EA044859B1 - PHARMACEUTICAL COMPOSITION FOR PEPTIDE DELIVERY - Google Patents

Description

Field of technology to which the invention relates

The present invention generally relates to the field of pharmaceuticals. In particular, the present invention relates to a pharmaceutical composition comprising a peptide in combination with a metal salt/complex and a reducing agent to provide protection, at least in part, of the peptide from proteolytic degradation when ingested.

Prerequisites for creating the invention

The description of the background to the invention includes information that may be useful in understanding the present invention. It is not an admission that any information provided herein constitutes prior art or relates to the invention currently claimed, or that any publication referred to directly or indirectly constitutes prior art.

Proteins and polypeptides are used as therapeutic agent(s), diagnostic agent(s), and the like. since ancient times, and even today their number is growing rapidly. However, their full potential has not yet been realized as their use is limited to parenteral administration only.

The oral route is a simple, convenient and most preferred route of administration of the therapeutic agent. However, degradation of peptides in the gastrointestinal tract prevents their absorption in an intact form. Thus, enzymatic degradation in the gastrointestinal tract and poor permeability through epithelial cells are the main reasons for their low oral bioavailability.

Previously, various approaches have been proposed for some time to improve the oral bioavailability of such proteins and polypeptides, such as the use of a large number of absorption enhancers and protease inhibitors, such as soy trypsin inhibitor, aprotinin, Bowman-Birk inhibitor, bacitracin, camostat mesylate and amastatin (Renukuntla J .et al., Int. J. Pharm., 2013, 447, 75-93; and application US 20070087957 A1). However, none of these protease inhibitors have succeeded as additives used in the delivery of commercially produced polypeptide drugs because they are toxic and may exhibit some side effects.

Here are some examples of protease inhibitors used for peptide delivery.

a) Soy (trypsin inhibitor) is one of the widespread allergens, and the number of people suffering from soy has been steadily increasing since the 1980s, which limits its use (Moroz L.A. et al., N. Engl. J. Med., 1980, 302, 1126-8; Focard T. et al., Allergy, 1999, 54, 261-5; Ramesh S., din Rev. Allergy Immunol., 2008, 34, 217-30). It causes immediate allergic reactions such as coughing, sneezing, runny nose, hives, diarrhea, facial swelling, shortness of breath, tongue swelling, difficulty swallowing, low blood pressure, excessive sweating, fainting, anaphylactic shock and even death.

b) Bowman (Birka inhibitor) is a soy derivative with high oral bioavailability even in the absence of an absorption enhancer, however it is reported to have undesirable systemic protease inhibition (systemic inhibition of serine proteases such as plasmin, which may increase the risk of thrombosis) after oral administration reception. In addition, Bowman can also lead to the formation of antibodies against itself (Wang KC et al., Nutr. Cancer, 2002, 43, 167-73).

c) Aprotinin is known to cause anaphylaxis at a rate of 1:200 upon initial use (Mahdi A.M. et al., 2004, 93, 842-58), and is also reported to be associated with a risk of acute renal failure, myocardial infarction, cardiac failure, stroke and encephalopathy in a patient with heart disease/surgery (Mangano D.T. et al., N. Engl. J. Med., 2006, 354, 353-65).

Because these protease inhibitors are associated with potential health risks, it is widely believed that the use of these protease inhibitors should be avoided. In addition to these limitations, they are associated with high production costs, heterogeneity, regulatory barriers, difficulties in achieving selective inhibition, and the need for high doses for effective activity (Renukuntla J. et al., Int. J. Pharm., 2013, 447 ), making their use unviable. Other protease inhibitors such as bacitracin (antibiotic activity), camostat mesylate (effective in the treatment of pancreatitis) or amastatin (antibacterial activity) are also associated with similar side effects (Renukuntla J. et al., Int. J. Pharm., 2013, 447, 75-93; and US Application Publication 20070087957 A1). European patent EP 3006045 B1 discloses a combination of trace elements such as copper or zinc with a pharmaceutically acceptable reducing agent, optionally in combination with a mucosal absorption enhancer, which results in a surprisingly high and beneficial oral bioavailability of various peptide or protein preparations. However, copper and zinc are associated with many metabolic pathways in mammals, and therefore their use in long-term therapy may lead to negative interactions.

Thus, there is a need to develop simple, safe, effective and cost-effective pharmaceutical compositions that can deliver peptide while protecting at least part of the peptides from proteolytic degradation when ingested. The present invention satisfies existing and other needs and eliminates the disadvantages of traditional

- 1 044859 tion pharmaceutical compositions and delivery methods.

Objectives of the invention

One object of the present invention is to provide a pharmaceutical composition that can overcome the disadvantages associated with existing prior art compositions.

Another object of the present invention is to provide a pharmaceutical composition for effective delivery of a peptide.

Another object of the present invention is to provide a pharmaceutical composition for oral delivery of a peptide.

Another object of the present invention is to provide a pharmaceutical composition that provides protection, at least in part, of an ingestible peptide from proteolytic degradation.

Another object of the present invention is to provide a pharmaceutical composition that increases the oral bioavailability of the peptide.

Another object of the present invention is to provide a pharmaceutical composition that is safe.

Another object of the present invention is to provide a pharmaceutical composition that is economical to manufacture.

Another object of the present invention is to provide a pharmaceutical composition that is easy to prepare.

Another object of the present invention is to provide a pharmaceutical composition that has a long shelf life.

Summary of the invention

The present invention generally relates to the field of pharmaceuticals. In particular, the present invention relates to a pharmaceutical composition comprising a peptide in combination with a metal salt/complex and a reducing agent to provide protection, at least in part, of the peptide from proteolytic degradation when ingested.

In one aspect of the present invention, there is provided a pharmaceutical composition comprising a pharmaceutically effective amount of at least one peptide; and a pharmaceutically acceptable amount of the combination of (a) at least one metal in the form of any salt thereof or a combination of a salt and a complex thereof; and (b) at least one reducing agent, wherein the at least one metal is selected from any one or combination of vanadium, chromium and manganese, and wherein the combination of (a) the at least one metal in the form of any salt or combination thereof salt and complex and (b) at least one reducing agent protects, at least in part, the at least one peptide from proteolytic degradation when ingested.

In one embodiment of the present invention, at least one metal is vanadium, wherein the pharmaceutical composition includes any vanadium salt or combination of a vanadium salt and a vanadium complex in an amount of from about 0.01 mg to about 15 mg per dosage unit. In one embodiment of the present invention, any of the vanadium salts and vanadium complex are selected separately from the group consisting of vanadium(V) oxide, sodium vanadate, vanadium sulfate, vanadyl sulfate, vanadium biguanide, bis(maltolato)oxavandium(IV), vanadium acetate, picolinate vanadyl and vanadyl citrate. In one embodiment of the present invention, at least one metal is chromium, wherein the pharmaceutical composition includes any chromium salt or combination of chromium salt and chromium complex in an amount of from about 0.02 mg to about 0.5 mg per dosage unit. In one embodiment of the present invention, any of the chromium salts and chromium complex are selected separately from the group consisting of chromium picolinate, chromium polynicotinate, chromium nicotinate, chromium chloride and chromium acetate. In one embodiment of the present invention, at least one metal is manganese, wherein the pharmaceutical composition includes any manganese salt or combination of a manganese salt and a manganese complex in an amount of from about 0.1 mg to about 10 mg per dosage unit. In one embodiment of the present invention, any of the manganese salts and the manganese complex are selected separately from the group consisting of manganese gluconate, manganese sulfate, potassium permanganate and manganese chloride.

In one embodiment of the present invention, at least one peptide has a molecular weight equal to or less than 60 kDa. In one embodiment of the present invention, at least one peptide is selected from the group consisting of insulin, insulin analog, insulin lispro, insulin PEGlispro, insulin aspart, insulin glulisine, insulin glargine, insulin detemir, NPH insulin, insulin degludec, B29K(N(ε )hexadecanedioyl-γ-L-GLu) A14E B25H des-B30 human insulin, B29K(N(ε)octadecanedioyl-γ-L-GLu-OEG-OEG) des-B30 human insulin, B29K(N(ε)octadecanedioyl-γ -L-GLu) A14E B25H des-B30 human insulin, B29K(N(ε)eicosandioyl

- 2 044859 γ-L-GLu) A14E B25H des-B30 human insulin, B29K(N(ε)octadecanedioyl-γ-L-GLu-OEG-OEG) A14E B25H des-B30 human insulin, B29K(N(ε)eicosanedioyl -γ-L-GLu-OEG-OEG) A14E B25H desB30 human insulin, B29K(N(e)eicosandioyl-γ-L-Glu-OEG-OEG) A14E B16H B25H des-B30 human insulin, B29K(N(ε) hexadecanedioyl-γ-L-GLu) A14E B16H B25H des-B30 human insulin, B29K(N(e)eicosandioyl-γ-L-Glu-OEG-OEG) A14E B16H B25H des-B30 human insulin, B29K(N(ε) octadecanedioyl) A14E B25H des-B30 human insulin, GLP-1, GLP-1 analog, acylated GLP-1 analog, diacylated GLP-1 analog, semaglutide, liraglutide, exenatide, lixisenatide, dual GLP-1 receptor and glucagon receptor agonist, amylin , amylin analog, pramlintide, somatostatin analog, octreotide, lanreotide, pasireotide, goserelin, buserelin, leptin, leptin analog, metreleptin, peptide YY, peptide YY analog, glatiramer, leuprolide, teriparatide, abaloparatide, tetracosactide, corticorelin, etelcalcetide , elcatonin, desmopressin , human growth hormone, human growth hormone analogue, glycopeptide antibiotic, glycosylated cyclic or polycyclic non-ribosomal peptide antibiotic, vancomycin, teicoplanin, telavancin, bleomycin, ramoplanin, decaplanin, bortezomib, cosyntropin, human chorionic gonadotropin, menotropin, sermorelin, luteinizing hormone-releasing hormone , somatropin, calcitonin, salmon calcitonin, pentagastrin, oxytocin, neseritide, anakinra, enfuvirtide, pegvisomant, dornase alfa, lepirudin, anidulafungin, eptifibatide, interferon alfacon-1, interferon alfa-2a, interferon alfa2b, interferon beta-1a, interferon beta- 1b, interferon gamma-1b, peginterferon alpha-2a, peginterferon alpha-2b, peginterferon beta-1a, fibrinolysin, vasopressin, aldesleukin, epoetin-alpha, darbepoetin-alpha, epoetin-beta, epoetin-delta, epoetin omega, epoetin zeta, filgrastim, interleukin-11, cyclosporine, glucagon, urokinase, biomycin, thyrotropin-releasing hormone, leucine-enkephalin, methionine-enkephalin, substance P, adrenocorticotropic hormone, parathyroid hormone and pharmaceutically acceptable salts thereof.

In one embodiment of the present invention, at least one peptide and at least one metal, in the form of any salt or combination of a salt thereof, and a complex thereof are present in a physically separated form in the pharmaceutical composition. In one embodiment of the present invention, the at least one peptide and the at least one metal, any salt or combination of a salt thereof, and a complex thereof are present in a physically separated form in separate compartments. In one embodiment of the present invention, the pharmaceutical composition is present in the form of any of the following: capsule-in-capsule and tablet-in-capsule.

In one embodiment of the present invention, at least one reducing agent is selected from any of the following or a combination of ascorbic acid, reduced glutathione, cysteine, uric acid, reducing sugar, glyceraldehyde, α-tocopherol, vitamin A, alipoic acid, dihydro-α-lipoic acid , glucose, galactose, lactose, maltose, thiol-containing compound, thiomer and pharmaceutically acceptable salts thereof. In one embodiment of the present invention, the pharmaceutical composition includes at least one reducing agent in an amount of from about 1 mg to about 1000 mg per dosage unit.

In one embodiment of the present invention, the pharmaceutical composition further includes at least one absorption or permeation enhancer, wherein the at least one absorption or permeation enhancer is present in an amount of from about 10 mg to about 1000 mg per dosage unit. In one embodiment of the present invention, the pharmaceutical composition is formulated in any solid oral dosage form and a liquid oral dosage form, provided that when the pharmaceutical composition is formulated in a liquid oral dosage form, the pharmaceutical composition includes water in an amount of less than about 5% by volume.

Various objects, features, aspects and advantages of the subject matter will become more apparent from the following detailed description of preferred embodiments taken together with the accompanying drawings, in which like numerals represent like components.

Brief description of drawings

Illustrated in the drawing is a graph depicting the concentration-time profile of insulin glargine (mU/L) from various formulations, in accordance with one embodiment of the present invention.

Detailed Description of Embodiments of the Invention

The following is a detailed description of embodiments of the present invention shown in the accompanying drawings. Embodiments of the present invention are presented in sufficient detail to enable the present invention to be fully disclosed. However, the proposed scope of information is not intended to limit possible variations in the embodiments of the present invention; rather, it is intended to cover all modifications, equivalents and alternatives consistent with the spirit and scope of the present invention as defined by the appended claims.

Each of the appended claims defines a separate invention that

- 3 044859 for the purposes of the offense is deemed to include equivalents of the various elements or limitations specified in the claims. Depending on the context, all of the following references to the present invention may, in some cases, only refer to certain specific embodiments of the present invention. In other cases, it will be recognized that references to the present invention will refer to the subject matter set forth in at least one, but not necessarily all, claims.

As used in the description herein and in the claims themselves, the singular content includes a reference to the plural unless the context clearly indicates otherwise.

All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly inconsistent with the context. The use of any and all examples or introductory words before an example (eg, such) provided herein with respect to certain embodiments of the present invention is intended only to better highlight the present invention and does not limit the scope of the present invention as otherwise claimed. No language used in this specification should be construed as indicating any non-claimed element that is essential to the practice of the present invention. The various terms used in this document are shown below. To the extent that a term used in a claim is not defined below, it shall be given the broadest definition that persons in the art have given that term as reflected in printed publications and issued patents at the time of filing of this application .

The present invention generally relates to the field of pharmaceuticals. In particular, the present invention relates to a pharmaceutical composition comprising a peptide in combination with a metal salt/complex and a reducing agent to provide protection, at least in part, of the peptide from proteolytic degradation when ingested. Serine proteases are ubiquitous in eukaryotes and cleave peptide bonds in which the main catalytic triad is serine, histidine and aspartic acid. Serine proteases mentioned in the present invention include trypsin, chymotrypsin, carboxypeptidase B and aminopeptidase M, which are responsible for the physiological functions of the body, in particular for degeneration (proteolytic degradation), i.e. for the hydrolysis of peptide bonds and amino acids. The present invention is directed to providing pharmaceutical composition(s) comprising a peptide in combination with a metal salt/complex and a reducing agent to provide protection, at least in part, of the peptide from proteolytic degradation when ingested.

Accordingly, in one aspect of the present invention there is provided a pharmaceutical composition comprising a pharmaceutically effective amount of at least one peptide; and a pharmaceutically acceptable amount of the combination of (a) at least one metal in the form of any salt thereof or a combination of a salt and a complex thereof; and (b) at least one reducing agent, wherein at least one metal is selected from any one or combination thereof of vanadium, chromium and manganese, and wherein the combination of (a) at least one metal in the form of any salt or combination thereof salt and complex and (b) at least one reducing agent provides protection, at least in part, of at least one peptide from proteolytic degradation when ingested.

In one embodiment of the present invention, at least one metal is vanadium, wherein the pharmaceutical composition includes any vanadium salt or combination of a vanadium salt and a vanadium complex in an amount of from about 0.01 mg to about 5 mg per dosage unit. In one embodiment of the present invention, any of the vanadium salts and vanadium complex are selected separately from the group consisting of vanadium(V) oxide, sodium vanadate, vanadium sulfate, vanadyl sulfate, vanadium biguanide, bis(maltolato)oxavandium(IV), vanadium acetate, picolinate vanadyl and vanadyl citrate. In one embodiment of the present invention, at least one metal is chromium, wherein the pharmaceutical composition includes any chromium salt or combination of chromium salt and chromium complex in an amount of from about 0.02 mg to about 0.5 mg per dosage unit. In one embodiment of the present invention, any of the chromium salts and chromium complex are selected separately from the group consisting of chromium picolinate, chromium polynicotinate, chromium nicotinate, chromium chloride and chromium acetate. In one embodiment of the present invention, at least one metal is manganese, wherein the pharmaceutical composition includes any manganese salt or combination of a manganese salt and a manganese complex in an amount of from about 0.1 mg to about 10 mg per dosage unit. In one embodiment of the present invention, any of the manganese salts and manganese complex are selected separately from the group consisting of manganese gluconate, manganese sulfate, potassium permanganate and manganese chloride.

In one embodiment of the present invention, at least one peptide has

- 4 044859 molecular weight equal to or less than 60 kDa. In one embodiment of the present invention, at least one peptide is selected from the group consisting of insulin, insulin analog, insulin lispro, insulin PEGlispro, insulin aspart, insulin glulisine, insulin glargine, insulin detemir, NPH insulin, insulin degludec, B29K(N(ε )hexadecanedioyl-γ-L-GLu) A14E B25H des-B30 human insulin, B29K(N(ε)octadecanedioyl-γ-L-GLu-OEG-OEG) des-B30 human insulin, B29K(N(ε)octadecanedioyl-γ -L-GLu) A14E B25H des-B30 human insulin, B29K(N(ε)eicosandioylγ-L-GLu) A14E B25H des-B30 human insulin, B29K(N(ε)octadecanedioyl-γ-L-GLu-OEG-OEG ) A14E B25H des-B30 human insulin, B29K(N(ε)eicosanedioyl-γ-L-GLu-OEG-OEG) A14E B25H desB30 human insulin, B29K(N(ε)eicosanedioyl-γ-L-Glu-OEG-OEG ) A14E B16H B25H des-B30 human insulin, B29K(N(ε)hexadecanedioyl-γ-L-GLu) A14E B16H B25H des-B30 human insulin, B29K(N(ε)eicosanedioyl-γ-L-Glu-OEG-OEG ) A14E B16H B25H des-B30 human insulin, B29K(N(ε)octadecanedioyl) A14E B25H des-B30 human insulin, GLP-1, GLP-1 analog, acylated GLP-1 analog, diacylated GLP-1 analog, semaglutide, liraglutide , exenatide, lixisenatide, dual GLP-1 receptor and glucagon receptor agonist, amylin, amylin analog, pramlintide, somatostatin analog, octreotide, lanreotide, pasireotide, goserelin, buserelin, leptin, leptin analog, metreleptin, peptide YY, peptide YY analog, glatiramer , leiprolid, teripipidide, abalparatide, tetracosactide, corticorelin, eelcalcetide, elkatonin, desmopressin, human growth hormone, an analogue of human growth hormone, glycopeptic antibiotic, glycosylated cyclic or polycicclic non -commercial peptide antibiotic, vancromezinin, teplainninin, bodybuilding , bleomycin, ramoplanin, Decoplanin, Bortesomib , cosyntropin, human chorionic gonadotropin, menotropin, sermorelin, luteinizing hormone-releasing hormone, somatropin, calcitonin, salmon calcitonin, pentagastrin, oxytocin, neseritide, anakinra, enfuvirtide, pegvisomant, dornase alfa, lepirudin, anidulafungin, eptifibatide, interferon alfacon-1, interferon alpha-2a, interferon alpha-2b, interferon beta-1a, interferon beta-1b, interferon gamma-1b, peginterferon alpha-2a, peginterferon alpha-2b, peginterferon beta-1a, fibrinolysin, vasopressin, aldesleukin, epoetin-alpha, darbepoetin alfa, epoetin beta, epoetin delta, epoetin omega, epoetin zeta, filgrastim, interleukin-11, cyclosporine, glucagon, urokinase, biomycin, thyrotropin releasing hormone, leucine enkephalin, methionine enkephalin, substance P, adrenocorticotropic hormone, parathyre oid hormone and pharmaceutically acceptable salts thereof.

