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US20030198666A1 - Oral insulin therapy - Google Patents

Oral insulin therapy Download PDF

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US20030198666A1
US20030198666A1 US10/237,138 US23713802A US2003198666A1 US 20030198666 A1 US20030198666 A1 US 20030198666A1 US 23713802 A US23713802 A US 23713802A US 2003198666 A1 US2003198666 A1 US 2003198666A1
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Prior art keywords
insulin
oral
concentration
cnab
dose
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Richat Abbas
Michael Goldberg
T. Woods
Steven Dinh
Ehud Arbit
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Emisphere Technologies Inc
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Individual
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Priority to US10/237,138 priority Critical patent/US20030198666A1/en
Priority to CA2471769A priority patent/CA2471769C/fr
Priority to AU2003226436A priority patent/AU2003226436B2/en
Priority to PCT/US2003/000337 priority patent/WO2003057170A2/fr
Priority to JP2003557529A priority patent/JP4659358B2/ja
Priority to US10/500,822 priority patent/US7429564B2/en
Priority to EP03729352.9A priority patent/EP1469812B1/fr
Assigned to EMISPHERE TECHNOLOGIES, INC. reassignment EMISPHERE TECHNOLOGIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WOODS, T. COOPER, ABBAS, RICHAT, DINH, STEVEN, ARBIT, EHUD, GOLDBERG, MICHAEL M.
Publication of US20030198666A1 publication Critical patent/US20030198666A1/en
Abandoned legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • This invention relates to the oral delivery of therapeutic proteins in a therapeutically effective amount to the bloodstream.
  • This invention further relates to oral administration of proteins as active agents as part of a therapeutic regimen.
  • This invention further relates to the oral administration of insulin in a therapeutically effective amount for the treatment of diabetes.
  • This invention further relates to compositions of a delivery agent and insulin for oral administration that facilitates insulin transport in a therapeutically effective amount to the bloodstream for the treatment of diabetes.
  • This invention further provides methods for the preparation of a composition comprising insulin for oral administration.
  • the present invention further relates to methods for reducing adverse effects on the vascular system that are associated with insulin therapy. More specifically, the present invention relates to methods that reduce the incidence of diseases associated with chronic administration of insulin. The present invention is also directed to oral pharmaceutical dosage forms that are administrable on a chronic basis to diabetics, in part to achieve such results.
  • Proteins, carbohydrates and other biological molecules are finding increasing use in many diverse areas of science and technology. For example, proteins are employed as active agents in the fields of pharmaceuticals, vaccines and veterinary products.
  • biological macromolecules are often severely limited by the presence of natural barriers of passage to the location where the active agent is required. Such barriers include the skin, lipid bi-layers, mucosal membranes, severe pH conditions and digestive enzymes.
  • Oral delivery of active agents is a particularly desirable route of administration, because of safety and convenience considerations.
  • oral delivery can eliminate the risk of infection through the use of needles and syringes.
  • oral delivery provides for more accurate dosing than multidose vials and can minimize or eliminate the discomfort that often attends repeated hypodermic injections.
  • biological macromolecules are large and are amphipathic in nature. More importantly, the active conformation of many biological macromolecules may be sensitive to a variety of environmental factors, such as temperature, oxidizing agents, pH, freezing, shaking and shear stress. In planning oral delivery systems comprising biological macromolecules as an active agent for drug development, these complex structural and stability factors must be considered.
  • pancreas One specific biological macromolecule, the hormone insulin, contributes to the normal regulation of blood glucose levels through its release by the pancreas, more specifically by the ⁇ -cells of a major type of pancreatic tissue (the islets of Langerhans). Insulin secretion is a regulated process which, in normal subjects, provides stable concentrations of glucose in blood during both fasting and feeding. Diabetes is a disease state in which the pancreas does not release insulin at levels capable of controlling glucose levels. Diabetes is classified into two types. The first type is diabetes that is insulin dependent and usually appears in young people. The islet cells of the pancreas stop producing insulin mainly due to autoimmune destruction and the patient must inject himself with the missing hormone.
  • Type 1 diabetic patients are the minority of total diabetic patients (up to 10% of the entire diabetic population).
  • type 2 is non-insulin dependent diabetes, which is caused primarily by the resistance of patients to their own insulin. This is the most common type of diabetes in the Western world. Close to 8% of the adult population of various countries around the world, including the United States, have Type 2 diabetes, and about 30% of these patients will need to use insulin at some point during their life span due to secondary pancreas exhaustion.
  • compositions of insulin that maintain protein tertiary structure so as not to alter physiological clinical activity and stability and do not require injections. It would also be desirable to provide compositions of insulin that could be orally administrable, e.g., absorbed from the gastrointestinal tract in adequate concentrations such that oral unit doses of insulin are bioavailable and bioactive after oral administration. Oral absorption allows delivery directly to the portal circulation.
  • Insulin exemplifies the problems confronted in the art in designing an effective oral drug delivery system for biological macromolecules.
  • the medicinal properties of insulin can be readily altered using any number of techniques, but its physicochemical properties and susceptibility to enzymatic digestion have precluded the design of a commercially viable delivery system.
  • a method of providing insulin without the need for injections has been a goal in drug delivery. Insulin absorption in the gastrointestinal tract is prevented by its large size and enzymatic degradation. It would be desirable to create an oral pharmaceutical formulation of a drug such as insulin (which is not normally orally administrable due to, e.g., insufficient absorption from the gastrointestintal tract), which formulation would provide sufficient absorption and pharmacokinetic/pharmacodynamic properties to provide the desired therapeutic effect.
  • a drug such as insulin (which is not normally orally administrable due to, e.g., insufficient absorption from the gastrointestintal tract), which formulation would provide sufficient absorption and pharmacokinetic/pharmacodynamic properties to provide the desired therapeutic effect.
  • Diabetes is the sixth leading cause of death in the United States and accounted for more than 193,000 deaths in 1997. However, this is an underestimate because diabetes contributes substantially to many deaths that are ultimately ascribed to other causes, such as cardiovascular disease. Complications resulting from diabetes are a major cause of morbidity in the population. For example, diabetic retinopathy is the leading cause of blindness in adults aged 20 through 74 years, and diabetic kidney disease accounts for 40% of all new cases of end-stage renal disease. Diabetes is the leading cause for amputation of limbs in the United States. Heart disease and strokes occur two to four times more frequently in adults with diabetes than in those who are healthy. Diabetes causes special problems during pregnancy, and the rate of congenital malformations can be five times higher in the children of women with diabetes.
  • Cardiovascular disease is responsible for up to 80% of the deaths of Type II diabetic patients. See, for example, Kirpichnikov et al., Trends Endocrinol Metab 12, 225-30 (2001); Garcia et al., Diabetes 23, 105-11 (1974); Haffner et al., N Engl J Med 339, 229-34 (1998); Sowers, Arch Intern Med 158, 617-21(1998); Khaw, K. T. et al., Bmj 322, 15-8 (2001).
  • Diabetics have a two- to four-fold increase in the risk of coronary artery disease, equal that of patients who have survived a stroke or myocardial infarction. See, for example, Haffner et al, N Engl J Med 339, 229-34 (1998); Sowers, Arch Intern Med 158, 617-21 (1998).
  • This increased risk of coronary artery disease combined with an increase in hypertensive cardiomyopathy manifests itself in an increase in the risk of congestive heart failure.
  • Stratton et al. Bmj 321, 405-12 (2000); Shindler, D. M. et al. Am J Cardiol 77, 1017-20 (1996).
  • These vascular complications lead to neuropathies, retinopathies and peripheral vascular disease. See Kirpichnikov et al., Trends Endocrinol Metab 12, 225-30 (2001).
  • There is a need for diabetes treatments that will decrease the prevalence of such vascular disease in diabetes patients.
  • compositions comprising a delivery agent and insulin for oral administration.
  • compositions of a delivery agent and insulin for oral administration that facilitates insulin transport in a therapeutically effective amount to the bloodstream for the treatment of diabetes, for the treatment of impaired glucose tolerance, for the purpose of achieving glucose homeostasis, for the treatment of early stage diabetes, and/or for the treatment of late stage diabetes.
  • vascular complications and resultant conditions such as, e.g., retinopathy, neuropathy, nephropathy
  • the invention is directed in part to an oral solid dosage form comprising a dose of unmodified insulin that achieves a comparable reduction in blood glucose concentration in human diabetic patients compared to a subcutaneous insulin injection in those patients, while providing a lower (e.g., 20% or greater) concentration of insulin in the peripheral blood circulation under acute, sub-acute and chronic conditions as compared to the peripheral blood insulin concentration obtained via the subcutaneous injection.
  • the invention is also directed in part to an oral solid dosage form comprising a dose of unmodified insulin that achieves a therapeutically effective reduction in blood glucose after oral administration to a human diabetic patient, and which maintains a physiological gradient, and in certain embodiments provides a ratio of portal vein to peripheral blood insulin concentration from about 2.5:1 to about 6:1, and preferably from about 4:1 to about 5:1.
  • the invention is further directed in part to an oral dosage form comprising a dose of unmodified insulin that achieves a therapeutically effective reduction in blood glucose after oral administration to human diabetic patients, the oral solid dosage form providing an insulin Tmax at a time point from about 0.25 to about 1.5 hours after oral administration to said patients, at least about 80% of the blood glucose concentration reduction caused by said dose of insulin occurring within about 2 hours after oral administration of said dosage form.
  • the invention is further directed in part to an oral dosage form comprising a therapeutically effective amount of unmodified insulin, said dosage form upon pre-prandial oral administration to human diabetic patients causing the mean plasma glucose concentration in said patients to be reduced for the first hour after oral administration relative to a mean baseline (fasted) plasma glucose concentration in said patients.
  • the invention is further directed in part to an oral dosage form comprising a therapeutically effective amount of unmodified insulin, said oral dosage form upon pre-prandial oral administration provides a mean plasma glucose concentration which does not vary by more than about 40% (and more preferably not more than 30%) for the first hour after oral administration, relative to a mean baseline (fasted) plasma glucose concentration in said patients, where a meal is eaten by said patients within about one half hour of oral administration of said dosage form.
  • the oral dosage form is solid, and is preferably provided incorporated within a gelatin capsule or is contained in a tablet.
  • the dose of unmodified insulin contained in the dosage form is from about 50 Units to about 600 Units (from about 2 to about 23 mg), preferably from about 100 Units (3.8 mg) to about 400 Units (15.3 mg) insulin, and most preferably from about 150 Units (5.75 mg) to about 300 Units (11.5 mg), based on the accepted conversion of factor of 26.11 Units per mg.
  • the dosage forms of the invention provide a T max for insulin at about 0.1 to about 1.5 hours, and more preferably by about 0.25 to about 0.5 hours, after oral administration.
  • the T max for insulin occurs at less than about 100 minutes after oral administration of the composition, preferably at less than about 45 minutes, more preferably at less than about 40 minutes, and still more preferably at about 22 minutes after oral administration of the composition.
  • the composition provides a T max for glucose reduction at about 0.25 to about 1.5 hours, more preferably by about 0.75 to about 1.0 hours, after oral administration.
  • the T max for glucose reduction occurs preferably at less than about 120 minutes, more preferably at less than about 80 minutes, and most preferably at about 45 minutes, after oral administration of the composition.
  • the dosage forms begin delivering insulin into the portal circulation (via absorption through the mucosa of the stomach) to achieve peak levels within about 30 minutes or less.
  • the dose of unmodified insulin in the absence of a delivery agent, is not adequately absorbed from the gastrointestinal tract when administered orally to render a desired effect.
  • the dose of insulin in the absence of a delivery agent, is not sufficiently absorbed when orally administered to a human patient to provide a desirable therapeutic effect but said dose provides a desirable therapeutic effect when administered to said patient by another route of adminstration.
  • the invention in such embodiments is further directed to an oral dosage form comprising a dose of unmodified insulin together with a pharmaceutically acceptable delivery agent in an amount effective to facilitate the absorption of said insulin, such that a therapeutically effective amount of said dose of insulin is absorbed from the gastrointestinal tract of human diabetic patients.
  • the pharmaceutical composition comprises from about 1 mg to about 800 mg of said delivery agent, preferably about 50 to about 600, more preferably from about 100 to about 300, most preferably about 200.
  • the composition provides a peak plasma delivery agent concentration C max from about 1,000 and about 150,000 ng/ml, and a T max at about 0.25 to about 1.5 hours, and more preferably by about 0.25 to about 0.75 hours, most preferably 0.5 hours, after oral administration.
  • a preferred delivery agent is identified via chemical nomenclature as 4-[(4-chloro, 2-hydroxybenzoyl)amino]butanoic acid.
  • the delivery agent is a sodium salt, preferably monosodium salt.
  • the same compound is identified by the alternative nomenclature monosodium N-(4-chlorosalicyloyl)-4-aminobutyrate, or by the short name “4-CNAB”.
  • the invention is further directed in part to a method of treatment of diabetes in humans, comprising administering one or more unit doses of the dosage forms described above and in further sections of the present specification.
  • the invention is further directed in part to a method of treatment of impaired glucose tolerance, achieving glucose homeostasis, early stage diabetes, and late stage diabetes in humans, comprising administering one or more unit doses of the dosage forms described above and in further sections of the present specification on a chronic basis.
  • the invention is also related to a method of orally treating mammals with an active agent (i.e., insulin) that is not sufficiently absorbed when orally administered to provide a desirable therapeutic effect but that provides a desirable therapeutic effect when administered by another route of adminstration, comprising orally administering said active agent together with a delivery agent which facilitates the absorption of insulin from the gastrointestinal tract, having one or more of the further characteristics set forth above.
  • an active agent i.e., insulin
  • the invention is further directed to a method of providing a therapeutically effective orally administrable unit dose of unmodified insulin, comprising combining from about 2 to about 23 mg of unmodified insulin with from about 100 to about 600 mg of a pharmaceutically acceptable delivery agent which facilitates absorption of said insulin from the gastrointestinal tract of human diabetic patients, and orally administering said unit dose to a human diabetic patient to provide a therapeutic effect.
  • the total weight of the unit dose is from about 102 mg to about 800 mg.
  • the present invention is also directed in part to a method of treating human diabetic patients comprising orally administering to human diabetic patients on a chronic basis an oral insulin treatment comprising a dose of unmodified insulin together with a delivery agent that facilitates the absorption of the dose of insulin from the gastrointestinal tract to provide a therapeutically effective reduction in blood glucose and a blood plasma insulin concentration that is reduced relative to the systemic blood insulin concentration of an equivalent therapeutically effective reduction in blood glucose concentration achieved by subcutaneous injection of insulin.
  • the invention is also directed to a method of reducing the incidence and/or severity of one or more disease states associated with chronic administration of insulin, comprising treating human diabetic patients via oral administration on a chronic basis with a therapeutically effective dose of a (preferably solid) pharmaceutical composition comprising a dose of unmodified insulin and a delivery agent that facilitates the absorption of said unmodified insulin from the gastrointestinal tract in an effective amount such that the pharmaceutical composition provides therapeutically effective control of mean blood glucose concentration and a mean systemic blood insulin concentration in diabetic patients that is reduced on a chronic basis relative to the mean systemic blood insulin concentration provided by chronic subcutaneous administration of insulin in an amount effective to achieve equivalent control of mean blood glucose concentration in a population of human diabetic patients.
  • a pharmaceutical composition comprising a dose of unmodified insulin and a delivery agent that facilitates the absorption of said unmodified insulin from the gastrointestinal tract in an effective amount such that the pharmaceutical composition provides therapeutically effective control of mean blood glucose concentration and a mean systemic blood insulin concentration in diabetic patients that is reduced on a chronic basis relative to
  • the invention is further directed to a method of treating diabetes and reducing the incidence and or severity of hyperinsulinemia associated with chronic dosing of insulin, comprising orally administering on a chronic basis to a diabetic patient a dose of insulin and a delivery agent that facilitates the absorption of the dose of insulin from the gastrointestinal tract to provide a therapeutically effective reduction and/or control in blood glucose and a mean systemic blood insulin concentration of the diabetic patient that is reduced relative to the mean systemic blood insulin concentration provided by subcutaneous injection of insulin in an amount effective to achieve equivalent reduction and/or control in a population of human diabetic patients.
  • Diabetic patient refers to humans suffering from a form of diabetes.
  • IGT impaired glucose tolerance
  • Diabetes is deemed to encompasses type 1 and type 2 diabetes, unless specifically specified otherwise.
  • Bio macromolecule biological polymers such as proteins and polypeptides.
