In one embodiment, the lipid construct is separated from the external medium using a filtration procedure using very fine micropore synthetic membranes. In another embodiment, the lipid construct is removed from the buffered medium containing free interferon using a filtration centrifuge device such as a Centricon device equipped with a suitable filter having a 100,000 molecular weight cutoff membrane, such as a NanoSep filter. . The concentration of interferon in the lipid construct is maintained because the associated interferon is no longer in equilibrium with the free interferon molecules located in the bulk phase medium removed from the construct. Free interferon that was in solution can be used to load other lipid constructs. Thus, the vector-derived process of concentrating interferon in the lipid construct is accomplished in one step in an essentially time-dependent procedure.

After the lipid construct is isolated from the bulk phase medium, the construct may range in size from approximately 0.0200 μm to 0.4000 μm in diameter. Lipid constructs generally include several particle sizes that follow a Gaussian distribution. The appropriate size of the lipid construct required to achieve the intended pharmacological efficacy can be selected from the lipid construct including the particle size of the Gaussian distribution by the hepatocyte binding receptor.

Lipid constructs comprising interferon, amphiphilic lipids and extended amphiphilic lipids are prepared using a micro-fluidization process that provides high shear forces to break down larger lipid constructs into smaller constructs. Amphiphilic lipid constituents of the lipid construct include 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetyl phosphate, 1,2-dipalmitoyl-sn-glycero-3- Phosphoethanolamine-N- (Cap biotinyl), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-force Poethanolamine-N- (succinyl), 1,2-dipalmitoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (sodium salt), triethylammonium 2,3- Diacetoxypropyl 2- (5-((3aS, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl) pentanamido) ethyl phosphate and their appropriate Derivatives (whose representative structures are shown in Table 3).

In one embodiment, the construct comprises a target molecule complex comprising a plurality of linked individual units formed by complexing a crosslinking component with a complex former. Typically, the target molecule complex is formed by complexing an aqueous buffered solution of a complex former with a selected metal compound, such as chromium chloride (III) hexahydrate. In one embodiment, the aqueous buffered solution of the complex former comprises a complex buffer, for example N- (2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid, in an aqueous buffered solution, for example 10 Prepared by dissolving to a final pH of pH 3.2 to 3.3 in mM sodium acetate buffer. The metal compound is added in excess in sufficient amount to form a complex with the isolable site of the complex former, and the reaction is allowed to precipitate for 24 to 96 hours at a temperature of 20 ° C. to 33 ° C. or the resulting complex precipitates from an aqueous buffered solution. Until you are done. The precipitated complex former, which demonstrates polymeric properties, is then isolated for later use. This complex is added to a mixture of amphipathic lipid molecules and extended amphipathic lipids prior to preparing the lipid construct.

Description of the invention-method of use

Patients with hepatitis are administered an effective amount of hepatocyte targeting composition comprising a mixture of free interferon and interferon associated with a water insoluble target molecule complex. In one embodiment, the interferon is delivered in HDV, although it may be ineffective by anti-viral agents such as ribavirin, acyclovir, double stranded DNA, oligonucleotides, protease inhibitors, reverse transcriptase inhibitors, and by themselves Mix in appropriate proportions with other possible anti-viral agents that may be effective. In one embodiment, the composition can be administered by the subcutaneous or oral route.

After administering the composition to the patient by subcutaneous injection, the in situ environment of physiological pH at the site of injection increases the pH which affects the morphology and chemical structure of the interferon associated with the free interferon and the water insoluble target molecule complex. Indicates. As the pH of the environment around the interferon increases, the interferon changes to a soluble form in the lipid construct and attaches to the lipid construct, which can travel through the circulation system to the liver.

After oral administration of a pharmaceutical composition comprising interferon associated with a target molecule complex, the interferon associated with the target molecule complex is absorbed into the body's circulatory system in the small intestine where it is exposed to blood at physiological pH. Lipid constructs are targeted for delivery to the liver. In one embodiment, the lipid construct is protected from hydrolase in the presence of cellulose acetate hydrogen phthalate in the construct. During oral administration, the protected lipid construct crosses the oral cavity and passes through the stomach to the small intestine, and the alkaline pH of the small intestine breaks down the cellulose acetate hydrogen phthalate barrier. Deprotected lipid constructs are absorbed into the circulatory system. This allows lipid structures to be delivered to the sinusoids of the liver. Receptor binding molecules, such as 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (Cap biotinyl) or other hepatocyte specific molecules mentioned above, are known that lipid structures bind to receptors. It then provides a means to be entrapped or endocytosis by hepatocytes. The interferon is then released from the lipid construct, which, when approaching the cellular environment, causes interferon to infect infectious viruses such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F, and G. It performs its designated function for acting as a substance against the virus of hepatitis and other hepatitis.

The lipid construct structures of the present invention provide materials useful for pharmaceutical use for administering interferon to a host. Accordingly, the structures of the present invention are useful as pharmaceutical compositions in combination with pharmaceutically acceptable carriers. Administration of the structures described herein may be by any mode of administration permitted for the interferon to be administered. These methods include oral, parenteral, nasal and other system or aerosol forms.

The amount of interferon administered will depend on the subject to be treated, the type and severity of the pain, the mode of administration and the judgment of the prescriber. The effective dosage range for the specific biologically active substance of interest depends on a variety of factors and is generally known to those skilled in the art, but some dosage guidelines may be generally defined. For most dosage forms, the lipid component will be suspended in an aqueous solution and generally does not exceed 4.0% (w / v) of the total formulation. The drug component of the formulation is most preferably less than 20% (w / v) of the formulation and generally exceeds 0.01% (w / v).