In one embodiment of the present invention, at least one peptide and at least one metal, in the form of any salt thereof or a combination of a salt and a complex thereof, are present in a physically separated form in the pharmaceutical composition. In one embodiment of the present invention, at least one peptide and at least one metal, in the form of any salt thereof or combination of a salt and a complex thereof, are present in physically separated form in separate compartments. In one embodiment of the present invention, the pharmaceutical composition is present in the form of any of the following: capsule-in-capsule and tablet-in-capsule.

In one embodiment of the present invention, at least one reducing agent is selected from any of the following or a combination of ascorbic acid, reduced glutathione, cysteine, uric acid, reducing sugar, glyceraldehyde, α-tocopherol, vitamin A, α-lipoic acid, dihydro-a- lipoic acid, glucose, galactose, lactose, maltose, thiol-containing compound, thiomer and pharmaceutically acceptable salts thereof. In one embodiment of the present invention, the pharmaceutical composition includes at least one reducing agent in an amount of from about 1 mg to about 1000 mg per dosage unit.

In one embodiment of the present invention, the pharmaceutical composition further includes at least one absorption or permeation enhancer, wherein the at least one absorption or permeation enhancer is present in an amount of from about 10 mg to about 1000 mg per dosage unit. In one embodiment of the present invention, the pharmaceutical composition is formulated in any solid oral dosage form and a liquid oral dosage form, provided that when the pharmaceutical composition is formulated in a liquid oral dosage form, the pharmaceutical composition includes water in an amount of less than about 5% by volume.

In one embodiment of the present invention, the peptide is any peptide or protein that is suitable for use as a therapeutic or diagnostic agent. In one embodiment of the present invention, the peptide is a linear peptide or a cyclic peptide. In one embodiment of the present invention, the peptide is a modified or derivatized peptide, such as a PEGylated peptide, or an acylated fatty acid peptide, or an acylated dibasic fatty acid peptide, or the like.

The peptides may be free of histidine residues and/or free of cysteine residues. It is generally preferred that the peptide be water soluble, especially at neutral pH (ie, about pH 7) and have at least one serine protease cleavage site, i.e. pep

- 5 044859 tide contains at least one amino acid residue susceptible or susceptible to cleavage by a serine protease (in particular, an intestinal serine protease such as trypsin, chymotrypsin, aminopeptidase, carboxypeptidase, elastase and/or dipeptidyl-4-peptidase), etc. P.

In one embodiment of the present invention, the peptide is selected from any of the following or a combination of insulin (in a preferred embodiment of the present invention, human insulin), an insulin analog, such as, but not limited to, a long-acting basal insulin analog, a protease-stabilized long-acting basal insulin analog , insulin lispro, insulin PEGlispro, insulin derivatives such as, A14E, B25H, B29K(N(ε)octadecanedioyl-gClu-OEG-OEG), des-B30 human insulin, insulin aspart, insulin glulisine, insulin glargine, insulin detemir, insulin NPH, insulin degludec, and insulin analogs/derivatives described in US Application No. 20140056953 A1, GLP-1, GLP-1 analog (acylated GLP-1 analog or diacylated GLP-1 analog), semaglutide, liraglutide, exenatide, lixisenatide, dual GLP-1 receptor and glucagon receptor agonist, amylin, amylin analog, pramlintide, somatostatin analog (octreotide, lanreotide or pasireotide), goserelin (goserelin acetate), buserelin, leptin, leptin analog (metreleptin), peptide YY (PYY), analog PYY peptide, glatiramer (glatiramer acetate), leuprolide, teriparatide, abaloparatide, tetracosactide, corticorelin, etelcalcetide, elcatonin, desmopressin, human growth hormone (hGH), human growth hormone analogue, glycopeptide antibiotic (glycosylated cyclic or polycyclic non-ribosomal peptide such as, vancomycin, teicoplanin, telavancin, bleomycin, ramoplanin or decaplanin), bortezomib, cosyntropin, human chorionic gonadotropin, menotropin, sermorelin, luteinizing hormone-releasing hormone (LHRH, also called gonadotropin-releasing hormone), somatropin, calcitonin (salmon calcitonin), pentagastrin, oxytocin, neseritide, anakinra, enfuvirtide, pegvisomant, dornase alfa, lepirudin, anidulafungin, eptifibatide, interferon alfacon-1, interferon alfa-2a, interferon alfa-2b, interferon beta-1a, interferon beta-1b, interferon gamma-1b , peginterferon alfa-2a (pegylated interferon alpha-2a), peginterferon alfa-2b (pegylated interferon alpha-2b), peginterferon beta-1a (pegylated interferon beta-1a), fibrinolysin, vasopressin, aldesleukin, epoetin alfa, darbepoetin alfa, epoetin beta, epoetin delta, epoetin omega, epoetin zeta, filgrastim, interleukin-11, cyclosporine, glucagon, urokinase, viomycin, thyrotropin-releasing hormone (TRH), leucine-enkephalin, methionine-enkephalin, substance P (CAS No. 33507-63- 0), adrenocorticotropic hormone (ACTH), parathyroid hormone (PTH) and their pharmaceutically acceptable salts. However, any other peptide molecule known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention.

In one embodiment of the present invention, for a human subject, the peptide is selected from any peptide or combination of an endogenous peptide, such as insulin or glucagon and the like. In a preferred embodiment of the present invention, an isoform of the corresponding human peptide is used that is recombinantly expressed or chemically synthesized. However, any other human peptide isoform known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention. In one embodiment of the present invention, the peptide is an insulin analogue. In one embodiment of the present invention, the insulin analog is selected from any of the following or a combination of insulin detemir, insulin glargine, insulin degludec and other insulin analogs obtained from human, porcine, fish. However, any other insulin analog/derivative known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention. In one embodiment of the present invention, a mixture of at least two peptides can be used. In one embodiment of the present invention, a mixture of human insulin and a GLP-1 agonist (eg, liraglutide, semaglutide, exenatide, or lixisenatide) is used. However, a mixture of any at least two peptides (including the peptides discussed above) known or understood by one skilled in the art can be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention. In one embodiment of the present invention, at least one peptide has a molecular weight equal to or less than 60 kDa. In one embodiment of the present invention, at least one peptide has a molecular weight equal to or less than 40 kDa. In one embodiment of the present invention, at least one peptide has a molecular weight equal to or less than 30 kDa. In one embodiment of the present invention, at least one peptide has a molecular weight equal to or less than 20 kDa. In one embodiment of the present invention, at least one peptide has a molecular weight equal to or less than 10 kDa. In one embodiment of the present invention, the at least one peptide has a molecular weight ranging from about equal to

- 6 044859 or greater than 300 Da to approximately equal to or less than 50 kDa. However, a peptide of any molecular weight range known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention.

In one embodiment of the present invention, the molecular weight of at least one peptide can be determined by any method, such as mass spectrometry (electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). MS)), gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)), hydrodynamic methods (gel filtration chromatography or gradient sedimentation) or static light scattering (for example, multi-angle light scattering (MALS)), known or understood by one skilled in the art in the art to achieve its desired purpose as set forth in the present invention, without departing from the scope and spirit of the present invention.

In one embodiment of the present invention, at least one metal is vanadium, wherein the pharmaceutical composition includes any vanadium salt or a combination of a vanadium salt and a vanadium complex. In one embodiment of the present invention, any vanadium salt or combination of vanadium salt and vanadium complex is selected separately from the group consisting of vanadium(IV), vanadium(V) and vanadium salt complex (V02) and vanadium anion in the salt/complex vanadata. In one embodiment of the present invention, the vanadium salt and vanadium complexes are selected from any of the following or a combination of vanadium(V) oxide, vanadium pentoxide, vanadium dioxide, sodium vanadate, vanadium sulfate, vanadyl sulfate, sodium metavanadate, vanadium tetrachloride, vanadium(V) oxychloride , vanadium trichloride, vanadyl chloride, vanadium trichloroxo, ammonium vanadate, vanadium ammonium oxide, vanadium monosulfide, vanadium sulfide, vanadium (IV) chloride, vanadium biguanide, bis(maltolato)oxavandium (IV), vanadium acetate, vanadyl picolinate, vanadium citrate silt, etc .P. In a preferred embodiment of the present invention, the vanadium salt and complex is vanadium (V). The advantage lies in the use of vanadium(V) salts and complexes due to their good solubility in water and better oxidation stability compared to vanadium(IV) salts and complexes. In one embodiment of the present invention, vanadium salts and complexes are a vanadium(IV) salt and/or complex, wherein the vanadium is part of the vanadate anion or part of the vanadyl cation. However, any vanadium salts or combination of vanadium salts and complexes known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention.

In one embodiment of the present invention, the pharmaceutical composition includes any vanadium salt or combination of a vanadium salt and a vanadium complex in an amount of from about 0.01 mg to about 15.0 mg per dosage unit.

In one embodiment of the present invention, the chromium salt and chromium complex are selected in a preferred embodiment of the present invention from a chromium(III) salt and/or complex. In one embodiment of the present invention, any chromium salt or combination of chromium salt and chromium complex is selected from chromium picolinate, chromium chloride, chromium nicotinate, chromium polynicotinate, chromium acetate, trivalent chromium, high chromium yeast, chromium 2-pyridine carboxylate, chromium tripicolinate, salts of 2-pyridinecarboxylic acid chromium, tris(picolinato)chromium, etc. In one embodiment of the present invention, the chromium salt and chromium complex are selected, in a more preferred embodiment of the present invention, from chromium picolinate, chromium polynicotinate, chromium nicotinate, chromium chloride, chromium acetate, and the like. However, any chromium salts or combination of chromium salts and complexes known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention.

In one embodiment of the present invention, the pharmaceutical composition includes any chromium salt or combination of chromium salt and chromium complex in an amount of from about 0.01 mg to about 50 mg per dosage unit, in a preferred embodiment of the present invention from about 0.02 mg to about 0 .5 mg per dose unit.

In one embodiment of the present invention, any manganese salt or combination of a manganese salt and a manganese complex is selected from manganese(II) salt and/or complex, manganese(III) salt and/or complex, manganese salt and/or permanganate complex (V02) . In one embodiment of the present invention, any manganese salts or combination of manganese salts and manganese complexes are selected from any of the following or a combination of manganese (II) sulfate (MnSO ₄ ), manganese (II) chloride (MnCl ₂ ), manganese (III) acetate, permanganate potassium, sodium permanganate, manganese gluconate, etc. In one embodiment of the present invention, the manganese salt and the manganese complex are selected, in a more preferred embodiment of the present invention, from a manganese(III) salt and/or complex. In one embodiment of the present invention, manganese(III) salts and/or complexes are selected from any of the following or a combination of manganese gluconate, sul.

- 7 044859 manganese veil, manganese chloride, etc. However, any manganese salts or combination of manganese salts and complexes known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention. In one embodiment of the present invention, the pharmaceutical composition includes any manganese salt or combination of a manganese salt and a manganese complex in an amount of from about 0.01 mg to about 50 mg per dosage unit, in a preferred embodiment of the present invention from about 0.1 mg to about 10 mg per dose unit.

In one embodiment of the present invention, any salts and complexes of vanadium, chromium and manganese known or understood by one skilled in the art can be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention.

In one embodiment of the present invention, vanadium salts and complexes are preferred over chromium and manganese salts and complexes because vanadium salts and complexes significantly improve the oral bioavailability of the peptides. In one embodiment of the present invention, chromium salts and complexes are preferred over manganese salts and complexes. In one embodiment of the present invention, the use of chromium salts and complexes is also advantageous because they are less toxic. In one embodiment of the present invention, the use of manganese salts and complexes is advantageous over vanadium and chromium salts because manganese salts and complexes are safe for humans even in high doses.

In one embodiment of the present invention, the reducing agent is selected from any of the following or a combination of ascorbic acid (in a preferred embodiment of the present invention from ascorbate such as sodium ascorbate), reduced glutathione (GSH), cystine, uric acid, reduced sugar (a reducing monosaccharide such as glucose, glyceraldehyde or galactose, or a reduced disaccharide such as lactose or maltose), mannitol, α-tocopherol, vitamin A, α-lipoic acid, dihydro-α-lipoic acid (DHLA), thiol-containing compounds, thiomer (includes Laffler thiomers F. et al., Future Med. Chem., 2012, 4, 2205-16), etc. In one embodiment of the present invention, mixtures of at least two reducing agents can be used, in a preferred embodiment of the present invention, ascorbate and reduced glutathione. However, any reducing agent(s) or combination of reducing agent(s) known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention.

In one embodiment of the present invention, the pharmaceutical composition includes a reducing agent in an amount of from about 1.0 mg to about 1000 mg per dosage unit, in a preferred embodiment of the present invention from about 50 mg to about 500 mg per dosage unit.

In one embodiment of the present invention, the pharmaceutical composition further includes at least one absorption enhancer (or permeation enhancer). It should be understood that the terms absorption enhancer and permeation enhancer, used herein interchangeably and synonymously throughout the description of the present invention, cover absorption enhancers and permeation enhancers as known or understood by one skilled in the art. In one embodiment of the present invention, administration of at least one absorption or permeability enhancer improves or facilitates mucosal absorption of the peptide in the gastrointestinal tract, especially if the peptide is large in size. In one embodiment of the present invention, the at least one absorption or permeation enhancer is selected from any of the following or a combination of a zwitterionic absorption enhancer or a non-ionic permeation enhancer. In one embodiment of the present invention, the at least one absorption enhancer is selected from any of the following or a combination of C _{8_20 -alkanoyl} carnitine (in a preferred embodiment of the present invention, lauroyl carnitine, myristoyl carnitine or palmitoyl carnitine; for example, lauroyl carnitine chloride , myristoyl carnitine chloride or palmitoyl carnitine chloride), salicylic acid (in a preferred embodiment of the present invention from a salicylate, such as sodium salicylate), a salicylic acid derivative (such as 3-methoxysalicylic acid, 5-methoxysalicylic acid or homovanillic acid, C ₈ _ ₂₀ -alkanoic acid acid (in a preferred embodiment of the present invention C8. ₂₀ -alkanoate, in a more preferred embodiment of the present invention caprate, caprylate, myristate, palmitate or stearate, such as sodium caprate, sodium caprylate, sodium myristate, sodium palmitate or sodium stearate), citric acid (in the preferred embodiment of the present invention from a citrate such as sodium citrate), an amino acid, an acylated fatty acid (any of the fatty acid acylated amino acids disclosed in patent application US 20140056953 A1, but not limited to, sodium lauroyl alaninate, N-dodecanoyl-L-αlanine, sodium lauroyl aspartate, K-dodecanoyl-L-asparagine, lau