  • biological macromolecules are also referred to as macromolecules.
  • Delivery agent refers to carrier compounds or carrier molecules that are useful in the oral delivery of therapeutic agents. “Delivery agent” may be used interchangeably with “carrier”.
  • Therapeutically effective amount of insulin an amount of insulin included in the oral dosage forms of the invention which are sufficient to achieve a clinically significant control of blood glucose concentrations in a human diabetic patient either in the fasting state or in the fed state effective, during the dosing interval.
  • Effective amount of delivery agent an amount of the delivery agent that promotes the absorption of a therapeutically effective amount of the drug from the gastrointestinal tract.
  • Organic solvents any solvent of non-aqueous origin, including liquid polymers and mixtures thereof.
  • Organic solvents suitable for the present invention include: acetone, methyl alcohol, methyl isobutyl ketone, chloroform, 1-propanol, isopropanol, 2-propanol, acetonitrile, 1-butanol, 2-butanol, ethyl alcohol, cyclohexane, dioxane, ethyl acetate, dimethylformamide, dichloroethane, hexane, isooctane, methylene chloride, tert-butyl alchohol, toluene, carbon tetrachloride, or combinations thereof.
  • Peptide a polypeptide of small to intermediate molecular weight, usually 2 or more amino acid residues and frequently but not necessarily representing a fragment of a larger protein.
  • Protein a complex high polymer containing carbon, hydrogen, oxygen, nitrogen and usually sulfur and composed of chains of amino acids connected by peptide linkages. Proteins in this application refer to glycoproteins, antibodies, non-enzyme proteins, enzymes, hormones and peptides. The molecular weight range for proteins includes peptides of 1000 Daltons to glycoproteins of 600 to 1000 kiloDaltons.
  • Reconstitution dissolution of compositions or compositions in an appropriate buffer or pharmaceutical composition.
  • Unit-Dose Forms refer to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. It is contemplated for purposes of the present invention that dosage forms of the present invention comprising therapeutically effective amounts of insulin may include one or more unit doses (e.g., tablets, capsules) to achieve the therapeutic effect.
  • unit doses e.g., tablets, capsules
  • Unmodified insulin means insulin prepared in any pharmaceutically acceptable manner or from any pharmaceutically acceptable source which is not conjugated with an oligomer such as that described in U.S. Pat. No. 6,309,633 and/or which not has been subjected to amphiphilic modification such as that described in U.S. Pat. Nos. 5,359,030; 5,438,040; and/or 5,681,811.
  • the phrase “equivalent therapeutically effective reduction” means that a maximal reduction of blood glucose concentration achieved by a first method of insulin administration (e.g. via oral administration of insulin in a patient(s)) is not more 20%, and preferably not more than 10% and even more preferably not more than 5% different from a maximal reduction of blood glucose concentration after administration by a second method (e.g., subcutaneous injection) in the same patient(s) or a different patient requiring the same reduction in blood glucose level.
  • a first method of insulin administration e.g. via oral administration of insulin in a patient(s)
  • a second method e.g., subcutaneous injection
  • AUC means area under the plasma concentration-time curve, as calculated by the trapezoidal rule over the complete dosing interval, e.g., 24-hour interval.
  • C max is the highest plasma concentration of the drug attained within the dosing interval.
  • T max is the time period which elapses after administration of the dosage form at which the plasma concentration of the drug attains the C max within the dosing interval.
  • multiple dose means that the human patient has received at least two doses of the drug composition in accordance with the dosing interval for that composition.
  • single dose means that the human patient has received a single dose of the drug composition and the drug plasma concentration has not achieved steady state.
  • mean when preceding a pharmacokinetic value (e.g., mean T max ) represents the arithmetic mean value of the pharmacokinetic value unless otherwise specified.
  • Bioavailability means the degree or ratio (%) to which a drug or agent is absorbed or otherwise available to the treatment site in the body. This is calculated by the formula Rel .
  • Bioavailability ⁇ ⁇ ( % ) Dose ⁇ ⁇ SC Dose ⁇ ⁇ Oral ⁇ AUC INS ⁇ Oral AUC INS ⁇ SC ⁇ 100
  • Biopotency means the degree or ratio (%) to which a drug or agent is effective to the treatment site in the body. This is calculated by the formula Rel .
  • Biopotency ⁇ ⁇ ( % ) Dose ⁇ ⁇ SC Dose ⁇ ⁇ Oral ⁇ AUC GIR ⁇ Oral AUC GIR ⁇ SC ⁇ 100
  • F rel means the relative bioavailability of insulin calculated by comparing dose corrected oral insulin AUC with the dose corrected SC insulin AUC.
  • AUC (0-x) means the area under the plasma concentration-time curving linear trapezoidal summation from time 0 to time x hours post-dose.
  • AUC (0-t) means the area under the plasma concentration-time curve using linear trapezoidal summation from time zero to time t post-dose, where t is the time of the last measurable concentration (C t ).
  • AUC %Extrap means the percentage of the total AUC (0-inf) obtained by extrapolation.
  • AUEC (0-x) means the area under the effect-time curve calculated using the linear trapezoidal summation from time 0 to the concentration at time x hours post-dose.
  • AUEC (0-t) means the area under the effect-time curve calculated using the linear trapezoidal summation from time 0 to the concentration at time t hours post-dose, where t is the time of the last measurable effect (E).
  • AURC (0-x) means the area under the response-time curve calculated using the linear trapezoidal summation from time zero to the concentration at time x (Baseline Subtracted AUEC).
  • AURC( 0 -t) means the area under the response-time curve calculated using the linear trapezoidal summation from time zero to the concentration at time t (Baseline Subtracted AUEC), where t is the time of the last measurable response (R).
  • C b means the maximum observed plasma insulin concentration prior to intervention for hypoglycemia.
  • CL/F means the apparent total body clearance calculated as Dose/AUC (0-inf) .
  • E b means the maximum observed effect (baseline subtracted) prior to intervention for hypoglycemia.
  • E max means the maximum observed effect (baseline subtracted).
  • MRT means the mean residence time calculated as the ratio of the Area Under the first moment of the plasma concentration-time curve (AUMC) and the area under the plasma concentration-time curve, (AUMC)/AUC (0-inf) .
  • R max means the maximum observed response (total response), i.e., minimum glucose concentration.
  • R b means the maximum observed response (total response) prior to hypoglycemic intervention.
  • t b means the time to reach insulin/glucose plasma concentration prior to hypoglycemic intervention.
  • t c means the time to reach glucose concentration change from baseline prior to hypoglycemic intervention.
  • t Rmax means the time to reach maximum response.
  • t Emax time of the maximum effect (obtained without interpolation).
  • t 1/2 means the terminal half-life calculated as ln(2)/K el .
  • V d /F means the apparent volume of distribution calculated as (CL/F)/K el .
  • FIG. 1 shows C-peptide measurements for insulin delivered orally and subcutaneously.
  • FIG. 2 shows the plasma insulin concentration versus time for administration of insulin subcutaneously and orally in the presence of 4-CNAB.
  • FIG. 3 shows the % decrease in plasma glucose versus time upon delivery of placebo and administering insulin subcutaneously and orally in the presence of 4-CNAB.
  • FIG. 4 shows C-peptide concentration versus time after oral dosing of 4-CNAB alone, Placebo and 150 U human insulin alone.
  • FIG. 5 shows the % decrease in C-peptide versus time after administering insulin subcutaneously and orally in the presence of 4-CNAB.
  • FIG. 10 shows plasma glucose versus time for patients from Group 1, following a minimum of 8 hour overnight fast, patients were given one capsule containing a mixture of insulin in a stepwise fashion (3 patients received 200 U insulin, 5 patients received 300 U insulin and 4 patients received 400 U insulin) and a fixed dose of 300 mg 4-CNAB as a delivery agent.
  • FIG. 11 shows plasma glucose versus time for patients from Group 2, patients had a standard meal (350 kcal) after a minimum of 8 hour overnight fast. Twenty minutes prior to the ingestion of food, the patients were administered a capsule contained 300 U or 400 U insulin (six patients received 300 U insulin and six patients received 400 U of insulin) and 300 mg of 4-CNAB.
  • FIG. 12 shows glucose versus time for the study of Type I diabetics and healthy subjects after dosing with oral insulin (300 IU insulin/400 mg 4-CNAB).
  • FIG. 13 shows a plot of the arithmetic means of blood glucose excursions (i.e., differences between pre-prandial and postprandial blood glucose concentrations) for all subjects.
  • FIG. 14 shows a comparison of blood glucose levels over a time period 180 minutes following single administration of insulin orally and subcutaneously (mean ⁇ SE).
  • FIG. 15 shows the serum insulin levels over a time period of 180 minutes following single administration orally and subcutaneously (mean ⁇ SE).
  • FIG. 16 shows Glucokinase and G6Pase mRNA expression compared to sham dosing.
  • FIG. 17 shows Fru-1, 6-P and 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase mRNA expression compared to sham dosing.
  • FIG. 18 shows PEPCK mRNA expression compared to sham dosing.
  • FIG. 19 shows Glycogen synthase MRNA expression compared to sham dosing.
  • FIGS. 20A and 20B show early response gene mRNA expression compared to sham dosing.
  • FIG. 21 shows insulin-like Growth Factor Binding Protein mRNA expression compared to sham dosing.
  • FIG. 22 shows Intracellular Adhesion Molecule-1 mRNA expression compared to sham dosing.
  • FIGS. 23A and 23B shows Cytokine mRNA expression compared to sham dosing.
  • FIG. 24 shows Lipid Peroxidation enzyme mRNA expression compared to sham dosing.
  • FIG. 25 shows Plasminogen Activator Inhibitors mRNA expression compared to sham dosing.
  • FIG. 26 shows NPY, TGF-beta, ICAM-1 and 12-LO mRNA expression compared to sham dosing.
  • FIG. 27 shows THY-1, VEGF-B and Integrin aE2 mRNA expression compared to sham dosing.
  • FIG. 28 shows a comparison of blood glucose levels over a time period 180 minutes following single administration of insulin orally and subcutaneously (mean ⁇ SE) in a Streptozotocin diabetic model.
  • FIG. 29 shows the serum insulin levels over a time period of 180 minutes following single administration orally and subcutaneously (mean ⁇ SE) in a Streptozotocin diabetic model.
  • compositions that are useful as delivery agents in the oral delivery of an active agent that is not generally considered by those skilled in the art to be administrable via the oral route, such as insulin.
  • Such compositions serve to make insulin bioavailable and absorbable through the gastrointestinal mucosa when orally administered.
  • the pharmaceutical composition includes insulin as the active agent.
  • insulin refers to insulin from a variety of sources. Naturally occurring insulin and structurally similar bioactive equivalents can be used. Insulin useful in the invention can be isolated from different species of mammal. For example, animal insulin preparations extracted from bovine or porcine pancreas can be used. Insulin analogues, derivatives and bioequivalents thereof can also be used with the invention. In addition to insulin isolated from natural sources, the present invention can use insulin chemically synthesizing using protein chemistry techniques such as peptide synthesis. Analogues of insulin are also suitable for the present invention.
  • the insulin used in the present invention may be obtained by isolating it from natural sources or by chemically synthesizing it using peptide synthesis, or by using the techniques of molecular biology to produce recombinant insulin in bacteria or eucaryotic cells. Analogs of insulin are also provided by the present invention. Insulin from other species of mammal may also be used in the present invention. The physical form of insulin may include crystalline and/or amorphous solid forms. In addition, dissolved insulin may be used. Other suitable forms of insulin, including, but not limited to, synthetic forms of insulin, are described in U.S. Pat. Nos. 4,421,685, 5,474,978, and 5,534,488, the disclosure of each of which is hereby incorporated by reference in its entirety.
  • the most preferred insulin useful in the pharmaceutical compositions and methods of the present invention is human recombinant insulin.
  • Human recombinant insulin can be prepared using genetic engineering techniques that are well known in the art. Recombinant insulin can be produced in bacteria or eucaryotic cells. Functional equivalents of human recombinant insulin are also useful in the invention.
  • Recombinant human insulin can be obtained from a variety of commercial sources. For example, insulin (Zinc, human recombinant) can be purchased from Calbiochem (San Diego, Calif.). Alternatively, human recombinant Zinc-Insulin Crystals: Proinsulin Derived (Recombinant DNA Origin) USP Quality can be obtained from Eli Lilly and Company (Indianapolis, Ind.).
  • insulin including insulin analogues (including but not limited to Insulin Lispro, Insulin Aspart, Insulin Glargine, Insulin Detemir) are deemed for the purposes of this specification and the appended claims are considered to be encompassed by the term “insulin.”
  • the present invention provides compositions of recombinant human zinc insulin and a delivery agent as a drug for oral administration of insulin in humans.
  • the active agent is not insulin but instead is an active agent of a biological nature suitable for use in the present invention including, but not limited to, proteins; polypeptides; peptides; hormones; polysaccharides, and particularly mixtures of muco-polysaccharides; carbohydrates; lipids; other organic compounds; and particularly compounds which by themselves do not pass (or which pass as only a fraction of the administered dose) through the gastro-intestinal mucosa and/or are susceptible to chemical cleavage by acids and enzymes in the gastro-intestinal tract; or any combination thereof.
  • proteins proteins
  • polypeptides peptides
  • hormones polysaccharides, and particularly mixtures of muco-polysaccharides
  • carbohydrates lipids
  • other organic compounds and particularly compounds which by themselves do not pass (or which pass as only a fraction of the administered dose) through the gastro-intestinal mucosa and/or are susceptible to chemical cleavage by acids and enzymes in the gastro-intestinal tract; or any combination thereof.
  • active agents of a biological nature include, but are not limited to, the following, including synthetic, natural or recombinant sources thereof: growth hormones, including human growth hormones (hGH), recombinant human growth hormones (rhGH), bovine growth hormones, and porcine growth hormones; growth hormone-releasing hormones; interferons, including ⁇ , ⁇ and ⁇ ; interleukin-1; interleukin-2; insulin, including porcine, bovine, human, and human recombinant, optionally having counter ions including sodium, zinc, calcium and ammonium; insulin-like growth factor, including IGF-1; heparin, including unfractionated heparin, heparinoids, dermatans, chondroitins, low molecular weight heparin, very low molecular weight heparin and ultra low molecular weight heparin; calcitonin, including salmon, eel, porcine and human; erythropoietin; atrial naturetic factor; antigens
  • the protein active agents have a molecular weight of less than or equal to 10,000 Daltons. In another embodiment of this invention, protein active agents have a molecular weight of about 6,000 Daltons. In another embodiment of this invention, protein active agents have a molecular weight of greater than or equal to 10,000 Daltons. According to an alternate embodiment of the present invention, protein active agents have a molecular weight that is greater than or equal to 20,000 Daltons. In a further embodiment, protein active agents have a molecular weight that is greater than or equal to 30,000 Daltons. According to an alternate embodiment, protein active agents have a molecular weight that is greater than or equal to 40,000 Daltons. According to another alternate embodiment, protein active agents have a molecular weight that is greater than or equal to 50,000 Daltons.
  • the oral dosage forms of the invention facilitate the oral delivery of insulin, and after insulin is absorbed into the bloodstream, the composition produces a maximal decrease in blood glucose in treated patients from about 20 to about 60 minutes after oral administration.
  • the pharmaceutical composition produces a maximal decrease in blood glucose in treated patients from about 30 to about 50 minutes post oral administration. More particularly, the pharmaceutical composition produces a maximal decrease in blood glucose in treated patients at about 40 minutes after oral administration.
  • human diabetic patients show a maximal decrease in blood glucose by at least 10% within one hour post oral administration. In another embodiment, human diabetic patients show a maximal decrease in blood glucose by at least 20% within one hour post oral administration, alternatively, at least 30% within one hour post oral administration.
  • Normal levels of blood glucose vary somewhat throughout the day and in relation to the time since the last meal.
  • One goal of the present invention is to provide oral compositions of insulin that facilitate achieving normal levels of blood glucose throughout the 24-hour daily cycle.
  • the pharmaceutical composition includes insulin or an insulin analog as the active agent and a delivery agent in an amount effective to achieve a fasting blood glucose concentration from about 90 to about 110 mg/dl.
  • the pharmaceutical composition includes insulin or an insulin analog as the active agent and a delivery agent in an amount effective to achieve a fasting blood glucose concentration from about 95 to about 105 mg/dl, more preferably, the subject manifests fasting blood glucose concentrations at about 100 mg/dl.
