Micro-particle fatty acid salt solid dosage formulations for therapeutic agents

[0001] The present invention claims priority from U.S. Provisional Patent Application No. 60 / 494,821, filed on August 13, 2003 by Opawale et al., Titled "Formulations of solid doses of fatty acid microparticles for therapeutic agents".

2. Field of the invention The invention relates to fatty acid salt microparticles and their use in pharmaceutical preparations, in particular pharmaceutical preparations for oral administration of polypeptide drugs.

3. BACKGROUND OF THE INVENTION [0003] Problems associated with oral administration of therapeutic agents such as polypeptides are well known in the pharmaceutical industry and various strategies are used in attempting to solve them. Delivery of therapeutic polypeptides through the gastrointestinal tract is difficult due to the presence of large amounts of polypeptide degrading enzymes in the stomach and intestines. In order to obtain a method of oral administration, the therapeutic polypeptide must both survive contact with gastrointestinal enzymes and be able to be transported through or next to intestinal enterocytes.

[0004] Examples of approaches for oral administration include 1) the use of enzyme inhibitors to reduce the rate of degradation of polypeptides and polypeptides in the gastrointestinal tract, 2) manipulation of pH to inactivate local digestive enzymes, 3) use of penetration enhancers to improve the absorption of polypeptides by increasing their paracellular and transcellular transport, 4) the use of nanoparticles as carrier particles to facilitate the absorption of intact substance by the intestinal epithelium, especially the Peyer's tuft, and to increase resistance to enzymatic degradation, and 5) the use of liquid emulsions to protect the drug from enzymatic degradation in the intestinal lumen.

[0005] Oral dosage forms adapted to administer conventional small molecule drugs have also been used in attempts to improve the administration of polypeptides orally. Except in cases where the polypeptide was chemically modified or where the proper absorption enhancer was used, the results were disappointing. To the knowledge of the inventors, only a few human clinical studies have taken place that have demonstrated adequate bioavailability and pharmacokinetics that suggest that commercialization of an orally delivered polypeptide is possible.

[0006] WO-A-94/08610 discloses a liquid composition in the form of a microemulsion containing oil, a mixture of surfactant, medium chain fatty acid salt, aqueous phase and biologically active agent. Importantly, the delivery system is in liquid form and gives no indication as to solid form for oral administration.

[0007] There is a need in the art for new approaches to formulating polypeptide drugs for oral administration that are an improvement over the current state of the art by incorporating more drugs into the bloodstream through the gastrointestinal tract.

4. Summary of the Invention [0008] The present invention provides a pharmaceutical composition comprising fatty acid salt particles with a size distribution characterized in that the particles have from 1 to 1000 microns in diameter, wherein the fatty acid salt contains at least one fatty acid selected from the group consisting of sodium salt capric acid and sodium lauric acid, wherein the composition further comprises a pegylated polypeptide drug and wherein the composition is in solid form for oral administration.

5. Brief description of the drawings [0009]

Figure 1 shows the amino acid sequence and structure of salmon calcitonin.

Figure 2 shows the amino acid sequence and structure of CT-025, a chemically modified form of salmon calcitonin.

Figure 3 shows the dissolution of sodium caprate released from three CT-025 tablet samples (see Fig. 2) formula A.

Figure 4 shows the dissolution of sodium laurate released from three samples of Formula A tablets.

Figure 5 shows the dissolution of sodium caprate released from three samples of formula B tablets CT-025.

Figure 6 shows the dissolution of sodium laurate released from three samples of Formula B tablets.

Figure 7 shows the dissolution of sodium caprate released from three samples of Formula C-025 tablets.

Figure 8 shows the dissolution of sodium laurate released from three samples of Formula C-025 tablets.

Figure 9 shows the dissolution of sodium caprate released from six placebo capsule samples.

Figure 10 shows the dissolution of sodium laurate released from six CT-025 capsule samples.

Figure 11 shows dissolution of sodium caprate released from six placebo capsule samples.

Figure 12 shows dissolution of sodium laurate released from six CT-025 capsule samples.

Figure 13 shows plasma CT-025 levels after oral administration to fasting beagle dogs of one tablet containing processed fatty acids and 430 μg CT-025 (equivalent to 320 μg salmon calcitonin).

Figure 14 shows salmon plasma calcitonin levels after oral administration to fasted beagle dogs of one tablet containing processed fatty acids and 320 μg salmon calcitonin.

6. Definitions [0010] The terminology used in the description of the invention is intended to describe only specific embodiments and is not intended to limit the scope of the invention. As used in the description of the invention and in the claims, the singular forms "the" "which" and "which" are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0011] As used herein, the following terms have the meanings indicated:

[0012] "Biologically active agent" means a therapeutic or pharmacological agent that is conjugated in a manner according to the present invention. The biologically active agent may be a small molecule, macromolecule, peptide, protein or nucleic acid. Examples of biologically active agents include, but are not limited to, those belonging to the following therapeutic categories: ACE inhibitors, antianginal drugs, anti-arrhythmic drugs, anti-asthma drugs, anti-cholesterol drugs, anticonvulsants, anti-depressants, anti-diarrheal preparations, antihistamines, antihypertensives, anti-infective agents, anti-inflammatory agents, anti-lipid agents, anti-manic attacks, anti-manic attacks, anti-stroke agents, anti-thyroid preparations, anti-cancer drugs, cough suppressants, anti-uricemia drugs, antiviral agents, acne drugs, alkaloids, amino acid preparations, anabolic drugs, painkillers, anesthetics, angiogenesis inhibitors, antacids, anti-rheumatic compounds, antibiotics, anticoagulants, anti-emetics, anti-obesity drugs, anti-parasitic agents antipsychotics, antipyretics, antispasmodics, anticoagulants, anxiolytics. appetite stimulants, appetite suppressants, beta-blockers, bronchodilators, cardiovascular agents, cerebral vasodilators, chelating agents, cholecystokinin antagonists, chemotherapeutics, cognitive activators, contraceptives, coronary dilators, antitussives, depressants hyperemia, deodorants, dermatological agents, diuretics, antidiabetic agents, emollients, enzymes, erythropoietics, expectorants, fertility enhancers, fungicides, gastrointestinal agents, growth regulators, hormone replacement agents, anti-hyperglycaemics, hypnotics, anti-hypoglycemics, laxatives, migraines, mineral supplements, mucolytics, dementia, neuroleptics, neuromuscular drugs, NSAIDs, nutritional supplements, peripheral vasodilators, prostaglandins, psychotropic agents, renin inhibitors, respiratory stimulants, steroids, stimulants. sympatolytic agents, thyroid preparations, sedatives, uterine relaxants, vaginal preparations, vasoconstrictors, vasodilators, dizziness agents, vitamins and wound healing agents. Other non-limiting examples of biological agents include coumarin, insulin, calcitonin, leu-enkephalin and met-enkephalin. [0013] "Effective amount" means the amount of a compound or blend that is sufficient to obtain the desired effect, which may be a therapeutic effect. The effective amount will vary depending on the age, general condition of the subject, severity of the condition being treated, the particular biologically active agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and similar factors within the knowledge and experience of the expert. in the field. Depending on the case, the "effective amount" in any particular case may be determined by anyone with typical skill in the field in relation to relevant documents and literature and / or by means of routine experimentation. (See, for example, Remington, Science and Practice of Pharmacy (20th ed., 2000)).

[0014] "Hydrolizable" means bonds that undergo hydrolysis under physiological conditions.

[0015] "Hydrophilic" means having a water-soluble trait, and the term "hydrophilic group" means a moiety that is hydrophilic and / or which, when bound to another chemical entity, increases the hydrophilicity of such chemical. Examples include, but are not limited to, sugars and polyalkylene groups such as polyethylene glycol. "Lipophilic" means having fat solubility characteristics such as fat and fat tissue accumulation, lipid solubility and / or ability to penetrate, interact with and / or pass through biological membranes, and the term "lipophilic group" means a moiety that is lipophilic, and / or which, when bound to another chemical entity, increases the lipophilicity of such chemical. "Amphiphilic" means hydrophilic and lipophilic, and the term "amphiphilic" means a moiety that is an amphiphilic substance, and / or which, after binding to a polypeptide or non-polypeptide drug, increases the amphiphilicity of the resulting conjugate, e.g., a PEG-fatty acid modifying moiety. sugar-fatty acid modifying moiety.

[0016] "Lower alkyl" means a substituted or unsubstituted, linear or branched alkyl residue having from one to six carbon atoms, for example C1, C2, C3, C4, C5 or C6. "Higher alkyl" means substituted or unsubstituted, linear or branched alkyl residues having six or more carbon atoms, for example C7, C8, C9, C10, C9, C10, C13, C14, C15, C16, C17, C18, C19, C20 , etc.

[0017] "Monodisperse" describes a mixture of compounds in which about 100 percent of the compounds in the mixture have the same molecular weight. "Substantially monodisperse" describes mixtures of compounds in which at least about 95 percent of these compounds in the mixture have the same molecular weight. "Purely monodisperse" describes mixtures of compounds in which about 100 percent of the compounds in the mixture have the same molecular weight and the same molecular structure. Thus, the pure monodisperse mixture is a monodisperse mixture, but the monodisperse mixture is not necessarily a pure monodisperse mixture. The term "essentially purely monodisperse"

describes mixtures of compounds in which at least about 95 percent of these compounds in the mixture have the same molecular weight and the same molecular structure. Thus, a substantially purely monodisperse mixture is a substantially monodisperse mixture, but a substantially monodisperse mixture is not always a substantially pure monodisperse mixture. the conjugated polypeptides used in the inventive blends are preferably monodisperse, essentially monodisperse, purely monodisperse or substantially pure monodisperse, but may also be polydisperse. "Polydisperse" means exhibiting a dispersion that is not monodisperse, essentially monodisperse, purely monodisperse or essentially purely monodisperse.

[0018] "Polyalkylene glycol" or PAG refers to substituted or unsubstituted linear or branched polyalkylene glycol polymers such as polyethylene glycol (PEG), polypropylene glycol (PPG) and polybutylene glycol (PBG) and combinations thereof (for example, linear or branched polymers comprising combinations of two or more different PAG subunits, such as two or more different PAG units selected from PEG, PPG subunits, PPG and PBG) and contains polyalkylene glycol monoalkylether. The term PAG subunit means one unit

PAG, for example, "PEG subunit" refers to a single polyethylene glycol unit, e.g. - (CH2CH2O) -, "PPG subunit" refers to a single polypropylene glycol unit, for example (CH2CH2CH2O) - and "PBG subunit" refers to to a single polypropylene glycol unit, for example - (CH2CH2CH2CH2O) - PAG and / or PAG subunits also include substituted PAG or PAG subunits, e.g. PAGs containing alkyl side chains such as methyl, ethyl or propyl side chains or carbonyl side chains as well as PAGs comprising one or more branched PAG subunits such as iso-PPG or iso-PBG.