Dosage forms or compositions containing the active ingredient in the range of 0.005% to 5% can be prepared in a balance prepared from a non-toxic carrier.

The exact composition of these agents can vary widely depending on the specific nature of the drug. However, such formulations generally comprise from 0.01% to 5%, preferably from 0.05% to 1% of active ingredient for high potency drugs and from 2% to 4% of active ingredient for intermediate active drugs.

The percentage of active compound contained in such parenteral compositions depends largely on the activity of the compound and the needs of the subject as well as the specific properties of the compound. However, 0.01% to 5% active ingredient percent in solution can be used, and the active ingredient percentage will be higher if the composition is solid and subsequently diluted to that percentage. Preferably the composition comprises 0.2% to 2.0% active agent in solution.

The pharmaceutical composition formulations described herein can be prepared by any method known or later developed in the pharmacological arts. In general, such methods include the step of bringing into association the active ingredient with the carrier or one or more other ingredients, followed by molding or packaging the product in the desired single or multiple dosage units, if desired or desired.

While the description of pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all kinds of animals. It is well understood that modifying a pharmaceutical composition suitable for administration to humans to make the composition suitable for administration to a variety of animals, and that any conventional veterinary pharmacologist can design and perform such modifications by routine experimentation alone. Can be. Subjects to which the pharmaceutical compositions of the present invention are administered include, but are not limited to, humans and other primates, mammals including commercially suitable mammals such as cattle, pigs, horses, sheep, cats, and dogs, and the like. .

Pharmaceutical compositions useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, parenteral, pulmonary, nasal, buccal, or other route of administration.

Pharmaceutical compositions of the present invention may be prepared, packaged, or sold in bulk, as a single unit dose or as a plurality of single unit doses. As used herein, “unit dosage” is the discrete amount of a pharmaceutical composition comprising a predetermined amount of active ingredient. The amount of active ingredient is generally equivalent to or equivalent to the dosage of the active ingredient administered to the subject, for example 1/2 or 1/3 of the dosage. However, the delivery of the active agents described herein may be as little as or less than 1/10, 1/100 or 1 / 1,000 of the dose generally administered because of the targeted properties of the interferon therapeutic.

The relative amounts of the active ingredients, pharmaceutically acceptable carriers, and any additional ingredients in the pharmaceutical compositions of the present invention will depend on the uniqueness, size and condition of the subject to be treated, and further on the route through which the composition is administered. For example, the composition may comprise 0.1% to 100% (w / w) active ingredient.

Formulations of the pharmaceutical compositions of the present invention suitable for oral administration include discrete solid dosage unit forms, including but not limited to tablets, hard or soft capsules, sachets, troches or lozenges, each containing a predetermined amount of active ingredient. Can be manufactured, packaged, or sold. Other agents suitable for oral administration include, but are not limited to, powdered or granular preparations, aqueous or oily suspensions, aqueous or oily solutions, or emulsions.

As used herein, "oily" liquids are liquids that contain carbon-containing liquid molecules and exhibit less polar character than water.

Tablets comprising the active ingredient can be prepared, for example, by pressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compacting powder or granular formulations in a suitable device with an active ingredient in free flowing form, such as, optionally, one or more of binders, lubricants, excipients, surfactants and dispersants. Molded tablets can be made by molding in a suitable device a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least a liquid sufficient to wet the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binders, lubricants, and the like. Known dispersants include, but are not limited to, potato starch and sodium starch glycolate. Known surface active agents include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, acacia, pregelatinized corn starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica and talc.

Tablets may be uncoated or coated by known methods to achieve sustained disintegration in the gastrointestinal tract of the subject to provide sustained release and absorption of the active ingredient. For example, tablets may be coated using materials such as glyceryl monostearate or glyceryl distearate. Further osmotic controlled release tablets can be formed by coating the tablets using, for example, the methods described in US Pat. Nos. 4,256,108, 4,160,452 and 4,265,874. Tablets may further comprise sweetening agents, flavoring agents, coloring agents, preservatives or some combination thereof in order to provide a pharmaceutical precise and tasteful formulation.

Hard capsules comprising the active ingredient may be prepared using physiologically degradable compositions such as gelatin. Such hard capsules contain the active ingredient and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, kaolin or cellulose acetate hydrogen phthalate.

Soft gelatin capsules comprising the active ingredient can be prepared using a physiologically degradable composition such as gelatin. Such soft capsules comprise the active ingredient which can be mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Liquid formulations of the pharmaceutical compositions of the present invention suitable for oral administration may be prepared, packaged, and sold in liquid form or in the form of a dry product for reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions can be prepared using conventional methods for obtaining suspensions of the active ingredient in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olives, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. . The liquid suspension may further comprise one or more additional ingredients, including but not limited to suspending, dispersing or wetting agents, emulsifiers, thickeners, preservatives, buffers, salts, flavors, colorants, and sweeteners. The oily suspension may further comprise a thickener. Known suspending agents include sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, tragacanth gum, acacia gum, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, and the like. There is, but is not limited to this. Known dispersants or wetting agents include, but are not limited to, natural phosphatides such as condensation products of lecithin, alkylene oxides and fatty acids, condensation products of alkylene oxides and long chain fatty alcohols, alkylene oxides and fatty acids and hexitols. Condensation products of partial esters, or partial esters derived from alkylene oxides with fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol mono, respectively) Oleate, and polyoxyethylene sorbitan monooleate), and the like. Known emulsifiers include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl para hydroxybenzoate, ascorbic acid, and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickeners for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. The liquid solution of the pharmaceutical composition of the present invention may comprise each component described for the liquid suspension, although it is understood that the suspending agent does not necessarily assist in dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olives, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. .