  • the present invention provides oral compositions of insulin that prevent or control very high levels of blood glucose from being sustained. More particularly, the present invention provides compositions which facilitate achieving normal levels of blood glucose after a meal has been consumed, i.e., post-prandial.
  • the pharmaceutical composition includes insulin as the active agent and a delivery agent in an amount effective to achieve a post-prandial blood glucose concentration from about 130 to about 170 mg/dl.
  • the pharmaceutical composition includes insulin or an insulin analog as the active agent and a delivery agent in an amount effective to achieve a post-prandial blood glucose concentration from about 140 to about 160 mg/dl, more preferably, the subject manifests fasting blood glucose concentrations at less than about 160 mg/dl.
  • the present invention provides pharmaceutical compositions for oral administration which includes insulin or an insulin analog as the active agent and a delivery agent in an amount effective to achieve pre-prandial (before a meal is consumed) blood glucose concentration from about 95 to about 125 mg/dl.
  • the present invention provides pharmaceutical compositions for oral administration which includes insulin or an insulin analog as the active agent and a delivery agent in an amount effective to achieve pre-prandial blood glucose concentration from about 100 to about 120 mg/dl.
  • the present invention provides pharmaceutical compositions for oral administration which include insulin as the active agent and a delivery agent in an amount effective to achieve blood glucose concentrations at 3 AM from about 70 to about 120 mg/dl.
  • the present invention provides pharmaceutical compositions for oral administration which include insulin or an insulin analog as the active agent and a delivery agent in an amount effective to achieve blood glucose concentrations at 3 AM from about 80 to about 120 mg/dl.
  • the pharmaceutical composition comprises insulin as the active agent and the compound 4-CNAB as a delivery agent to facilitate the oral delivery of insulin, and after insulin is absorbed into the bloodstream, the composition produces a maximal decrease in C peptide concentration in treated patients from about 80 and about 120 minutes post oral administration. More particularly, the composition produces a maximal decrease in C peptide concentration in treated patients from about 90 and about 110 minutes post oral administration.
  • Absorption of insulin can be detected in subjects treated with the pharmaceutical compositions of the present invention by monitoring the plasma levels of insulin after treatment.
  • the time it takes for an active agent to reach a peak in the bloodstream (T max ) may depend on many factors such as the following: the nature of the unit dose, i.e., solid, liquid, tablet, capsule, suspension; the concentration of active agent and delivery agent in the GI tract; the feeding state of the subject; the diet of the subject; the health of the subject and the ratio of active agent to the delivery agent.
  • the pharmaceutical composition includes the compound 4-CNAB as the delivery agent and insulin as the active agent
  • the composition provides a peak plasma insulin concentration from about 0.1 to about 1 hour after oral administration.
  • the composition provides a peak plasma insulin concentration from about 0.2 to about 0.6 hours after oral administration. In a preferred embodiment, the composition provides a peak plasma insulin concentration from about 0.3 to about 0.4 hours after oral administration. In another embodiment, the composition provides a peak plasma insulin concentration within about 1 hour after oral administration.
  • the pharmaceutical composition comprises insulin as the active agent and the compound 4-CNAB as a delivery agent to facilitate the oral delivery of insulin, and after insulin is absorbed into the bloodstream, the plasma insulin levels in treated patients peak at about 20 minutes post oral administration with a second peak at about 105 minutes.
  • the compositions of the present invention include an active agent (e.g., insulin) and a delivery agent that serves to render the active agent orally absorbable through the mucosa of the stomach.
  • an active agent e.g., insulin
  • a delivery agent that serves to render the active agent orally absorbable through the mucosa of the stomach.
  • the delivery agents used in the invention have the following structure:
  • X is one or more of hydrogen, halogen, hydroxyl or C 1 -C 3 alkoxy, and R is substituted or unsubstituted C 1 -C 3 alkylene, substituted or unsubstituted C 1 -C 3 alkenylene.
  • the delivery agents of the invention preferably have the following structure:
  • X is halogen
  • R is substituted or unsubstituted C 1 -C 3 alkylene, substituted or unsubstituted C 1 -C 3 alkenylene.
  • the pharmaceutical composition includes a delivery agent wherein X is chlorine and R is C 3 alkylene.
  • the pharmaceutical composition includes the compound 4-[(4-chloro, 2-hydroxybenzoyl)amino]butanoic acid as a delivery agent for the oral delivery of insulin, preferably the monosodium salt thereof.
  • the delivery agents may be in the form of the carboxylic acid or salts thereof.
  • Suitable salts include, but are not limited to, organic and inorganic salts, for example alkali-metal salts, such as sodium, potassium and lithium; alkaline-earth metal salts, such as magnesium, calcium or barium; ammonium salts; basic amino acids, such as lysine or arginine; and organic amines, such as dimethylamine or pyridine.
  • the salts are sodium salts.
  • the salts may be mono- or multi-valent salts, such as monosodium salts and di-sodium salts.
  • the salts may also be solvates, including ethanol solvates, and hydrates.
  • suitable delivery agents that can be used in the present invention include those delivery agents described U.S. Pat. Nos. 5,650,386, 5,773,647, 5,776,888, 5,804,688, 5,866,536, 5,876,710, 5,879,681, 5,939,381, 5,955,503, 5,965,121, 5,989,539, 5,990,166, 6,001,347, 6,051,561, 6,060,513, 6,090,958, 6,100,298, 5,766,633, 5,643,957, 5,863,944, 6,071,510 and 6,358,504, the disclosure of each of which is incorporated herein by reference. Additional suitable delivery agents are also described in International Publications Nos.
  • Salts of the delivery agent compounds of the present invention may be prepared by methods known in the art.
  • sodium salts may be prepared by dissolving the delivery agent compound in ethanol and adding aqueous sodium hydroxide.
  • the compounds described herein may be derived from amino acids and can be readily prepared from amino acids by methods known by those with skill in the art based upon the present disclosure and the methods described in International Publications Nos. WO 96/30036, WO 97/36480, WO 98/34632 and WO 00/07979, and in U.S. Pat. Nos. 5,643,957 and 5,650,386, the disclosure of each of which is incorporated herein by reference.
  • the compounds may be prepared by reacting the single amino acid with the appropriate acylating or amine-modifying agent, which reacts with a free amino moiety present in the amino acid to form amides.
  • Protecting groups may be used to avoid unwanted side reactions as would be known to those skilled in the art.
  • the delivery agents may also be prepared by the methods of International Patent Application No. PCT/US01/21073, the disclosure of which is incorporated herein by reference.
  • the delivery agents may also be prepared by alkylation of the appropriate salicylamide according to the methods of International Publication No. WO 00/46182, the disclosure of which is incorporated herein by reference.
  • the salicylamide may be prepared from salicylic acid via the ester by reaction with sulfuric acid and ammonia.
  • poly amino acids and peptides comprising one or more of these compounds may be used.
  • An amino acid is any carboxylic acid having at least one free amine group and includes naturally occurring and synthetic amino acids.
  • Poly amino acids are either peptides (which are two or more amino acids joined by a peptide bond) or are two or more amino acids linked by a bond formed by other groups which can be linked by, e.g., an ester or an anhydride linkage.
  • Peptides can vary in length from dipeptides with two amino acids to polypeptides with several hundred amino acids.
  • the delivery agent compound may be purified by recrystallization or by fractionation on one or more solid chromatographic supports, alone or linked in tandem.
  • Suitable recrystallization solvent systems include, but are not limited to, ethanol, water, heptane, ethyl acetate, acetonitrile, methanol and tetrahydrofuran and mixtures thereof. Fractionation may be performed on a suitable chromatographic support such as alumina, using methanol/n-propanol mixtures as the mobile phase; reverse phase chromatography using trifluoroacetic acid/acetonitrile mixtures as the mobile phase; and ion exchange chromatography using water or an appropriate buffer as the mobile phase.
  • anion exchange chromatography preferably a 0-500 mM sodium chloride gradient is employed.
  • the delivery agent passes though the mucosal barriers of the GI tract and is absorbed into the blood stream where it can be detected in the plasma of subjects.
  • the level of delivery agent in the bloodstream as measured in the plasma is dose-dependent.
  • the delivery agent facilitates the absorption of the drug (active agent) administered therewith (either in the same dosage form, or simultaneously therewith), or sequentially (in either order, as long as both the delivery agent and the drug are administered within a time period which provides both in the same location, e.g., the stomach, at the same time).
  • a peak plasma concentration (C max ) of the delivery agent achieved after oral administration is preferably from about 10 to about 250,000 ng/ml, after oral administration, preferably from about 100 to about 125,000, and preferably the peak plasma concentration of the delivery agent is from about 1,000 to about 50,000 ng/ml, after oral administration. More preferably, the peak plasma concentration of the delivery agents of the present invention is from about 5,000 to about 15,000 ng/ml, after oral administration.
  • the time it takes for the delivery agent to reach a peak in the bloodstream may depend on many factors such as the following: the nature of the unit dose, i.e., solid, liquid, tablet, capsule, suspension; the concentration of delivery agent in the GI tract; the feeding state of the subject; the diet of the subject; the health of the subject and the ratio of delivery agent to the active agent.
  • the delivery agents of the present invention are rapidly absorbed from the gastrointestinal tract when orally administered in an immediate release dosage form, and preferably provide a peak plasma concentration within about 0.1 to about 8 hours after oral administration, and preferably at about 0.1 to about 3 hours after oral administration.
  • the T max of the delivery agent occurs at about 0.3 to about 1.5 hours after oral administration. In certain embodiments, the delivery agent achieves a (T max ) within about 2 hours after oral administration, and most preferably, within about 1 hour after oral administration.
  • the amount of delivery agent necessary to adequately deliver an active agent into the blood stream of a subject needing the therapeutic effect of that active agent may vary depending on one or more of the following; the chemical nature of the active agent; the chemical structure of the particular delivery agent; the nature and extent of interaction from about the active agent and delivery agent; the nature of the unit dose, i.e., solid, liquid, tablet, capsule, suspension; the concentration of delivery agent in the GI tract; the feeding state of the subject; the diet of the subject; the health of the subject and the ratio of delivery agent to the active agent.
  • the amount of the delivery agent preferred for the pharmaceutical composition is from about 1 mg to about 2,000 mg delivery agent, more preferably from about 1 mg to about 800 mg of said delivery agent, more preferably from about 50 mg to about 700 mg of said delivery agent, even more preferably from about 70 mg to about 700 mg of said delivery agent, still more preferably from about 100 to about 600 mg.
  • the delivery agent is 4-CNAB. Since the amount of delivery agent required to deliver a particular active agent is variable and the amount of active agent required to produce a desired therapeutic effect is also a variable, the ratio of active agent to delivery agent may vary for different active agent/delivery agent combinations. In certain preferred embodiments of the invention where the oral pharmaceutical composition includes insulin as the active agent and the delivery agent is the compound 4-CNAB, the amount of the delivery agent included in the pharmaceutical composition may be from about 100 mg to about 600 mg of said delivery agent.
  • the pharmaceutical composition includes insulin as the active agent and the delivery agent is the monosodium salt of 4-CNAB, the ratio of insulin [Units] to delivery agent [mg] ranges from 10:1 [Units/mg] to 1:10 [Units/mg], preferably, the ratio of insulin [Units] to delivery agent [mg] ranges from 5:1 [Units/mg] to 0.5:1 [Units/mg].
  • Preferred insulin doses in a single administration are about 5 to about 1000 insulin units USP, preferably from about 50 to about 400, more preferably from about 150 to about 400, and still more preferably from about 150 to about 300 units.
  • the optimum ratio of insulin to delivery agent can vary depending on the delivery agent. Optimizing the ratio of insulin to delivery agent is within the knowledge of one skilled in the art.
  • the composition provides a peak plasma delivery agent concentration within about 0.1 to about 3 hours after oral administration.
  • the peak plasma concentration of delivery agent attained is from about 8,000 to about 37,000 ng/ml.
  • a preferred embodiment of the invention provides methods for reducing the incidence of vascular disease associated with chronic dosing of insulin.
  • the methods in a preferred embodiment comprise treating human diabetic patients on a chronic basis with an oral and a delivery agent or pharmaceutically acceptable salt thereof that facilitates the absorption of insulin from the gastrointestinal tract (i.e., bioavailable).
  • the delivery agent may be used directly by mixing one or more such agents with the active agent (e.g., unmodified insulin) prior to administration.
  • the delivery agent and active agent may be mixed in dry powder form or wet granulated together.
  • other pharmaceutically acceptable excipients may be added.
  • the mixture may be then tableted or placed into gelatin capsules containing a unit dose of the active agent and the delivery agent.
  • the delivery agent/active agent mixture may be prepared as an oral solution or suspension.
  • the delivery agent and active agent do not need to be mixed together prior to administration, such that, in certain embodiments, the unit dose of active agent (with or without other pharmaceutically acceptable excipients) is orally administered without the delivery agents of this invention, and the delivery agent is separately orally administered (with or without other pharmaceutically acceptable excipients) before, after, or simultaneously with the active agent.
  • the oral dosage forms of the present invention are solid.
  • the unmodified insulin in dry powder form is stable, and in certain preferred embodiments is simply mixed in a desirable ratio with the delivery agent.
  • the dry powder mixture may then be filled into gelatin capsules, with or without optional pharmaceutical excipients.
  • the unmodified insulin in dry powder form may be mixed with the delivery agent together with optional pharmaceutical excipients, and the mixture may be tableted in accordance with standard tableting procedures known to those having ordinary skill in the art.
  • the present invention also provides methods for treating human diabetic patients with active agents that are not inherently bioavailable, such as for example treating diabetics with insulin. More particularly, the present invention provides method of treating humans with an oral dosage form of a pharmaceutical composition, wherein the pharmaceutical composition includes the following: first, an active agent or a pharmaceutically acceptable salt thereof, which is not orally bioavailable when dissolved or suspended in aqueous solution, wherein the active agent provide a therapeutic effect when administered to a subject by another means (e.g., via subcutaneous injection); and, second, an effective amount of a delivery agent or a pharmaceutically acceptable salt thereof, which renders the active agent orally absorbed (e.g., bioavailable).
  • the method comprises the following steps: first, contacting the active agent (e.g., insulin) with said delivery agent, and thereafter orally administering the pharmaceutical composition.
  • the method comprises administering the insulin and the delivery agent in such a manner that the insulin and delivery agent contact each other in-vivo (e.g., in the stomach), such that the delivery agent is available to facilitate absorption of the insulin through the stomach mucosa.
  • the dosage forms of the present invention may be produced by first dissolving the active agent and delivery agents into one solution or separate solutions.
  • the solvent will preferably be an aqueous solution, but organic solvents or aqueous organic solvent mixtures may be used when necessary to solubilize the delivery agent. If two solutions are used, the proportions of each necessary to provide the correct amount of either active agent or delivery agent are combined and the resulting solution may be dried, by lyophilization or equivalent means.
  • the oral dosage form may be dried and rehydrated prior to oral administration.
  • the administration mixtures may be prepared, e.g., by mixing an aqueous solution of the delivery agent with an aqueous solution of the active ingredient, such as insulin, just prior to administration.
  • the delivery agent and the biologically or chemically active ingredient can be admixed during the manufacturing process.
  • the solutions may optionally contain additives such as phosphate buffer salts, citric acid, acetic acid, gelatin, and gum acacia.
  • Stabilizing additives may be incorporated into the delivery agent solution. With some drugs, the presence of such additives promotes the stability and dispersibility of the agent in solution.
  • the stabilizing additives may be employed at a concentration ranging from about 0.1 and 5% (W/V), preferably about 0.5% (W/V).
  • Suitable, but non-limiting, examples of stabilizing additives include gum acacia, gelatin, methyl cellulose, polyethylene glycol, carboxylic acids and salts thereof, and polylysine.
  • the preferred stabilizing additives are gum acacia, gelatin and methyl cellulose.
  • the amount of active agent is an amount effective to accomplish the purpose of the particular active agent.
  • the amount in the composition is a therapeutically effective dose, i.e., a pharmacologically or biologically effective amount.
  • the amount can be less than a pharmacologically or biologically effective amount when the composition is used in a dosage unit form, such as a capsule, a tablet or a liquid, because the dosage unit form may contain a multiplicity of delivery agent/biologically or chemically active agent compositions or may contain a divided pharmacologically or biologically effective amount.
  • the total effective amounts can then be administered in cumulative units containing, in total, pharmacologically or biologically or chemically active amounts of biologically or pharmacologically active agent.