[0019] The term "pharmaceutically acceptable" means, when referring to an ingredient such as a salt, carrier, excipient or diluent of the composition of the present invention, that it is an ingredient that (i) is compatible with other ingredients of the composition, including be combined with the compositions of the present invention, without rendering the composition unsuitable for the intended purpose and (ii) is suitable for use in the subjects described herein without causing undesirable side effects (such as toxicity, irritation and allergic reaction). Side effects are "excessive" when their risk outweighs the benefits provided by the composition.

[0020] "Polypeptide drug" means a polypeptide having therapeutic activity when administered to a patient or resulting in metabolites having therapeutic activity, and derivatives or conjugates of such polypeptides.

[0021] "Polypeptide" means a peptide or protein.

[0022] "Prodrug" or "pure prodrug" means a biologically active agent that is chemically derivatized such that (i) retains some, all or no biological activity of the parent drug compound, and (ii) is metabolized by the patient by the patient to form the parent compound the drug. In the context of a prodrug of an oligomerized polypeptide or a pure prodrug, all of the oligomer is removed in vivo to form a biologically active unconjugated polypeptide.

[0023] The terms "treat" or "treatment" refer to any type of treatment that produces a modulating effect that may, for example, have a beneficial effect on a patient suffering from a disorder, disease or condition, including improvement of the patient's condition (e.g. (one or more symptoms), delaying the progression of the condition, preventing or delaying the occurrence of this disorder and / or changing clinical parameters, disease or condition etc. which are well known in the art and / or improving to normal physiological functioning.

7. Detailed Description of the Invention [0024] The present invention provides mixtures of microparticles of fatty acid salts containing these salts. Microparticles of fatty acid salts are particularly useful in the preparation of solid-dose preparations for the oral administration of therapeutic agents, in particular polypeptide drugs, such as polypeptide conjugates. The fatty acid salt microparticle mixtures of the present invention are surprisingly able to facilitate the delivery of a pharmaceutically effective amount of a polypeptide through the gastrointestinal tract to the systemic circulation. Without wishing to be bound by theory, the inventors believe that microparticles of fatty acid salts dissolve quickly in the intestine, create a microenvironment that protects polypeptides from enzymatic attack and helps transport polypeptides through the intestinal epithelium.

7.1. Microparticles of Fatty Acid Salt [0025] The invention uses microparticles of fatty acid salts useful in the formation of therapeutic compounds. The inventors have surprisingly found that increasing the surface area to volume ratio of fatty acid salts, e.g., by reducing salt particle size, significantly increases their ability to facilitate the delivery of polypeptide drugs across the intestinal mucosa. Preferably, the dissolution rate is higher than the dilution rate.

[0026] Salts of fatty acids useful according to the invention are sodium capric acid and sodium lauric acid. Other examples of fatty acid salts include, but are not limited to, short, medium and long chain fatty acids, such as, for example, medium chain fatty acid salts such as butyric (butanoic) acid, caproic acid (hexanoic acid), caprylic acid (octanoic acid), capric acid (decanoic acid), lauric acid (dodecanoic acid), mystic acid (tetradecanoic acid), palmitic acid (hexadecanoic acid), palmitoleic acid (9hexadecenoic acid), stearic acid (octadecanoic acid), oleic acid (9-octadecenoic acid), linoleic acid (9,12-octadecadienoic acid), alpha-linolenic acid (ALA), (9,12,15-octadecatrienic acid) ), gamma-linolenic acid (GLA) (6,9,12-octadecatrienoic acid), peanut acid (eicosanoic acid), gadoleic acid (9-eicosenic acid), arachidonic acid (AA) (5,8,11,14-eicosatetraenoic acid) ), EPA (5,8,11,14,175 eicosapentaenoic acid), behenic acid (docosanic acid), erucic acid (13-docosenoic acid), DHA (4,7,10,13,16,19-docosahexaenoic acid), lignoceric acid (tetracosanoic acid).

[0027] To ensure an adequate dissolution / dilution ratio, salts are provided in particle sizes much smaller than currently available particle sizes. Salts of fatty acids may be in a mixture in which at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% or about 100% of the particles are from about 1 to about 1000 microns in diameter. Salts of fatty acids may be in a composition wherein at least about

10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% of the particles have from about 5 up to about 500 microns in diameter. Salts of fatty acids may be in a mixture in which at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98% , 99% or about 100% of the particles are from about 10 to about 100 microns in diameter. Salts of fatty acids may be in a mixture in which at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% or about 100% of the particles are from about 20 to about 80 microns in diameter. In addition, fatty acid salts may be included in a blend in which the average particle size of the fatty acid salt particles is from about 1 to about 1000 microns in diameter, preferably from about 5 to about 500 microns in diameter, more preferably from about 10 to about 100 microns in diameter , preferably about 20 to about 80 microns in diameter.

7.2. Formulations [0028] Microparticles of fatty acid salts are used to prepare solid oral dosage forms such as tablets and capsules for the use of oral administration of therapeutic agents. While this work focuses on the use of these new mixtures for the administration of polypeptide drugs, it should be understood that the mixtures are also useful for the formulation of small molecule drugs. The mixtures generally contain microparticles of the fatty acid salt of the present invention and a pegylated polypeptide drug. However, a wide range of other excipients may also be included in the formulations of the present invention, as long as such excipients do not eliminate the beneficial effects provided by the use of fatty acid salt microparticles. The formulations of the invention are particularly useful for providing drugs based on polypeptides, especially chemically modified polypeptide drugs, such as conjugated polypeptides.