- 8 044859 sodium royl aspartic acid, N-dodecanoyl-L-aspartic acid, sodium lauroyl cysteinate, N-dodecanoyl-L-cysteine, sodium lauroyl glutamic acid, N-dodecanoyl-L-glutamic acid, sodium lauroyl glutamate, N-dodecanoyl -L-glutamine, sodium lauroyl glycinate, N-dodecanoyl-bglycine, sodium lauroyl histidinate, N-dodecanoyl-b-histidine, sodium lauroyl isoleucinate, N-dodecanoyl-b-isoleucine, sodium lauroyl leucinate, N-dodecanoyl-b-leucine, lauroyl sodium methioninate, N-dodecanoyl-L-methionine, sodium lauroyl phenylalaninate, N-dodecanoyl-L-phenylalanine, sodium lauroyl prolinate, N-dodecanoyl-L-proline, sodium lauroyl serinate, N-dodecanoyl-L-serine, sodium lauroyl threonate , N-dodecanoyl-L-threonine, sodium lauroyl tryptophanate, N-dodecanoyl-L-tryptophan, sodium lauroyl tyrosinate, N-dodecanoyl-L-tyrosine, sodium lauroyl valinate, N-dodecanoyl-L-valine, sodium lauroyl sarcosinate, N -dodecanoyl-L-sarcosine, sodium capric alaninate, N-decanoyl-L-alanine, sodium capric aspartate, N-decanoyl-L-asparagine, sodium capric aspartic acid, Ndecanoyl-L-aspartic acid, sodium capric cysteate, N-decanoyl -L-cysteine, sodium capric glutamic acid, N-decanoyl-L-glutamic acid, sodium capric glutamate, Ndecanoyl-L-glutamine, sodium capric glycinate, N-decanoyl-L-glycine, sodium capric histidinate, N-decanoyl-L -histidine, sodium capric isoleucinate, N-decanoyl-L-isoleucine, sodium capric leucinate, N-decanoyl-L-leucine, sodium capric methioninate, N-decanoyl-L-methionine, sodium capric phenylalaninate, N-decanoyl-L-phenylalanine , sodium capric prolinate, Ndecanoyl-L-proline, sodium capric serinate, N-decanoyl-L-serine, sodium capric threonate, Ndecanoyl-L-threonine, sodium capric tryptophanate, N-decanoyl-L-tryptophan, sodium capric tyrosinate, N -decanoyl-L-tyrosine, sodium capric valinate, N-decanoyl-L-valine, sodium capric sarcosinate, N-decanoyl-L-sarcosine, sodium oleoyl sarcosinate, sodium N-decylleucine, sodium stearoyl glutamate (Amisoft HS-11 P) , monosodium myristoyl glutamate (Amisoft MS-11), monosodium lauroyl glutamate (Amisoft LS-11), monosodium cocoyl glutamate (Amisoft CS-11), sodium cocoyl glycinate (Amilite GCS-11), sodium Ndecylleucine, sodium cocoyl glycine, cocoyl glutamate sodium, sodium lauroyl alaninate, Ndodecanoyl-L-alanine, sodium lauroyl aspartate, N-dodecanoyl-L-asparagine, sodium lauroyl aspartic acid, N-dodecanoyl-L-aspartic acid, sodium lauroyl cysteate, N-dodecanoyl-L-cysteine, lauroyl glutamic acid sodium acid, N-dodecanoyl-L-glutamic acid, sodium lauroyl glutamate, N-dodecanoyl-L-glutamine, sodium lauroyl glycinate, N-dodecanoyl-L-glycine, sodium lauroyl histidinate, N-dodecanoyl-L-histidine, lauroyl isoleucinate sodium, N-dodecanoyl-L-isoleucine, sodium lauroyl leucinate, N-dodecanoyl-L-leucine, sodium lauroyl methioninate, N-dodecanoyl-Lmethionine, sodium lauroyl phenylalaninate, N-dodecanoyl-L-phenylalanine, sodium lauroyl prolinate, Ndodecanoyl- L-proline, sodium lauroyl serinate, N-dodecanoyl-L-serine, sodium lauroyl threonate, N-dodecanoyl-L-threonine, sodium lauroyl tryptophanate, N-dodecanoyl-L-tryptophan, sodium lauroyl tyrosinate, N-dodecanoyl-L-tyrosine, Sodium lauroyl valinate, N-dodecanoyl-b-valine, N-dodecanoyl-b-sarcosine, sodium capric alaninate, N-decanoyl-b-alanine, sodium capric aspartate, N-decanoyl-b-asparagine, sodium capric aspartic acid, N-decanoyl-b -aspartic acid, sodium capric cysteate, N-decanoyl-L-cysteine, sodium capric glutamic acid, Ndecanoyl-L-glutamic acid, sodium capric glutamate, N-decanoyl-L-glutamine, sodium capric glycinate, N-decanoyl-L- glycine, sodium capric histidinate, N-decanoyl-L-histidine, sodium capric isoleucinate, N-decanoyl-L-isoleucine, sodium capric leucinate, N-decanoyl-L-leucine, sodium capric methioninate, N-decanoyl-L-methionine, capric phenylalaninate sodium, Ndecanoyl-L-phenylalanine, sodium capric prolinate, N-decanoyl-L-proline, sodium capric serinate, N-decanoyl-L-serine, sodium capric threonate, N-decanoyl-L-threonine, sodium capric tryptophanate, N- decanoyl-L-tryptophan, sodium capric tyrosinate, N-decanoyl-L-tyrosine, sodium capric valinate, N-decanoyl-L-valine, sodium capric sarcosinate, sodium oleoyl sarcosinate and pharmaceutically acceptable salts of any of the above compounds, such as, C _{8_20 -} alkanoyl sarcosinate (lauroyl sarcosinate, such as sodium lauroyl sarcosinate) or one of the 20 standard proteinogenic α-amino acids that is acylated with a _{C8_20 -} alkanoic acid, an alkylsaccharide ( _{C1_20} alkylsaccharide, such as _{C8_ 20-} alkyl polysaccharide such as Multitrope™ 1620-tQ-(MV) or n-octyl-beta-D-glucopyranoside, or n-dodecyl-beta-D-maltoside), cyclodextrin (α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-b β-cyclodextrin, hydroxypropyl β-cyclodextrin or sulfobutylether β-cyclodextrin), sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC), thiomer (includes thiomers disclosed in Laffler F. et al., Future Med . Chem., 2012, 4, 2205-16), chelated calcium compounds (ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), sodium citrate or polyacrylic acid), Cremophor EB (Colliphor EB; CAS No. 61791-12-6) , chitosan, N,N,N-trimethyl chitosan, benzalkonium chloride, bestatin, cetylpyridinium chloride, cetyltrimethylammonium bromide, C ₂ _ ₂₀ -alkanol (for example, ethanol, decanol, lauryl alcohol, myristyl alcohol or palmityl alcohol), C ₈ _ ₂₀ -alkenol (e.g. oleyl alcohol), C ₈ - ₂₀ alkenoic acid (e.g. oleic acid), dextran sulfate, diethylene glycol monoethyl ether (transcutol), 1-dodecyl acyclo-heptan-2-one (Azone®), ethyl caprylate, glyceryl monolaurate, lysophosphatidylcholine, menthol, C ₈ _ ₂₀ -alkylamine, and C ₈ _ ₂₀ -alkenylamine (for example, oleylamine),

- 9 044859 phosphatidylcholine, poloxamer, polyethylene glycol monolaurate, polyoxyethylene, polypropylene glycol monolaurate, polysorbate (polysorbate 80), deoxycholate (sodium deoxycholate), sodium glycocholate, sodium glycodeoxycholate, sodium lauryl sulfate (SDS), taurocholate (e.g. sodium taurocholate), taurodeoxycholate ( taurodeoxycholate sodium), sucrose laurate, sulfoxide (( _{C1.10 -} αalkyl)-(C1.10 _- alkyl)-sulfoxide such as decylmethyl sulfoxide or dimethyl _sulfoxide ), cyclopentadecalactone, 8-(N-2-hydroxy-5-chloro -benzoyl)-aminocaprylic acid (5-CNAC), dodecyl-2-N,Ndimethylamino propionate (DDAIP), D-a-tocopheryl polyethylene glycol-1000 succinate (TPGS) and pharmaceutically acceptable salts of the above compounds and the like. In one embodiment of the present invention, a mixture of any of at least two absorption enhancers, including the absorption enhancers described above, can be used. However, any combination of absorption enhancer(s) known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention. In one embodiment of the present invention, the pharmaceutical composition optionally includes an absorption or permeation enhancer in an amount of from about 10 mg to about 1000 mg per dosage unit, in a preferred embodiment of the present invention from about 50 mg to about 500 mg per dosage unit.

In one embodiment of the present invention, the pharmaceutical composition is formulated such that if the pharmaceutical composition is added to ten milliliters of a 5% HCl solution, it will neutralize the acid and generate a pH value greater than about 6. In one embodiment of the present invention, the pharmaceutical composition formulated in such a way that if the pharmaceutical composition is added to ten milliliters of an aqueous solution, it will generate a pH value of 6 to 9.

In one embodiment of the present invention, a pharmaceutical composition comprising a pharmaceutically effective amount of at least one peptide having a molecular weight equal to or less than 50 kDa, at least any vanadium salt or combination of a vanadium salt and a vanadium complex, at least one reducing agent, and optionally an absorption enhancer administered orally to provide protection, at least in part, of at least one peptide from proteolytic degradation upon oral administration.

In one embodiment of the present invention, a pharmaceutical composition comprising a pharmaceutically effective amount of at least one peptide having a molecular weight equal to or less than 50 kDa, at least any chromium salt or combination of a chromium salt and a chromium complex, at least one reducing agent, and optionally an absorption enhancer administered orally to provide protection, at least in part, of at least one peptide from proteolytic degradation upon oral administration. In one embodiment of the present invention, a pharmaceutical composition comprising a pharmaceutically effective amount of at least one peptide having a molecular weight equal to or less than 50 kDa, at least any manganese salt or combination of a manganese salt and a manganese complex, at least one reducing agent, and optionally an absorption enhancer administered orally to provide protection, at least in part, of at least one peptide from proteolytic degradation upon oral administration.

In one embodiment of the present invention, the pharmaceutical composition further includes, optionally, any pharmaceutically acceptable excipients or a combination of at least one pharmaceutically acceptable excipient, such as, but not limited to, a carrier, diluent, filler, disintegrant, lubricant, binder, colorant, pigment , stabilizer, preservative, antioxidant and/or solubility enhancer. In one embodiment of the present invention, the pharmaceutical composition optionally further includes at least one pharmaceutically acceptable additive, such as vitamin E, histidine, microcrystalline cellulose (MCC), mannitol, starch, sorbitol and/or lactose. In one embodiment of the present invention, pharmaceutical compositions can be formulated using any methods known or understood by one skilled in the art to achieve their desired purpose as set forth in the present invention, without departing from the scope and spirit of the present invention.

In one embodiment of the present invention, the at least one solubility enhancer is selected from any poly(ethylene glycol) or combination of poly(ethylene glycol), including poly(ethylene glycol) having a molecular weight in the range of about 200 to about 5000 Da, ethylene glycol, propylene glycol, nonionic surfactants, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate, phospholipids, lecithin, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, cyclodextrins, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl -β- cyclodextrin, hydroxyethyl-γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, dihydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, sulfobutylether-γ-cyclodextrin, glucosyl-α-cyclodextrin, glucosyl-β

- 10 044859 cyclodextrin, diglucosyl-b-cyclodextrin, maltosyl-a-cyclodextrin, maltosyl-b-cyclodextrin, maltosyl-b-cyclodextrin, maltotriosyl-b-cyclodextrin, maltotriosyl-b-cyclodextrin, dimaltosyl-b-cyclodextrin, methyl-b -cyclodextrin, carboxyalkyl thioesters, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, vinyl acetate copolymers, vinylpyrrolidone, sodium lauryl sulfate, sodium dioctyl sulfosuccinate, etc. However, any combination of solubility enhancer(s) known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention. In one embodiment of the present invention, the pharmaceutical composition is formulated in a dosage form for oral administration, in the preferred embodiment of the present invention for oral administration. In one embodiment of the present invention, at least one peptide, at least one metal in the form of any salt or combination of a salt thereof, and a complex thereof of at least one reducing agent and an optional absorption enhancer are administered orally.

In one embodiment of the present invention, the oral pharmaceutical dosage form is selected from any of the following or a combination of tablets (coated or uncoated tablets), capsules (soft gelatin capsules, hard gelatin capsules, HPMC capsules or HPMC capsules), capsule-in-capsule , tablet-in-capsule, lozenges, trochets, vaginal suppositories, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medical chewing gums, chewable tablets, effervescent tablets, dosage forms, consisting of many particles, etc. However, any oral pharmaceutical dosage form(s) or combination of oral pharmaceutical dosage form(s) known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention. In one embodiment of the present invention, the tablets may include any excipients or combination of excipients, such as, but not limited to, microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, disintegrants such as starch (in a preferred embodiment of the present invention corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granular binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, gum acacia, lubricants, such as magnesium stearate, stearic acid, glyceryl behenate and talc. However, any excipient(s) or combination of excipient(s) known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention.

In one embodiment of the present invention, the capsule may include any excipients or combination of excipients, such as, but not limited to, lactose, starch, cellulose or high molecular weight polyethylene glycols. However, any excipient(s) or combination of excipient(s) known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention. In one embodiment of the present invention, the aqueous suspensions and/or elixirs may include any excipients or combination of excipients, such as, but not limited to, sweeteners or flavoring agents, coloring agents or dyes, emulsifying and/or suspending agents and diluents such as water, ethanol, propylene glycol and glycerin. However, any excipient(s) or combination of excipient(s) known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention.

In one embodiment of the present invention, the pharmaceutical composition is formulated in any solid oral dosage form and a liquid oral dosage form, provided that when the pharmaceutical composition is formulated in a liquid oral dosage form, the pharmaceutical composition includes water in an amount of less than about 5% by volume, in in a preferred embodiment of the present invention less than about 3% by volume, in a more preferred embodiment of the present invention less than about 1% by volume, in an even more preferred embodiment of the present invention less than about 0.5% by volume and in an even more preferred embodiment of the present of the invention is less than about 0.1% by volume, but in an even more preferred embodiment, without water. In one embodiment of the present invention, such a liquid oral dosage form is particularly advantageous because it provides improved storage stability. In an alternative embodiment of the present invention, such a liquid oral dosage form

- 11 044859 can be obtained immediately before administration and long-term storage should be avoided.

The amount of vanadium, chromium and/or manganese used in accordance with embodiments of the present invention is well below the recommended daily intake levels for these micronutrients and may therefore be considered safe. In addition, vanadium, chromium and/or manganese in combination with a reducing agent have an inhibitory effect on serine proteases in the gastrointestinal tract, but do not exhibit a systemic effect, which provides a further increase in safety compared to the protease inhibitors discussed above. Moreover, vanadium, chromium or manganese, as well as reducing agents such as ascorbate or reduced glutathione, can be offered at significantly lower production costs than the protease inhibitors discussed above, which have previously been proposed for the oral delivery of peptide or protein drugs. The actual dosage that will be most appropriate for a particular subject will generally be determined by the attending physician. The specific dose level and dosing frequency for any given subject may vary and will depend on various factors, including the potency of the particular peptide or protein drug used, the metabolic stability and duration of action of that compound, age, body weight, general health, gender, diet, regimen and the time of administration, the rate of excretion, the drug combination, the severity of the specific health condition and the specific subject receiving therapy. Ultimately, the exact dosage will be determined at the discretion of the treating physician or veterinarian. The subject or patient to be treated, such as the subject in need of treatment or prophylaxis, may be an animal (eg, non-human animal), vertebrate, mammal, rodent (eg, guinea pig, hamster, rat, mouse), murine ( e.g. mouse), canid (e.g. dog), felid (e.g. cat), pig (e.g. pig), equid (e.g. horse), primate, ape (e.g. monkey or ape), ape (eg, monkey, baboon), ape (eg, gorilla, chimpanzee, orangutan, gibbon) or human. It is also contemplated in the context of the present invention that it is necessary to treat animals that are of economic or agronomic importance. Non-limiting examples of agronomically important animals include sheep, cattle and pigs, while cats and dogs, for example, may be considered animals of economic importance. In a preferred embodiment of the present invention, the subject/patient is a mammal; in a more preferred embodiment of the present invention, the subject/patient is a human or non-human mammal (such as a guinea pig, hamster, rat, mouse, rabbit, dog, cat, horse, monkey, ape, monkey, baboon, gorilla, chimpanzee, orangutan , gibbon, sheep, cattle or pig); in the most preferred embodiment of the present invention, the subject/patient is a human.

In one embodiment of the present invention, at least one peptide, at least one metal in the form of any salt or combination of a salt thereof, and a complex thereof of at least one reducing agent and an optional absorption enhancer may be administered simultaneously/parallel or sequentially. In one embodiment of the present invention, when administered sequentially, at least one metal, in the form of any salt or combination of a salt thereof, and a complex of at least one reducing agent may be administered first, followed by the peptide and an optional absorption enhancer (for example, after at least about 5 minutes after the first administration, in a preferred embodiment of the present invention from about 5 minutes to about 3 hours after the first administration, in a more preferred embodiment of the present invention from about 10 minutes to about 1 hour after the first administration), which is a special advantage if the peptide is insulin (human insulin). In one embodiment of the present invention, at least one metal, in the form of any salt or combination of a salt thereof, and a complex thereof of at least one reducing agent and an optional absorption enhancer may be administered first, followed by administration of the peptide (e.g., after at least about , 5 minutes after the first administration, in a preferred embodiment of the present invention from about 5 minutes to about 3 hours after the first administration, in a more preferred embodiment of the present invention from about 10 minutes to about 1 hour after the first administration), which also represents It is advantageous if the peptide is insulin (human insulin). In one embodiment of the present invention, the at least one metal is selected from any one or combination of vanadium, chromium, and manganese.

In one embodiment of the present invention, when administered simultaneously, at least one metal, in the form of any salt or combination of a salt thereof, and a complex thereof, at least one reducing agent may be administered first followed by the peptide and an optional absorption enhancer included in the same pharmaceutical composition, or at least two different/separate pharmaceutical compositions, or at least two different/separate compartments of the same pharmaceutical dosage form. In one embodiment, on

- 12 044859 of the present invention at least one metal is selected from any of the following or a combination: vanadium, chromium and manganese. In one embodiment of the present invention, at least one peptide and at least one metal, in the form of any salt or combination of a salt thereof, and a complex thereof are present in a physically separated form in the pharmaceutical composition. In one embodiment of the present invention, the pharmaceutical dosage form includes at least two separate compartments that are physically separated from each other (eg, through a physical separating layer). In one embodiment of the present invention, the pharmaceutical dosage form includes a layer of physical separation between at least one peptide, at least one metal in the form of any salt or combination of a salt thereof, and a complex thereof. In one embodiment of the present invention, at least one peptide is present only in the first compartment and at least one metal, in the form of any salt or combination of a salt thereof, and a complex thereof is/are present only in the second compartment of the pharmaceutical dosage form. In one embodiment of the present invention, the reducing agent is present in either the first compartment, the second compartment, both the first and second compartments, or the third compartment of the pharmaceutical dosage form. In one embodiment of the present invention, the at least one metal is selected from any one or combination of vanadium, chromium, and manganese.

In one embodiment of the present invention, the pharmaceutical composition is present in any one of capsule-in-capsule and tablet-in-capsule pharmaceutical dosage forms, wherein the pharmaceutical composition includes at least one peptide having a molecular weight equal to or less than about 50 kDa , which is present in the first compartment of the pharmaceutical dosage form;

at least one metal in the form of any salt thereof or a combination of a salt and a complex thereof, which is present in the second compartment of the pharmaceutical dosage form; and a reducing agent that is present in the first compartment and/or in the second compartment of the pharmaceutical dosage form.

In one embodiment of the present invention, the present invention provides a pharmaceutical dosage form (e.g., a multiparticulate dosage form) comprising at least one peptide having a molecular weight equal to or less than about 60 kDa that is present in a first compartment of the pharmaceutical dosage form ;

a reducing agent that is present in the second compartment of the pharmaceutical dosage form; and at least one metal in the form of any salt thereof or a combination of a salt and a complex thereof, which is present(s) in the third compartment of the pharmaceutical dosage form.

In one embodiment of the present invention, the pharmaceutical dosage form is a capsule-in-capsule or multiparticulate dosage form. In one embodiment of the present invention, in a capsule-in-capsule dosage form, the larger outer capsule (the contents of which will be released first) contains at least one metal in the form of any salt or combination of a salt thereof and a complex thereof, and a reducing agent, and the smaller inner capsule (the contents of which will be released later) contains a peptide. In one embodiment of the present invention, the at least one metal is selected from any one or combination of vanadium, chromium, and manganese.