  • the total amount of active agent, and particularly insulin, to be used can be determined by those skilled in the art. However, it has surprisingly been found that with some biologically or chemically active agents, the use of the presently disclosed delivery agents provides extremely efficient delivery.
  • the amount of delivery agent in the present composition is a delivery effective amount and can be determined for any particular delivery agent/active agent combination by methods known to those skilled in the art.
  • the oral dosage forms of the present invention containing a mixture of the active agent, e.g., insulin and the delivery agent, e.g., 4-CNAB or separately containing the active agent and the delivery agent, may include additional materials known to those skilled in the art as pharmaceutical excipients. Any excipient or ingredient, including pharmaceutical ingredients or excipients.
  • Such pharmaceutical excipients include, for example, the following: Acidifying agents (acetic acid, glacial acetic acid, citric acid, fumaric acid, hydrochloric acid, diluted hydrochloric acid, malic acid, nitric acid, phosphoric acid, diluted phosphoric acid, sulfuric acid, tartaric acid); Aerosol propellants (butane, dichlorodifluoro-methane, dichlorotetrafluoroethane, isobutane, propane, trichloromonofluoromethane); Air displacements (carbon dioxide, nitrogen); Alcohol denaturants (denatonium benzoate, methyl isobutyl ketone, sucrose octacetate); Alkalizing agents (strong ammonia solution, ammonium carbonate, diethanolamine, diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, sodium hydroxide, trolamine); Anticaking agents (see glidant); Antifo
  • Insulin absorbed in the gastrointestinal tract mimics the physiology of insulin secreted by the pancreas because both are released into the portal vein and carried directly to the liver. Absorption into the portal circulation maintains a peripheral-portal insulin gradient that regulates insulin secretion. In its first passage through the liver, roughly 60% of the insulin is retained and metabolized, thereby reducing the incidence of peripheral hyper-insulinism, a factor in diabetes related systemic complications. A feared and not uncommon complication of insulin treatment and other oral antidiabetic agents is hypoglycemia.
  • the present invention relates in part to a method of treating human diabetics via the chronic oral administration of insulin together with a drug delivery agent that enhances the absorption of insulin (e.g., from the duodenum) such that a therapeutically effective control and/or reduction in blood glucose is achieved while effecting a reduction in the systemic blood insulin concentration (serum insulin level) on a chronic basis required to achieve the reduction in blood glucose concentration, e.g., relative to the serum insulin level required to achieve therapeutic efficacy via subcutaneous injection of insulin.
  • a drug delivery agent that enhances the absorption of insulin (e.g., from the duodenum) such that a therapeutically effective control and/or reduction in blood glucose is achieved while effecting a reduction in the systemic blood insulin concentration (serum insulin level) on a chronic basis required to achieve the reduction in blood glucose concentration, e.g., relative to the serum insulin level required to achieve therapeutic efficacy via subcutaneous injection of insulin.
  • the oral dosing method of the present invention shifts the site of insulin entry back to the portal vein.
  • the effect of this route of dosing is two fold.
  • a greater control of glucose may be achieved.
  • Various studies have shown that intraportal delivery of insulin can yield a comparable control of glucose at infusion rates lower than those required by peripheral administration. (Stevenson, R. W. et al., Insulin infusion into the portal and peripheral circulations of unanaesthetized dogs, Clin Endocrinol (Oxf) 8, 335-47. (1978); Stevenson, R. W.
  • the physiologic ratio of blood insulin concentration in the portal vein as compared to systemic (peripheral) blood insulin concentration is greater than about 2:1.
  • administration of insulin to human diabetic patients has been found to shift this ratio of portal vein insulin blood concentration to systemic insulin blood concentration to about 0.75:1.
  • the ratio of concentration of unmodified insulin in the portal circulation to systemic circulation approaches the normal physiological ratio, e.g., from about 2:1 to about 6:1.
  • the present invention provides a method of attenuating and/or reducing the incidence of diseases associated with exposure to systemic hyperinsulinemia by the oral administration to a patient a dosage form in accordance with the invention comprising unmodified insulin, preferably along with a suitable drug delivery agent that facilitates the absorption of insulin from the gastrointestinal tract of the patient in a therapeutically effective amount, for treatment of diabetes.
  • a dosage form in accordance with the invention comprising unmodified insulin, preferably along with a suitable drug delivery agent that facilitates the absorption of insulin from the gastrointestinal tract of the patient in a therapeutically effective amount, for treatment of diabetes.
  • compositions of the invention can attenuate and/or reduce the incidence of cardiovascular disease associated with chronic dosing of insulin. It is believed that orally administering insulin with the compositions of the invention will decrease the complications associated with vascular disease by lowering the systemic vasculature's exposure to insulin that is greater than normal physiological levels. With a first passage through the liver, roughly 50% of the insulin is retained and metabolized, thereby reducing the incidence of peripheral hyper-insulinemia.
  • the invention provides a method of treating diabetics comprising orally administering to diabetic patients on a chronic basis an oral insulin treatment comprising a dose of insulin together with a delivery agent which facilitates the absorption of the dose of insulin from the gastrointestinal tract to provide a therapeutically effective reduction in blood glucose and a peak serum insulin concentration that is reduced relative to the peak serum insulin concentration of an equivalent therapeutically effective reduction in blood glucose concentration achieved by subcutaneous injection of insulin.
  • this method can result in the reduction of the incidence of a disease state associated with chronic insulin administration, which disease states include, for example, cardiovascular diseases.
  • Cardiovascular diseases include, for example, congestive heart failure, coronary artery disease, neuropathy, nephropathy, retinopathy, arteriopathy, atherosclerosis, hypertensive cardiomyopathy and combinations thereof.
  • the invention provides a method of reducing the incidence and/or severity of one or more disease states associated with chronic administration of insulin comprising treating diabetic patients via oral administration on a chronic basis of a therapeutically effective dose of a pharmaceutical composition which comprises insulin and a delivery agent that facilitates the absorption of insulin from the gastrointestinal tract, such that the pharmaceutical composition provides a therapeutically effective reduction in blood glucose and a peak serum insulin concentration of the diabetic patient that is reduced relative to the peak serum insulin concentration of an equivalent therapeutically effective reduction in blood glucose concentration achieved by subcutaneous injection of insulin.
  • Disease states associated with chronic administration of insulin for which the incidence and/or severity can be reduced by the method described herein include, for example, cardiovascular diseases, such as congestive heart failure or coronary artery disease. Other such disease states include, for example, neuropathy, nephropathy, retinopathy, arteriopathy, atherosclerosis, hypertensive cardiomyopathy and combinations thereof.
  • the method of reducing the incidence and/or severity of one or more disease states associated with chronic administration of insulin can provide for a reduced expression of genes associated with vascular disease as compared to the level of expression of genes associated with vascular disease resulting from an equivalent reduction in blood glucose concentration achieved in a population of patients via subcutaneous injection of insulin.
  • the genes associated with vascular disease can include, for example, early response genes, genes associated with cytokines, genes associated with adhesion molecules, genes associated with lipid peroxidation, genes associated with thrombosis and combinations thereof.
  • Early response genes can include, for example, c-myc, jun B, Egr-1, Ets-1 and combinations thereof.
  • the methods provided herein relating to oral administration of insulin and oral administration of insulin on a chronic basis provide for obtaining plasminogen activator inhibitor concentrations that are lower as compared to the plasminogen activator inhibitor concentrations resulting from an equivalent therapeutically effective reduction in blood glucose concentration achieved by subcutaneous injection of insulin. These methods also provide for obtaining pro-inflammatory cytokine concentrations that are lower than pro-inflammatory cytokine concentrations resulting from an equivalent therapeutically effective reduction in blood glucose concentration achieved by subcutaneous injection of insulin.
  • the invention provides a method of treating diabetes and reducing the incidence and or severity of hyperinsulinemia associated with chronic dosing of insulin, comprising orally administering on a chronic basis to a diabetic patient a dose of insulin and a delivery agent that facilitates the absorption of the dose of insulin from the gastrointestinal tract to provide a therapeutically effective reduction in blood glucose and a peak serum insulin concentration of the diabetic patient that is reduced relative to the peak serum insulin concentration of an equivalent therapeutically effective reduction in blood glucose concentration achieved by subcutaneous injection of insulin.
  • the invention provides a method of screening a drug for vascular injury associated with route of administering the drug, comprising administering a drug to a first test animal parenterally, administering the drug to a second test animal orally, and comparing the expression of early response genes selected from the group consisting of c-myc, c-fos, Jun B, Erg-1 and combinations thereof for the first and second test animal, wherein an increase in the expression of one or more early response genes is indicative of vascular injury.
  • the step of measuring the change in expression is done using gene chip analysis and can comprise measuring the changes in mRNA expression.
  • the invention provides a method of reducing the incidence of and/or the severity of disease states or of vascular diseases associated with chronic insulin administration to diabetics, comprising orally administering an oral insulin treatment comprising a dose of insulin together with a delivery agent that facilitates the absorption of said insulin from the gastrointestinal tract on a chronic basis to diabetic patients to reduce blood glucose levels in said diabetic patients by a desired amount, such that the concentration of insulin circulating in the blood of said diabetic patients as a result of insulin treatment is reduced relative to the peak serum insulin concentration of an equivalent therapeutically effective reduction in blood glucose concentration achieved by subcutaneous injection of insulin.
  • the invention provides a method for reducing the incidence of, the severity of, or the incidence and severity of vascular diseases associated with chronic insulin therapy in diabetics, comprising orally administering an oral insulin treatment comprising a dose of insulin together with a delivery agent that facilitates the absorption of said insulin from the gastrointestinal tract on a chronic basis to diabetic patients to reduce blood glucose levels in said diabetic patients by a desired amount, such that the concentration of insulin circulating in the blood of said diabetic patients as a result of insulin treatment is reduced relative to the peak serum insulin concentration of an equivalent therapeutically effective reduction in blood glucose concentration achieved by subcutaneous injection of insulin.
  • the invention provides a method of attenuating processes resulting from the reaction to a mild injurious stimulus in multiple areas of the response to increases in mRNA during insulin treatment, comprising orally administering an oral insulin treatment comprising a dose of insulin together with a delivery agent that facilitates the absorption of said insulin from the gastrointestinal tract on a chronic basis to diabetic patients to reduce blood glucose levels in said diabetic patients by a desired amount, such that the concentration of insulin circulating in the blood of said diabetic patients as a result of insulin treatment is reduced relative to the peak serum insulin concentration of an equivalent therapeutically effective reduction in blood glucose concentration achieved by subcutaneous injection of insulin.
  • the invention provides a method of treating diabetic patients, comprising orally administering an oral insulin treatment comprising a dose of insulin together with a delivery agent that facilitates the absorption of said insulin from the gastrointestinal tract on a chronic basis to diabetic patients to reduce blood glucose levels in said diabetic patients by a desired amount, such that the concentration of insulin circulating in the blood of said diabetic patients as a result of said oral insulin treatment is not substantially greater than normal physiological levels.
  • Impairment to the vascular system is believed to be the reason behind conditions such as microvascular complications or diseases (retinopathy (lesions in the small blood vessels and capillaries supplying the retina of the eye); neuropathy (impairment of the function of the autonomic nerves, leading to abnormalities in the function of the gastrointestinal tract and bladder, and also loss of feeling in lower extremities); nephropathy (lesions in the small blood vessels and capillaries supplying the kidney, which may lead to kidney disease)); or macrovascular complications or diseases (e.g., cardiovascular disease; etc.).
  • retinopathy lesions in the small blood vessels and capillaries supplying the retina of the eye
  • neuropathy immas in the function of the autonomic nerves, leading to abnormalities in the function of the gastrointestinal tract and bladder, and also loss of feeling in lower extremities
  • nephropathy lesions in the small blood vessels and capillaries supplying the kidney, which may lead to kidney disease
  • macrovascular complications or diseases e.g., cardiovascular disease; etc.
  • Hyperinsulinemia is caused by the administration of insulin in a location (and manner) which is not consistent with the normal physiological route of delivery.
  • insulin is released from the pancreas into the portal vein, which transfers the insulin to the liver.
  • the liver utilizes a large portion of the insulin which it receives from the portal circulation.
  • Glucose is the principal stimulus to insulin secretion in humans.
  • Glucose enters the ⁇ cell by facilitated transport, and is then phosphorylated by glucokinase.
  • Expression of glucokinase is primarily limited to cells and tissues involved in the regulation of glucose metabolism, such as the liver and the pancreatic ⁇ cells.
  • Insulin circulates in blood as the free monomer, and its volume distribution approximates the volume of extracellular fluid.
  • the concentration of insulin in portal blood is, e.g., about 2-4 ng/ml
  • the systemic (peripheral) concentration of insulin is, e.g., about 0.5 ng/ml, in normal healthy humans, translating into, e.g., a 5:1 ratio.
  • the ratio is changed to about 0.75:1.
  • the liver does not receive the necessary concentrations of insulin to adequately control blood glucose.
  • the present invention provides a method of administering insulin and pharmaceutical compositions useful for administering insulin such that the insulin is bioavailable and absorbable from the gastrointestinal tract and such that the incidence of vascular diseases normally associated with chronic dosing of insulin is attenuated.
  • the delivery agents of the invention enable insulin to be orally absorbable through the mucosa of the stomach.
  • the delivery agent passes though the mucosal barriers of the gastrointestinal tract and is absorbed into the blood stream where it can be detected in the plasma of subjects.
  • the level of delivery agent in the bloodstream as measured in the plasma is dose-dependent.
  • the delivery agent facilitates the absorption of insulin administered therewith (either in the same dosage form, or simultaneously therewith), or sequentially (in either order, as long as both the delivery agent and insulin are administered within a time period which provides both in the same location, e.g., the stomach, at the same time).
  • oral administration of insulin in particular using the delivery agents disclosed herein, effectively reduces the incidence of vascular and other disease states that are associated with traditional dosing of insulin, i.e., subcutaneously.
  • compositions of the invention comprise a combination of insulin and a delivery agent in a suitable pharmaceutical carrier or excipient as understood by practitioners in the art.
  • the means of delivery of the pharmaceutical composition can be, for example, a capsule, compressed tablet, pill, solution, freeze-dried, powder ready for reconstitution or suspension suitable for administration to the subject.
  • the pharmaceutical compositions and method of the present invention provide a number of advantages in addition to convenience, acceptance and patient compliance. Insulin absorbed in the gastrointestinal tract mimics the physiology of insulin secreted by the pancreas because both are released into the portal vein and carried directly to the liver. Absorption into the portal circulation maintains a peripheral-portal insulin gradient that regulates insulin secretion.
  • the present invention comprises pharmaceutical compositions and method for oral insulin delivery that enable achieving low blood glucose without having high levels of systemic insulin.
  • the methods and pharmaceutical compositions provide the pharmacokinetic parameters set forth in U.S. Provisional Applications Nos. 60/346,746 and 60/347,312, the disclosure of each of which is incorporated herein by reference.
  • the amount of delivery agent necessary to adequately deliver insulin into the blood stream of a subject needing the therapeutic effect of insulin can vary depending on one or more of the following; chemical structure of the particular delivery agent; the nature and extent of interaction of insulin and the delivery agent; the nature of the unit dose, i.e., solid, liquid, tablet, capsule, suspension; the concentration of delivery agent in the GI tract, the feeding state of the subject, the diet of the subject, the heath of the subject and the ratio of delivery agent to insulin.
  • the oral dosage forms of the present invention comprise a mixture of insulin and a delivery agent, e.g., 4-CNAB, or separately containing insulin and the delivery agent.
  • a delivery agent e.g., 4-CNAB
  • the oral dosage forms described herein are orally administered as described herein in combination with an additional therapy to treat diabetes, impaired glucose tolerance, or to achieve glucose homeostasis, said additional therapy comprising, for example, an additional drug such as sulfonylurea, a biguanide, an alpha-glucosidase, insulin delivered via a different pathway (e.g., parenteral insulin), and/or an insulin sensitizer.
  • an additional therapy comprising, for example, an additional drug such as sulfonylurea, a biguanide, an alpha-glucosidase, insulin delivered via a different pathway (e.g., parenteral insulin), and/or an insulin sensitizer.
  • the oral dosage forms described herein reduce the likelihood of hypoglycemic events, mainly because of two reasons: (a) one cannot hyperinsulinize the liver, because even under hyperinsulinemia the liver uptake of glucose will be unchanged. Unlike the peripheral tissue, the liver will only cease producing endogenous insulin and not sequester additional glucose; and (b) the short peak of insulin (e.g., as shown in the appended examples) shows that even if insulin were to reach high peripheral levels, the peak drops precipitously.