[0029] In some embodiments, the formulations of the present invention may contain a bile salt, which in some embodiments may be an unconjugated bile salt, in which the primary side chain contains one carboxyl group which is in its end position and which is unsubstituted (e.g. cholate, ursodeoxycholate, chenodeoxycholate and deoxycholate). In other embodiments, the blends of the present invention may include a conjugated bile salt, which is a bile salt in which the primary side chain contains a carboxyl group which is substituted, e.g., an amino acid derivative bonded through a nitrogen atom to a carboxyl group (e.g., taurocholate, glycocholate, taurodeoxycholate and glycodeoxycholate). Mixtures of various unconjugated and / or conjugated bile salts can also be used.

[0030] In other embodiments, the mixtures and / or preparations of the present invention in particular do not contain any bile salts, or do not contain individual bile salts. For example, the compositions and preparations of the present invention may include any one or more bile salts in any combination, and may also exclude one or more other bile acid in any combination. The formulation according to the invention may contain taurocholate and / or exclude cholate. Any specific bile salt may be included, and any particular bile salt may be specifically excluded, and therefore, all of the listed combinations of included and excluded bile salts are within the scope of the present invention.

[0031] In embodiments of the present invention, a wide range of optional ingredients known in the art for use in pharmaceutical preparations may also be included. Examples include, but are not limited to, surfactants, buffering ingredients, coating agents, disintegrators, bulking agents, lubricants, emulsifying agents, tablet binding agents, fillers, wetting agents such as those well known in the art.

[0032] Although optional, surfactants may be useful in the compositions of the invention to increase the solubility of fatty acids, which may improve the dissolution / dilution ratio. Examples of useful surfactants are, but are not limited to, acacia, lecithin, sodium lauryl sulfate, glyceryl monostearate, mono and di-glycerides, oleic acid, poloxamer, polysorbates, sorbitan esters and trolamine. When present, surfactants can be included in an amount that ranges from> 0% to about 20% by weight of the pharmaceutical composition. More preferably, the surfactants may be included in an amount that is from> 0% to about 5% by weight of the pharmaceutical composition. Most preferably, the surfactants are included in an amount that ranges from> 0% to about 2% by weight of the pharmaceutical composition.

[0033] The formulations of the present invention may also contain a buffer. Preferred buffers are pharmaceutically acceptable. The buffers used in the formulations of the invention preferably have a pHod of about 5 to about 10, more preferably higher than 7.0 to about 10.0, more preferably higher than 7.0 to about 9.0, and most preferably from about 7.6 to about 8.0. High pH, ​​as described herein, has the advantage of being able to temporarily neutralize the low pH of the stomach, and thus inactivate gastric enzymes such as pepsin. In addition, a high pH buffer system as described herein maintains a favorable pH for fatty acids in solution. The use of the preparation according to the invention creates conditions in the gastrointestinal tract that favor the protection of conjugated or unconjugated polypeptide drugs and increase the penetration of these polypeptide drugs when combined with the fatty acid salt microparticles of the present invention.

[0034] Examples of suitable buffer ingredients for use in the pharmaceutical mixtures of the invention include, but are not limited to, acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, lactic acid, potassium citrate, potassium metaphosphate, potassium dihydrogen phosphate, sodium acetate , sodium citrate, dibasic sodium phosphate and monobasic sodium phosphate.

[0035] In some embodiments, the formulation of the invention is in a dosage form such as a tablet that is not enteric coated, and in some embodiments, the dosage form is enteric coated. In other embodiments, the formulation of the invention does not contain an adhesive, and in some embodiments, the adhesive is included. One or more disintegrants, lubricants, bulking agents, fillers may or may not be present.

[0036] The active ingredients used in the pharmaceutical compositions of the invention may be contained in nanoparticles. Particles in the nanometer size range are absorbed intact through the intestinal epithelium, especially the Peyer's patches, and travel to places such as the liver, spleen, and other tissues. Polypeptide drugs and / or small molecules encapsulated in nanoparticles may be less susceptible to enzymatic degradation. Once in the bloodstream, the particles can provide sustained release of the drug.

7.3. Active Ingredients [0037] The active ingredients are pegylated polypeptide drugs. Preferred polypeptide drugs include calcitonin, oligomer conjugated calcitonin, pegylated calcitonin, natriuretic brain peptide, oligomer-conjugated natriuretic brain peptide, pegylated natriuretic brain peptide, insulin, oligomer conjugated insulin, GL-1 pegylated insulin and / or pegylated GLP-1. One preferred group of drugs are those that are mainly absorbed via the paracellular route.

[0038] Other polypeptide drugs useful in the present invention include, but are not limited to, adenosine deaminase, adrenocorticotropic hormone, arginase, arginine deaminase, asparaginase, chymotrypsin, endorphins, enkephalins, erythropoietin, glucagon-like peptide-like , hypothalamic releasing factors, insulin, interferon, natriuretic peptides (e.g. brain natriuretic peptide and vestibular natriuretic peptide), non-naturally occurring opioids, oxytocin, papain, parathyroid hormone, prolactin, ribonuclease, somatomedin, somatostatin, somatotropin, superoxide dismutase, thyroid stimulating hormone and thyroid hormone, tyrotinated hormone and tryptone version .

[0039] Preferred polypeptides of the invention may have a size in the range of from about 300 to about 10,000,000 Dalton, more preferably in the range of from about 1,000 to about 50,000 Dalton, and most preferably in the range of from about 1,000 to about 10,000 Dalton.