Powder and granule formulations of the pharmaceutical formulations of the invention can be prepared using known methods. Such formulations may be administered directly to the subject, or the formulations may be used to form a tablet, fill a capsule, or add an aqueous or oily vehicle to prepare an aqueous or oily suspension or solution. Each of these agents may further comprise one or more of a dispersing or wetting agent, suspending agent and preservative. These formulations may also include additional excipients such as fillers and sweeteners, flavors or colorants.

Pharmaceutical compositions of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination thereof. Such compositions are derived from a combination of one or more emulsifiers, for example natural gums such as acacia gum or tragacanth gum, naturally occurring phosphatides such as soy or lecithin phosphatides, fatty acids and hexitol anhydrides. Esters or partial esters such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical disruption of the subject's tissue and any route of administration of the pharmaceutical composition via disruption in the tissue. Accordingly, parenteral administration includes, but is not limited to, administration of pharmaceutical compositions by injection of the composition, application of the composition via surgical incisions, and application of the composition through tissue-permeable non-surgical wounds, and the like. In particular, parenteral administration is considered to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and renal dialysis infusion techniques.

Formulations of pharmaceutical compositions suitable for parenteral administration include the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multiple dose containers containing a preservative. Preparations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable delayed-release or biodegradable preparations. Such formulations may further comprise one or more additional ingredients, including but not limited to suspending, stabilizing or dispersing agents. In one embodiment of the formulation for parenteral administration, the active ingredient is dried (ie, sterile) for reconstitution with a suitable vehicle (eg, sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Powder or granules).

Pharmaceutical compositions may be prepared, packaged, or sold in the form of sterile injectable aqueous or oily suspensions or solutions. Such suspensions or solutions can be formulated according to the known art and may comprise further ingredients, in addition to the active ingredient, such as the dispersing, wetting or suspending agents described herein. Such sterile injectable preparations may be prepared using, for example, water or a solvent such as 1,3-butane diol or a nontoxic parenterally acceptable diluent. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and nonvolatile oils such as synthetic mono- or diglycerides. Other parenteral administrable formulations that may be useful include those that include the active component in microcrystalline form in the lipid construct formulation or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts.

Pharmaceutical compositions of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations may include dry particles comprising the active ingredient and having a diameter in the range of about 0.5 to about 7 μm, preferably in the range of about 1 to about 6 μm. Such compositions may be prepared using a device comprising a dry powder reservoir, in which a stream of propellant may be directed to disperse the powder, or low boiling point in a self-propelled solvent / powder-dispensing vessel, such as a sealed vessel. In the case of administration using a device comprising the active ingredient dissolved or suspended in propellant, it is conveniently in the form of a dry powder. Preferably, such powders comprise particles in which at least 98% by weight of the particles have a diameter of greater than 0.5 μm and at least 95 number% of the particles have a diameter of less than 7 μm. More preferably, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% of the particles have a diameter of less than 6 μm. The dry powder composition preferably comprises a solid fine powder diluent such as sugar and is conveniently provided in unit dosage form.

Low boiling propellants generally include liquid propellants having a boiling point of less than 65 ° F. at ambient pressure. In general, the propellant may comprise 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as liquid nonionic or solid anionic surfactants or solid diluents, preferably having the same order of particle size as the particles comprising the active ingredient.

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold in an optionally sterile aqueous or dilute alcohol solution or suspension containing the active ingredient, conveniently administered using any nebulization or atomization device. can do. Such formulations may further comprise one or more additional ingredients, including but not limited to flavoring agents such as saccharin sodium, volatile oils, buffers, surface active agents, or preservatives such as methylhydroxybenzoate. . The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 μm.

The formulations described herein useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the present invention.

Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle of about 0.2 to 500 μm. Such formulations are administered by the method of performing respiration, i.e. by rapid inhalation through the nose from a container of powder held near the nare.

Formulations suitable for non-administration may include, for example, as little as about 0.1% (w / w) and as much as 75% (w / w) active ingredients, and may further include one or more additional ingredients described herein. have.

Pharmaceutical compositions of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges prepared using conventional methods, for example 0.1 to 20% (w / w) of active ingredient, orally soluble or degraded. Possible compositions and optionally one or more additional ingredients described herein are balanced. Alternatively, formulations suitable for buccal administration may comprise aerosolized or particulated solutions or suspensions or powders comprising the active ingredient. Such powdered, aerosolized or micronized formulations, when dispersed, preferably have an average particle or droplet size in the range of about 0.1 to about 200 μm and may further comprise one or more additional ingredients described herein.

Pharmaceutical compositions of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of ophthalmic preparations comprising 0.1% to 1.0% (w / w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such loadings may further include buffers, salts, or one or more other additional ingredients described herein. Other useful agents that can be ophthalmically administered include those containing the active ingredient in microcrystalline form or in a lipid construct preparation.