  • the present invention comprising compositions of insulin and the delivery agent 4-CNAB was evaluated for safety and toxicity in a nonclinical program that included pharmacological screening, pharmacokinetic profiling, and toxicity assessments in rats and monkeys.
  • animal physiological responses to 4-CNAB alone and to Insulin/4-CNAB were comparable.
  • Pharmacokinetic studies in mice, rats and monkeys have shown that 4-CNAB is absorbed rapidly following oral administration, and subsequently cleared from the body.
  • 4-CNAB did not demonstrate potential activity in any of the primary molecular targets evaluated in receptor binding screening assays.
  • Four genotoxicity studies have been conducted with 4-CNAB, with no positive findings. Based on 14-day oral repeated dose toxicity studies, the NOAEL (No-Adverse Effect Level) was estimated to be 500 mg/kg in Sprague-Dawley rats, and 400 mg/kg in rhesus monkeys.
  • the insulin for the subcutaneous injection was HUMULIN® R injection 100 U/ml insulin from Eli Lilly and Company (Indianapolis, Ind.).
  • 4-CNAB for the human dosings (Monosodium N-(4-chlorosalicyloyl)-4-amino-butyrate) was made under good manufacturing practices (GMP) conditions by Regis Technologies, Inc. (Morton Grove, Ill.) according to the methods of International Publication No. WO 00/46182 except that the starting material 4-chlorosalicylic acid (purchased from Ihara Chemical Industry Co. Inc, Ltd., Tokyo, Japan and Aapin Chemicals Ltd., Oxfordshire, UK) was used and converted to the amide via a methyl ester using 0.14 equivalents sulfuric acid in methanol and then about 4 equivalents ammonia in methanol.
  • the alkylating agent used was ethyl-4-bromobutyrate.
  • the monosodium salt of 4-CNAB was made according to the following method on a 40 kilogram scale.
  • the flask was equipped with an overhead stirrer, a thermocouple temperature read out, a reflux condenser and a heating mantle, and was placed under nitrogen.
  • Reagent grade acetone 13 L was added to the reactor and the mixture was agitated.
  • the 4-CNAB/acetone mixture was heated to 50° C. to dissolve any solids. A hazy brown solution was achieved.
  • the 50° C. solution was pumped through a warm pressure filter (dressed with Whatman #1 filter paper, 5 microns, 18.5 sq. in. area) into a clean 22 L reactor to remove sodium chloride and other insolubles.
  • the pressure dropped across the filter to about 20 psig at the end of filtration.
  • the reactor containing the clear yellow filtrate was agitated and heated. At 50° C. the reactor was removed from heat.
  • All capsules containing 200 mg 4-CNAB and 150 insulin units USP were prepared as follows. First, the total amount of delivery agent material necessary for filling the delivery agent alone capsules and the delivery agent plus insulin composition capsules was prepared by weighing 3160 g of 4-CNAB. The 3160 g 4-CNAB was then milled in a Quadro comil, model 197S mill with screen number 2A 050 G 037 19 136 (1270 micron). Next, 1029 g of the milled 4-CNAB was passed through a #35 mesh screen. Then, the pass through screened material was transferred into a 4 quart shell and blended using for example, a V blender, at 25 rpm for 10.2 minutes. The resultant blended material was used to fill capsules.
  • a Fast Cap Capsule Filler was used with a size 3 Fast Cap Encapsulation tray.
  • the empty capsules weighed approximately 48 mg each and were filled with an average fill weight of 205.6 mg of 4-CNAB alone.
  • the dose of the delivery agent alone capsules was 205.6 mg.
  • the insulin compositions were prepared by first dispensing 31.8 g of recombinant human zinc crystalline insulin (Potency 26.18 Units per mg) (proinsulin derived (recombinant DNA origin) USP quality) from Eli Lilly and Company (Indianapolis, Ind.) into an appropriately sized plastic bag. Next, sequential 30 g additions of the milled and screened 4-CNAB were added to the bag until approximately 510 g had been added. The bag was thoroughly mixed after each 30 g addition of 4-CNAB by shaking and inversion. In order to add and mix the next 532.5 g of 4-CNAB, the 541.8 g mixture of insulin and 4-CNAB was transferred to a V blender and mixed again at 25 rpm for 10.2 minutes.
  • the final capsules contained an average of 5.7 mg insulin (equivalent to 150 units insulin) and 200.5 mg of 4-CNAB or a ratio of 1:57.3, insulin: 4-CNAB. Multiple samples of the final blend were run on HPLC to verify uniformity and were found to be uniform.
  • Example 2 a single center, double-blind, randomized placebo-controlled study of escalating single oral doses of 4-CNAB capsules and escalating single doses of Insulin/4-CNAB capsules was undertaken in healthy male volunteers.
  • each capsule contained 200 mg 4-CNAB.
  • each Insulin/4-CNAB capsule contained 150 Insulin Units USP and 200 mg 4-CNAB.
  • Group 1 and Group 2 treatment 2 capsules were made per Example 1c.
  • Group 2, treatments 3 and 4 capsules and all Group 3 capsules were extemporaneously prepared at the pharmacy department of Medeval Ltd. (Manchester, England).
  • the placebo contained Methocel E15 Premium LV (hydroxypropylmethylcellulose).
  • Capsules were administered with 240 mL water. After each dosing, safety, pharmacokinetic, and pharmacodynamic parameters were measured and evaluated before proceeding to the next dose level.
  • the concentration of 4-CNAB in plasma was determined using a combination of liquid chromatography and Mass spectrometry assay known as LC-MS/MS.
  • the method involves protein precipitation followed by separation on liquid chromatography using a Hypersil BDS column and a mobile phase consisting of methanol and acetate buffer. The eluting peaks were quantified by MS/MS.
  • the equipment used for the determination of 4-CNAB in plasma comprises of an Agilent 1100 modular HPLC system with a Micromass Quattro Micro MS/MS detector. HPLC and AR grade chemicals and reagents were used throughout the study.
  • Calibration standards were prepared in duplicate for each batch by adding diluted standard solutions of 4-CNAB in solvent to blank plasma, to give a range of concentrations of approximately 10.6, 20.6, 42.9, 75.2, 116.4, 174.6, 238.1, 317.5, 402.2 and 497.5 ng/ml. Calibration curves were produced for each batch by the method of weighted (1/y 2 ) least squares linear regression on the 4-CNAB to internal standard peak area and the theoretical concentrations.
  • Quality control standards were prepared in duplicate for each batch by adding diluted standard solutions of 4-CNAB in solvent to plasma, to give a range of concentrations of approximately 30.1, 93.6, 258.7 and 443.4 ng/ml. Quality control standards were prepared from different primary standards (i.e. separate weighings) to those used for the preparation of the calibration standards. The concentrations of 4-CNAB in the quality control standards was calculated by reference to the appropriate calibration curve.
  • insulin that has been prepared according to the present invention can be absorbed by the gastrointestinal (GI) tract of human subjects and produces a decrease in plasma glucose levels in human subjects.
  • GI gastrointestinal
  • the present invention provides insulin in a form that can be orally administered, absorbed through the GI tract and remains bioavailable and bioactive.
  • Compositions prepared according to the present invention produce clinically relevant magnitudes of responses in human patients at reasonable doses. The response of the human subjects was reproducible and dose dependent response profiles were obtained.
  • Table 5 shows the peak plasma concentration of the delivery agent as C max (average of all subjects in treatment group).
  • the T max the time for the delivery agent to reach a peak concentration in the bloodstream, is also shown.
  • the area under the curve (AUC) is also shown.
  • a single-center, open-label, randomized, active controlled, 3-period crossover study was performed in 10 patients with type 2 diabetes.
  • Male subjects between the ages of 35 and 70 years, inclusive with Type 2 diabetes mellitus as defined by the American Diabetes Association (1998 Diabetes Care, 21: S5-S 19) for more than one year were chosen.
  • Body Mass Index was less than 36 kg/m 2 .
  • Stable glycemic control was Hb AlC ⁇ 11%.
  • Patients were off all oral hypoglycemic agents 24 hours prior to each study dosing day and off any investigational drug for at least four weeks.
  • the blood samples were centrifuged at 3000 rpm for a period of fifteen minutes at a temperature between 2° C.-8° C., within one hour of sample collection. Using a plastic pipette and without disturbing the red cell layer, the plasma from the collection tube was pipetted in pre-labeled polypropylene tubes for each analysis of plasma insulin, C-peptide, and plasma glucose (approximately 0.3-0.5 ⁇ l each). The samples were stored at ⁇ 20° C. until analysis.
  • Pharmacokinetic and pharmacodynamic data were statistically analyzed for subjects that received at least the first two treatments (visit 1 to 4) and for subjects who completed all three treatment visits (visit 1 to 6).
  • Pharmacodynamic parameters used for analysis were the maximum glucose infusion rate after application of the study drugs (GIR max ), the time to maximum glucose infusion rate (t GIRmax ) time to half-maximal GIR values before GIR max (early t 50% ), and the area under the glucose infusion rate versus time curves from 0 to 60, 120, 240 and 360 minutes after dosing and from 180 to 360 min post dose (AUC GIR 0-1 h , AUC GIR 0-2 h , AUC GIR 0-4 h , AUC GIR 0-6 h , AUC GIR 3-6 h , respectively).
  • GIR max , t max and early t 50% were calculated by fitting a polynomial function (6th order) to each individual's GIR profile after subtraction of the mean baseline GIR. Areas under the curve (AUCs) were calculated from the raw data using the trapezoidal rule. Pharmacokinetic parameters were calculated accordingly but without curve fitting.
  • Pharmacokinetic parameters determined included the maximum plasma insulin concentration (C max ), time to maximum insulin concentration (T max ) and the area under the plasma insulin concentration versus time curve from 0 to 1,2 and 6 hours after application of the study drugs (AUC INS 0-1 h , AUC INS 0-2 h , AUC INS 0-4 h and AUC INS 0-6 h respectively).
  • the calculation of AUCs from time of dosing until return to the baseline concentration (AUC INS 0-t′ ) was omitted as in some patients insulin concentrations did not return to baseline measurement within 360 minutes post dosing.
  • the pharmacokinetic and pharmacodynamic data obtained were used for comparative analysis of the treatments with 15 IU s.c. insulin and 300 IU oral insulin.
  • the oral treatment patients showed a faster and higher rise in insulin concentrations and showed a faster onset of action than the s.c treatment (AUC 0-1 oral 300 vs. s.c. 15: 2559 ⁇ 1831 vs. 542 ⁇ 296 mU.mL-1 .min, p ⁇ 0,01; C max oral 300 vs. s.c. 15: 93 ⁇ 71 vs. 33 ⁇ 11 ⁇ /U/ml, p ⁇ 0,01; T max oral 300 vs. s.c. 15: 27 ⁇ 9 vs. 161 ⁇ 83 min, p ⁇ 0,01).
  • pharmacodynamic results showed a significantly faster onset of action of the oral treatment (AUC 0-1 oral 300 vs. s.c. 15 173 ⁇ 86 vs. 27 ⁇ 32, p ⁇ 0,01; T GIRmax oral 300 vs. s.c. 15 40 ⁇ 16 vs. 255 ⁇ 108, p ⁇ 0,01; early t 50% oral 300 vs. s.c. 15 13 ⁇ 6 vs. 150 ⁇ 87, p ⁇ 0,01).
  • the maximum glucose infusion showed no statistically significant difference.
  • FIG. 1 shows a plot of C-peptide [nmol/l] vs. time for 15 IU s.c., 300 IU oral and 150 IU oral. As shown in FIG. 1, C-peptide measurements showed no significant changes during the treatment periods.
  • Delivery agents 1-3 were investigated for their ability to penetrate the GI mucosa. The plasma concentrations of each delivery agent was measured in human subjects after oral administration of delivery agent loaded capsules as a measure of each delivery agents penetration efficiency. See Tables 9 and 10. TABLE 9 Structures of Delivery Agents 1-3 Delivery Agent 1 (SNAC) Delivery Agent 2 (SNAD) Delivery Agent 3 (4-CNAB)
  • Blood sampling for plasma delivery agent concentration determination (2 mL in sodium heparin tube) were drawn 15 minutes before dosing, and at 5, 10, 15, 30, and 45 minutes and 1, 1.5, 2, 3, 4, 6, 8, and 12 hours post-dose (14 samples per treatment) for delivery agent measurements in all treatment groups.
  • delivery agents 1-3 were compared for the ability to efficiently transport insulin across the GI mucosa in a biologically active form by determining the relationship between delivery agent dose, insulin dose and the glucose response. See Table 12. The effective dose of delivery agent necessary to deliver a therapeutic dose of insulin and produce a therapeutic effect was measured. See Table 12. The therapeutic effect was determined by the ability of the delivery agent/insulin combination to lower serum glucose by at least 10% within one hour post administration. TABLE 12 Effective Clinical Dose of Delivery Agent in Humans Delivery Agent Delivery Agent X N Dose (Mg) 1 (SNAC) H 7 2400 2 (SNAD) H 9 1500 3 (4-CNAB) Cl 3 200
  • delivery agent 3 with X as chlorine and n equal to 3 alkyl was approximately 12 fold more efficient in facilitating insulin transit across the GI mucosa in a biologically active form than was delivery agent 1 having n equal to 7 alkyl.
  • delivery agent no. 3 was 7.5 fold more efficient in facilitating transport of insulin across the GI mucosa in a biologically active form than was delivery agent 2 having n equal to 5 alkyl. See Table 12.
  • delivery agent 3 is efficient enough at facilitating transport of biologically active insulin to allow packaging of a therapeutically effective dose of insulin plus delivery agent into a single capsule.
  • a double-blind, randomized placebo-controlled study was done in order to assess the safety and tolerability of escalating single oral doses of 4-CNAB capsules and insulin/4-CNAB capsules.
  • the objectives of this study were to (1) evaluate the safety and tolerability of single oral doses of 4-CNAB and of Insulin/4-CNAB capsules in healthy subjects, and (2) assess the pharmacokinetic (PK) of 4-CNAB and insulin.
  • PK pharmacokinetic
  • the volunteers were all healthy, adult male volunteers between 18 and 50 years of age, inclusive, and were determined to be in good health, within the permissible deviations (+/ ⁇ 15%) of ideal weight. Laboratory values (hematology, serum chemistries, and urinalysis) obtained during screening were within normal ranges.
  • the subjects first completed a screening period between 2 and 21 days prior to study start. The evening before each treatment period, subjects entered the clinical research center for check-in assessments and to begin a pre-dose fast of at least 8 hours. Study data was collected through to 12 hours post-dose, and the subjects remained in the unit under observation for a further 12 hours for Groups 2 and 3.
  • PK pharmacokinetic
  • PD pharmacodynamic
  • PD parameters were computed for plasma glucose concentration change from baseline: AUEC (0-2) , AUEC (0-t) , E max (baseline subtracted), t Emax , percent change from baseline and concentrations (E c ) and corresponding time (t c ) immediately prior to intervention for hypoglycemia.
  • the recombinant human insulin used in the nonclinical studies discussed in the above examples commercially obtained from Calbiochem (San Diego, Calif.).
  • the recombinant human insulin used in the clinical studies is Zinc-Insulin Crystals Human: Proinsulin Derived (Recombinant DNA Origin) USP Quality obtained from Eli Lilly and Company (Indianapolis, Ind.). No modifications or processing were done to the bulk drug substance in the manufacture of the clinical Recombinant Human Insulin/4-CNAB oral dosage form.
  • Insulin is administered parenterally, usually by subcutaneous injection. It is not absorbed to any extent through the gastrointestinal tract, presumably due to its size and potential for enzymatic degradation.
  • Monosodium N-(4-chlorosalicyloyl)-4-aminobutyrate (4-CNAB) is a novel compound discovered by Emisphere Technologies, Inc. When combined with insulin, 4-CNAB has been shown to enhance the gastrointestinal absorption of Human Insulin following oral administration to rats and primates.
  • 4-CNAB was previously evaluated in a non-clinical program that included pharmacological screening, pharmacokinetic profiling, and toxicity assessments in rats and monkeys. In general, animal physiological responses to 4-CNAB alone and to Insulin/4-CNAB were comparable. Pharmacokinetic studies in mice, rats and monkeys have shown that 4-CNAB is absorbed rapidly following oral administration, and subsequently cleared from the body. 4-CNAB did not demonstrate potential activity in any of the primary molecular targets evaluated in receptor binding screening assays. Four genotoxicity studies have been conducted with 4-CNAB, with no positive findings.