In one embodiment of the present invention, the dosage form is selected from any of the following or a combination: a modified-release dosage form (such as a dosage form (capsule, multiparticulate dosage form, or tablet) having an enteric coating), a dosage form ( capsule, multiparticulate dosage form or tablet) coated with Eudragit L30D55 or Eudragit FS30D, acid-resistant capsule such as HPMCP capsules (commonly known in the market as AR Caps®) and the like. However, any dosage form known or understood by one skilled in the art may be used to achieve its desired purpose as set forth in the present invention without departing from the scope and spirit of the present invention.

Example

The serine proteases trypsin, chymotrypsin, carboxypeptidase B and aminopeptidase M, responsible for the proteolytic degradation of peptide bonds and amino acids, were tested specifically for their oxidative inactivation by a combination of metal ion(s) and reducing agent(s).

Quantitative analysis of enzymatic activity. Quantitative analysis of the enzymatic activity of each enzyme in the presence of its specific substrate was carried out at a specific wavelength using a UV spectrophotometer and this served as a negative control. Enzyme activity was calculated as follows:

Units ———solid =

Units/ml enzyme mg solid/mg enzyme

- 13 044859

Quantitative enzyme inhibition assay. Incubation was performed with an inhibitor of each enzyme and served as a positive control.

Incubation with metal ion(s) and reducing agent(s). Incubation of enzymes with a combination of metal salt(s) and reducing agent(s) in a 96-well microtiter plate was carried out for a period of time in the presence of substrates to study their oxidative inactivation. Enzyme inactivation in the presence of metal ion(s) and reducing agent(s) was compared with the initial enzyme activity in the presence of substrate as a negative control and in the presence of an inhibitor as a positive control.

pH measurement. The pH change of enzymes after incubation with metal salt(s) and reducing agent(s) was monitored using a Hanna combination pH electrode.

Zymogram. The treated enzymes were subjected to zymography, which is an electrophoretic method for detecting hydrolytic enzymes based on the substrate repertoire of the enzyme, i.e. the substrate for the enzyme was incorporated into the resolving gel during the preparation of the acrylamide gel, and the degeneration of the substrate by the enzyme was monitored.

Conducting quantitative analysis using kits. To confirm inactivation, quantitative analysis was carried out using a fluorescent protease detection kit and a trypsin activity quantitative assay kit.

Materials.

In table 1A below sets forth materials used to evaluate the effectiveness of various metal salts and metal complexes, optionally in combination with at least one reducing agent, in inhibiting serine protease enzyme(s).

Table 1A

Material used

No. p/p Enzymes Substrates Inhibitors Metal salts (tested) Will you restore (tested) Kits for carrying out quantitative and other analysis Actively Yes pH 1. Human trypsin Na-6en ₃ oin-L-arginine ethyl ester (BAEE) 4Amidinophenyl methanesulfonyl fluoride hydrochloride (Serine protease (inhibitor for Trypsin/Chymotrypsin) Copper chloride Copper carbonate Copper sulfate Zinc sulfate Zinc chloride Zinc acetate Vanadium (IV) sulfate Vanadium (V) oxide Sodium vanadate Potassium permanganate Manganese glycerophosphate Manganese gluconate Chromium chloride Chromium picolinate Sodium ascorbate Reduced glutathione Uric acid Mannitol Benzohydroxamic acid Cysteine Piperine Kit for other assays for fluorescent detection of proteases and Kit for other assays for trypsin activity Combined Nappa pH electrode No-ocihoh.t-Larginine-7amido-4msilku marin hydrochloride 3.4- dichloroisocoumar in (serine protease inhibitor - for Trypsin/Chymotrypsin

- 14 044859

2. Chymothrips in humans Alanine-alanine-phenylalanine-7amido-4methylcoumarin 4- amidinophenyl ethanesulfonyl fluoride hydrochloride (Serine protease (inhibitor - for Trypsin/Chymotrypsin) Kit for other quantitative analysis for fluorescent detection of proteases S-benzoyl-b- tyrosine amidobenzoic acid sodium salt 3.4- dichloroisocoumar in (serine protease inhibitor - for Trypsin/Chymotrypsin 3. Carboxyl eptidase B human Gippuril- Lysine Ethylenediamine tetraacetic acid disodium salt dihydrate K-benzoyl-b- tyrosine amidobenzoic acid 4. Aminopept idaza M pig L-leucine-p- Pitroapilid 4.1 Lleucythiol, oxidized dihydrochloride N-succinylalanine-alanineprolinephenylalanine-7amido-4methylcoumarin

In table 1B below are combinations of metal salts/metal complexes with reducing agent(s) that were evaluated to inhibit serine protease enzyme(s).

- 15 044859

Table 1B

Combination of metal salts and complexes with reducing agents that have been evaluated for enzyme inactivation

No. p/p Reducing agent (5 µM) Metal salt (1 mM/L) 1. Sodium ascorbate Copper chloride/sodium ascorbate Copper sulfate/sodium ascorbate Zinc sulfate/sodium ascorbate vanadium (IV) sulfate/sodium ascorbate vanadium(V) oxide Sodium vanadate Potassium permanganate/sodium ascorbate Manganese gluconate/sodium ascorbate Chromium picolinate/sodium ascorbate 2. Reduced glutathione Copper chloride/reduced glutathione vanadium sulfate (1U)/reduced glutathione vanadium (V) oxide/reduced glutathione Sodium vanadate/reduced glutathione Manganese glycerophosphate/reduced glutathione Chromium Chloride/Reduced Glutathione 3. Uric acid Copper sulfate/uric acid Sodium Vanadate/Uric Acid Manganese gluconate/uric acid Chromium Picolinate/Uric Acid Piperine/absent 4. Mannitol vanadium sulfate (1U)/mannitol Zinc sulfate/mannitol vanadium sulfate (1U)/mannitol Manganese glycerophosphate/mannitol Chromium Picolinate/Mannitol 5. Benzohydroxamic acid Copper sulfate/benzohydroxamic acid vanadium (IV) sulfate/ benzohydroxamic acid vanadium (V) oxide/benzohydroxamic acid Sodium vanadate/benzohydroxamic acid Manganese glycerophosphate/benzohydroxamic acid

- 16 044859

Quantitative assay of trypsin enzymatic activity using Nα-benzoyl-L-arginine ethyl ester (BAEE) A 200 units/mL solution of trypsin in cold HCl solution and a 0.25 mM substrate solution of BAEE were prepared separately and incubated. The above solutions were mixed by inversion to form a reaction mixture and the increase in absorbance at A ₂₅₃ was recorded (a minimum of 4 data points for 1 min will be used here) for the blank solution (without enzyme) and the test (reaction mixture) solution. And ₂₅₃ /min will be obtained using the maximum linear speed for both the blank and the test solution.

Calculation of trypsin content in 3 ml of the test sample:

UNITS BAEE/ml enzyme = ^(AA ^^'^a Test - AA ₂₅₃ / _M minute Blank) x (df) x (3)

0.808 where df=dilution factor;

3=total volume of sample tested for trypsin content (in ml);

0.1=total volume of enzyme (in ml);

0.808=extinction coefficient of Nα-benzoyl-L-arginine at 253 nm.

Determination of trypsin inactivation in the presence of inhibitors. Trypsin (1 mM/L) was incubated with specific (known) inhibitors (shown in Table 1A) and a metal salt/reducing agent combination (shown in Table 2 below) separately. Inactivation with inhibitors served as a positive control.

table 2

Combinations of metal salts/complexes with reducing agents to inactivate proteolytic enzymes

No. p/p Restorers Metal salts Name Concentration Name Concentration 1 Sodium ascorbate 1 mM Copper chloride 5 µM 2 Sodium ascorbate 1 mM Copper sulfate 5 µM 3 Sodium ascorbate 1 mM Zinc sulfate 5 µM 4 Sodium ascorbate 1 mM Vanadium sulfate 5 µM 5 Sodium ascorbate 1 mM Vanadium oxide 5 µM 6 Sodium ascorbate 1 mM Sodium vanadate 5 µM 7 Sodium ascorbate 1 mM Potassium permanganate 5 µM 8 Sodium ascorbate 1 mM Manganese gluconate 5 µM 9 Refurbished 1 mM Chromium picolinate 5 µM 10 Refurbished 1 mM Copper chloride 5 µM eleven Refurbished 1 mM Vanadium sulfate 5 µM 12 Refurbished 1 mM Vanadium oxide 5 µM 13 Refurbished 1 mM Sodium vanadate 5 µM 14 Refurbished 1 mM Chromium chloride 5 µM 15 Uric acid 1 mM Copper sulfate 5 µM 16 Uric acid 1 mM Sodium vanadate 5 µM 17 Uric acid 1 mM Manganese gluconate 5 µM 18 Uric acid 1 mM Chromium picolinate 5 µM 19 Mannitol 1 mM Zinc sulfate 5 µM 20 Mannitol 1 mM Vanadium sulfate 5 µM 21 Mannitol 1 mM Chromium picolinate 5 µM 22 Benzohydroxamic 1 mM Copper sulfate 5 µM 23 Benzohydroxamic 1 mM Vanadium sulfate 5 µM 24 Benzohydroxamic 1 mM Vanadium oxide 5 µM 25 Benzohydroxamic 1 mM Sodium vanadate 5 µM 26 Benzohydroxamic 1 mM Chromium chloride 5 µM 27 Cysteine 1 mM Copper chloride 5 µM 28 Cysteine 1 mM Vanadium oxide 5 µM 29 Cysteine 1 mM Manganese gluconate 5 µM thirty Cysteine 1 mM Chromium picolinate 5 µM 31 Piperine 1 mM Piperine/absent 5 µM 32 Inhibitors 1 mM

Determination of oxidative inactivation (activity and pH) of trypsin in the presence of a combination of metal ions and reducing agents.

For a reaction mixture with a volume of 200 μl, approximately 10 μl of trypsin was incubated in a buffer solution with a combination of metal salts and reducing agents (presented in Table 2 under serial numbers 1-31) at the appropriate concentration at 37°C in microtiter plates with 96 wells

- 17 044859 (Table 3 below provides detailed information on the use of a specific combination of the combinations presented under serial numbers 1-31 in Table 2) for 5, 15 and 30 minutes followed by the addition of the appropriate substrate in a volume of 90 μl (presented in Table 1A).

Enzyme activity was measured spectrophotometrically in a microplate reader and compared with quantitative analysis of the original activity. pH was measured using a Hanna combined pH electrode (enzyme reaction was performed in triplicate). In table 4 below shows the enzymatic activity of trypsin after specified periods of time after treatment with the combination of metal salt/complex and the reducing agent evaluated.

Table 3

Distribution of the metal salt/complex combination and the reducing agent evaluated in 96-well microtiter plates

A Control Control Control 1 1 2 2 2 3 3 3 IN 4 4 4 5 5 5 6 6 6 7 7 7 WITH 8 8 8 9 9 9 10 10 10 eleven eleven eleven D 12 12 12 13 13 13 14 14 14 15 15 15 E 16 16 16 17 17 17 18 18 18 19 19 19 F 20 20 20 21 21 21 22 22 22 23 23 23 G 24 24 24 25 25 25 26 26 26 27 27 27 N 28 28 28 29 29 29 thirty thirty thirty 31 31 31

Table 4

Enzymatic activity of trypsin

Reducing agents and metal salts Time 5 minutes 15 minutes 30 min Sodium ascorbate: Copper chloride 89.65517241 66.66666667 45.71428571 Sodium Ascorbate: Copper Sulfate 89.65517241 40.25 40 Sodium Ascorbate: Zinc Sulfate 55.17241379 54.16666667 57.14285714 Sodium Ascorbate: Vanadium Oxide 41.37931034 31.25 17.14285714 Sodium Ascorbate: Vanadium Sulfate 82.75862069 20.83333333 14.28571429 Sodium ascorbate:Sodium vanadate 27.5862069 54.16666667 31.42857143 Sodium ascorbate:Potassium permanganate 165.5172414 20.83333333 76 Sodium ascorbate:!'manganese luconate 27.5862069 29.16666667 17.14285714 Reduced Glutathione:Chromium Picolinate 55.17241379 45.83333333 71.42857143 Reduced Glutathione: Copper Chloride 158.6206897 83.33333333 80.57142857 Reduced glutathione: Su l fat vanadium 188.2758621 41.66666667 37.14285714 Reduced Glutathione: Vanadium Oxide 20.68965517 95.83333333 48.57142857 Sodium Vanadate/Sodium Vanadate 108.0482759 58.33333333 20 Reduced Glutathione:Chromium Chloride 48.27586207 16.91666667 5.714285714 Uric acid: Copper sulfate 179.3103448 12.5 5.714285714 Uric acid: Sodium vanadate 68.96551724 29.16666667 26.74285714 Uric acid: Manganese gluconate 55.17241379 37.5 37.14285714 Uric Acid Chromium Lycolinate 103.4482759 33.33333333 22.85714286 Mannitol: Zinc sulfate 158.6206897 70.83333333 62.85714286 Mannitol: Vanadium sulfate 103.4482759 95.83333333 80 Mannitol:Chromium Picolinate 89.65517241 83.33333333 37.14285714

- 18 044859

Benzohydroxamic acid: Copper sulfate 110.3448276 4.166666667 57.14285714 Benzohydroxamic acid: Vanadium sulfate 43.2137931 29.16666667 28.57142857 Benzohydroxamic acid: Vanadium oxide 158.6206897 87.5 45.71428571 Benzohydroxamic acid: Sodium vanadate 131.0344828 66.66666667 2.857142857 Benzohydroxamic acid:Chromium chloride 68.96551724 66.66666667 20 Cysteine: Copper Chloride 117.2413793 50 40 Cysteine: Vanadium Oxide 186.2068966 16.66666667 14.28571429 Cysteine Manganese tluconate 55.17241379 58.33333333 11.42857143 Cysteine:Chromium Picolinate 186.2068966 25 20 Piperine 172.4137931 66.66666667 45.71428571

Table 5A-5C presented below herein represent pH measurements for trypsin at various time intervals, i.e. after 5, 15 and 30 minutes.

Table 5A

Trypsin pH measurement at 5 min intervals

1 2 3 4 5 6 7 8 9 10 AND 12 A 3.5 3.45 3.5 2.5 2.45 1.7 2.5 2.4 2.5 2.6 2.5 2.7 IN 2.5 2.6 2.3 2.2 2.5 2.8 2.2 2.3 2.6 2.4 2.2 2.2 WITH 2.5 2.2 2.5 2.5 2.5 2.5 2.5 2.5 2.8 2.5 2.5 2.5 D 2.6 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.8 2.4 2.5 2.5 E 2.5 2.52 2.6 2.5 2.6 2.5 2.6 2.6 2.6 2.6 2.6 2.6 F 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 G 2.4 2.4 2.5 2.2 2.2 2.2 2.2 2.2 2.2 2 2 2.8 N 2.5 2.2 2.2 2 2 2 2 2 2 2.2 2.2 2.2

Table 5 B

Trypsin pH measurement at 15 min intervals

Table 5C

Trypsin pH measurement at 30 min intervals

1 2 3 4 5 6 7 8 9 10 AND 12 A 3.5 3.45 3.5 2.5 1.6 2.3 2.4 2.4 2.5 2.6 2.9 2.7 IN 2.6 2.6 2.3 2.2 2.5 2.8 2.2 2.3 2.6 2.4 2.2 2.7 WITH 2.7 2.2 2.5 2.1 2.5 2.5 2.5 2.5 2.8 2.5 2.5 2.5 D 2.1 2.4 2.6 2.1 2.5 2.5 2.5 2.5 2.8 2.4 2.5 2.5 E 2.5 2.52 2.6 2.5 2.6 2.5 2.6 2.6 2.6 2.6 2.6 2.6 F 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 G 2.4 2.4 2.5 2.2 2.2 2.2 2.2 2.2 2.2 2 2 2.8 N 2.5 2.2 2.2 2 2 2 2 2 2 2.2 2.2 2.2

Quantitative assay for the enzymatic activity of chymotrypsin using alanine-phenylalanine-7-amido-4-methylcoumarin.

Approximately 2 units of chymotrypsin in cold HCl solution, 1.18 mM substrate solution, 2 M calcium chloride solution, and 80 mM Tris HCl buffer solution were prepared separately. Substrate solution and calcium chloride solution were added to 3 ml of buffer solution and mixed by inversion at a temperature of approximately 25 °C to form a blank reaction mixture and a test reaction mixture (Table 6). Next, the HCl solution was added to the blank reaction mixture, and the enzymatic solution was added to the test reaction mixture, immediately mixed by inversion and the increase in absorbance was recorded at A ₂₅₆ for 3-5 minutes. A ₂₅₆ /min will be obtained for both the blank reaction and the test reactions using the maximum linear speed over a minute interval using at least 4 data values.

Table 6

Scheme for obtaining a blank and test solution

Reagents Blank solution (ml) Test solution (ml) Buffer solution 1.42 1.42 Substrate solution 1.40 1.40 CaC12 solution 0.08 0.08 Mixed by inversion followed by raising the temperature to 25°C HC1 solution 0.10 - Enzyme solution — 0.10

Calculation (for alanine-alanine-phenylalanine-7-amido-4-methylcoumarin substrate):

DA25b/minute Test subject - DA25b/minute Idle) x (3) x (df) (83.4) X (0.10)

Units ------enzyme = ml where 3=volume (ml) of the reaction mixture;

df=dilution factor;

83.4=millimolar extinction coefficient of the substrate at a wavelength of 256 nm;

0.10=volume (ml) of the test sample used in the quantitative analysis.

Determination of chymotrypsin inactivation in the presence of inhibitors. Chymotrypsin (1 mM/L) was incubated with specific (known) inhibitors (presented in Table 1A above) and a combination of metal salts and reducing agents (presented in Table 2 above) separately. Inactivation with inhibitors served as a positive control. Determination of oxidative inactivation (activity and pH) of chymotrypsin in the presence of a combination of metal ions and reducing agents.

For a reaction mixture with a volume of 200 μl, approximately 10 μl of chymotrypsin was incubated in a buffer solution with a combination of metal salts and reducing agents (presented in Table 2 under serial numbers 1-31) in the appropriate concentration at 37°C in microtiter plates with 96 wells (in Table 2). Table 3 above provides details of the use of a specific combination of the combinations presented under serial numbers 1-31 in Table 2) for 5, 15 and 30 minutes followed by the addition of the appropriate substrate in a volume of 90 μl (shown in Table 1A). Activity was measured spectrophotometrically in a microplate reader and compared with quantitative analysis of baseline activity. pH was measured using a Hanna combined pH electrode (enzyme reaction was performed in triplicate). In table 7 below shows the enzymatic activity of chymotrypsin after specified periods of time after treatment with the combination of salt/metal complex and the reducing agent evaluated.