[0040] In a preferred embodiment, the polypeptide drug is a polypeptide conjugated to a modifying group having a structure selected from

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[0041] Each of the above groups may, for example, be conjugated to human insulin at a nucleophilic residue, e.g., A1, B1 or B29, or any combination of the above.

[0042] In another embodiment, the polypeptide drug is conjugated to a modifying residue of formula:

-X-R¹-Y-PAG-ZR² where,

X, Y and Z are independently selected binding groups and each is optionally present, and X, when present, is linked to insulin via a covalent bond, at least one of R¹ and R² is present, and is lower alkyl, optionally it may contain a carbonyl group,

R² is a drip group. iPAG is a linear or branched carbon chain containing one or more alkylene glycol residues and optionally containing one or more additional residues selected from the group consisting of -S-, -O-, -N-, and -C (O) - and where the modifying residue has a maximum of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 atoms heavy.

[0043] In embodiments of the invention, one or more of X, Y and

Z may be absent. In addition, if present, X, Y and / or Z is independently selected from -C (O) -, -O-, -S- and -N-. In one embodiment, Z is -C (O) -.

[0044] In some embodiments, R¹ is lower alkyl and R² is 0. In other embodiments, R² is lower alkyl and R¹ is 0.

[0045] The modifying residue may, for example, be conjugated to human insulin at the nucleophilic residue, for example A1, B1 or B29, or any combination of the above.

[0046] In another embodiment, the modifying residue may contain a linear or branched substituted carbon chain residue having a backbone of 330 atoms selected from the group consisting of -C, -C-, -O-, = O, -S-, -N-, -Si-. Heavy atoms usually include one or more carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -O-, 16

S-, -N- i = O, preferably -O-, N- i = O, more preferably -O- i = O. Carbon and non-carbon heavy atoms are usually present in a ratio of at least 1 carbon atom for each non-carbon heavy atom, preferably at least 2 carbon atoms for each non-carbon heavy atom, more preferably at least 3 carbon atoms for each non-carbon heavy atom. Carbon and oxygen atoms are usually present in a ratio of at least 1 carbon atom for each oxygen atom, preferably at least 2 carbon atoms for each oxygen atom, more preferably at least 3 carbon atoms for each oxygen atom. The modifying residue may contain one or more capping groups, such as branched or linear C 1-6, branched or linear or carbonyl. The modifying residue usually contains hydrogen atoms and one or more hydrogen atoms can be substituted by fluorine (which is a heavy atom but should not be treated as a heavy atom in the above formula). The modifying residue may, for example, be conjugated to human insulin at a nucleophilic residue, e.g., A1, B1 or B29, or any combination of the above. [0047] In other embodiments of the present invention, the polypeptide drug may be native insulin, unconjugated bioactive insulin analogue and / or conjugated insulin. In specific embodiments, the polypeptide drug may be as follows:

Insulin B29 Lys- (NH-CO (CH2) 5 (OCH2CH2) 7OCH3) where the -NH- group is nitrogen ε of the Lys residue.

[0048] In particular embodiments of the present invention, the polypeptide drug may be native calcitonin, an unconjugated bioactive calcitonin analog and / or conjugated calcitonin. In specific embodiments, the polypeptide drug may be as follows:

Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys (NH-CO (CH₂) 7 (OCH₂CH₂) 7OCH₃)

Leu Ser Gly Glu Leu His Lys (NH-CO (CH₂MOCH₂CH₂) Toch₃) Leu Gin Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro.

where the -NH- group is nitrogen ε lysines.

[0049] Other conjugated polypeptides useful in the mixtures of the present invention can be found in the following patents: patent

USA 6,303,569, issued on October 16, 2001 to Enzon, Inc., under the title "Trialkyl17 lock-facilitated polymeric prodrugs of amino-containing bioactive agents"; U.S. Patent 6,214,330, issued April 10, 2001 Enzon, Inc., under the title "Coumarin and related aromatic-based polymeric prodrugs"; U.S. Patent No. 6,113,906, issued September 5, 2000, Enzon, Inc., entitled "Water-soluble non-antigenic polymer linkable to biologically active material"; U.S. Patent 5,985,263, issued November 16, 1999 Enzon, Inc., titled "Substantially pure histidine-linked protein polymer conjugates"; U.S. Patent 5,900,402, issued May 4, 1999 Enzon, Inc., titled "Method of reducing side effects associated with administration of oxygen-carrying proteins"; U.S. Patent 5,681,811, issued October 28, 1997 Protein Delivery, Inc., titled "Conjugation-stabilized therapeutic agent compositions, delivery and diagnostic formulations comprising same, and method of making and using the same"; U.S. Patent 5,637.749, issued June 10, 1997 Enzon, Inc., titled "Aryl imidate activated polyalkylene oxides"; U.S. Patent 5,612,460, issued March 18, 1997 Enzon, Inc., under the title "Active carbonates ofpolyalkylene oxides for modification of polypeptides"; U.S. Patent 5,567,422, issued October 22, 1996 Enzon, Inc., titled "Azlactone activated polyalkylene oxides conjugated to biologically active nucleophiles"; U.S. Patent 5,405,877, granted

11.04.1995 Enzon, Inc., under the title "Cyclic imide thione activated polyalkylene oxides"; and US Patent 5,359,030, issued on October 25, 1994 Protein Delivery, Inc., titled "Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same," and International

Publication Patent WO / 2004/047871, entitled "Modified naturetic compounds, conjugates, and uses thereof." Additional examples of conjugated polypeptides useful in the mixtures of the present invention can be found in the following US patent applications. U.S. Patent Application 09 / 134.803, filed August 14

1998; U.S. Patent Application 10 / 018,879, filed December 19, 2001; U.S. Patent Application 10 / 235,381, filed September 5, 2002; U.S. Patent Application 10 / 235,284, filed September 5, 2002; and US Patent Application 09 / 873,797, filed June 4, 2001. The present invention specifically contemplates mixtures containing each type and species of therapeutic compound described in the above-mentioned patents and patent applications.