  • NOAEL No Adverse Effect Level
  • a subcutaneous (s.c.) insulin treatment group was added to allow comparison of the combined treatment against an existing standard treatment and an oral insulin alone treatment group was also included to further evaluate the effect of 4-CNAB on oral insulin absorption.
  • This study evaluated the safety and tolerability of single oral doses of 4-CNAB and of Insulin/4-CNAB capsules in healthy subjects and assessed the PK of 4-CNAB and Insulin and the effect of study drug on blood glucose.
  • This study was a single center, double-blind, partially randomized placebo-controlled study of escalating single oral doses of 4-CNAB capsules, escalating single doses of Insulin/4-CNAB capsules, a single oral dose of insulin and a s.c. administration of insulin in healthy male volunteers.
  • Group 1 The 29 volunteers were divided into three groups. In Group 1, there were four separate treatments as shown in the following Table 13. For each treatment, seven subjects received active treatment and 2 received placebo, according to the pre-prepared randomization code. Randomization was stratified such that any individual subject received placebo only on a single occasion or not at all. TABLE 13 Group 1 Treatments (4-CNAB alone - 4 escalating doses) 4-CNAB only # of Subjects & # Placebo Treatment: 4-CNAB Dose Subjects # of Capsules Treatment 1 7 subjects 400 mg 2 2 Treatment 2 7 Subjects 800 mg 2 4 Treatment 3 7 Subjects 1400 mg 2 7 Treatment 4 7 Subjects 2000 mg 2 10
  • Group 2 there were three separate treatments as shown in the following Table 14. For each treatment 8 subjects received active treatment and two received placebo, according to the pre-prepared randomization code. TABLE 14 Group 2 Treatments (Insulin/4-CNAB - 2 escalation doses and SC insulin dose) # of Subjects & Insulin/4-CNAB Insulin/4-CNAB Dose # Placebo # Treatment (Unit Insulin/mg 4-CNAB) Subjects of Capsules Treatment 1 8 Subjects (10/0) 2 0 (SC only) Treatment 2 8 Subjects (150/200) 2 1 Treatment 3 8 Subjects (100/600) 2 4
  • Group 3 there were four separate treatments as shown in the following Table 15. For each treatment 8 subjects received active treatment and two received placebo, according to the pre-prepared randomization code. TABLE 15 Group 3 Treatments (Insulin/4-CNAB - 3 escalation doses) # of Subjects & Insulin/4-CNAB Insulin/4-CNAB Dose # Placebo # Treatment: (Unit Insulin/mg 4-CNAB) Subjects of Capsules Treatment 1 8 Subjects (100/300) 2 2 Treatment 2 8 Subjects (100/450) 2 3 Treatment 3 8 Subjects (150/100) 2 1 Treatment 4 8 subjects (150/0) 2 1
  • Capsules were administered with 240 mL water. A washout period of at least 72 hr was observed between doses. After each dosing, safety data (i.e. vital signs, blood glucose, and available 4-CNAB plasma concentrations) were collected and evaluated before proceeding to the next dose level.
  • safety data i.e. vital signs, blood glucose, and available 4-CNAB plasma concentrations
  • Group 3 there were 3 escalating oral doses of Insulin/4-CNAB (100 Units/300 mg, 100 Units/450 mg and 150 Units/100 mg) and one SC dose of 150 Units of insluin. Each subject received either all four of these treatments or three of these treatments and one placebo treatment.
  • the 4-CNAB alone and Insulin/4-CNAB capsules were prepared by AAIPharma Inc., Wilmington N.C., and shipped prior to the study start.
  • the 4-CNAB used for the capsules was manufactured under cGMP compliance.
  • the Insulin used to prepare the capsules was Zinc-Insulin Crystals Human: Proinsulin Derived (Recombinant DNA Origin) USP Quality obtained from Eli Lilly and Company (Indianapolis, Ind.). Insulin used for the SC dosing was provided by Medeval Ltd.
  • This insulin was zinc-insulin crystals human: proinsulin derived (recombinant DNA origin) equivalent to Humulin R (The trade name is Humulin S injection 100 Units/mL).
  • Both 4-CNAB capsules and Insulin/4-CNAB capsules were provided in high-density polyethylene (HDPE) bottles. Each bottle contained 40 capsules and a polyester coil, had a heat-induction seal and a child-resistant cap, and was stored frozen at or below minus 10° C. On the day of dosing, the appropriate number of bottles were removed from the freezer and brought to room temperature (between 15 and 30° C.) for about one hour. Once the bottles were at room temperature, the caps were opened and the seals broken. The polyester coils were removed and the capsules dispensed. Capsules were used within 4 hours of dispensing. Unopened bottles were not left at room temperature for more than 4 hours. Each bottle was intended for single use, and the unused capsules were not to be used for subsequent dosing.
  • HDPE high-den
  • Placebo capsules consisted of Size 3 hard gelatin capsules filled with 200 mg of Methocel E15 Premium LV. Stability data were not required for this extemporaneously prepared placebo as the placebo capsules only contained Methocel, which is a stable excipient. The placebo capsules were stored at room temperature between 15 and 30° C. in a dry place.
  • Each 4-CNAB capsule contained 200 mg of 4-CNAB and was stored frozen at or below minus 10° C. The bottle was brought to room temperature (between 15 and 30° C.) before opening and was not left bottle at room temperature for more than 4 hours.
  • Each Insulin/4-CNAB capsule contained 150 Insulin Units USP of Human insulin and 200 mg of 4-CNAB. Each capsule was stored frozen at or below minus 10°C., was brought to room temperature (between 15 and 30° C.) before opening, and was not left at room temperature for more than 4 hours
  • the subjects were assigned a randomization number consecutively in the order of their inclusion into the study from 0 to 030 after they had given their informed consent and had successfully completed screening tests. Thirty subjects were to be enrolled into three separate groups. Within each treatment period of Group 1, seven subjects were randomized to receive active treatment and two subjects to receive placebo. In Groups 2 and 3, eight subjects were randomized to receive active treatment and two subjects to receive placebo. In each group any individual received placebo only on one occasion or not at all. Subjects who withdrew from the study were not replaced. From the screening visit until the allocation of a randomization number, the subjects were identified by their initials and date of birth.
  • the doses selected for this study were based on results of pre-clinical toxicology studies of 4-CNAB and Insulin conducted in Sprague-Dawley rats and in rhesus monkeys to determine the no effect adverse event level (NOAEL).
  • NOAEL no effect adverse event level
  • the estimated NOAEL level of 4-CNAB in monkeys was 400 mg/kg.
  • the highest proposed dose of 4-CNAB in man 2000 mg [28.5 mg/kg in 70 kg man]) was 12-16 fold lower than the NOAEL in monkeys.
  • an insulin dose of 15 U/kg in combination with a 4-CNAB dose of 1200 mg/kg was associated with a single hypoglycemic episode but no effects were observed at 15 U/kg in combination with lower doses. Taking this information into account, 15 insulin Units USP was chosen as the starting dose of insulin in man for this study. Escalating doses would only be administered once all safety and tolerability data had been reviewed.
  • study medication capsules or SC dose
  • the capsules were administered with 240 mL of water with subjects in an upright position.
  • the total administration time did not exceed 2.5 minutes.
  • the SC dose of insulin solution or placebo (saline) was injected in the abdominal wall as a single bolus administration.
  • Each treatment period lasted between 12 and 24 h.
  • Subjects also refrained from grapefruit, grapefruit juice, grapefruit containing products, Seville oranges and marmalade during the 24 hours prior to dosing and throughout the study periods.
  • No alcohol was allowed for 24 hours prior to admission and while resident in the Clinical Unit.
  • Non-smokers or smokers who smoked up to five cigarettes a day were recruited. Smoking was not allowed while resident in the Clinical Unit.
  • Subjects were asked to avoid strenuous physical activity and contact sports from 48 hours prior to Day -1 until the end of the residential period.
  • Plasma glucose was measured based on a timed-endpoint method using a BECKMAN Synchron CX system that mixes exact proportions of reagents that catalyse the phosphorylation of glucose.
  • the Synchron CX measures changes in the absorbance spectrum at 340 nm at a fixed time interval. The change in absorbance is directly proportional to the concentration of glucose in the sample.
  • Plasma C-peptide was measured using a DELFIA C-peptide kit, based on a solid phase, two-site fluoroimmunometric assay, which used the direct sandwich technique in which two monoclonal antibodies are directed against antigenic determinants on the C-peptide molecule.
  • Reagent dissociates europium ions from the labeled antibody, which form fluorescent chelates with the reagent. The fluorescence is directly proportional to the concentration of C-peptide in the sample.
  • Blood samples for plasma glucose, insulin and C-peptide analyses were stored between 2° C. and 8° C. immediate after sampling and prior to centrifugation. The samples were centrifuged at 3000 rpm for a period of 15 minutes at a temperature between 2° C. and 8° C., within one hour of sample collection. Using a plastic pipette and without disturbing the red cell layer, the plasma from the collection tube was pipetted into pre-labelled polypropylene tubes without delay. The samples were stored at ⁇ 70° C. until analysis.
  • Total blood volume (including study screening and safety assessment) collected from each subject for the entire study did not exceed 625 mL for subjects in Group 3, and 487 mL for subjects in Group 2, and 380 mL for subjects in Group 1.
  • Plasma glucose measurements were used to compute various pharmacodynamic parameters. The following parameters were planned to be derived for 4-CNAB and insulin: AUC (0-t) , AUC (0-inf) , K el , t 1/2 , C max , t max , CL/F, Vd/F, MRT and F rel .
  • the following PD parameters were computed for plasma glucose and plasma glucose change from baseline: AUEC (0-t) (baseline subtracted), AURC (0-t) (total response), E max (baseline subtracted), R max (total response) and t Emax , (obtained without interpolation).
  • Plasma glucose concentrations were also to be presented as percent change (decrease or increase) from the baseline, where baseline was taken as the pre-dose concentration.
  • Glucose ⁇ ⁇ % ⁇ ⁇ Change ⁇ ⁇ from ⁇ ⁇ Baseline ( Glucose ⁇ ⁇ Conc . ⁇ - ⁇ Baseline ⁇ ⁇ Glucose ⁇ ⁇ Conc . Baseline ⁇ ⁇ Glucose ⁇ ⁇ Conc . ) ⁇ 100
  • Subjects first completed a screening period between 2 and 21 days prior to study start. The evening before each treatment period, subjects entered the clinical research center for check-in assessments and to begin a pre-dose fast of at least 8 hours. Study data was collected through 12 hr post-dose. Subjects from Groups 2 and 3 remained under observation in the unit for a further 12 hours.
  • Safety assessments included physical examinations, medical history, vital signs, 12-lead electrocardiogram (ECG) monitoring, laboratory evaluations and checking for adverse events (AEs).
  • Activity parameters included blood glucose, insulin, C-peptide, and 4-CNAB plasma concentration measurements.
  • Blood sampling for PK/pharmacodynamic (PD) parameters was done throughout the study.
  • An 18-gauge IV line was situated in each arm prior to dosing.
  • One IV line was used for blood sampling, and the second IV line was to be used for potential infusion of 20% glucose (Group 2 and 3 only) in case of hypoglycaemia.
  • Blood samples (2 mL in sodium heparin tubes) were drawn at specified timepoints described in Section 9.7.2 for blood glucose, plasma glucose, insulin and C-peptide, and 4-CNAB measurements in all treatment groups.
  • Subject 15 displayed a baseline glucose concentration of 1.2 mmol/L. This was considered unreasonably low and the baseline value for this treatment in this subject taken to be the same as the concentration measured at the subsequent time point (5.2 mmol/L).
  • the plasma concentration-time profiles for 4-CNAB were evaluated in those subjects who received 4-CNAB or Insulin/4-CNAB treatments. Insulin, C-peptide and glucose concentration-time profiles following administration of all treatments were evaluated for the 20 subjects in Groups 2 and 3.
  • PK parameters for 4-CNAB, insulin and C-peptide corrected insulin and PD parameters for glucose concentration change from baseline, respectively were calculated for subjects whether they required hypoglycemic rescue or not. Concentrations from subjects who required food/drink due to hypoglycemia were excluded from descriptive statistics. PK and PD parameters were summarized separately for these subjects, except following SC insulin where descriptive statistics were provided for all 8 subjects who required intervention with food/drink due to hypoglycemia.
  • C max levels of 4-CNAB and exposure (AUCs) generally increased with 4-CNAB doses, as both C max and AUC values increased in a dose-dependent manner for the 400 mg and 800 mg doses.
  • C max ranged between 22315 ⁇ 11456 ng/mL and 135199 ⁇ 86565 for doses of 400 mg and 2000 mg, respectively.
  • the time of maximum 4-CNAB concentration was consistent across all doses with median values ranging between 0.50-0.75 h.
  • Mean elimination half-life values for 4-CNAB were variable and ranged between 5.90 and 15.3 h due to the variability in the terminal elimnation phase and difficulty in estimating the elimination rate constants. However, the MRT values were more consistent and ranged from 1.4 to 1.8 hours.
  • c Value corresponds to AUC (0-t) for 6 h sampling schedule.
  • NA not applicable; either 2 subjects or less.
  • Mean C max insulin values in non-hypoglycemic subjects following 100 Units Insulin/300 mg 4-CNAB, 100 Units Insulin/450 mg 4-CNAB and 100 Units Insulin/600 mg 4-CNAB were 69.9 ⁇ 21.4 pmol/L, 112.4 ⁇ 53.1 pmol/L and 120.1 ⁇ 64.2 pmol/L, respectively.
  • the times of peak insulin concentrations ranged between approximately 0.1 and 0.4 h for all Insulin/4-CNAB combined treatments.
  • the insulin concentration was lowest following 150 USP Units oral insulin alone indicating no absorption of insulin when oral administration of insulin alone was administered.
  • Mean C max insulin values for all subjects were highly variable. Following 100 Units Insulin/300 mg 4-CNAB, 100 Units Insulin/450 mg 4-CNAB and 100 Units Insulin/600 mg 4-CNAB mean C max values were 91.0 ⁇ 63.4 pmol/L, 152.5 ⁇ 123.5 pmol/L, and 159.3 ⁇ 125.6 pmol/L, respectively. The median times of peak insulin concentrations ranged between approximately 0.25 and 0.4 h for all Insulin/4-CNAB combined treatments.
  • c Value corresponds to AURC (0-t) for 6 h sampling schedule.
  • d Value corresponds to AUEC (0-t) for 6 h sampling schedule.
  • subjects required rescue treatment on twenty occasions, i.e. food/drink such as a chocolate bar, banana or apple juice, in order to raise their blood glucose.
  • food/drink such as a chocolate bar, banana or apple juice
  • 12 were following 10 Units SC insulin and 3 were following 150 Units Insulin/200 mg 4-CNAB.
  • C-peptide corrected plasma insulin increased with increasing doses of 4-CNAB demonstrating effective oral delivery and absorption of human insulin.
  • a single-center, open-label, randomized, active controlled, 3-period crossover study was conducted in ten 10 patients with type 2 diabetes in order to compare the pharmacodynamic (PD) and pharmacokinetic (PK) characteristics of an oral insulin formulation with SC regular insulin using the glucose clamp technique and in order to get a first impression about the metabolic effect of oral insulin in the main target population.
  • PD pharmacodynamic
  • PK pharmacokinetic
  • the glucose clamp technique was applied to compare the time-action profiles of the orally applied insulin in comparison to SC regular insulin. This method utilizes negative feedback from frequent blood glucose sample values to adjust a glucose infusion to maintain a defined and constant blood glucose level. The glucose infusion rate therefore becomes a measure of the pharmacodynamic effect of any insulin administered.
  • a primary objective of this study was to compare the PK and PD effect of two doses of an oral insulin capsule formulation (300 U Insulin/400 mg 4-CNAB in 2 capsules, and 150 U Insulin/200 mg 4-CNAB in one capsule) with that of 15 U SC injected regular insulin. Relative bioavailability and biopotency of the two oral formulations vs. SC injection was determined, inter-subject variability was investigated for selected PD and PK parameters.
  • the oral treatment provided was Insulin/4-CNAB (Monosodium N-(4-chlorosalicyloyl)-4-aminobutyrate (4-CNAB).
  • the insulin used to prepare the capsules was Zinc-Insulin Crystals Human: Proinsulin Derived (Recombinant DNA Origin) USP Quality obtained from Eli Lilly and Company (Indianapolis, Ind.).