As used herein, “additional ingredients” include excipients, surface active agents, dispersants, inert diluents, granulating and disintegrating agents, binders, lubricants, sweeteners, flavoring agents, coloring agents, preservatives, physiologically degradable compositions, eg For example gelatin, aqueous vehicles and solvents, oily vehicles and solvents, suspending agents, dispersing or wetting agents, emulsifiers, thickeners, buffers, salts, thickeners, fillers, emulsifiers, antioxidants, antibiotics, antifungal agents, stabilizers, and pharmaceutically acceptable Or one or more of the possible polymeric or hydrophobic materials. Other "additional ingredients" that can be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed., 1985, Remington's. Pharmaceutical Sciences , Mack Publishing Co., Easton, PA.

Typically dosages of the compositions of the invention that can be administered to animals, preferably humans, range from 1 μg to about 100 g per kg body weight of the animal. The exact dosage administered will depend on any number of factors including but not limited to the type of disease state to be treated and the type of animal, the age of the animal and the route of administration. Preferably, the dosage of the composition will vary from about 1 mg to about 10 g per kg body weight of the animal. More preferably, the dosage will vary from about 10 mg to about 1 g per kg body weight of the animal.

The composition may be administered frequently to the animal as several daily administrations or the composition may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently. For example, up to once every several months or even up to once a year. The frequency of administration will be very obvious to the skilled person and will vary depending on any number of factors including, but not limited to, the type and severity of the disease to be treated, the type and age of the animal, and the like.

The invention also includes a kit comprising a composition of the invention and instructions describing the administration of the composition to mammalian tissue. In another embodiment, the kit comprises a (preferably sterile) solvent suitable for dissolving or suspending the composition of the invention prior to administering the composition to the mammal.

As used herein, “instructions” include publications, records, schemes or any other expression media that can be used to demonstrate the utility of the proteins of the invention in kits for alleviating the various diseases or disorders cited herein. . Optionally or alternatively, the instructions may describe one or more methods of alleviating a disease or disorder in a cell or tissue of a mammal. Instructions for kits of the present invention may be attached, for example, to a container containing a component of the invention or may be retained with a container containing a component of the invention. Alternatively, the instructions may be retained separately from the container with the intention of causing the instructions and the composition to be used cooperatively by the recipient.

Pharmaceutical compositions useful in practicing the present invention may be administered to deliver a dosage equivalent to a standard dosage of interferon.

While the description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all kinds of animals. It is well understood that modifying a pharmaceutical composition suitable for administration to humans to make the composition suitable for administration to a variety of animals and designing such modifications with only routine experimentation, even if there is a pharmacologist with ordinary skill in veterinary medicine And can be performed. Subjects contemplated for administration of the pharmaceutical compositions of the invention include, but are not limited to, humans and other primates, companion animals, and other mammals.

Pharmaceutical compositions useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral or injectable routes of administration.

The relative amounts of the active ingredients, pharmaceutically acceptable carriers, and any additional ingredients in the pharmaceutical compositions of the present invention will depend on the uniqueness, size and condition of the subject to be treated, and further on the route through which the composition is administered.

Experimental Example

In the following the invention is described with reference to the following examples. These examples are provided for illustrative purposes only, and the present invention should not be construed as being limited to these examples in any way, but rather is understood to cover any and all variations that become apparent as a result of the teachings provided herein. .

The materials and methods used in the experiments presented in this Experimental Example are described below.

Experimental Example  1.Pharmaceutical Composition 1

Lipid constructs include amphipathic lipid 1,2-dstearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetyl phosphate, 1,2-dstearoyl-sn-glycero-3-phospho Ethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (succinyl), 1,2-dipalmitoyl-sn-glycero-3- [phospho-rac -(1-glycerol)] (sodium salt), a mixture of extended amphipathic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (Cap biotinyl) and interferon do.

Experimental Example  2. Pharmaceutical Composition 2

Lipid constructs include amphipathic lipid 1,2-dstearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetyl phosphate, 1,2-dstearoyl-sn-glycero-3-phospho Ethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (succinyl), 1,2-dipalmitoyl-sn-glycero-3- [phospho-rac -(1-glycero)] (sodium salt), interferon-alpha, extended amphipathic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (Cap biotinyl), And / or polychrome-poly (bis)-[N- (2,6- (diisopropylphenyl) carbamoylmethyl) imino] diacetic acid]. Extended Amphiphilic Lipid 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (Cap Biotinyl) and Polychrome-poly (bis)-[N- (2,6- ( Diisopropylphenyl) carbamoylmethyl) imino diacetic acid] was added to the lipid constructs at levels of 1.68 wt% ± 0.5 wt% and 1.2 wt% ± 0.5 wt%, respectively.

Experimental Example  3. Pharmaceutical Composition 3

Lipid constructs include amphiphilic lipid 1,2-dstearoyl-sn-glycero-3-phosphocholine (12.09 g), cholesterol (1.60 g), dicetyl phosphate (3.10 g), polychrom-poly (bis )-[N- (2,6- (diisopropylphenyl) carbamoylmethyl) imino] diacetic acid] (0.20 g) and a mixture of interferon-alpha. The mixture was added to the aqueous medium and the total mass was 1200 g.

Experimental Example  4. Preparation of Lipid Constructs Containing Interferon-alpha

The lipid construct is formed by preparing a mixture of amphiphilic lipid molecules and extended amphipathic lipids, preparing a lipid construct from a mixture of amphiphilic lipid molecules and extended amphiphilic lipids, and complexing interferon-alpha to the lipid construct. It became.