7.4. Preparation of the preparation [0050] The preparations according to the invention are intended to cause the rapid dissolution of fatty acids at the absorption site. With this in mind, the method of preparing the formulations of the present invention typically includes the step of providing fatty acid salts with reduced particle size, as described herein.

[0051] The formulations of the present invention may be processed as a lyophilized powder that contains microparticles of the fatty acid salts of the present invention with buffering components. [0052] The formulations of the present invention may be processed as a spray dried powder that contains microparticles of the fatty acid salts of the present invention, with or without buffering ingredients and other formulation ingredients.

[0053] The formulations of the present invention may be processed in the form of dry granulation or dry powder mix or wet granulation, which contains microparticles of the fatty acid salts of the present invention, with or without buffering ingredients and other formulation ingredients.

[0054] Fatty acid salts having a size range according to the invention can be provided by milling using to reduce particle size any suitable device that is known in the art. These finely powdered fatty acids may form the basis of solid oral dosage forms such as capsules or tablets.

[0055] Active ingredients and other ingredients may be combined with the above mixtures by methods known in the art.

7.5. Assay [0056] The formulations of the invention preferably have a high dissolution rate. The release rate can be determined as follows: Use a USP 2 dissolution apparatus (paddles) at 50 rpm in 500 ml pH 7.4 phosphate buffer.

USP 26, &lt;711&gt; DISSOLUTION Test Description:

[0057] This test is used to determine compliance with the dissolution requirements stated in the individual monograph for the dosage form in tablet or capsule form. From the types of apparatus described therein, use apparatus 2. For hard or soft gelatin capsules and gelatin-coated tablets that do not meet the dissolution specifications, repeat the test as follows. In the case where in an individual monograph water or a carrier with a pH value lower than 6.8 is specified as the medium, the same specified medium can be used with the addition of purified pepsin, which results in activity

750000 units or less per 1000 ml. For media with a pH of 6.8 or higher, pancreatin may be added to obtain no more than 1750 USP units of protease activity per 1000 ml. USP &lt;11&gt; reference standards (dissolution calibrator, disintegration). (Dissolution calibrator, no disintegration) are used to calibrate the device.

[0058] Device 2: The kit consists of the following: a closed tank made of glass or other inert transparent material, engine, metal drive shaft, mixing element. The vessel is partially immersed in a suitable water bath of a convenient size or placed in a heating jacket. The water bath or heating jacket allows the temperature inside the vessel to be maintained within the range of 37 ± 0.5 ° C during the test and keeps the fluid in a continuous, smooth motion. No part of the device, including the environment in which the device has been placed, causes shocks or vibrations, beyond those generated by the smoothly rotating mixing element. A device that allows the sample and mixing element to be observed during the test is advisable. The vessel is cylindrical with a semi-spherical bottom and has the following dimensions and volumes: for a nominal volume of 1 liter, the height is between 160 mm and 210 mm, and the inner diameter is 98 mm to 106 mm. The sides are turned up at the top. A fitted cover can be used to delay evaporation.

[0059] The shaft and mixing element are made of stainless steel, type 316 or equivalent. The shaft is positioned so that its axis at any point is not tilted more than 2mm from the vertical axis of the vessel, rotates gently and without significant vibration. A speed control device is used that allows you to select the rotation speeds of the drive shaft and maintain a certain mixing speed. A blade consisting of a blade and a shaft serves as a mixing element.

[0060] The term "high dissolution rate" as used herein means that during the test as described herein, the fatty acid release rate of the present invention in a buffer at pH 7.4 is high enough to release at least about 25% of the fatty acids to solution for up to ten minutes. In other embodiments, the dissolution rate may be at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% fatty acids in solution for up to ten minutes.

7.6. Methods of Treatment [0061] The invention provides a method of treating a patient in need of such treatment by administering to the patient an effective amount of the mixture of the invention. The preparation may be contained in a pharmaceutically acceptable carrier.

The invention particularly relates to methods of treating osteoporosis or pain (e.g., pain associated with osteoporosis or peripheral pain) in a patient in need of such treatment, comprising the oral administration of an effective amount of a preparation according to the invention containing native calcitonin, unconjugated bioactive calcitonin analog, conjugated calcitonin and / or the following relationship:

Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys (NH-CO (CH2) 7 (OCH2CH2) 7OCH3) Leu Ser Gly Glu Leu His Lys GNH-COfCHjHOCHiC ^ bOCTT) Leu Gin Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro.

where -NH- groups are ε lysine nitrogen.

[0063] For other uses, there are methods for treating diabetes and / or hyperglycemia in a subject in need thereof comprising orally administering to the patient an effective amount of the mix of the present invention containing native insulin, unconjugated bioactive insulin analogue, conjugated insulin and / or

Insuline B29 Lys- (NH-CO (CH2) 5 (OCH2CH2) 7OCH3) where the -NH- groups are nitrogen ε of the Lys residue; or insulin conjugated to a modifying residue having a structure selected from the following:

<img file="PL1660047T3_D0002.tif" />

[0064] Each of the above groups may, for example, be conjugated to human insulin at nucleophilic residues, e.g., A1, B1 or B29, or any combination of the above.