  • Each Insulin/4-CNAB capsule contained 150 Insulin Units USP and 200 mg 4-CNAB, and was prepared by AAI Pharma, Inc., Wilmington, N.C. Two tablets were given to those who received the 300 U oral Insulin/400 mg 4-CNAB treatments.
  • Insulin/4-CNAB capsules were provided in HDPE bottles, each of which contained 40 capsules and a polyester coil. Each bottle had a heat-induction seal and a child-resistant cap, and were stored frozen at or less than minus 10° C. On the day of dosing, the appropriate number of capsules was removed from the freezer and brought to room temperature (between 15 and 30° C.) for about one hour. Capsules were used within four hours of dispensing, and unopened bottles were not left at room temperature for more than four hours.
  • the SC injection was U-100 human regular insulin (Humulin® R from Eli Lilly and Company, at a dose of 15 U, supplied in 1.5-mL cartridges-100 units/mL, provided by Profil.
  • the Insulin was stored in the refrigerator within a temperature range of 5-8° C.
  • each subject was randomized to one of the two possible treatment sequences.
  • Each subject received one of the two treatments during a glucose clamp procedure: an oral treatment (treatment A) of 300 U oral Insulin/400 mg 4-CNAB (in 2 capsules) or one SC treatment (treatment B) of 15 U regular SC insulin.
  • Visit 3 the subjects received the alternative treatment A or B, i.e., the one they did not receive in Visit 2, in conjunction with a glucose clamp procedure according to their randomization sequence. Only subjects having received both treatments by the end of Visit 3 were regarded as completers. A final examination (Visit 4) was performed after Visit 3, preferably immediately after the glucose clamp procedures were completed, but no longer than 14 days after Visit 3.
  • the SC insulin dose of 15 U was selected to fall within a range typical for type 2 diabetic patients.
  • the oral dose of 150 U insulin (combined with 200 mg 4-CNAB) estimated to be equivalent to the SC dose was a 10-fold scale-up compared with the SC dose, based on previous investigational studies.
  • the oral dose of 300 U insulin (combined with 400 mg 4-CNAB) was a 20-fold scale-up compared with the SC dose.
  • the clamp level was adjusted by a variable intravenous (IV) insulin infusion and glucose infusion rate during a 6 hrs baseline period.
  • IV intravenous
  • the insulin infusion was set to a rate of 0.2 mU/kg/min, which rate was not changed until the end of the experiment.
  • exogenous insulin was administered by oral administration or by SC injection.
  • the PD response elicited by the study medication was registered for another 6 hrs. No food intake was allowed during this period, but water could be consumed as desired.
  • the primary PD endpoint of the study was the area under the glucose infusion rate curve (AUC GIR ) in the first hr after drug administration (AUC GIR 0-1 h ).
  • the secondary endpoints for pharmacodynamic assessment were the following parameters: Maximum glucose infusion rate (GIR max ), time to GIR max (t GIRmax ), area under the glucose infusion rate curve in defined time-intervals (AUC GIR 0-2 h , AUC GIR 0-3 h , AUC GIR 0-4 h , AUC GIR 0-5 h , AUC GIR 0-6 h ), time to early and late half-maximum glucose infusion rate (early and late T GIR 50% ), and maximum reduction of C-peptide concentrations.
  • the secondary endpoints for pharmacokinetic assessment were the following parameters: Maximum plasma insulin concentrations (C INSmax ), time to C INSmax (t INSmax ), area under the insulin concentration curves in defined time-intervals (AUC INS 0-1 h , AUC INS 0-2 h , AUC INS 0-3 h AUC INS 0-4 h , AUC INS 0-5 h AUC INS 0-6 h ). Inter-subject variability was investigated for selected PD and PK parameters.
  • blood samples were collected for the determination of plasma insulin concentrations, plasma C-peptide and plasma glucose concentration. Sampling occurred from 6 hrs before dosing and continued for 6 hrs after the dose was administered. Blood samples were collected via a venous cannula. Blood samples were collected relative to the administration of the study drug (1) prior to study of drug administration at ⁇ 1 and ⁇ 0.5 hrs, (2) immediately after study drug administration (time 0), and (3) post administration of the study drug at 10, 20, 30, 40, 50 min, and 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 5, and 6 hrs
  • the glucose clamp procedure was performed using a Biostator (glucose controlled insulin infusion system—GCIIS, Life Science Instruments, Elkhart, Ind., USA).
  • GCIIS glucose controlled insulin infusion system
  • a 17-gauge PTFE catheter was inserted into an antecubital vein for blood sampling and kept patent with 0.15 mol/L saline.
  • a dorsal hand vein or lateral wrist vein of the same arm was cannulated in retrograde fashion for insertion of an 18-gauge PTFE double lumen catheter connected to the glucose sensor of the Biostator.
  • the catheterized hand was placed in a hot box and warmed to an air temperature of 55° C.
  • a third vein was cannulated with an 18-gauge PTFE catheter to infuse a glucose solution (20% in water).
  • an insulin solution (regular human insulin in 0.15 mol/L saline diluted with 2 mL of the patient's blood per 100 mL) was continuously infused by means of a syringe pump (Perfusor Secura FT, Braun, Melsungen, Germany).
  • Plasma concentrations of insulin were determined by a good laboratory practice (GLP) validated microparticle enzyme immunoassay (MEIA). TABLE 29 Summary of Plasma Insulin Concentrations (pmol/L) 300 U 150 U 15 U SC oral oral Time Point N Mean SD N Mean SD N Mean SD Time 0 10 4.6 7.3 10 9.2 9.9 8 0.3 0.9 10 minutes 10 5.7 10.8 10 50.3 62.4 8 59.6 44.7 20 minutes 10 13.7 18.3 10 429.6 474.6 8 188.3 162.6 30 minutes 10 41.2 32.5 10 409.5 268.1 8 192.5 250.6 40 minutes 10 94.9 63.1 10 366.2 258.2 8 114.3 158.9 50 minutes 10 109.4 75.6 10 214.2 185.8 8 79.8 121.7 60 minutes 10 116.4 63.2 10 122.1 108.2 8 50.2 69.5 75 minutes 10 137.4 86.5 10 48.2 37.3 8 30.3 38.6 90 minutes 10 116.5 50.9 10 20.6 28.2 8 19.5
  • baseline corrected values were used, i.e., pre-dose baseline values were subtracted, and in case of a negative result, the value was set to zero.
  • Table 30 below shows a Summary of C-Peptide Levels (nmol/L): TABLE 30 Summary of C-Peptide Levels 15 U SC 300 U oral 150 U oral Time Point N Mean SD N Mean SD N Mean SD ⁇ 60 minutes 10 1.02 0.42 10 0.95 0.37 8 0.86 0.33 ⁇ 30 minutes 10 1.05 0.40 10 0.98 0.36 8 0.86 0.30 Time 0 10 1.04 0.39 10 0.99 0.30 8 0.95 0.35 10 minutes 10 1.05 0.42 10 1.00 0.31 8 1.00 0.38 20 minutes 10 1.07 0.47 10 1.01 0.34 8 1.02 0.37 30 minutes 10 1.09 0.46 10 1.02 0.38 8 1.01 0.34 40 minutes 10 1.11 0.47 10 1.02 0.36 8 0.97 0.28 50 minutes 10 1.05 0.43 10 1.01 0.36 8 0.99 0.36 60 minutes 10 1.04 0.39 10 0.97 0.35 8 1.00 0.36 75 minutes 10 1.07 0.45 10 1.00 0.41 8 0.99 0.34 90 minutes 10 1.03 0.44 10 1.04 0.38 8 0.92 0.30 105 minutes 10 1.
  • the primary PD endpoint of the study was the area under the glucose infusion rate curve (AUC GIR ) in the first hr after drug administration (AUC GIR 0-1 h ).
  • PK and PD data were statistically analyzed for subjects that received at least the first 2 treatments (Visit 1 to 4) and for subjects who completed all three treatment visits (Visit 1 to 6).
  • PD parameters used for analysis were the maximum glucose infusion rate after application of the study drugs (GIR max ), the time to maximum glucose infusion rate (t GIRmax ), time to half-maximum GIR values before GIR max (early t GIR 50% ), time to half-maximum GIR values after GIR max (late t GIR 50% ), and the area under the glucose infusion rate versus time curves from 0 to 60, 120, 180, 240, 300, and 360 min after dosing, and from 180 to 360 min post dose (AUC GIR 0-1 h , AUC GIR 0-2 h , AUC GIR 0-3 h AUC GIR 0-4 h , AUC GIR 0-5 h , AUC GIR 0-6 h , AUC GIR 3-6 h , respectively).
  • GIR max , t GIRmax and early and late t GIR 50% were calculated by fitting a polynomial function (6th order) to each individual's GIR profile after subtraction of the mean baseline GIR. Areas under the curve (AUCs) were calculated from the raw data using the trapezoidal rule.
  • PK parameters were calculated using non-compartmental methods. PK parameters determined included the maximum plasma insulin concentration (C INSmax ), time to maximum insulin concentration(t INSmax ), and the area under the plasma insulin concentration versus time curves from 0 to 1, 2, 3, 4, 5 and 6 hrs after application of the study drugs (AUC INS 0-1 h , AUC INS 0-2 h , AUC INS 0-3 h , AUC INS 0-4 h , AUC INS 0-5 h and AUC INS 0-6 h , respectively). The calculation of AUCs from time of dosing until return to baseline concentration (AUC INS 0-t′ ) was omitted as in some patients insulin concentrations did not return to baseline measurement within 360 min post dosing.
  • C INSmax maximum plasma insulin concentration
  • t INSmax time to maximum insulin concentration
  • Safety data included AEs, laboratory data, vital signs, physical examinations, and ECGs. Vital signs (systolic and diastolic blood pressure, respiration rate, heart rate, and body temperature) for each treatment group were summarized at baseline (defined as the ⁇ 30 min time point) and at the end of each study day.
  • PK and PD data were analyzed for the 10 patients who received the study treatments A and B (oral 300 U Insulin/400 mg 4-CNAB, SC 15 U regular insulin) and had evaluable data. PK and PD data were also analyzed for the amended group of 8 patients who received the second oral study treatment (treatment C: oral 150 U Insulin/200 mg 4-CNAB). All included patients with treatment received at least 2 treatments (one oral and one SC treatment) as planned in the protocol.
  • the pharmacokinetic and pharmacodynamic parameters discussed here are the averages and standard deviations of the individual values.
  • the oral dose of 300 U Insulin/400 mg 4-CNAB showed a faster and higher rise in plasma insulin concentrations indicating a faster onset of action than the SC treatment (AUC INS 0-1 h oral 300 U vs. SC 15 U: 2559 ⁇ 1831 vs. 542 ⁇ 296 ⁇ U ⁇ mL ⁇ 1 ⁇ min, p ⁇ 0.01; C INS max oral 300 U vs. SC 15 U: 93 ⁇ 71 vs. 33 ⁇ 11 ⁇ U/mL, p ⁇ 0.01; t INSmax oral 300 U vs. SC 15 U: 27 ⁇ 9 vs. 161 ⁇ 83 min, p ⁇ 0.01).
  • Relative bioavailability and biopotency of the two oral insulin doses in comparison to the SC administration were calculated as defined hereinabove.
  • Relative bio-availability of oral insulin is listed in the Table 33.
  • Respective values for bio-potency for oral insulin are listed in Table 34. TABLE 33 Summary of Relative Bioavailability of Insulin Time interval n Mean SD SEM Max Min Median Bioavailability (%): 300 U oral vs.
  • Relative biopotency (based on PD results) of 300 U oral Insulin/400 mg 4-CNAB was as high as 54.9 ⁇ 91.9% in the first hr after application, and 31.0 ⁇ 89.4% over 6 hrs. Respective values for bioavailability (based on PK results) were 43.7 ⁇ 60.5%, and 2.7 ⁇ 3.0%.
  • the unexpected increase in mean relative biopotency for the time intervals 0-4, 0-5 and 0-6 hrs accounts for Patient 101 whose values were calculated only for these three time periods and which were up to 100-fold higher than those found for the other patients (177.45%, 447.03%, and 285.54%, respectively).
  • the oral dose of 150 U Insulin/200 mg 4-CNAB also showed a faster rise in plasma insulin concentrations compared to the SC treatment (AUC INS 0-1 h oral 150 U vs. SC 15 U: 1100 ⁇ 1221 vs. 542 ⁇ 296 ⁇ U ⁇ mL ⁇ 1 ⁇ min; t INSmax oral 300 U vs. SC 15 U: 23 ⁇ 7 vs. 161 ⁇ 83 min), whereas the observed maximum plasma concentrations were similar for both treatments (C INSmax oral 150 U vs. SC 15 U: 38 ⁇ 39 vs. 33 ⁇ 11 ⁇ U/mL).
  • GIR results for the oral 150 U insulin dose showed a faster onset of the PD effect (AUC GIR 0-1 h oral 150 U vs. SC 15 U: 58 ⁇ 40 vs. 27 ⁇ 32 mg/kg; t GIRmax oral 150 U vs. SC 15 U: 132 ⁇ 146 vs. 255 ⁇ 108 min; early t 50% oral 150 U vs. SC 15 U: 104 ⁇ 141 vs. 150 ⁇ 87 min).
  • the maximum glucose infusion rate was lower after the oral than after the SC treatment (GIR max . oral 150 U vs. SC 15 U: 2.1 ⁇ 0.9 vs. 3.6 ⁇ 1.8 mg/kg/min).
  • Insulin/4-CNAB seems to be a very attractive candidate for pre-prandial (before meal) insulin therapy in type 1 and type 2 diabetic patients.
  • the diabetic volunteers were divided into two groups—in one group, 12 patients were studied on fasting state, and in a second group, 12 patients were studied before and during standard meal. Every patient served as his own control and was tested without getting the insulin-4-CNAB mixture.
  • the delivery agent 4-CNAB was supplied by Emisphere Technologies Inc., of Tarrytown, N.Y., and was stored at room temperature desiccated until use. Recombinant Human Zinc Insulin was shipped directly by Eli Lilly and Company, and was stored at ⁇ 20 C. Standard capsules were made of gelatin, size 00C, and were prepared by a pharmacist at Hadassah Medical Organization.
  • a catheter was inserted into the antecubital arm vein of each patient. Blood was withdrawn at baseline twice, 5 to 10 min. apart, and at timed intervals after the administration of the capsule. In group 1, the fasting group, blood was withdrawn during the first hour every 5 minutes and thereafter every 10-20 minutes. In group 2, the standard meal group, blood was withdrawn during the first two hours every 10 minutes and thereafter every 20 minutes. In both groups, blood samples were withdrawn until blood glucose reached basal levels. All plasma samples were analyzed for glucose, insulin, C-peptide and the delivery agent 4-CNAB. Blood glucose levels were measured in real time using two Elite Glucometers (Bayer corporation, Elkhart, Indianapolis, Ind., USA).
  • Plasma glucose concentrations were measured, using an Enzymatic UV test of Roche Diagnostics (Roche Diagnostics Indianapolis, Ind., USA). Plasma insulin and C-peptide were determined using radio-immunoassay kits produced by Linco Research, Inc. St. Charles, Mo., USA.
  • the C-peptide levels were measured in order to evaluate the extent of enteral absorption of insulin.
  • the absorption of insulin caused a drop in the C-peptide levels, particularly in the standard meal group, indicating a decrease in endogenous insulin secretion due to the absorption of the insulin and the resulting hypoglycemia.
  • Plasma insulin levels were elevated in most of the subjects in the fasting group. These levels were not always followed by a reduction in the glucose levels.
  • the study consisted of an eligibility screening period, three study periods and a follow-up exam at the conclusion of the last period.
  • the three study periods included the following: administration of single doses of 4-CNAB/insulin followed by fasting (treatment A), followed by an ADA breakfast 20 min after dosing (treatment B), and followed by an ADA breakfast 20 min after dosing (treatment C).
  • the study was conducted using an open label, randomized, crossover design with an interval of at least 7 days between treatments.
  • the patients fasted overnight.
  • the type 1 diabetics were randomized to treatment A or treatment B in periods 1 and 2. In period 3 all diabetics received treatment C.
  • a total of 8 type 1 diabetic patients were enrolled.
  • As a control group 2 healthy volunteers were enrolled.
  • the study consisted of an eligibility screening period, one study period and a follow-up exam at the conclusion of the period.
  • the healthy control subjects were not receiving any medication but served as a control for the effect of breakfast on insulin production.
  • Blood sampling and safety assessments followed the same schedule as for the diabetics.
  • the healthy control subjects received the standard ADA breakfast at the same time as the type 1 diabetics in one study period (treatment D).