Amphiphilic lipid molecules and mixtures of extended amphipathic lipids were prepared using the following procedure. The mixture of lipid components [total mass is 8.5316 g] of the lipid construct comprises lipid 1,2-dstearoyl-sn-glycero-3-phosphocholine (5.6881 g), cholesterol crystalline (0.7980 g), Dicetyl phosphate (1.5444 g), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (Cap biotinyl) (0.1436 g), 1,2-distearoyl-sn -Glycero-3-phosphoethanolamine (0.1144 g), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (succinyl) (0.1245 g) and 1,2- An aliquot of dipalmitoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (sodium salt) (0.1186 g) was prepared in combination.

A 100 ml solution of chloroform: methanol (2: 1 v: v) was dehydrated on 5.0 g of molecular sieve. The mixture of lipid components of the lipid construct was placed in a 3 liter flask and 45 ml of chloroform / methanol solution was added to the lipid mixture. The solution was placed in a flask on a rotary evaporator with a water bath of 60 ° C. ± 2 ° C. and spun slowly. The chloroform: methanol solution was removed under vacuum on a rotary evaporator using an inhaler for approximately 45 minutes, followed by removal of residual solvent using a vacuum pump for approximately two hours, and a solid lipid compound formed. The dried mixture of lipids may be stored at approximately −20 ° C. to 0 ° C. for an undetermined time in the freezer.

Lipid constructs were prepared from a mixture of amphiphilic lipids and extended amphiphilic lipids using the following procedure. The lipid mixture was mixed with approximately 600 ml of 28.4 mM sodium phosphate (monobasic-dibasic) buffer at pH 7.0. Vortex was formed in the lipid mixture and the mixture was then hydrated in a heated water bath at 0 ° C. ± 4 ° C. for 30 minutes.

The M-110 EHI microfluidizer was preheated to 70 ° C. ± 10 ° C. using SWI with pH 6.5-7.5. The suspension of hydrated target complex was transferred to the microfluidizer and the suspension of the hydrated target molecule complex was passed through the microfluidizer once to microfluidize at approximately 9000 psig. After passing through the microfluidizer, an unfiltered sample (2.0-5.0 ml) of fluidized suspension was collected for particle size analysis using unimodal distribution data from Coulter N-4 plus particle size analyzer. Prior to all particle size determinations, samples were diluted using 0.2 μm filtered SWI adjusted to pH 6.5-7.5. Particle size was required to range from 0.020 to 0.40 μm. If the particle size was outside this range, the suspension was passed back to the microfluidizer at approximately 9000 psig and the particle size was analyzed again until the particle size requirement was reached. Microfluidized target molecule complexes were collected in sterile containers.

The microfluidized target molecule complex was maintained at 60 ° C. ± 2 ° C. but filtered twice through a sterile 0.8 μm + 0.2 μm gang filter attached to a 5.0 ml syringe. An aliquot of the filtered suspension was analyzed to determine the particle size range of the particles in the suspension. The particle size range of the final 0.2 μm filtered sample is in the range of 0.0200 to 0.2000 μm, as determined from the unimodal distribution output from the particle size analyzer.

Interferon is loaded into the construct by reverse loading of the construct using the method described in US Pat. No. 5,104,661, which is incorporated herein by reference.

Experimental Example  5. How to use

The efficacy of HDV-interferon alpha was evaluated in a mouse model with genetic marker response to the liver effect of interferon. C57B16 mice were obtained from the Jackson Laboratory, and breeding colonies were established at the Cleveland MetroHealth Center, Cleveland, Ohio. Mice were obtained from these breeding colonies. Two groups of mice were treated, namely test group and control group. The test group received interferon + HDV but the control group received only interferon. HDV-interferon consisted of 100 mcg HDV for 10 mcg interferon alpha. HDV was supplied by Hepasome Pharmaceuticals and Roeferon was a source of interferon alpha. The HDV and interferon alpha were allowed to equilibrate for 12 hours prior to injection into mice. Mice from these two groups received 100,000 U / kg body weight. To test the timing of the response to IFN, roferon was injected subcutaneously in mice. Mice were sacrificed at 6 hours after dosing. The spleen and liver of the sacrificed rats were obtained for analysis.

Interferon-stimulated responses that induce double stranded RNA dependent protein kinase (PKR) genes were used as markers of interferon liver tissue transfer. The assay used real-time quantitative PCR (polymerase chain reaction) to assay the level of PKR messenger ribonucleic acid (mRNA). Oligonucleotide primers corresponding to introns extending to the exon sequence of mouse PKR mRNA were designed using Oligo V6 software and the sequence is specific by processing the sequence by blast search in NCBI for genomic and mRNA mouse sequences It was confirmed. More than 30 primer pairs were designed as well as two pairs were selected for the experiment. The state of the selected pair was optimized using a continuous temperature and magnesium concentration gradient. RNA was extracted from the liver and spleen of the animal and then reverse transcribed using our proprietary mixing ratio for random hexamer and oligo-dT and M-MLV RT. The resulting cDNA was subjected to semi-quantitative PCR and a 6 hour time point was selected for the HDV experiment. Two sets of mice (three each) were injected with either IFN alone or HDV-IFN in saline. Mice were sacrificed after 6 hours and RNA was extracted from the liver and spleen and subjected to RT reaction. Real-time quantitative PCR was performed on cDNAs generated using cybr green technology. A comparison of the levels of PKR expression levels between liver and spleen in HDV-IFN and IFN treated mice was performed.