Table 7

Enzymatic activity of chymotrypsin

Reducing agent and metal salts Time 5 minutes 15 minutes 30 min Sodium ascorbate: Copper chloride 36.84210526 thirty 25.80645161 Sodium ascorbate:Copper sulfate 168.4210526 85 106.4516129 Sodium Ascorbate:Zinc Sulfate 47.36842105 55 25.80645161 Sodium Ascorbate: Vanadium Oxide 78.94736842 70 38.70967742 Sodium ascorbate: Vanadium sulfate 89.47368421 65 51.61290323

- 20 044859

Sodium ascorbate:Sodium vanadate 100 95 16.12903226 Sodium ascorbate:Potassium permanganate 163.1578947 130 9.677419355 Sodium ascorbate: Manganese gluconate 78.94736842 70 9.677419355 Reduced glutathione: Chromium picolinate 21.05263158 thirty 25.80645161 Refurbished glutathione: copper chloride 47.36842105 45 45.16129032 Reduced glutathione: Vanadium sulfate 115.7894737 85 25.80645161 Reduced glutathione: Vanadium oxide 105.2631579 65 45.16129032 Sodium Vanadate/Sodium Vanadate 105.2631579 55 16.12903226 Reduced glutathione: Chromium chloride 105.2631579 40 41.93548387 Uric acid: Copper sulfate 68.42105263 20 25.80645161 Uric acid: Vanadate sodium 63.15789474 20 9.677419355 Uric acid: Gluconate manganese 89.47368421 85 67.74193548 Uric acid: Picolinate chromium 68.42105263 65 9.677419355 Mannitol: Zinc Sulfate 100 80 51.61290323 Mannitol: Vanadium Sulfate 68.42105263 35 54.83870968 Mannitol:Chromium Picolinate 115.7894737 40 19.35483871 Benzohydroxamic acid: Copper sulfate 152.6315789 80 35.48387097 Benzohydroxamic acid: Vanadium sulfate 142.1052632 BY 32.25806452 Benzohydroxamic acid: Vanadium oxide 100 80 56.77419355 Benzohydroxamic acid: Sodium vanadate 115.7894737 45 12.90322581 Benzohydroxamic acid:Chromium chloride 89.47368421 55 58.06451613 Cysteine: Copper Chloride 89.47368421 85 70.96774194 Cysteine: Vanadium Oxide 115.7894737 140 32.25806452 Cysteine Manganese Gluconate 63.15789474 65 29.03225806 Cysteine:Chromium Picolinate 84.21052632 70 22.58064516 Piperine 84.21052632 80 35.48387097

Table 8A-8C below represent pH measurements for chymotrypsin at various time intervals, i.e. after 5, 15 and 30 minutes.

-21 044859

Table 8A

pH measurement for chymotrypsin at 5 min intervals________

1 2 3 4 5 6 7 8 9 1 eleven 12 A 1.68 1.66 1.65 1.7 1.75 1.76 1.7 1.72 1.69 1.78 1.77 1.65 IN 1.65 1.77 1.7 1.76 1.75 1.78 1.75 1.7 1.67 1.7 1.69 1.7 WITH 1.7 1.7 1.68 1.7 1.69 1.67 1.7 1.7 1.78 1.7 1.68 1.67 D 1.67 1.67 1.67 1.67 1.7 1.7 1.7 1.7 1.78 1.7 1.7 1.7 E 1.67 1.67 1.67 1.67 1.7 1.7 1.7 1.7 1.67 1.7 1.7 1.7 F 1.67 1.67 1.7 1.7 1.7 1.67 1.67 1.67 1.7 1.7 1.7 1.7 G 1.66 1.66 1.66 1.66 1.6 1.67 1.65 1.68 1.65 1.67 1.65 1.68 N 1.65 1.67 1.67 1.65 1.67 1.68 1.68 1.69 1.69 1.67 1.65 1.63

Table 8B

pH measurement for chymotrypsin at 15 min intervals

1 2 3 4 5 6 7 8 9 10 eleven 12 A 1.68 1.66 1.65 1.68 1.65 1.68 1.7 1.77 1.65 1.75 1.65 1.65 IN 1.65 1.69 1.7 1.7 1.68 1.78 1.75 1.7 1.67 1.7 1.69 1.7 WITH 1.7 1.7 1.68 1.7 1.69 1.67 1.7 1.7 1.78 1.7 1.68 1.67 D 1.67 1.67 1.67 1.67 1.7 1.7 1.7 1.68 1.69 1.7 1.7 1.7 E 1.67 1.67 1.67 1.67 1.7 1.7 1.7 1.7 1.67 1.7 1.7 1.7 G 1.67 1.67 1.7 1.7 1.7 1.67 1.67 1.67 1.7 1.7 1.7 1.7 G 1.66 1.66 1.66 1.66 1.6 1.67 1.65 1.68 1.69 1.67 1.65 1.68 N 1.65 1.67 1.67 1.65 1.67 1.68 1.68 1.69 1.69 1.67 1.65 1.63

Table 8C

pH measurement for chymotrypsin at 30 min intervals

1 2 3 4 5 6 7 8 9 10 eleven 12 A 1.68 1.66 1.65 1.69 1.65 1.7 1.77 1.77 1.69 1.68 1.68 1.66 IN 1.65 1.69 1.7 1.7 1.68 1.78 1.75 1.7 1.67 1.7 1.69 1.7 WITH 1.7 1.7 1.68 1.7 1.69 1.67 1.7 1.7 1.78 1.7 1.68 1.67 D 1.67 1.67 1.67 1.67 1.7 1.7 1.7 1.68 1.69 1.7 1.7 1.7 E 1.67 1.67 1.67 1.67 1.7 1.7 1.7 1.7 1.67 1.7 1.7 1.7 G 1.67 1.67 1.7 1.7 1.7 1.67 1.67 1.67 1.7 1.7 1.7 1.7 G 1.66 1.66 1.66 1.66 1.6 1.67 1.65 1.68 1.69 1.67 1.65 1.68 N 1.65 1.67 1.67 1.65 1.67 1.68 1.68 1.69 1.69 1.67 1.65 1.63

Quantitative analysis of carboxypeptidase B enzymatic activity using hippuryl-arginine.

4 units of carboxypeptidase B in cold deionized water, 1 mM solution of hippuryl-arginine in 25 mM Tris buffer solution containing 100 mM sodium chloride (pH 7.65) were prepared separately at a temperature of approximately 25°C. 3 ml of reaction mixtures (test and blank) were prepared using the above obtained solutions in accordance with table. 9 at 25°C. Calculations:

UNITS/ML of enzyme = ( ^YES 254 nm/min Test - YES 254 nm/min Blank)(3)(<10 (0.36)(0..1) where 3=volume (ml) of the reaction mixture;

df=dilution factor;

0.36=millimolar extinction coefficient of the substrate at a wavelength of 254 nm;

0.10=volume (ml) of the test sample used in the quantitative analysis.

Table 9

Scheme for obtaining test and blank solutions

Reagents Test solution (ml) Idle solution (ml) A solution of hippuryl-arginine in Tris buffer solution containing sodium chloride 2.90 2.90 Deionized water — 0.10 Carboxypeptidase B solution 0.10 —

Determination of carboxypeptidase inactivation in the presence of inhibitors.

Carboxypeptidase (1 mM/L) was incubated separately with a specific (known) inhibitor (presented in Table 1A above) and a combination of metal salts/reducing agents (presented in

- 22 044859 table. 2 above). Inactivation with inhibitors served as a positive control.

Determination of oxidative inactivation (activity and pH) of carboxypeptidase in the presence of a combination of metal ions and reducing agents.

For a reaction mixture with a volume of 200 μl, approximately 10 μl of carboxypeptidase was incubated in a buffer solution with a combination of metal salts and reducing agents (presented in Table 2 under serial numbers 1-31) in the appropriate concentration at 37°C in microtiter plates with 96 wells (in Table 2). Table 3 above provides details of the use of a specific combination of the combinations presented under serial numbers 1-31 in Table 2) for 5, 15 and 30 minutes followed by the addition of the appropriate substrate in a volume of 90 μl (shown in Table 1A). Enzyme activity was measured spectrophotometrically in a microplate reader and compared with quantitative analysis of the original activity. pH was measured using a Hanna combined pH electrode (enzyme reaction was performed in triplicate). In table 10 below shows the enzymatic activity of carboxypeptidase after specified periods of time after treatment with the combination of salt/metal complex and the reducing agent evaluated.

Table 10

Enzymatic activity of carboxypeptidase

Reducing agent and metal salts Time 5 minutes 15 minutes 30 min Sodium ascorbate: Copper chloride 43.01886792 21.58490566 58.11320755 Sodium ascorbate:Copper sulfate 79.24528302 87.54716981 41.50943396 Sodium Ascorbate:Zinc Sulfate 120.0113208 33.20754717 33.20754717 Sodium Ascorbate: Vanadium Oxide 34.67169811 29.05660377 0 Sodium ascorbate: Vanadium sulfate 89.13962264 66.41509434 66.41509434 Sodium ascorbate:Sodium vanadate 61.01886792 58.11320755 58.49056604 Sodium ascorbate:Potassium permanganate 83.39622642 58.11320755 41.50943396 Sodium ascorbate Manganese gluconate 49.81132075 62.26415094 66.41509434 Reduced glutathione: Chromium picolinate 65.09433962 29.05660377 20.75471698 Refurbished glutathione: copper chloride 100 58.11320755 75.09433962 Refurbished Glutathione: Vanadium Sulfate 91.69811321 33.20754717 0 Refurbished Glutathione: Vanadium Oxide 79.24528302 45.66037736 20.75471698 Sodium Vanadate/Sodium Vanadate 49.43396226 41.50943396 37.35849057 Refurbished glutathione: chromium chloride 79.24528302 4.150943396 58.49056604 Uric acid: Copper sulfate 100 62.26415094 49.81132075 Uric acid: Sodium vanadate 83.77358491 53.96226415 29.05660377 Uric acid: Gluconate manganese 95.8490566 45.66037736 45.66037736

- 23 044859

Uric acid: Picolinate chromium 62.26415094 50.18867925 45.66037736 Mannitol: Zinc Sulfate 74.71698113 62.26415094 41.50943396 Mannitol: Vanadium Sulfate 56.22641509 37.35849057 37.35849057 Mannitol Chromium Picolinate 79.24528302 58.11320755 58.11320755 Benzohydroxamic acid: Copper sulfate 91.32075472 83.39622642 Benzohydroxamic acid: Vanadium sulfate 45.66037736 24.90566038 16.60377358 Benzohydroxamic acid: Vanadium oxide 79.24528302 79.24528302 49.81132075 Benzohydroxamic acid: Sodium vanadate 41.50943396 33.20754717 24.90566038 Benzohydroxamic acid:Chromium chloride 54.33962264 24.90566038 12.45283019 Cysteine: Copper Chloride 87.54716981 41.50943396 16.60377358 Cysteine: Vanadium Oxide 33.20754717 25.94339623 25.94339623 Cysteine: Manganese gluconate 49.81132075 74.71698113 62.26415094 Cysteine Chromium Picolinate 70.94339623 41.50943396 6.918238994 Piperine 104.5283019 91.69811321 62.26415094

Table PA-PS represents the pH measurement for carboxypeptidase at various time intervals, i.e. after 5, 15 and 30 minutes.

IA table

pH measurement for carboxypeptidase at 5 min intervals

1 ¹² 3 4 5 6 7 8 9 10 eleven 12 A 7.77 7.78 7.76 7.76 7.65 7.77 7.77 7.78 7.76 7.65 7.78 7.7 IN 7.78 7.8 7.67 7.78 7.79 7.69 7.72 7.67 7.74 7.75 7.76 7.78 WITH 7.79 7.76 7.75 7.76 7.78 7.7 7.6 7.8 7.7 7.5 7.67 7.7 D 7.8 7.76 7.7 7.6 7.72 7.6 7.9 7.7 7.7 7.7 7.8 7.8 E 7.76 7.6 7.78 7.7 7.6 7.6 7.8 7.6 7.7 7.76 7.8 7.7 F 7.7 7.8 7.7 7.7 7.7 7.8 7.8 7.7 7.7 7.7 7.7 7.7 G 7.7 7.8 7.71 7.75 7.8 7.8 7.7 7.78 7.7 7.7 7.7 7.7 N 7.67 7.6 7.6 7.76 7.8 7.7 7.7 7.7 7.7 7.7 7.7 7.7

Table IV

pH measurement for carboxypeptidase at 15 min intervals

1 2 3 4 5 6 7 8 9 10 eleven 12 A 7.77 7.7 7.7 7.7 7.75 7.77 7.77 7.78 7.76 7.65 7.78 7.7 IN 7.78 7.8 7.67 7.78 7.79 7.69 7.72 7.67 7.74 7.75 7.76 7.78 WITH 7.79 7.76 7.75 7.76 7.78 7.7 7.6 7.8 7.7 7.5 7.67 7.7 D 7.8 7.76 7.7 7.6 7.72 7.6 7.9 7.7 7.7 7.7 7.7 7.7 E 7.76 7.6 7.78 7.7 7.6 7.6 7.8 7.6 7.7 7.76 7.8 7.7 F 7.7 7.8 7.7 7.7 7.7 7.8 7.8 7.7 7.7 7.7 7.7 7.7 G 7.7 7.8 7.71 7.75 7.8 7.8 7.7 7.78 7.7 7.7 7.7 7.7 N 7.67 7.6 7.6 7.76 7.8 7.7 7.7 7.7 7.7 7.7 7.7 7.7

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Table 11C

pH measurement for carboxypeptidase at 30 min intervals_____

1 2 3 4 5 6 7 8 9 10 AND 12 A 7.77 7.7 7.7 7.8 7.75 7.77 7.77 7.78 7.76 7.65 7.78 7.7 IN 7.78 7.9 7.67 7.78 7.79 7.69 7.72 7.67 7.74 7.75 7.76 7.78 WITH 7.79 7.8 7.75 7.76 7.78 7.7 7.6 7.8 7.7 7.5 7.67 7.7 D 7.8 7.76 7.7 7.6 7.72 7.6 7.9 7.7 7.7 7.7 7.8 7.6 E 7.76 7.6 7.78 7.7 7.6 7.6 7.8 7.6 7.7 7.76 7.8 7.7 F 7.7 7.8 7.7 7.7 7.7 7.8 7.8 7.7 7.7 7.7 7.7 7.7 G 7.7 7.8 7.71 7.75 7.8 7.8 7.7 7.78 7.7 7.7 7.7 7.7 N 7.67 7.6 7.6 7.76 7.8 7.7 7.7 7.7 7.7 7.7 7.7 7.7

Conduct a quantitative analysis of the enzymatic activity of aminopeptidase M using L-leucine-p-nitroanilide.

A 1 mM solution of tricine (prepared in 100 ml of deionized water, reagent A) and a 50 mM solution of L-leucine-p-nitroanilide in absolute methanol (reagent B) were prepared separately. An approximately 10 mM solution of L-leucine-p-nitroanilide (sodium leucine, reagent C) was prepared by adding 0.1 mL of reagent B to 4.9 mL of reagent A. Separately, 200 mM of tricine-buffered solution in deionized water was prepared ( reagent D) and 200 mM tricine buffer solution with 0.05% (w/v) BSA pH 8.0 at 25°C (reagent E). 0.04 units/ml of aminopeptidase in reagent E (enzyme solution, reagent F) was obtained. Reagent C (leucine sodium, 2.0 ml), reagent D (200 mM tricine buffer solution, 1.0 ml) and deionized water (7.0 ml) were pipetted and mixed in a container by vortex flow to obtain the reaction mixture (reagent G). Reagent G, reagent E and reagent F were immediately mixed by inversion to obtain the test and blank solutions as indicated in the table. 12, and an increase in ΔΑ ₄₀₅ ημ was recorded for approximately 5 minutes. A ΔΑ _4Ο5 nm/min was obtained using the maximum linear velocity for both the test and blank solutions. Calculations:

(DA405nm/min Test - DA405nm/ ^min Idle)(1)(4G)

Units/ml enzyme = (0.36)(0.1) where 1=total volume (ml) of quantitative analysis;

df=dilution factor;

10.8=millimolar extinction coefficient of 1 p-nitroaniline at A ₄₀₅ nm;

0.1=volume (in milliliters) of enzyme used.

Table 12

Scheme for obtaining the test and blank solution____________

Reagents Test solution (ml) Blank solution (ml) Reaction mixture (Reagent G) 0.90 0.90 Reagent F (Enzyme solution) 0.10 Reagent E — 0.10

Determination of aminopeptidase inactivation in the presence of inhibitors.

Aminopeptidase (1 mM/L) was individually incubated with a specific inhibitor (presented in Table 1A above) and a combination of metal salts/reducing agents (presented in Table 2 above). Inactivation with inhibitors served as a positive control.

Determination of oxidative inactivation (activity and pH) of aminopeptidase in the presence of a combination of metal ions and reducing agents.

For a reaction mixture with a volume of 200 μl, approximately 10 μl of aminopeptidase was incubated in a buffer solution with a combination of metal salts and reducing agents (presented in Table 2 under serial numbers 1-31) in the appropriate concentration at 37°C in microtiter plates with 96 wells (in Table 2). Table 3 above provides details of the use of a specific combination of the combinations presented under serial numbers 1-31 in Table 2) for 5, 15 and 30 minutes followed by the addition of the appropriate substrate in a volume of 90 μl (shown in Table 1A). Enzyme activity was measured spectrophotometrically in a microplate reader and compared with quantitative analysis of the original activity. pH was measured using a Hanna combined pH electrode (enzyme reaction was performed in triplicate). In table 13 below shows aminopeptidase enzymatic activity after specified periods of time following treatment with the salt/metal complex combination and the reducing agent evaluated.