8. Examples [0065] These techniques have been applied to various native polypeptides and chemically modified polypeptides.

8.1. Preparation of calcitonin and CT-025 (modified calcitonin) preparations

8.1.1. Grinding of sodium caprate and sodium laurate [0066] Salmon calcitonin and CT-025 preparations (see Fig. 2) were prepared as follows:

<td>Additional substance</td><td>% w / w</td>

<td>Sodium caprate</td><td> 53.4%</td>

<td>Sodium Laurate</td><td> 46.6%</td>

1. Mix together sodium caprate and sodium laurate (preferably in a 1: 1 molar ratio).

2. Pass through a milling jet mill until fine particle size is achieved. Fine particle size is a particle size that is less than or is in the range 50-100 microns.

3. The resulting material is a premix for further processing

8.1.2. Preparation A [0067]

<td>Additional substance</td><td>% w / w</td><td>mg / tablet</td>

<td>CT-025 (See Fig. 2)</td><td> 036</td><td> .43</td>

<td>Sodium caprate / sodium laurate premix</td><td> 50</td><td> 450</td>

<td>mannitol</td><td> 34.5</td><td> 310.5</td>

<td>sorbitol</td><td> 10</td><td> 90</td>

<td>Croscarmellose sodium</td><td> 5</td><td> 45</td>

<td>Sodium stearyl fumarate, NF</td><td> .5</td><td> 4.5</td>

<td>SUM</td><td> 100</td><td> 900.43</td>

Preparation preparation [0068]

1. CT-025 (See Fig. 2) was removed from the freezer and allowed to thaw for 1 hour at room conditions and then equilibrated in an open container for an additional 2 hours.

2. Prepare a 1% acetic acid solution: pipette 2 mL of glacial acetic acid to 200 mL of the required amount and make up to volume with purified water, then shake well.

3. Weigh CT-025 and transfer to a 100 ml Pyrex bottle. Add 45 ml of 5% acetic acid solution and mix to dissolve. It is a granulation solution.

4. Mix the premix of sodium caprate / sodium laurate with all mannitol, sorbitol and croscarmellose sodium in a Key granulator with a 5 L bowl.

5. Start the granulator and set the rotor and chipper to the appropriate speed to obtain proper mixing.

6. Add the granulation solution from step 2. Rinse the solution container with 1% acetic acid in 10 ml aliquots to a total of 60-65 mL of granulation fluid.

7. Granulate for about 20 minutes.

8. Transfer the particles to a tray lined with aluminum foil.

9. Dry the particles in the oven at 37 ° C for 16-24 hours.

The target moisture content is &lt;3%.

10.1 Pass the dried particles through a # 12 screen.

11. Add sodium stearyl fumarate and particles to an 8-quarter-type V mixer. Mix for 2 minutes and then unload.

12. Using a suitable tablet press with the right equipment to prepare a 900 mg capsule-shaped tablet, compact the granules into tablets.

8.1.3. Preparation B [0069]

<td>Additional substance</td><td>% w / w</td><td>mg / tablet</td>

<td>CT-025 (See Fig. 2)</td><td> .048</td><td> .430</td>

<td>Sodium caprate / sodium laurate premix</td><td> 32.94</td><td> 221.17</td>

<td>mannitol</td><td> 46.27</td><td> 310.67</td>

<td>Additional substance</td><td>% w / w</td><td>mg / tablet</td>

<td>sorbitol</td><td> 13.41</td><td> 90.04</td>

<td>Croscarmellose sodium</td><td> 6.71</td><td> 45.05</td>

<td>Sodium stearyl fumarate, NF</td><td> .67</td><td> 4.5</td>

<td>SUM</td><td> 100</td><td> 671.86</td>

[0070] Preparation of the preparation was done as above.

8.1.4. Preparation C [0071]

<td>Additional substance</td><td>% w / w</td><td>mg / tablet</td>

<td>CT-025 (See Fig. 2)</td><td> .036</td><td> .43</td>

<td>Sodium caprate / sodium laurate premix</td><td> 49.55</td><td> 442.0</td>

<td>mannitol</td><td> 34.81</td><td> 310.5</td>

<td>sorbitol</td><td> 10.09</td><td> 90.0</td>

<td>Croscarmellose sodium</td><td> 5.04</td><td> 45.0</td>

<td>Sodium stearyl fumarate, NF</td><td> .5</td><td> 4.5</td>

<td>SUM</td><td> 100</td><td> 892.43</td>

[0072] Preparation of the preparation was carried out as above.

8.1.5. Preparation D [0073]

<td>Additional substance</td><td>% w / w</td><td>mg / tablet</td>

<td>Salmon calcitonin</td><td> .036</td><td> 0.37</td>

<td>Sodium caprate / sodium laurate premix</td><td> 49.55</td><td> 442.0</td>

<td>mannitol</td><td> 34.81</td><td> 310.5</td>

<td>sorbitol</td><td> 10.09</td><td> 90.0</td>

<td>Additional substance</td><td>% w / w</td><td>mg / tablet</td>

<td>Croscarmellose sodium</td><td> 5.04</td><td> 45.0</td>

<td>Sodium stearyl fumarate, NF</td><td> .50</td><td> 4.5</td>

<td>SUM</td><td> 100</td><td> 892.37</td>

[0074] Salmon calcitonin was removed from the freezer and allowed to thaw for 1 hour at room conditions, then equilibrated in an open container for an additional 2 hours, and preparation was done as above.