PKR results are shown in FIG. 12. 12A shows relative expression levels in liver and spleen from mice receiving interferon alpha. The spleen was chosen as a surrogate for evaluating systemic delivery. Relative expression levels in the spleen were compared to relative expression levels in the liver. The relative expression level in the spleen was approximately twice the relative expression level in the liver. 12B shows relative expression levels in liver and spleen from mice administered HDV in addition to interferon alpha. The relative expression level in the liver was approximately twice the relative expression level in the spleen. The relative expression level in the liver of mice treated with HDV-interferon was approximately twice the relative expression level in the liver of mice treated with interferon alone.

The effect of HDV targeting on hepatic PKR activation by interferon alpha in a mouse model is shown in FIG. 13. Interferon alone provided an approximately 5-fold increase in PKR activation relative to baseline. HDV-interferon provided an approximately 15-fold increase in PKR activation relative to baseline and an approximately 3-fold increase relative to interferon alone. Interferon activity in liver tissue was significantly increased by delivering interferon with HDV.

While the present invention has been disclosed in connection with specific embodiments, it is clear that one of ordinary skill in the art can plan other embodiments and variations of the present invention without departing from the true spirit and scope of the invention. The appended claims are intended to cover all such embodiments and equivalent variations.

Claims (56)

A lipid construct comprising at least one interferon, an amphipathic lipid and an extended amphipathic lipid, wherein the extended amphipathic lipid comprises a proximal, midterm and distal portions, wherein the proximal portion comprises an extended amphipathic lipid. Wherein the distal portion targets the construct to a receptor presented by hepatocytes, and the medial portion connects the proximal and distal portions. The method of claim 1, wherein the interferon is interferon-alpha, interferon alpha-1a, pegylated interferon alpha-1a, interferon-alpha-n1, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n3 , Interferon alphacon-1, interferon n-3, PEG-interferon alpha 2a, PEG-interferon alpha 2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, interferon gamma-1a, interferon gamma-1b, A lipid construct selected from the group consisting of PEGylated interferon beta-1a, PEGylated interferon beta-1b, derivatives thereof, and combinations of any of the aforementioned interferons. A lipid construct comprising one or more anti-viral agents, amphiphilic lipids, and extended amphipathic lipids that are not interferon or interferon derivatives, wherein the extended amphipathic lipids include a proximal, mid, and distal portions, wherein the proximal portion is An extended amphipathic lipid to the construct, the distal portion targeting the construct to a receptor presented by hepatocytes, and the medial portion connecting the proximal and distal portions. The lipid construct of claim 1, wherein the active ingredient comprises at least one interferon and at least one anti-viral agent that is not an interferon or an interferon derivative. The lipid construct of claim 1, further comprising at least one active ingredient in an insoluble form associated with the lipid construct. The method of claim 1 wherein the amphiphilic lipid is 1,2- distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetyl phosphate, 1,2-dipalmitoyl-sn-glycerol- [3 -Phospho-rac- (1-glycero)], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3- A lipid construct comprising at least one lipid selected from the group consisting of phosphoethanolamine-N- (succinyl), derivatives thereof, and mixtures of any of the above compounds. The method of claim 1, wherein the proximal portion of the extended amphipathic lipid comprises at least one but not more than two long chain acyl hydrocarbon chains bonded to the backbone, wherein each hydrocarbon chain consists of a saturated hydrocarbon chain and an unsaturated hydrocarbon chain. A geological construct that is independently selected from the group. 8. The lipid construct of claim 7, wherein the backbone comprises glycerol. The method according to claim 1, wherein the distal portion of the extended amphipathic lipid is biotin, biotin derivative, iminobiotin, iminobiotin derivative, biocitin, biocitin derivative, iminobiocitin, iminobiocitin derivative, And at least one member selected from the group consisting of hepatocyte specific molecules that bind to receptors on hepatocytes. The extended amphipathic lipid of claim 1, wherein the extended amphiphilic lipid is N-hydroxysuccinimide (NHS) biotin, sulfo-NHS-biotin, N-hydroxysuccinimide long chain biotin, sulfo-N-hydroxysuccinimide long chain Biotin, D-Biotin, Biocitin, Sulfo-N-hydroxysuccinimide-SS-Biotin, Biotin-BMCC, Biotin-HPDP, Iodoacetyl-LC-Biotin, Biotin-Hyrazide, Biotin-LC-Hydra Gide, Biocithin Hydrazide, Biotin Cadaberine, Carboxybiotin, Photobiotin, ρ-Aminobenzoyl Biocitin Trifluoroacetate, ρ-Diazobenzoyl Biocitin, Biotin DHPE, Biotin-X-DHPE, 12- ((Biotinyl) amino) dodecanoic acid, 12-((biotinyl) amino) dodecanoic acid succinimidyl ester, S-biotinyl homocysteine, biocytin-X, biocytin x-hydrazide, biotinethylenediamine, Biotin-XL, Biotin-X-on Tylenediamine, Biotin-XX Hydrazide, Biotin-XX-SE, Biotin-XX, SSE, Biotin-X-Cadaberine, α- (t-BOC) Biocitin, N- (Biotinyl) -N '-( Iodoacetyl) ethylenediamine, DNP-X-biocitin-X-SE, biotin-X-hydrazide, norbiotinamine hydrochloride, 3- (N-maleimidylpropionyl) biocitin, ARP, biotin -1-sulfoxide, biotin methyl ester, biotin-maleimide, biotin-poly (ethyleneglycol) amine, (+) biotin 4-amidobenzoic acid sodium salt, biotin 2-N-acetylamino-2-deoxy-β -D-glucopyranoside, biotin-α-DN-acetylneuramid, biotin-α-L-fucoside, biotin lacto-N-bioside, biotin-Lewis-A trisaccharide, biotin-Lewis-Y Tetrasaccharide, Biotin-α-D-Mannopyranoside, Biotin 6-O-Phospho-α-D-Mannopyranoside and Polychrome-Poly (bis)-[N- ( 2,6- (diisopropylphenyl) carbamoylmethyl) imino] diacetic acid. The lipid construct of claim 1, wherein the median of the extended amphiphilic lipid comprises a thio-acetyl triglycine polymer or derivative thereof, wherein the extended amphiphilic lipid molecule extends out of the surface of the lipid construct. The method of claim 1, further comprising one or more active ingredients associated with the water-insoluble target molecule complex, wherein the complex comprises a plurality of linked individual units, wherein the individual units are a. A crosslinking component selected from the group consisting of transition elements, internal transition elements, adjacent elements of transition elements, and mixtures of any of the above elements, and b. Complex-forming components But comprising a chromium target molecule complex when the transition element is chromium. 