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Table 13

Enzymatic activity of aminopeptidase

Reducing agent and metal salts Time 5 minutes 15 minutes 30 min Sodium ascorbate: Copper chloride 100 98.06034483 91.31455399 Sodium ascorbate:Copper sulfate 92.57142857 81.89655172 91.07981221 Sodium Ascorbate:Zinc Sulfate 109.1428571 105.1724138 101.6431925 Sodium Ascorbate: Vanadium Oxide 113.4285714 109.9137931 80.51643192 Sodium ascorbate: Vanadium sulfate 99.42857143 99.13793103 99.06103286 Sodium ascorbate:Sodium vanadate 101.7142857 90.94827586 73.94366197 Sodium ascorbate:Potassium permanganate 100 99.56896552 88.2629108 Sodium ascorbate: Manganese gluconate 104.8571429 100.4310345 100.4694836 Refurbished glutathione: chromium picolinate 92.57142857 87.5 79.10798122 Refurbished glutathione: copper chloride 96 94.39655172 88.96713615 Refurbished Glutathione: Vanadium Sulfate 96 86.42241379 83.09859155 Refurbished Glutathione: Vanadium Oxide 102 98.27586207 100.4694836 Refurbished glutathione: sodium vanadate 96 95.04310345 84.50704225 Refurbished glutathione: chromium chloride 108.2857143 95.68965517 89.20187793 Uric acid: Copper sulfate 94.51428571 93.53448276 97.18309859 Uric acid: Sodium vanadate 102.8571429 101.7241379 99.29577465 Uric acid: Gluconate manganese 98.57142857 95.68965517 93.42723005 Uric AcidLicolinate chromium 93.42857143 93.53448276 86.61971831 Mannitol: Zinc Sulfate 105.1428571 90.0862069 96.24413146 Mannitol: Vanadium Sulfate 106.5714286 101.7241379 99.06103286 Mannitol:Chromium Picolinate 94 87.71551724 101.8779343 Benzohydroxamic acid: Copper sulfate 89.71428571 75.86206897 74.64788732 Benzohydroxamic acid: Vanadium sulfate 84 70.68965517 75.11737089 Benzohydroxamic acid: Vanadium oxide 87.42857143 85.12931034 77.9342723 Benzohydroxamic acid: Sodium vanadate 64.51428571 88.36206897 79.81220657

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Benzohydroxamic acid:Chromium chloride 96.85714286 67.88793103 78.16901408 Cysteine: Copper Chloride BY 113.362069 114.5539906 Cysteine: Vanadium Oxide 93.71428571 88.57758621 88.2629108 Cysteine: Manganese gluconate 88 88.36206897 88.73239437 Cysteine Chromium Picolinate 105.4285714 90.94827586 64.31924883 Piperine 108 100.4310345 80.51643192

In table 14A-14C presented below herein show pH measurements for aminopeptidase at various time intervals, i.e. after 5, 15 and 30 minutes.

Table 14A

pH measurement for aminopeptidase at 5 min intervals

1 2 3 4 5 6 7 8 9 10 eleven 12 A 8.11 8.15 8.16 8.16 8.13 8.13 8.11 8.13 8.13 8.13 8.11 8.13 IN 8.13 8.1 8.11 8.12 8.09 8.09 8.1 8.1 8.09 8.11 8.1 8.12 WITH 8.11 8.12 8.09 8.1 8.11 8.12 8.09 8.11 8.11 8.12 8.09 8.11 D 8.11 8.11 8.12 8.12 8.11 8.11 8.1 8.11 8.1 8.12 8.12 8.12 E 8.11 8.11 8.12 8.1 8.11 8.11 8.12 8.11 8.09 8.11 8.1 8.11 F 8.12 8.12 8.12 8.11 8.12 8.09 8.11 8.1 8.1 8.1 8.1 8.1 G 8.11 8.11 8.12 8.11 8.11 8.1 8.11 8.11 8.12 8.1 8.1 8.1 N 8.1 8.1 8.1 8.1 8.12 8.12 8.1 8.1 8.12 8.12 8.12 8.12

Table 14B

pH measurement for aminopeptidase at 15 min intervals

1 2 3 4 5 6 7 8 9 10 eleven 12 A 8.11 8.15 8.16 8.16 8.13 8.13 8.11 8.13 8.13 8.13 8.11 8.13 IN 8.13 8.1 8.11 8.12 8.13 8.14 8.1 8.1 8.11 8.11 8.1 8.12 WITH 8.11 8.12 8.12 8.1 8.11 8.12 8.13 8.11 8.11 8.12 8.15 8.11 D 8.11 8.11 8.12 8.12 8.11 8.11 8.1 8.11 8.1 8.12 8.12 8.12 E 8.11 8.11 8.12 8.1 8.11 8.11 8.12 8.11 8.14 8.11 8.1 8.11 F 8.12 8.12 8.12 8.11 8.12 8.14 8.11 8.1 8.1 8.1 8.1 8.1 G 8.11 8.11 8.12 8.11 8.11 8.1 8.11 8.11 8.12 8.1 8.1 8.1 N 8.1 8.1 8.1 8.1 8.12 8.12 8.1 8.1 8.12 8.12 8.12 8.12

Table 14C

pH measurement for aminopeptidase at 30 min intervals ____

1 2 3 4 5 6 7 8 9 10 eleven 12 A 8.11 8.15 8.16 8.16 8.13 8.13 8.11 8.13 8.13 8.13 8.11 8.13 IN 8.13 8.1 8.11 8.12 8.15 8,.12 8.1 8.1 8.09 8.11 8.1 8.12 WITH 8.11 8.12 8.15 8.1 8.11 8.12 8.09 8.11 8.11 8.12 8.09 8.11 D 8.11 8.11 8.12 8.12 8.11 8.11 8.1 8.11 8.1 8.12 8.12 8.12 E 8.11 8.11 8.12 8.1 8.11 8.11 8.12 8.11 8.09 8.11 8.1 8.11 F 8.12 8.12 8.12 8.11 8.12 8.09 8.11 8.1 8.1 8.1 8.1 8.1 G 8.11 8.11 8.12 8.11 8.11 8.1 8.11 8.11 8.12 8.1 8.1 8.1 N 8.1 8.1 8.1 8.1 8.12 8.12 8.1 8.1 8.12 8.12 8.12 8.12

Based on the experiments performed and the data presented above, we can conclude that the highest level of inhibition of enzymatic activity (proteolytic degradation) was observed when using the following combinations of reducing agents and metal salts: sodium ascorbate-vanadium oxide; benzohydroxamic acid-vanadium sulfate; vanadium mannitol sulfate; uric acid manganese gluconate; reduced chromium glutathione chloride and reduced vanadium glutathione oxide.

Bioavailability studies.

To provide protection from the acidic pH of the intragastric environment and degradation by proteolytic enzymes, capsule-in-capsule formulations have been prepared, wherein the enteric-coated capsule can protect the peptide from the intragastric environment, MIRA granules present in the outer capsule can inactivate proteolytic enzymes, and permeation enhancers may promote/increase peptide absorption across the intestinal epithelial membrane.

Preparation of capsule-in-capsule formulations.

Preparation of granules containing reducing agent(s) and metal salt(s).

In table 15A-15F below present various reducing agent(s) and metal salt(s) containing bead formulations (hereinafter referred to as MIRA beads) prepared for bioavailability assessment.

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Table 15A

MIRA 1 granule formula (rat studies)

No. Ingredients Quantity per capsule 1 Refurbished glutathione 12 mg 2 Chromium picolinate 0.03 mg 3 Microcrystalline cellulose 101 12 mg 4 Croscarmellose sodium 1.5 mg 5 Mannitol 4.47 mg 6 HPMS-E-5 In sufficient quantity

Total 30 mg

Table 15B

MIRA 2 granule formula (rat studies)

No. Ingredients Quantity per capsule 1 Sodium ascorbate 30 mg 2 Vanadium oxide 0.03 mg 3 Microcrystalline cellulose 101 12 mg 4 Mannitol 5.47 mg 5 Croscarmellose sodium 2.5 mg 6 HPMS-E-5 In sufficient quantity

Total 50 mg

Table 15C

MIRA 3 granule formula (rat studies)

No. Ingredients Quantity per 1 Uric acid capsule 3 mg 2 Sodium vanadate 0.03 mg 3 Microcrystalline 12 mg 4 I'm pulp 101 Mannitol 3.97 mg 5 Croscarmellose 1 mg 6 sodium HPMS-E-5 In sufficient Total quantity 20 mg

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Table 15D

MIRA 4 granule formula (rat studies)

No. Ingredients Quantity per 1 Sodium ascorbate capsule 30 mg 2 Manganese gluconate 0.03 mg 3 Microcrystalline 12 mg 4 I'm pulp 101 Mannitol 5.47 mg 5 Croscarmellose 1.5 mg 6 sodium HPMS-E-5 In sufficient Total quantity 50 mg

Table 15E

MIRA 5 kibble formula (canine studies)

No. Ingredients Quantity per capsule 1 Sodium ascorbate 100 mg 2 Vanadium sulfate 0.1 mg 3 Microcrystalline cellulose 101 50 mg 4 Croscarmellose sodium 10 mg 5 HPMS-E-5 In sufficient quantity Total 160.1 mg Table 15F

MIRA 2 kibble formula (canine studies)

No. Ingredients Quantity per capsule 1 Sodium ascorbate 100 mg 2 Vanadium oxide 0.1 mg 3 Microcrystalline cellulose 101 50 mg 4 Croscarmellose sodium 10 mg 5 HPMS-E-5 In sufficient quantity Total 160.1 mg

Preparation of MIRA granules.

MIRA granules were prepared according to the procedure described below.

A) Preparation of the binder solution: 0.3% w/v. HPMC E-5 solution was prepared by dissolving 75.0 mg of HPMC E-5 in 25.0 mL deionized (D.I.) water.

B) To prepare the powder mixture: All the ingredients were added one at a time in a suitable vessel and then mixed thoroughly using a plastic bag to ensure homogeneity of the mixture.

C) Preparation of granules: To create granules, manual granulation was used by adding dropwise 2.0 mL of the binder solution (2 ml was required to granulate 2.5 mg of powder mixture).

D) Drying of granules: The resulting granules were dried at 40°C for approximately 12 hours in a dry-heat oven.

E) Sieving of granules: The dried granules were passed through a No. 40 stainless steel sieve, collected in a suitable glass container and stored at room temperature.

(During the entire granulation procedure, temperature: 23°C and humidity: 39% relative humidity were noted.)

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Preparation of granules containing peptide(s).

In table 16A-16I below are various peptide containing bead formulations (hereinafter referred to as PA beads) prepared for bioavailability assessment.

Table 16A

Formula for Insulin Glargine Granules RA 1 (rat studies)

No. Ingredients Quantity per capsule 1 Chenodeoxycholic acid 30 mg 2 Insulin glargine 0.06 mg 3 Microcrystalline cellulose 101 15 mg 4 Mannitol 2.44 mg 5 Croscarmellose sodium 2.5 mg 6 HPMS-E-5 In sufficient quantity Total 50 mg

Table 16B

Formula for Insulin Glargine Granules RA 2 (rat studies)

No. Ingredients Quantity per 1 Labrazol ALF capsule 5 mg 2 Insulin glargine 0.06 mg 3 Microcrystalline 35 mg 4 I'm pulp 101 Mannitol 7.44 mg 5 Croscarmellose 2.5 mg 6 sodium HPMS-E-5 In sufficient quantity

Total 50 mg

Table 16С

Formula for octreotide acetate granules PA 1 (rat studies)

No. Ingredients Quantity per capsule 1 Chenodeoxycholic acid 30 mg 2 Octreotide acetate 0.3 mg 3 Microcrystalline cellulose 101 15 mg 4 Mannitol 2.2 mg 5 Croscarmellose sodium 2.5 mg 6 HPMS-E-5 In sufficient quantity Total 50 mg

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Table 16D

Teriparatide Granule Formula RA 1 (Rat Studies)

No. Ingredients Quantity per capsule 1 Chenodeoxycholic acid 30 mg 2 Teriparatide 0.12 mg 3 Microcrystalline cellulose 101 15 mg 4 Mannitol 2.38 mg 5 Croscarmellose sodium 2.5 mg 6 HPMS-E-5 In sufficient quantity

Total 50 mg

Table 16E

Teriparatide Granule Formula RA 3 (Rat Studies)

No. Ingredients Quantity per 1 Piperine capsule 3 mg 2 Teriparatide 0.12 mg 3 Microcrystalline 20 mg 4 I'm pulp 101 Mannitol 5.38 mg 5 Croscarmellose 1.5 mg 6 sodium HPMS-E-5 In sufficient Total quantity 30 mg

Table 16F

Formula for liraglutide sodium granules RA 2 (canine studies)

No. Ingredients Quantity per capsule 1 Liraglutide sodium 12 mg 2 Labrazol ALF 40 mg 3 Microcrystalline cellulose 101 100 mg 4 Croscarmellose sodium 10 mg 5 HPMS-E-5 In sufficient quantity Total 162 mg

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Table 16G

Formula for sodium liraglutide granules RA 3+4 (canine studies)

No. Ingredients Quantity per capsule 1 Liraglutide sodium 12 mg 2 Piperine 10 mg 3 Solutol HS 15 25 mg 4 Microcrystalline cellulose 101 100 mg 5 Croscarmellose sodium 10 mg 6 HPMS-E-5 In sufficient quantity Total 157 mg Table 16H

Formula for leuprolide acetate granules RA 2 (canine studies)

No. Ingredients Quantity per capsule 1 Leuprolide acetate 1.25 mg 2 Labrazol ALF 40 mg 3 Microcrystalline cellulose 101 88.75 mg 4 Croscarmellose sodium 10 mg 5 HPMS-E-5 In sufficient quantity Total 140 mg

Table 16I

Formula for leuprolide acetate granules RA 2+3 (canine studies) No. Ingredients Quantity per capsule 1 Leuprolide acetate 1.25 mg 2 Labrazol ALF 30 mg 3 Piperine 10 mg 4 Microcrystalline cellulose 101 88.75 mg 5 Croscarmellose sodium 10 mg 6 HPMS-E-5 In sufficient quantity

Total 140 mg

Peptide granule granulation procedure.

A) Preparation of the binder solution: 0.3% w/v. HPMC E-5 solution was prepared by dissolving 75.0 mg of HPMC E-5 in 25.0 mL deionized (D.I.) water.

B) Preparation of powder mixture: All ingredients except liquid excipients were accurately weighed and mixed in a plastic bag for 5.0 min.

C) Addition of Binder: A weighed amount of liquid excipients along with the peptide was added to the HPMC E-5 (0.03%) binder solution. The resulting mixture was added dropwise to carry out wet granulation.

D) Drying of granules: The granules were dried in a vacuum desiccator over a layer of silica gel overnight.

E) Sieving of granules: The dried granules were passed through a No. 40 stainless steel sieve, collected in a suitable glass container and stored at room temperature.

(During the entire granulation procedure, temperature: 22°C and humidity: 35% relative humidity were noted.)

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Capsule filling.

MIRA beads and peptide beads were filled into capsules manually using a weighing scale. In table 17A and 17B below show the size of capsules used to fill capsules for rat studies and dog studies, respectively.

Table 17A

Size of capsules used for filling

capsules for rat studies No. Granules Size 1 Insulin glargine + Chenodeoxycholic acid 3 2 Insulin glargine + Labrazole 3 3 Octreotide acetate + Chenodeoxycholic acid 3 4 Reduced Glutathione + Chromium Picolinate 1 5 Sodium Ascorbate + Vanadium Oxide 0 6 Uric acid + Vanadate sodium 4 7 Teriparatide + Piperine 3 8 Teriparatide + Chenodeoxycholic acid 3 9 Sodium Ascorbate + Manganese Gluconate 0 Table 17B

Size of Capsules Used to Fill Canine Research Capsules

No. Granules Size 1 Sodium Ascorbate + Vanadium Sulfate 00 (Enteric coating) 2 Sodium Ascorbate + Vanadium Oxide 00 (Enteric coating) 3 Liraglutide sodium + Solutol + Piperine 3 4 Liraglutide sodium + Labrazole 3 5 Leuprolide acetate + Labrazole 3 6 Leuprolide acetate + Labrazole + Piperine 3

Packaging and storage of capsules.

Capsules were packaged in plastic bags and transferred to HDPE containers with silica gel packets to control humidity.

Preparation of batches of placebo granules.

A placebo batch was prepared using sunset yellow and blue dyes to understand disintegration and release of granules from capsules (visually). The dried granules were filled into size 3 capsules and disintegration time was determined on a disintegration meter (Electrolab) using guide discs. The disintegration time was found to be 3 ± 1 min in water and phosphate buffer (pH 6.8) at 37 ± 0.2°C. Placebo granule release study (capsule-in-capsule) Outer capsule: size 0, yellow granules. Inner capsule: size 4, blue granules.

The release study was carried out in a disintegration apparatus (Electrolab Mumbai) at 37±0.5°C in 900 ml of 0.1 N HCl (for 2 hours) and pH 6.8 phosphate buffer.

From visual observation it can be concluded that the outer capsules with enteric acid

- 33 044859 coatings remain intact in 0.1 N HCl (stable in intragastric media), but begin to disintegrate in phosphate buffer with pH 6.8 within 3 minutes.

The inner capsules began to disintegrate after 8 min and were completely dissolved within 13 min. Similarly, release of MIRA beads can occur within 3 min of exposure to alkaline pH, followed by complete release of the peptide within 13 min.

Evaluation of capsules.

Procedure for quantitative analysis of insulin glargine, octreotide acetate and teriparatide for rat studies - 50/30 mg granules (removed from one capsule) were accurately weighed and dissolved in the mobile phase. The dispersion was sonicated for 5 min in a sonication bath, filtered through a 0.22 μm syringe filter, and injected into the HPLC system. A calibration curve for all APIs was generated using multiple dilutions in their respective mobile phases suggested by USP 2017. Quantitative analyzes were performed and percentage drug content was calculated using the calibration curves.

Table 18

Results of quantitative analyzes for each composition (studies on rats)

No. Code Compound Square Quantity 1 PA1 Insulin glargine + (mAU*S) 1664 ny analysis (%) 105.2 2 PA2 chenodeoxycholic acid Insulin glargine + labrazole 1860 115.8 3 PA1 Octreotide acetate + 1638 24.5 4 PA1 chenodeoxycholic acid Teriparatide + 2203 98.02 5 ONCE chenodeoxycholic acid Teriparatide + piperine 2385 106.1 Stability studies of insulin glargine capsules.

Table 19

Composition of insulin glargine granules

No. Ingredient Quantity per Quantity per capsule capsules Batch I Batch II 1 Insulin glargine 0.12 mg 0.12 mg 2 Henodeoxycholic 30 mg - acid 3 Labrazol - 30 mg 4 Microcrystalline 15 mg 20 mg cellulose 5 Croscarmellose 2.5 mg 1.5 mg sodium 6 Mannitol 2.38 mg 5.38 mg 7 Binder In sufficient In sufficient substance (HRMSE- 5) quantity quantity total weight 50 mg 50 mg

Capsule filling. Mode: manual filling; capsule size: 2; mass of filled granules: according to the formula presented above.