8.1.6. Preparation E (capsule) [0075]

<td>Additional substance</td><td>% w / w</td><td>mg / tablet</td>

<td>CT-025 (SEE FIG. 2)</td><td> 0</td><td> 0</td>

<td>Sodium caprate</td><td> 23.1</td><td> 155.07</td>

<td>Sodium Laurate</td><td> 26.5</td><td> 3.95</td>

<td>mannitol</td><td> 34.8</td><td> 5.18</td>

<td>sorbitol</td><td> 10.1</td><td> 1.50</td>

<td>Croscarmellose sodium</td><td> 5.0</td><td> 0.74</td>

<td>Sodium stearyl fumarate, NF</td><td> 0.5</td><td> 0.07</td>

<td>SUM</td><td> 100</td><td> 671.3</td>

[0076] Preparation of the preparation

1. 1. Mix together sodium caprate and sodium laurate. Pass 10 through a grinding jet mill.

2. 2. Add the premix from step 1 to all mannitol, sorbitol, croscarmellose sodium and sodium stearyl fumarate, then mix.

3. 3. Place 671.3 mg of the mixture in a hard gelatin capsule size 15 000.

8.1.7. Formulation F (capsule) unground [0077]

<td>Additional substance</td><td>% w / w</td><td>mg / tablet</td>

<td>CT-025 (SEE FIG. 2)</td><td> 0</td><td> 0</td>

<td>Sodium caprate</td><td> 23.1</td><td> 155.07</td>

<td>Sodium laurate premix</td><td> 26.5</td><td> 3.95</td>

<td>mannitol</td><td> 34.8</td><td> 5.18</td>

<td>sorbitol</td><td> 10.1</td><td> 1.50</td>

<td>Croscarmellose sodium</td><td> 5.0</td><td> 0.74</td>

<td>Sodium stearyl fumarate, NF</td><td> 5.0</td><td> 0.07</td>

<td>SUM</td><td> 100</td><td> 671.3</td>

[0078] Preparation of the preparation

1. Mix together sodium caprate and sodium laurate. (Do not grind).

2. Add the premix from step 1 to all mannitol, sorbitol, croscarmellose sodium and sodium stearyl fumarate, then mix.

3. Place 671.3 mg of the mixture in a hard gelatin capsule size

000.

8.2. Investigation of Calcitonin Formulations [0079] Tablet and capsule formulations were prepared using the formulas and processing steps described above. Figures 3-8 show dissolution results showing that sodium capric acid and sodium lauric acid are easily released from various tablet formulations in which fatty acids are ground to optimal particle sizes.

[0080] Figure 3 shows the dissolution of sodium caprate released from three CT-025 tablet samples (see Fig. 2) of formula A.

[0081] Figure 4 shows the dissolution of sodium laurate released from three CT-025 tablet samples of formula A.

[0082] Figure 5 shows the dissolution of sodium caprate released from three CT-025 Formula B tablet samples.

[0083] Figure 6 shows the dissolution of sodium laurate released from three samples of Formula B tablets.

[0084] Figure 7 shows the dissolution of sodium caprate released from three CT-025 Formula C tablet samples.

[0085] Figure 8 shows the dissolution of sodium laurate released from three CT-025 Tablet C formula samples.

[0086] Encapsulated formulations were prepared from ground fatty acids (Formula E, Figures 9 and 10) and unground ground fatty acids (Formula F, Figures 11 and 12). The dissolution results are clearly slower when the fatty acids are not ground. Figure 9 shows the dissolution of sodium caprate released from three placebo capsule samples in which the fatty acids were ground. Figure 10 shows the solubility of sodium laurate released from three CT-025 capsule samples in which the fatty acids were not ground.

[0087] Figure 11 shows the dissolution of sodium caprate released from six placebo capsule samples.

[0088] Figure 12 shows the dissolution of sodium laurate released from six CT-025 capsule samples.

[0089] One tablet containing CT-025 (320 μg calcitonin equivalent per tablet) (Figure 13, 371 = Formula B; 372 = Formula C) or salmon calcitonin (320 μg calcitonin equivalent per tablet) (Figure 14, 373 = Formula D ) with ground fatty acids was given to fasting beagle dogs. The dogs were starved overnight and dosing took place in the morning. Blood was collected from the jugular vein after 15, 30, 60, 90 and 120 minutes. Blood sample analyzes were performed to calculate the plasma concentration of either CT-025 or salmon calcitonin in equivalents of salmon calcitonin.

[0090] Figure 13 shows plasma plasma CT-025 levels after oral administration to fasted beagle dogs of one tablet containing processed fatty acids and 430 μg CT-025 (equivalent to 320 μg salmon calcitonin).

[0091] Figure 14 shows salmon plasma calcitonin levels after oral administration to fasting beagle dogs of one tablet containing processed fatty acids and 320 μg salmon calcitonin.

[0092] The invention has been described with reference to its preferred embodiments described herein. The invention may be carried out in various forms and should not be construed as limited to the specific embodiments set forth herein. Rather, these embodiments are presented to make this disclosure accurate and complete and to fully convey the scope of the invention to those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention.

9. References [0093] AT Florence, The oral absorption of micro- and nanoparticulates: neither exceptional nor unusual, Pharm. Res., 14 (1997), 259-266.

[0094] Ermak et al, Uptake and transport of copolymer biodegradable microspheres by rabbit Peyer's patch M cells, Cell Tissue Res., 279 (1995),

433-436.

[0095] Sakuma et al, Design of nanoparticles composed of graft copolymers for oral peptide delivery, Adv. Drug Del. Rev., 47 (2001), 21-37.