13. The lipid construct of claim 12, further comprising one or more active ingredients not associated with the target molecule complex. 13. The lipid construct of claim 12, wherein the crosslinking component is chromium. 13. The lipid construct of claim 12, wherein the complex forming component comprises poly (bis)-[(N- (2,6-diisopropylphenyl) carbamoylmethyl) iminodiacetic acid]. 52. The hepatocyte-targeting composition of claim 51, wherein the lipid component comprises a mixture of 1,2-dstearoyl-sn-glycero-3-phosphocholine, cholesterol and dicetyl phosphate. The lipid construct of claim 1, wherein the distal component of the extended amphiphilic lipid comprises a nonpolar derivatized benzene ring or heterobicyclic ring structure. The lipid construct of claim 1, comprising a positive charge, a negative charge, or a combination thereof. The lipid construct of claim 1, wherein the extended amphipathic lipid comprises one or more carbonyl moieties positioned at a distance of about 13.5 mm 3 or less from the distal distal end. The lipid construct of claim 1, wherein the extended amphipathic lipid comprises one or more carbamoyl moieties comprising secondary amines. The lipid construct of claim 1, wherein the extended amphipathic lipid comprises chromium charged at the median point. The lipid construct of claim 1, further comprising cellulose acetate hydrogen phthalate. a. Producing a mixture comprising an amphipathic lipid and an extended amphipathic lipid, b. A lipid construct comprising at least one interferon, an amphipathic lipid and an extended amphipathic lipid, wherein the extended amphipathic lipid comprises a proximal, midterm and distal portions, wherein the proximal portion connects the extended amphipathic lipid to the structure. Forming a suspension of the lipid construct in an aqueous medium, wherein the distal portion targets the construct to a receptor presented by hepatocytes and the distal portion connects the proximal and distal portions, and c. Loading the active ingredient into the lipid construct Including, the method for producing the lipid construct. The method of claim 23, wherein the step of loading the active ingredient into the lipid construct comprises an equilibrium support and a non-equilibrium support. The method of claim 23, wherein the step of loading the active ingredient into the lipid construct comprises adding a solution containing the free active ingredient to the mixture of lipid constructs in an aqueous medium and maintaining the active ingredient in contact with the mixture until equilibrium is reached. Which method. The method of claim 25, d. After the mixture reaches equilibrium, the last active ingredient is submerged in the lipid construct and the free active ingredient containing solution is removed from the construct. Further comprising wherein the structure further contains one or more active ingredients associated therewith. The method of claim 25, e. In a group consisting of rapid filtration procedures, centrifugation, filter centrifugation, and chromatography using streptavidin agarose affinity-resin gels having affinity for ion-exchange resins or biotin, iminobiotin, or derivatives thereof. Removing the free active ingredient containing solution from the lipid construct containing one or more active ingredients associated therewith through the selected process Further comprising. The method of claim 23, wherein f. Adding to the lipid construct a chromium complex comprising a plurality of linked individual units Further comprising. The method of claim 23, wherein g. Adding cellulose acetate hydrogen phthalate to the lipid construct Further comprising. The method of claim 23, wherein h. Recovering from the process at least one substance selected from the group consisting of an active ingredient, an ion-exchange resin and a streptavidin agarose affinity gel Further comprising. a. Combining at least one active ingredient with a lipid construct comprising a plurality of non-covalent multi-dentate binding sites, and b. Administering to the patient the construct containing the active ingredient A method of increasing the bioavailability of one or more active ingredients in a patient, comprising. 32. The method of claim 31, further comprising adjusting the isoelectric point of the one or more active ingredients. The active ingredient according to claim 31, wherein the active ingredient is interferon-alpha, interferon alpha-1a, PEGylated interferon alpha-1a, interferon-alpha-n1, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n3, interferon Alphacon-1, interferon n-3, PEG-interferon alpha 2a, PEG-interferon alpha 2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, interferon gamma-1a, interferon gamma-1b, PEGylation Interferon beta-1a, pegylated interferon beta-1b, derivatives thereof, and combinations of any of the aforementioned interferons. 32. The method of claim 31, wherein the active ingredient is an anti-viral agent that is not an interferon or an interferon derivative. The method of claim 31, wherein the active ingredient comprises one or more interferons and one or more anti-viral agents that are not interferon or interferon derivatives. 