Storage of capsules and granules. Temperature: 25±3°C; Humidity: relative humidity 35±5%; container: HDPE 60 cu.m. cm for capsules/transparent glass bottle with rubber cap for granules.

Observation. From the equation (y=30.977x-292.26, obtained from HPLC data), it was found that the content of insulin glargine present in the composition was 105.2 and 115.8% for batch I and batch II, respectively.

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Stability studies (75 days). Both batches were stored at room temperature 25°C for 85 days and quantitative assays were repeated.

Observation. From the equation (y=30.977x-292.26, obtained from HPLC data), it was found that the content of insulin glargine present in the composition is 97.25 and 91.63% for batch I and batch II, respectively, as can be seen from the table . 20 below.

Table 20

Comparative analysis of stability studies

Compound Area (0 Quantitative Area (85 Quantitative days) th analysis (0 days) th analysis (85 days) days) Insulin 1664 mAU*s 105.2% 1515.3mAU*s 97.25% glargine + Henodeoxychol pile acid Insulin 1860mAU*s 115.8% 1410.9 mAU*s 91.63%

glargine +

Labrazole (Batch II)

Stability studies of teriparatide.

Table 21

Composition of teriparatide granules

No. Ingredient Quantity per Quantity per 1 Teriparatide capsule Batch I 0.12 mg capsules Batch II 0.12 mg 2 Henodeoxycholic 30 mg - 3 acid Piperine — 3 mg 4 Microcrystalline 15 mg 20 mg 5 cellulose Croscarmellose 2.5 mg 1.5 mg 6 sodium Mannitol 2.38 mg 5.38 mg 7 Binder In sufficient In sufficient substance (HRMSE- quantity quantity 5) total weight 50 mg 30 mg

Capsule filling. Mode: manual filling; capsule size: 2; mass of filled granules: according to the formula presented above.

Storage of capsules and granules. Temperature: 25±3°C; Humidity: relative humidity 35±5%; container: HDPE 60 cu.m. cm for capsules/transparent glass bottle with rubber cap for granules.

Observation. From the equation (y=18.924x-22.539 obtained from HPLC data), the content of teriparatide acetate present in the formulation was found to be 98.02 and 106% for batch I and batch II, respectively.

Stability studies (75 days). Both batches were stored at room temperature 25°C for 75 days and quantitative assays were repeated.

Observation. From the equation (y=18.924x-22.539 obtained from HPLC data), the content of teriparatide acetate present in the formulation was found to be 92.88 and 89.76% for batch I and batch II, respectively.

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Table 22

Comparative analysis of stability studies

Compound Area (0 days) Quantitative analysis (0 days) Area (75 days) Quantitative analysis (75 days) Teriparatide + 2203 mAU*s 98.02% 2087 mAU*s 92.88% Chenodeoxycholic acid (Batch I) Teriparatide + 2385 mAU*s 106.0% 2016mAU*s 89.76%

piperine (Batch II)

Procedure for quantitative analysis of leuprolide acetate for rat studies.

The calibration curve for leuprolide acetate was obtained using HPLC; approximately 112 mg and 104 mg of beads (equivalent to 100 μg) were accurately weighed and diluted with 1 ml of mobile phase. This gives a dispersion with a theoretical concentration of 100 µg/mL for leuprolide acetate. The dispersion was vortexed for 2 min and then filtered through a syringe filter with a pore size of 0.2 μm. Then, 20.0 μL of this filtered solution was injected into the HPLC system to determine the peptide content.

Observation. From the equation (y = 42.67x - 76.447, obtained from HPLC studies for the peptide), it was found that for leuprolide acetate, the % peptide content is (A) leuprolide acetate + labrazole granules ALF (PA 2): 97.45%; and (B) leuprolide acetate + labrazole ALF + piperine granules (PA 2+3): 105.11%.

Dissolution study of leuprolide capsules.

A formulation containing Labrazole ALF with leuprolide (PA 2) was selected for dissolution studies (based on the results of quantitative analysis).

Scheme 1: use of a dialysis membrane. Medium: phosphate buffer solution pH 6.80; volume: 10 ml; output volume: 400 µl; shaking speed: 100 rpm; granule filling: 140 mg (equivalent to 1 capsule); Dialysis membrane specification: HIMEDIA LM395-30MT; pore size: 25 nm; average flat width: 29.31 mm; average diameter: 17.5 mm.

Table 23

Leuprolide release study using a dialysis membrane

Time Square o/ /0 Releases 5 0 Leuprolide is not released through the dialysis membrane 10 0 15 0 20 0 25 0

Scheme 2: use of a rotating basket (USP TYPE 1). Medium: phosphate buffer solution pH 6.80; volume: 25 ml; output volume: 400 µl; shaking speed: 100 rpm; granule filling: 140 mg per gelatin capsule.

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Table 24

In vitro release data for regimen 2

Time (min) Area hell concentration (µg/ml) concentration (µg/0.5 ml) Error concentration (µg/25 ml) CDR %CDR 0 0 0 0 0 0 0 0 5 214.9 6.82791188 2 3.41395594 1 3.4139559 170.697797 174.111753 13.92894 10 400.76 11.1836653 4 5.59183266 9 5.5918327 279.591633 5 285.183466 22.81468 15 279.59 8.34396531 5 4.17198265 8 13.177771 208.599132 9 221.776904 17.74215 20 366.37 10.3777126 8 5.18885633 9 18.366628 259.442817 277.809445 22.22476 thirty 370.09 10.4648933 7 5.23244668 4 23.599074 261.622334 2 285.221408 22.81771 45 330 9.52535739 4 4.76267869 7 28.361753 238.133934 8 266.495688 21.31966 60 456.86 12.4984063 7 6.24920318 7 34.610956 312.460159 4 347.071116 27.76569 90 321.25 9.32029528 9 4.66014764 5 39.271104 233.007382 2 272.278486 21.78228

Scheme 3: use of a rotating basket (USP TYPE 1). Medium: phosphate buffer solution pH 6.80; volume: 30 ml; output volume: 400 µl; shaking speed: 500 rpm; granule filling: 140 mg per gelatin capsule.

Table 25

In vitro release data for regimen 3

Time (min) Square concentration (µg/ml) Concentration (µg/0.4 ml) Error concentration (µg/30 ml) CDR %CDR 0 0 0 0 0 0 0 0 5 229.95 7.1806187 02 2.8722474 81 2.8722475 215.41856 1 218.29080 9 17.46326 10 215.206 6.8350831 97 2.7340332 79 2.7340333 205.05249 59 207.78652 9 16.62292 20 258,028 7.8386454 18 3.1354581 67 8.7417389 235.15936 25 243.90110 1 19.51209 thirty 218.98 6.9235294 12 2.7694117 65 11.511151 207.70588 24 219.21703 3 17.53736 45 236.02 7.3228732 13 2.9291492 85 14.4403 219.68619 64 234.12649 6 18.73012 60 337.56 9.7025310 52 3.8810124 21 18.321312 291.07593 16 309.39724 4 24.75178

Scheme 4: using a rotating basket (USP TYPE 1). Medium: phosphate buffer solution pH 6.80; volume: 30 ml; output volume: 400 µl; shaking speed: 500 rpm; granule filling: 140 mg per hypromellose capsule (size 0).

- 37 044859

Table 26

In vitro release data for regimen 4

Time (min) Area hell concentration (µg/ml) concentration (µg/0.4 ml) Error concentration (µg/30 ml) CDR %CDR 0 0 0 0 0 0 0 0 uh 0 1.791586595 0.716634638 0.716634 6 53.74759784 54.4642325 4.35713 9 15 343.5 9.841738927 3.936695571 3.936695 6 295.2521678 299.188863 23.9351 1 thirty 416.63 11.55558941 4.622235763 9.275566 346.6676822 355.943248 28.4754 6 45 527.55 14.15507382 5.662029529 14.93759 6 424.6522147 439.58981 35.1671 8 90 428.46 11.83283337 4.733133349 19.67072 9 354.9850012 374.65573 29.97 246

Diagram 5: Using a rotating basket (USP TYPE 1). Medium: phosphate buffer solution pH 6.80; volume: 30 ml; output volume: 500 µl; shaking speed: 100 rpm; granule filling: 140 mg without capsules directly into the basket.

Table 27

In vitro release data for regimen 5

Time (min) Square concentration (µg/ml) concentration (µg/0.5 ml) Error concentration (µg/30 ml) CDR %CDR 0 0 0 0 0 0 0 0 5 1491.97 36.7569018 18.3784509 18.378451 1102.70705 4 1121.08551 89.68684 10 1550.89 38.1377314 3 19.0688657 1 19.068866 1144.13194 3 1163.20081 93.05606 15 1280.8 31.8079915 6 15.9039957 8 53.351312 954.239746 9 1007.59106 80.60728 20 1422 35.1171080 4 17.5585540 2 70.909866 1053.51324 1 1124.42311 89.95385

Scheme 6: using a magnetic stirrer. Medium: phosphate buffer solution pH 6.80; volume: 30 ml; output volume: 500 µl; shaking speed: 200 rpm; granule filling: 140 mg in gelatin capsule size 2.

Table 28

In vitro release data for regimen 6

Time (min) Square concentration (µg/ml) concentration (µg/0.5 ml) Error concentration (µg/30 ml) CDR %CDR 0 0 0 0 0 0 0 0 5 1761.8 43.080548 39 21.540274 2 21.540274 1292.4164 52 1313.9567 3 105.1165

-38044859

10 1771.8 43.314905 09 21.657452 54 21.657453 1299.4471 53 1321.1046 1 105.6884 15 1750 42.804007 5 21.402003 75 64.59973 1284.1202 25 1348.7199 6 107.8976 20 1736.9 42.497000 23 21.248500 12 85.848231 1274.9100 07 1360.7582 4 108.8607

Scheme 7: using a magnetic stirrer. Medium: phosphate buffer solution pH 6.80; volume: 30 ml; output volume: 500 µl; shaking speed: 200 rpm; granule filling: 140 mg in gelatin capsule size 2.

Table 29

In vitro release data for regimen 7

Time (min) Area hell concentration (µg/ml) concentration (µg/0.5 ml) Error concentration (µg/30 ml) CDR %CDR 0 0 0 0 0 0 0 0 2.5 1440.6 35.5530114 8 17.7765057 4 17.776506 1066.59034 5 1084.36685 86.74935 5 1670.9 40.9502460 7 20.4751230 4 20.475123 1228.50738 2 1248.98251 99.9186 10 1603 39.3589641 4 19.6794820 7 57.931111 1180.76892 4 1238.70004 99,096 15 1638.4 40.1885868 3 20.0942934 1 78.025404 1205.65760 5 1283.68301 102.6946

Based on the data presented in table. 24-29, it can be noted that leuprolide was not released from the granules through the dialysis membrane (Scheme 1); a rotating basket (USP TYPE 1) was used for scheme 2, and the rotation of the basket was not able to generate sufficient rotational motion to move/rotate the material in the medium and, therefore, the material along with the gelatin settled, ultimately negatively affecting the release; Scheme 3 was carried out by increasing the number of revolutions per minute of the basket (100-500), but even after increasing the number of revolutions per minute of the basket, the % CDR was found to be 24.75%; scheme 4 was carried out with an increased number of revolutions per minute and replacing the gelatin capsule with a hypromellose capsule, and the % CDR was found to be 29.97%; scheme 5 was carried out in a rotating basket without a capsule, the % CDR was found to be 90%, which confirms that the material (gelatin/hypromellose) can increase the viscosity and slow down the release of leuprolide from the granules; Schemes 6 and 7 were carried out using a magnetic stirrer to generate suitable rotation of the media during quantitative analysis, and the % CDR was found to be 108.8 and 102.6%, respectively; All experimental schemes were carried out at 37°C, however, temperature changes negatively affect the disintegration of the capsule, the disintegration time at 25°C was 12 minutes, and the disintegration time at 37°C was less than 2 minutes.

Quantitative analysis of sodium liraglutide using HPLC.

Test 1.

Procedure. Approximately 10.0 mg of granules, PA 2 (Table 16F) and PA 3+4 (Table 16G) were diluted with 10.0 ml of HPLC diluent (10% ACN in D.I. water). This gives us a dispersion with a theoretical concentration of 74 µg/mL for sodium liraglutide. The dispersion was sonicated for 5.0 min in an ultrasonic bath and then filtered through a syringe filter with a pore size of 0.2 μm. Then, 20.0 μL of this filtered solution was injected into the HPLC system to determine the peptide content.

Observation. From the equation (y=79.283x-571.72, obtained as a result of HPLC studies for the peptide), it was found that the content for the composition of sodium liraglutide with Labrazole ALF granules (PA 2) is 44.4 and 25.4% for the composition of liraglutide sodium with piperine and solutol HS-15 (RA 3+4).

(a) Quantitative analysis for the formulation of sodium liraglutide with piperine and HS-15 solutol: 67.39% was vortexed for 2 min.

(b) Quantitative analysis for the formulation of sodium liraglutide with Labrazole ALF granules: 80.43% was vortexed for 2 min.

(c) Quantitative analysis for the composition of sodium liraglutide with piperine and HS-15 solutol: 95.66% was vortexed for 5 min.

- 39 044859 (d) Quantitative analysis for the formulation of sodium liraglutide with Labrazole ALF granules: 96.99% vortexed for 5 min.

Study of the solubility of liraglutide.

Medium: phosphate buffer solution pH 6.80; volume: 30 ml; output volume: 500 µl; shaking speed: 200 rpm.

Table 30

In vitro release data for liraglutide PA 2 (Table 16F)

Time (min) Square concentration (µg/ml) concentration (mcg/1ml) Error concentration (µg/100ml) CDR %CDR 0 0 0 0 0 0 0 0 5 5050 70.90700402 35.45350201 35.45350201 7090.700402 7126.153904 59.38461587 7.5 5085.23 71.35136158 35.67568079 35.67568079 7135.136158 7170.811839 59.75676532 10 7804.21 105.6459771 52.82298853 123.9521713 10564.59771 10688.54988 89.07124899 15 9447.27 126.3699658 63.18498291 187.1371542 12636.99658 12824,13374 106.8677811 20 9912.43 132.2370496 66.11852478 253.255679 13223.70496 13476.96063 112.3080053 thirty 9912.43 132.2370496 66.11852478 319.3742038 13223.70496 13543.07916 112.858993

Table 31

In vitro release data for liraglutide RA 3+4 (Table 16G)

Time (min) Square concentration (µg/ml) concentration (µg/1ml) Error concentration tion (µg/100m k) CDR %CDR 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 7.5 6823.8 93.2800222 46.6400111 46.6400111 9328.00222 9374.64223 1 78.1220185 9 10 7692.74 104.240001 52.1200005 98.7600116 10424.0001 10522.7601 1 87.6896676 15 7778.6 105.322957 52.6614785 151.421490 1 10532.2957 10683.7171 9 89.0309765 9 20 7987.41 107.956686 8 53.9783434 205.399833 5 10795.6686 8 11001.0685 1 91.6755709 5 thirty 8131.53 109.774478 8 54.8872393 8 260.287072 9 10977.4478 8 11237.7349 93.6477912

Observation. The % CDR was found to be 112.85 and 93.64% for liraglutide RA 2 and RA 3+4, respectively. It was found that more than 50% of sodium liraglutide is released within 5-7 minutes.

Quantitative determination of glucose and insulin glargine levels in the blood plasma of STZ-induced diabetic rats.

The levels of glucose and insulin glargine in blood plasma were quantified after administration of several formulations of insulin glargine into the midjejunum of STZ-induced Sprague-Dawley rats with diabetes mellitus.

Test composition I. Insulin glargine (Lantus®) - appearance: injection solution in a pre-filled pen syringe; concentration: 100IU/ml; storage condition: 2-8°C; dose: 0.2 units/kg; route: subcutaneous.

Test composition II. Composition of Insulin glargine - MIRA 1 (reduced glutathione/chromium picolinate from Table 15A) + permeability enhancer 1 (PA 1 from Table 16A) (oral solution in TRIS buffer solution); dose: 1.7 units/animal; route: middle part of the jejunum.

Test composition III. Composition of Insulin glargine - RA 2 (from Table 16B) and MIRA 2 (sodium ascorbate/vanadium oxide from Table 15B) (oral solution in TRIS buffer solution); dose: 1.7 units/animal; route: middle part of the jejunum.

Observations. No deaths were observed following subcutaneous or midjejunal administration of insulin glargine. The observed clinical symptoms are normal. Regarding sampling time points, during dosing, blood collection time points are located

- 40 044859 were applied at 0, 20, 40, 60, 120 and 150 minutes after dosing. —100 µl Blood was collected into an Eppendorf prefilled with Na-EDTA from a puncture of the retro-orbital sinus. The blood was centrifuged at 5000 rpm for 5 min at 4°C to obtain plasma. Glucose was measured immediately after blood collection using a glucometer.

Table 32

Effect of treatment with different compounds on STZ-induced diabetic rats

Groups Quantities 0 % inhibition (min) 20 40 60 120 150 Carrier 2 L6 -1.6 -2.2 -1.5 -1.8 STZ + Insulin glargine (0.2 U/kg) 3 22.9 25.5 22 39 17.2 STZ + Test composition II 3 -23.2 -15.1 -14.7 -13.2 -15.4 STZ + Test composition III 3 -3.5 -13.1 0.8 8.8 10

Table 33

Bioavailability of insulin glargine

Groups Quantity AUC±SEM Carrier 2 67705 ± 2965 STZ + Insulin glargine (0.2 U/kg) 3 39408 ± 8561 STZ + Test composition II 3 80133 ±5168 STZ + Test composition III 3 57315 ±3474

ELISA study.

Source. Invitron Ltd Catalog No. MBS495369.

Principle. This glargine ELISA is a two-step immunoassay using a monoclonal antibody immobilized in microtiter wells and a soluble horseradish peroxidase (HRP)-labeled antibody.

The plasma sample is incubated in a microtiter well together and after a washing step, the antibody-HRP conjugate solution is added. After the second incubation, a further washing step occurs to remove unbound antibody-HRP conjugate before measurement. Enzyme substrate is added to each well and after a short incubation, another reagent is added to stop the reaction. The intensity of color appearing in each well is quantified in a microtiter plate reader set to record transmitted light at a wavelength of 450 nm (kit protocol: Manufacturer's recommended protocol was used, Catalog No. MBS495369).