32. The composition of claim 31 wherein the lipid construct is interferon, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetyl phosphate, 1,2-dipalmitoyl-sn-glycero- [3-phospho-rac- (1-glycerol)], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and 1,2-dipalmitoyl-sn-glycero-3 Phosphoethanolamine-N- (succinyl) or derivatives thereof and hepatocyte receptor binding molecules. a. Binding the lipid construct to a streptavidin agarose affinity gel of pH 9.5 or higher via a lipid comprising an iminobiotin or an iminobiotin derivative to remove the construct from a bulk phase medium, b. Separating the structure from the bulk phase medium, and c. Adjusting the pH of the affinity gel aqueous mixture to pH 4.5 to release the construct from the affinity gel Wherein the released construct contains at least one insoluble active ingredient, wherein the construct is one wherein the insoluble active ingredient is redissolved under physiological pH conditions in the host upon administration to a warm blooded host. A method of forming a sustained release composition that increases the bioavailability of the compound. A lipid construct comprising at least one interferon, amphipathic lipids, and extended amphipathic lipids, wherein the extended amphipathic lipids comprise a proximal, midterm, and distal portions, wherein the proximal portion connects the extended amphipathic lipids to the construct and A method of treating a hepatitis patient, comprising administering to the hepatitis patient an effective amount of a lipid construct that distal targets the construct to a receptor presented by hepatocytes and the medial connects the proximal and distal ends. 39. The method of claim 38, wherein the patient is at least one hepatitis patient selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F, and hepatitis G. The method of claim 38, wherein the active ingredient is interferon-alpha, interferon alpha-1a, PEGylated interferon alpha-1a, interferon-alpha-n1, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n3, interferon Alphacon-1, interferon n-3, PEG-interferon alpha 2a, PEG-interferon alpha 2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, interferon gamma-1a, interferon gamma-1b, PEGylation Interferon beta-1a, PEGylated interferon beta-1b, derivatives thereof, and combinations of any of the aforementioned interferons. The method of claim 38, wherein the active ingredient is an anti-viral agent that is not an interferon or an interferon derivative. The method of claim 38, wherein the active ingredient comprises at least one interferon and at least one anti-viral agent that is not an interferon or an interferon derivative. The composition of claim 38, wherein the lipid construct further comprises a target molecule complex, wherein the complex comprises a plurality of linked individual units, wherein the linked individual units are a. A crosslinking component selected from the group consisting of transition elements, internal transition elements, adjacent elements of transition elements, and mixtures of any of the above elements, and b. Complex-forming components But when the transition element is chromium, a chromium target molecule complex is formed. The method of claim 38, wherein the lipid construct further comprises one or more active ingredients not associated with the target molecule complex. The method of claim 38, wherein the route of administration is selected from the group consisting of oral, parenteral, subcutaneous, pulmonary and buccal. Liver of a virus infected patient is administered to a virus infected patient by administering a lipid construct of various sizes comprising one or more active ingredients, amphiphilic lipids and extended lipids, wherein the extended lipids comprise a hepatocyte receptor binding moiety. In which at least one active ingredient increases delivery to hepatocytes. 47. The virus of claim 46, wherein the patient has at least one hepatitis A selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F, or hepatitis G; Infected with a combination of hepatitis virus. 47. The method according to claim 46, wherein the active ingredient is interferon-alpha, interferon alpha-1a, PEGylated interferon alpha-1a, interferon-alpha-n1, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n3, interferon Alphacon-1, interferon n-3, PEG-interferon alpha 2a, PEG-interferon alpha 2b, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, interferon gamma-1a, interferon gamma-1b, PEGylation Interferon beta-1a, pegylated interferon beta-1b, derivatives thereof, and combinations of any of the aforementioned interferons. 47. The method of claim 46, wherein the active ingredient is an anti-viral agent that is not an interferon or an interferon derivative. 47. The method of claim 46, wherein the active ingredient comprises at least one interferon and at least one anti-viral agent that is not an interferon or an interferon derivative. 47. The method of claim 46, further comprising providing a three-dimensional structural array of lipid molecules that prevents the hydrolase from accessing the active ingredient in the lipid construct, thereby protecting the active ingredient in the lipid construct from hydrolysis. How to do. 47. The method of claim 46, further comprising adding cellulose acetate hydrogen phthalate to the lipid construct and reacting with the individual lipid molecules. 47. The method of claim 46, further comprising producing the active ingredient in an insoluble dosage form within the lipid construct. A lipid construct, a physiological buffer, an applicator, and instructions for use, wherein the lipid construct comprises one or more interferons, amphiphilic lipids, and extended amphipathic lipids, the extended amphipathic lipids being proximal, median A proximal and distal portion, wherein the proximal portion connects the extended amphipathic lipids to the construct, the distal portion targets the construct to receptors presented by hepatocytes, and the proximal portion connects the proximal and distal portions. Kits for the treatment of infected mammals. 55. The kit of claim 54, further comprising at least one active ingredient. 55. The patient of claim 54, wherein the patient is infected with one or more viruses of hepatitis selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F, and hepatitis G. Kit.

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