Procedure. Bring all kit components and samples to room temperature before use. Collect the required number of coated strips in the plate holder. For strips not used immediately, they can be stored in a sealed plastic bag with silica gel desiccant. Ensure that the remaining spaces in the plate holder are filled with uncoated strips to ensure even heat transfer during incubation. Pipette 100 µl of sample buffer solution into each well. Pipette 25 µl of standard or sample solution into the appropriate wells. It is recommended that all standard and reference solutions be used in duplicate. Attach the plate sealer and incubate for 2 hours at room temperature (18-22°C). Remove the plate sealer and perform 3 wash cycles using chilled* working wash buffer (300 µL each cycle) using an automatic plate washer. Pipette 100 µl of the antibody conjugate working solution into each well. Attach the plate sealer and incubate for another 4 hours at 4°C (2-8°C). Remove the plate sealer and perform 3 wash cycles using chilled* working wash buffer in an automatic plate washer. Add 100 µl of substrate solution to each well. Incubate for 15 minutes at room temperature (18-22°C) in the dark. Add 100 µl stopping solution to each well. Measure light transmittance in a microtiter plate reader set at 450 nm, with background subtraction measured at OD 620/650 nm if possible. Illustrated in the drawing is a graph depicting the concentration-time profile of insulin glargine (mU/L) from various formulations.

The test formulation insulin glargine-MIRA 1 (reduced glutathione/chromium picolinate) plus penetration enhancer 1 (PA 1) was found to exhibit a relative bioavailability of 9.25%, and the test formulation insulin glargine-PA 2 and MIRA 2 (ac

-41 044859 sodium corbate/vanadium oxide) exhibits a relative bioavailability of 28.86%.

Quantitative determination of leuprolide levels in canine plasma using an ELISA kit.

Background information on the tested composition: luprodex (depot). Concentration: each vial will contain 3.75 mg of leuprolide acetate; date of manufacture: November 2017; expiration date: October 2020; storage condition: store at room temperature (below 25°C); do not freeze; number of bottles: 1 bottle with diluent.

Test composition FB: MIRA 5 (Table 15E) + RA 2 (Table 16H) - appearance: hard gelatin capsule with a white cap and white body; concentration: each 300 mg capsule contains 1.25 mg of leuprolide acetate; date of manufacture: April 21, 2018; expiration date: no data; storage condition: store at room temperature (below 25°C); number of capsules of the test substance: 70 capsules (1 bottle).

Test composition H: MIRA 2 (Table 15F)+PA 2+3 (Table 16I) - appearance: hard gelatin capsule with a white cap and white body; concentration: each 300 mg capsule contains 1.25 mg of leuprolide acetate; date of manufacture: April 19, 2018; expiration date: no data; storage condition: store at room temperature (below 25°C); number of capsules of the test substance: 70 capsules (1 bottle).

Table 34

Study plan

Number of animals Route of administration Compound Dose Group 1 2 Subcutaneous Reference Leuprorelin, as a label (one implant at the beginning of the program) Group 2 2 Oral Composition H (MIRA 2) Leuprorelin 1.25 mg, one capsule once a day for 30 days Group 3 2 Oral FB Composition (MIRA 5) Leuprorelin 1.25 mg, one capsule once a day for 30 days

During the dosing period, dogs (Canis familiaris, Beagle breed) were fasted (water allowed) overnight for approximately 12 hours before and 4 hours after dosing. Following drug administration, all animals were monitored for adverse clinical signs for up to 720 hours post-dose. The body weight of the dogs used in the study was recorded before dosing began.

Time points for sampling.

Approximately 2 mL of blood sample from each dog for the subcutaneous and oral dosing routes was collected at the following time points: 0, 1, 2, 6, 12, 24, 48, 72, 96, 120, 240, 312, 360, 480, and 720 h (15 points in total) from the jugular vein into labeled K2EDTA-coated tubes.

ELISA study data.

Source: Catalog No. S-1174 (Des-Gly10, D-Leu6, Pro-NHEt9)-LHRH (Leuprolide).

Recruitment protocol: Manufacturer's recommended protocol (catalog no. S1174) was used.

Results: Test formulation FB was found to exhibit a relative bioavailability of 56.53%, and test formulation H was found to exhibit a relative bioavailability of 16%.

Quantitative determination of liraglutide plasma levels in dogs using an ELISA kit.

Test composition I: liraglutide - appearance: pre-filled injection solution

- 42 044859 line syringe pen; concentration: 6 mg/ml; date of manufacture: 02/2017; expiration date: 07/2019; storage condition: 2-8°C; dose: 0.6 mg/dog; route: subcutaneous.

Test composition II (FA): MIRA 5 (Table 15E) + RA 2 (Table 16F) - dose: 12 mg (one capsule)/dog; route: oral.

Test composition III (G): MIRA 5 (Table 15E) + RA 3+4 (Table 16G) - dose: 12 mg (one capsule)/dog; route: oral.

Table 35

Study plan

Number of animals Route of administration Compound Dose Period 1 2 Subcutaneous Composition I Liraglutide 0.6 mg/dog 4-5 days of hatching Period 2 2 Oral Composition II FA 12 mg (one capsule)/dogs A 4-5 days of hatching Period 3 2 Oral Composition III G 12 mg (one capsule)/dogs A

No deaths were observed following subcutaneous or oral administration of liraglutide. The observed clinical symptoms are normal. The body weight of the dogs used in the study was recorded before dosing began. During the dosing period, dogs (Canis lupus familiaris, Beagle breed) were fasted overnight (water allowed) for approximately 12 hours before and 4 hours after dosing. During dosing, blood collection time points were 0, 20, 30, 60, 120, 180, 240, 480 min after dosing. 2 ml of Blood was collected from the jugular vein into labeled K2EDTA coated tubes. Glucose was measured immediately after blood collection using a glucometer.

ELISA study data.

Source: Krishgen BioSystems Catalog No. KBI5020 Ver2.0.

Recruitment protocol: Manufacturer's recommended protocol (catalog no. KBI5020) Ver2.0 was used.

(1) Identify wells for diluted standard, blank, and sample solution. Prepare 5 time point wells for standard solution, 1 well for blank solution. Add 50 µl of each diluted standard (see reagent preparation), blank and sample solution to the corresponding wells, respectively. And then immediately add 50 µl of liraglutidebiotin to each well. Gently shake the plate (microplate shaker recommended). Cover with a tablet sealer. Incubate for 1 hour at 37°C. Liraglutide Biotin may appear opaque. Warm to room temperature and stir gently until the solution is smooth.

(2) Aspirate the solution and wash 350 µL of wash solution 1x each well using a spray bottle, multi-channel pipette, multi-channel pipette or car wash, and leave for 1-2 minutes. Completely remove any remaining liquid from all wells by snapping the plate onto absorbent paper. Repeat 3 times. After the final wash, remove any remaining wash buffer by aspiration or decantation. Turn the plate over and blot it with absorbent paper.

(3) Add 100 μL streptavidin-HRP working solution to each well. Incubate for 30 min at 37°C after covering with a plate sealer.

(4) Repeat the aspiration/flushing process a total of 5 times as described in step 2.

(5) Add 90 µL of substrate solution to each well. Cover with a new tablet sealer. Incubate for 10-20 minutes at 37°C (no more than 30 minutes). Keep away from light. When adding substrate solution, the liquid will turn blue.

(6) Add 50 µL stopping solution to each well. When adding stopping solution, the liquid will turn yellow. Mix the liquid by tapping the edge of the plate. If the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

(7) Remove any water drops or fingerprints from the bottom of the tablet and make sure it is

- 43 044859 There are no bubbles on the surface of the liquid. Then start the microplate reader and immediately take a measurement at 450 nm.

Test formulation FA (Mira 5 plus liraglutide+labrazole) was found to exhibit a relative bioavailability of 3.82%, and test formulation G (Mira 5 plus liraglutide+solutol+piperine) was found to exhibit a relative bioavailability of 3. 57%.

Quantitative determination of octreotide levels in rat plasma using an ELISA kit.

Test composition I: octreotide - appearance: solution for injection; concentration: 0.1 mg/ml; storage condition: 2-8°C; dose: 10 units/kg; route: subcutaneous.

Test composition II: MIRA 3 (Table 15C)+PA 1 (Table 16C) for dosing into the distal small intestine (ileum) of an anesthetized Sprague-Dawley rat; dose: 144 mcg/animal; route: distal small intestine (ileum). The animals were fed during the experiment.

Table 36

Study plan

Groups Quantity Description of dose Octreotide 3 subcutaneously MIRA 3 + RA 1 3 MIRA3 and PA1 mixed with Tris buffer solution (2 ml/kg)

No deaths were observed following subcutaneous or distal small bowel (ileum) administration of octreotide. The observed clinical symptoms are normal. During dosing, blood collection time points were 0, 7, 15, 30, 45, 60, and 90 min postdose. ~100 μl of Blood was collected into an Eppendorf prefilled with Na-EDTA from a puncture of the retroorbital sinus. The blood was centrifuged at 5000 rpm for 5 min at 4°C to obtain plasma. ELISA Study Data

Source: Peninsula Laboratories International, Inc, Catalog No. S-1341.0001.

Kit protocol: we used the protocol recommended by the manufacturer (catalog No. S-1341.0001) - add 25 μl of antiserum (in ELISA buffer solution) to each well of the immunoplate. Add 25 µl of ELISA buffer solution to the empty wells; Incubate at room temperature for 1 hour. Add 50 µl of standard or sample solution (in diluent). Do not rinse the plate before adding. Add 50 µl of diluent to the empty wells. Incubate at room temperature for 2 hours. Shorter preincubations may result in decreased sensitivity. Rehydrate the Bt indicator (in ELISA buffer) and add 25 µl/well. Incubate at 4°C overnight. For best results, return to room temperature before continuing. Wash the immunoplate 5 times with 300 µl/well ELISA buffer solution. Be very careful not to allow cross-contamination between wells in the first wash/dispense cycle. With each rinse cycle, dry the contents of the tablet with a quick, oscillating motion of your wrist, then gently pat the top of the tablet dry with paper towels. Dispense 300 µl of ELISA buffer solution into each well and shake gently for at least a few seconds. Thorough rinsing is necessary. Add 100 µl/well streptavidin-HRP. Evacuate or centrifuge the SAHRP vial to collect any liquid contents at the bottom of the vial. Dilute 1/200 in ELISA buffer solution (60 µl/12 ml) and vortex. Add 100 µl to all wells, including empty wells. Incubate at room temperature for 1 hour. Wash the immunoplate 5 times (see step 7). Add 100 µl/well TMB solution. Add to all wells, including empty ones. Incubate at room temperature (usually 30-60 minutes). You can read the developed blue color at 650 nm and use the data for your calculations. Stop reactions by adding 100 µl 2 N HCl per well. Read absorbance at 450 nm for 10 min. The test formulation MIRA 3 (uric acid: sodium vanadate)+PA 1 was found to exhibit a relative bioavailability of 0.41%.

Quantitative determination of teriparatide levels in rat plasma using an ELISA kit.

Test composition I: teriparatide. Appearance: solution for injection; concentration: 600 units/2.4 ml; storage condition: 2-8°C; dose: 10 units/animal; route: subcutaneous test formulation II: MIRA 4 (Table 15D) plus PA 1 (Table 16D) was administered into the distal small intestine (ileum) at a dose of 240 μg/animal in anesthetized rats; dose: 240 mcg/animal; route: distal small intestine (ileum).

Test composition III: MIRA 1 (Table 15A) + RA 3 (Table 16E) was administered into the distal small intestine (ileum) at a dose of 240 μg/abdomen in Sprague-Dawley rats that had undergone

- 44 044859 pain relief; dose: 240 mcg/animal; route: distal small intestine (ileum).

Table 37

Study plan

Groups Quantity Description of dose Teriparatide 3 subcutaneously MIRA 4 + RA 1 3 MIRA 4 and PA 1 mixed with Tris buffer solution (2 ml/kg) MIRA 1 + RA 3 3 MIRA 1 and PA 3 mixed with Tris buffer solution (2 ml/kg)

The animals were fed during the experiment. No deaths have been observed following subcutaneous or distal small bowel (ileum) administration of teriparatide. The observed clinical symptoms are normal. During dosing, blood collection time points were 0, 7, 15, 30, 45, 60, and 90 min postdose. ~100 μl of Blood was collected into an Eppendorf prefilled with Na-EDTA from a puncture of the retro-orbital sinus. The blood was centrifuged at 5000 rpm for 5 min at 4°C to obtain plasma.

ELISA study data.

Source: Immutopics, Catalog #60-3900.

Kit Protocol: Using the manufacturer's recommended protocol (cat. no. 60-3900), place a sufficient number of streptavidin-coated strips in the holder to run PTH standard solutions, control solutions, and unknown sample solutions. Pipette 150 µl of standard, control or sample solution into the designated or designated well. Freeze remaining standard and control solutions as soon as possible after use. Pipette 50 µl of antibody working solution consisting of 1 part HRP antibody and 1 part biotinylated antibody into each well. Cover the plate with one plate sealer and then cover with aluminum foil to avoid exposure to light. Incubate the plate at room temperature for 3 hours on a horizontal rotator set at 180-220 rpm. Remove the aluminum foil and tablet sealer. Using an automatic microtiter plate washer, aspirate the contents of each well. Wash each well five times by dispensing 350 µL of working wash solution into each well and then aspirate the contents completely. A suitable suction device can also be used. Pipette 200 µl of HRP ELISA substrate into each well. Cover the plate again with the plate sealer and aluminum foil. Incubate at room temperature for 30 minutes on a horizontal rotator set at 180-220 rpm; Remove the aluminum foil and tablet sealer. Read the absorbance at 620 nm (see note) for 5 min in the microtiter plate reader against the wells of the 0 pg/mL standard solution as a blank solution. Immediately pipette 50 µl of ELISA stopping solution into each well. Mix on a horizontal rotator for 1 minute. Read absorbance at 450 nm for 10 min in a microtiter plate reader against a reagent blank of 200 µL substrate and 50 µL stopping solution. If dual wavelength correction is available, then set the measurement wavelength to 450 nm and the reference wavelength for absorbance used in step #9.

The test formulation MIRA 4 (sodium ascorbate: manganese gluconate) plus PA 1 was found to exhibit a relative bioavailability of 0.89%. The test formulation MIRA 1 (reduced glutathione/chromium picolinate) plus PA 3 was found to exhibit a relative bioavailability of 0.89%.

Although the foregoing describes various embodiments of the present invention, other and additional embodiments of the present invention may be developed without departing from its basic scope. The scope of the present invention is defined by the claims, which are presented below. The present invention is not limited to the described embodiments, versions or examples, which are included to enable a person of ordinary skill in the art to make and use the present invention in combination with information and knowledge available to a person of ordinary skill in the art.

Advantages of the invention

The present invention provides a pharmaceutical composition that can overcome the disadvantages associated with existing prior art compositions.

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Claims (13)

The present invention provides a pharmaceutical composition for effective delivery of a peptide. The present invention provides a pharmaceutical composition for oral delivery of a peptide. The present invention provides a pharmaceutical composition that protects at least part of the peptide from proteolytic degradation when ingested. The present invention provides a pharmaceutical composition that increases the bioavailability of the peptide. The present invention provides a pharmaceutical composition that is safe. The present invention provides a pharmaceutical composition that is cost effective, easy to prepare and has a long shelf life. CLAIM 1. A pharmaceutical composition containing a pharmaceutically effective amount of at least one peptide selected from the following group: insulin, liraglutide, octreotide, teriparatide, leuprolide; and a pharmaceutically acceptable amount of the combination of (a) at least one metal in the form of a salt thereof or a combination of a salt and a complex thereof; and (b) at least one reducing agent selected from the following group: sodium ascorbate, reduced glutathione, uric acid, mannitol, benzohydroxamic acid, cysteine, piperine, and any combination thereof, characterized in that said at least one metal is selected from any of the following or combinations thereof: vanadium(V) oxide, sodium vanadate, vanadium sulfate, vanadyl sulfate, vanadium biguanide, bis(maltolato)oxavandium(IV), vanadium acetate, vanadyl picolinate, vanadyl citrate, chromium picolinate, chromium polynicotinate, chromium nicotinate , chromium chloride, chromium acetate, manganese gluconate, manganese sulfate, potassium permanganate and manganese chloride, wherein said pharmaceutical composition additionally contains at least one absorption enhancer selected from the following group: chenodeoxycholic acid, labrazole ALF, piperine, solutol HS 15 - and any combination thereof. 2. The pharmaceutical composition according to claim 1, characterized in that said at least one metal is selected from any of the following or a combination thereof: vanadium (V) oxide, sodium vanadate and vanadium sulfate, wherein the pharmaceutical composition contains at least one specified metal in an amount from 0.01 to 15 mg per dose unit. 3. The pharmaceutical composition according to claim 1, characterized in that said at least one metal is selected from any of the following or a combination thereof: vanadium (V) oxide, sodium vanadate and vanadium sulfate. 4. The pharmaceutical composition according to claim 1, characterized in that said at least one metal is selected from any of the following or a combination thereof: chromium picolinate and chromium chloride, wherein the pharmaceutical composition contains at least one of said metal in an amount of from 0. 02 to 0.5 mg per dose unit. 5. The pharmaceutical composition according to claim 1, characterized in that said at least one metal is selected from any of the following or a combination thereof: chromium picolinate and chromium chloride. 6. The pharmaceutical composition according to claim 1, characterized in that said at least one metal is selected from any of the following or a combination thereof: potassium permanganate and manganese gluconate, wherein the pharmaceutical composition contains at least one of said metal in an amount of from 0. 1 to 10 mg per dose unit. 7. The pharmaceutical composition according to claim 1, characterized in that said at least one metal is selected from any of the following or a combination thereof: manganese gluconate and potassium permanganate. 8. Pharmaceutical composition according to claim 1, characterized in that said at least one peptide has a molecular weight equal to or less than 60 kDa. 9. The pharmaceutical composition according to claim 1, characterized in that said at least one peptide and said at least one metal in the form of a salt thereof or a combination of a salt and a complex thereof are present in a physically separated form in said pharmaceutical composition. 10. The pharmaceutical composition according to claim 1, characterized in that said at least one peptide and said at least one metal in the form of a salt thereof or a combination of a salt and a complex thereof are present in separate compartments. 11. The pharmaceutical composition according to claim 1, characterized in that said pharmaceutical composition is present in the form of any of the following: capsule-in-capsule and tablet-in-capsule. 12. The pharmaceutical composition according to claim 1, characterized in that said pharmaceutical composition contains at least one reducing agent in an amount of 1 to 1000 mg per dose unit. 13. Pharmaceutical composition according to claim 1, characterized in that at least one absorption enhancer is present in an amount of from 10 to 1000 mg per dose unit. -