  • This study tested the effect of a standard ADA breakfast administered 30 or 20 minutes after dosing on the absorption and pharmacokinetics of 4-CNAB/insulin administered as oral capsules.
  • a control group of healthy subjects received a standard ADA breakfast in one period to measure the amount of insulin produced for this breakfast in healthy control subjects.
  • the type I diabetic patientss were taken off their regular long-acting insulin 24 hrs prior to dosing and their glucose levels were controlled prior to dosing by overnight insulin infusion.
  • the type I diabetics stopped their regular long acting insulin 24 hrs prior to dosing. They were allowed to use their immediate acting insulin up to their entry into the clinic around 15:00 hrs on day 1. They received 4-6 units (depending on their weight) of regular insulin subcutaneously (s.c.) at approximately 17:30 hrs on day 1. Thirty minutes after the administration of s.c. insulin, the diabetics received a standard dinner. Between 20:30 and 21:00 hrs, the diabetics received a snack. At approximately 21:00 hrs on day-1 an i.v. infusion of insulin was started at the infusion rate indicated in Table 38. The composition of the insulin infusion and the infusion rate were dependent on the patient's weight and blood glucose concentration as described in Tables 37 and 38. TABLE 37 Composition of insulin infusate Insulin (U/L) Patient weight (kg) 80 60-65 88 65-70 96 70-75 104 75-80 112 80-85 124 85-90 140 90-95 180 >95
  • Infusion rate was adjusted, if necessary, based on the results of blood glucose measurements done every 60 minutes.
  • a blood sample (one drop) for assessment of real-time blood glucose using a Glucocard® was taken from an indwelling cannula.
  • the blood glucose concentration was adjusted to remain between 6 and 8 mmol/l.
  • the insulin infusion was stopped 30 minutes before drug administration at approximately 09:00 hrs on day 1. At times when no insulin was needed, only normal saline is administered.
  • the 4-CNAB Sodium N-[4-(4-chloro-2-hydroxybenzoyl)amino]butyrate
  • Emisphere Technologies, Inc. of Tarrytown, N.Y. in 400 mg strength oral capsules.
  • Glucose stabilization prior to dosing was done with Actrapid, manufactured by Novo Nordisk and having an active compound of insulin, at 100 U/ml strength, via i.v. infusion.
  • the insulin for subcutaneous injection was Actrapid, manufactured by Novo Nordisk, at 100 U/ml strength.
  • the type I diabetics stopped their regular long acting insulin 24 hrs prior to dosing. They were allowed to use their immediate acting insulin up to their entry into the clinic around 15:00 hrs on day-1. They received 4-6 units of regular insulin s.c. at approximately 17:30 hrs on day-1. Thirty minutes after the administration of s.c. insulin, the diabetics received a standard dinner (see section 13.4). Between 20:30 and 21:00 hrs, the diabetics received a snack then they were fasted until the next morning. At approximately 21:00 hrs an i.v. infusion of insulin was started. The insulin infusion was stopped 30 minutes before drug administration at approximately 09:00 hrs on day 1.
  • the healthy control subjects did not receive any medication but received a standard ADA breakfast at the same time as the diabetics (approximately 09:30 hrs on day 1) after an overnight fast.
  • the controls may resume normal meals after the 3-hr blood sample has been taken.
  • Methylxanthine-containing beverages or food were not allowed from 48 hours (2 days) prior to entrance into the clinical research center and during the study.
  • Blood samples for Pharmacokinetic analysis of 4-CNAB and insulin were drawn 30, 15 and 5 minutes prior to and at 10, 20, 30, 40, 50, 60, 75, 90, 105, 120, 150 and 180 minutes after drug administration (15 samples per subject per period).
  • the blood samples (6 mL each) were taken via an indwelling Venflon® catheter or by direct venepuncture into sodium heparin-containing tubes.
  • the blood samples were centrifuged at 1500 ⁇ g for fifteen minutes at a temperature between 2° C. and 8° C., within one hour of sample collection.
  • the total volume of about 450 mL (type 1 diabetics) or about 180 mL (healthy control subjects) blood was taken during the study.
  • Blood samples for plasma glucose will be drawn at: 30, 15 and 5 min prior to and at 10, 20, 30, 40, 50, 60, 75, 90, 105, 120, 150 and 180 minutes after drug administration (15 samples per subject per period). Samples will be drawn into 2 ml lithium heparin tubes. The blood samples were centrifuged at 1500 ⁇ g for fifteen minutes at a temperature between 2° C.-8° C., within one hour of sample collection.
  • Blood samples for real-time glucose were drawn every 60 min during i.v. insulin infusion on day ⁇ 1 and day 1, at pre-dose and at 30, 60, 90, 120, 150 and 180 after each drug administration on day 1 (10 samples per subject per period).
  • the blood samples were taken from the indwelling canula (with obturator), one drop per assessment.
  • the samples were analyzed for glucose in real time using a Glucocard®.
  • the pharmacokinetic parameters to be determined or calculated from the plasma concentration time data for 4-CNAB and insulin are C max (maximum plasma concentration), t max (time to attain maximum plasma concentration), k el (elimination rate constant), t 1/2 (elimination half-life), AUC last (area under the plasma concentration-time curve up to time t, where t is the last time point with concentrations above the lower limit of quantitation (linear trapezoidal rule)), AUC 0- ⁇ (total AUC after extrapolation from time t to time infinity, where t is the last time point with a concentration above the lower limit of quantitation), AUC last +c last /k el , and %AUC extra (percentage of estimated part for the calculation of AUC 0- ⁇ : (AUC 0- ⁇ ⁇ AUC last )/AUC 0- ⁇ )*100%)).
  • a blood sample (one drop) was analyzed for real-time glucose every 60 min. during the time of insulin infusion and at pre-dose, 30, 60, 90, 120, 150, 180 and 240 min after each drug administration on day 1 (8 samples per subject per period).
  • a blood sample (one drop) will be analyzed for real-time at pre-dose, 30, 60, 90, 120, 150, 180 and 240 min after each drug administration on day 1 (8 samples per subject per period).
  • Blood samples for plasma glucose will be drawn at: 30, 15 and 5 min prior to and at 10, 20, 30, 40, 50, 60, 75, 90, 120, 150, 180, 210, and 240 min after drug administration (16 samples per subject per period).
  • pre-dose refers to the time that the group of diabetics receives 4-CNAB/insulin. The healthy control subjects do not receive medication.
  • a primary objective of this study was to compare the effect of an oral insulin formulation (300 U insulin combined with 400 mg 4-CNAB in 2 capsules, each capsule containing 150 U insulin/200 mg 4-CNAB) with that of 12 U subcutaneous (s.c.) injected short acting insulin [Humalog® injection 100 U/ml from Eli Lilly and Company] on postprandial blood glucose excursions.
  • the postprandial blood glucose excursions were assessed after a standardized breakfast intake.
  • each subject was randomized to one of six possible treatment sequences, such that each received one of six possible treatments at each treatment visit day, as determined by the randomization sequence. Subjects were randomly assigned to one of six sequence groups:
  • the 4-CNAB used for the capsules was manufactured under GMP compliance.
  • the Insulin used to prepare the capsules was Zinc-Insulin Crystals Human: Proinsulin Derived (Recombinant DNA Origin) USP Quality obtained from Eli Lilly and Company (Indianapolis, Ind.).
  • the Insulin/4-CNAB capsules contained 150 Insulin Units USP and 200 mg 4-CNAB.
  • the insulin/4-CNAB capsules were prepared by aaiPharma Inc., Wilmington N.C.
  • Insulin/4-CNAB capsules were provided in HDPE bottles, each of which contained 40 capsules and a polyester coil. Each bottle had a heat-induction seal and a child-resistant cap, and were stored frozen at or less than minus 10° C. On the day of dosing, the appropriate number of capsules was removed from the freezer and brought to room temperature (between 15 and 30° C.) for about one hour. Capsules were used within four hours of dispensing, and unopened bottles were not left at room temperature for more than four hours.
  • exogenous insulin was administered by oral insulin administration or by subcutaneous injection at two of the three experimental days.
  • the pharmacodynamic response elicited was studied by measurements of blood glucose concentrations in 5 minute intervals for another 6 hours, and no food intake was allowed during this period, although water was consumed as desired.
  • Plasma samples for the determination of plasma insulin concentrations, 4CNAB and c-peptide were collected at defined intervals (see Table 39). Plasma samples were stored at approximately ⁇ 20° C. (4CNAB at ⁇ 70° C.) until determination by immunoassay is performed. After the end of the sampling period, the study subjects were released from the clinic.
  • the pharmacodynamic endpoint was the area under the blood glucose excursion curve (AUC BG ) in the first two hours after meal intake (AUC BG 0-2 h ).
  • AUC BG blood glucose excursion curve
  • TBG max time to BG max
  • AUC BG 0-1 h Area under the blood glucose excursion curve in defined time-intervals
  • AUC BG 0-1 h Area under the blood glucose excursion curve in defined time-intervals
  • AUC BG 0-1 h AUC BG 0-2 h
  • AUC BG 0-3 h AUC BG 0-4 h
  • AUC BG 0-6 h maximal absolute blood glucose concentrations
  • BGabs max time to BGabs max
  • Plasma insulin concentrations were be subjected to appropriate pharmacokinetic analyses. Parameters to be determined include maximum plasma concentrations (C max ), time to maximum plasma concentrations (t max ), and the area under the plasma concentration versus time curve from the time of dosing until a return to the baseline measurement (AUC 0-t′ ), where t′ is the time that the level of plasma insulin concentration returns to the baseline. In addition, other pharmacokinetic parameters, such as half-life (t 1/2 ), elimination rate constant ( ⁇ z ) and partial AUC values, may be calculated if considered appropriate. Parameters will be calculated for each individual subject enrolled within the study.
  • the delivery agent 4-CNAB was prepared by Emisphere Technologies, Tarrytown, N.Y. Insulin (Zinc, Human Recombinant) was purchased from Calbiochem (San Diego, Calif.). The Insulin potency was approximately 26 USP units/mg. Insulin was stored as a lyophilized solid at ⁇ 20° C. In solution, it was stored as frozen aliquots (15 mg/mL) that were subjected to only one freeze-thaw cycle.
  • An aqueous insulin stock solution was prepared (at pH 7.5) with a final insulin concentration of approximately 15 mg/mL. Delivery agents were dissolved in water with subsequent additions of sodium hydroxide or hydrochloric acid to both dissolve the delivery agent and to titrate the dosing solution to pH 7.5-8.5. The required amount of insulin was added to the delivery agent solution before dosing.
  • Insulin levels in the rats were assayed using the Insulin ELISA Test Kit [DSL, Webster, Tex. Cat. #DSL-10-1600]. The assay covered the range 3.125 to 250 mU/mL. Blood glucose levels in the rats were measured using a glucometer, One-Touch Basic Blood Glucose Monitoring System, manufactured by Lifescan Inc. (Milpitas, Calif.).
  • Insulin p.o., 1 mL/kg; 0.5 mg/kg
  • Insulin and Carrier p.o., 1 mL/kg; 0.5 mg/kg Insulin and 200 mg/kg 1182)
  • Insulin s.c., 0.05 mg/kg
  • FIG. 14 shows a graph of blood glucose (mg/mL) over time following a single administration with subcutaneous and oral delivery. This figure shows that the administration of insulin orally, using 4-CNAB as a carrier, yielded approximately 95% of the glucose depression seen with the traditional subcutaneous dosing.
  • FIG. 15 which shows the serum insulin (mU/mL) over time using the administrations of FIG. 13, the serum insulin C max required to achieve this depression however, in the orally administered animals was approximately 30% of those receiving subcutaneous injections. The T max was also about 15 minutes later for the subcutaneous sample.
  • the depression in blood glucose was likely eliminated by the continued administration of anesthesia in order to continue blood sampling.
  • Glycolysis/Gluconeogenesis occurs through three main cycles that can be driven in both a glycolytic and gluconeogenic direction. From a glycolytic standpoint, the first cycle is the Glu/Glu-6-Pase Cycle, which converts glucose to Glc-6-P. This is followed by the Fru-6-P/Fru-1, 6-P 2 Cycle, and the Pyruvate/PEPCK Cycle.
  • FIG. 16 shows Glucokinase and G6Pase mRNA expression compared to sham dosing. As shown in FIG. 16, despite the lower serum insulin levels, the orally dosed animals showed a 2-fold higher level of hexokinase II at 120 minutes. Direct comparison of the arrays from the orally dosed and subcutaneously dosed animals indicates a 2.8-fold higher mRNA level at 30 minutes.
  • the bi-functional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase serves as a switch between gluconeogenesis and glycolysis. Insulin administration has been shown to drive increases in this enzyme. Granner et al., J Biol Chem 265, 10173-6. (1990); Lemaigre et al.,. Biochem J 303, 1-14. (1994); Denton. et al., Adv Enzyme Regul 36, 183-98 (1996).
  • FIG. 4 shows Fru-1, 6-P and 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase mRNA expression compared to sham dosing. In our studies and as shown in FIG. 17, this enzyme showed a nearly identical pattern of expression between the two routes of administration with no significant differences in gene expression being observed.
  • the enzyme Fructose 1,6-bisphosphatase catalyzes the conversion of Fru-1, 6-P 2 to Fru-6-P, the gluconeogenesis side of this cycle. This mRNA is induced by diabetes and starvation and reduced by insulin administration. As shown in FIG. 17 and similar to 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, the pattern of expression for this enzyme is nearly identical in both sets of test animals.
  • Phosphoenolpyruvate carboxykinase is a key enzyme in the gluconeogenesis pathway converting oxaloacetate to phosphoenolpyruvate. It is known to be down regulated by insulin. See, for example, Granner et al., J Biol. Chem. 265, 10173-6. (1990); Lemaigre et al,. Biochem J 303, 1-14. (1994); Denton, R. M. et al., Adv Enzyme Regul 36, 183-98 (1996); Gabbay et al., J Biol Chem 271, 1890-7 (1996).
  • FIG. 18 shows PEPCK mRNA expression compared to sham dosing. As shown in FIG. 18, little difference is seen in the expression levels of this mRNA.
  • Insulin is also known to increase the rate of glucose conversion to glycogen. This is performed by linking glucose-1-P molecules into a branched chain. This chain then serves as a store of glucose to be utilized in hypoglycemic states.
  • Glycogen Synthase enzyme is responsible for extending the chain of glucose molecules. Administration of insulin is known to up-regulate this enzyme. Vestergaard et al., Dan Med Bull 46, 13-34 (1999).
  • FIG. 19 shows glycogen synthase mRNA expression compared to sham dosing. As shown in FIG. 19, oral dosing and subcutaneous dosing produced nearly identical patterns of expression in the levels of this enzyme, with oral dosing yielding nearly twice the increase in mRNA at 120 minutes.
  • Glycogen Synthase Kinase 3 is involved in the inhibition of glycogen synthesis through the phosphorylation of glycogen synthase. As shown in FIG. 6, the expression pattern of this enzyme in the two dosing samples was very similar, exhibiting an initial decrease that returns to sham levels at 120 and 180 minutes. Subcutaneous dosing achieved a slightly stronger down regulation at 60 minutes; however, this difference was not seen in the direct comparison between the two GeneChips.
  • Glycogen Phosphorylase is responsible for the breakdown of the glycogen chain. Insulin is known to normalize phosphorylase levels in diabetic animals. In our studies, as shown in FIG. 19, a dramatic difference in the levels of this enzyme was observed.
  • the oral dosing achieved an immediate decrease in mRNA levels that slowly increased to sham levels at 180 minutes.
  • the subcutaneous dosing yielded an early up regulation that was reversed dramatically at 120 minutes and returned to near sham levels at 180 minutes. The differences seen between the oral and subcutaneous dosings were observed in comparison to sham as well as to each other.
  • vascular diseases are commonly described as a response to injury.
  • the vessel is exposed to a stimulus (injury) that leads to a progression of responses designed to repair damage to the vessel wall.
  • This injury may be in several forms, including oxidative stress, mechanical stress, viral infection and changes in shear stress.
  • the injury itself is variable, the response to injury has many common aspects.
  • Early response genes are up regulated leading to the transcription of genes for cellular migration and proliferation as well as the recruitment of inflammatory cells to the site of injury. As the response continues, enzymes that lead to matrix remodeling will be expressed.
  • the result is generally the thickening of the arterial wall through smooth muscle proliferation and atherosclerotic plaque formation.
  • the clinical result is the arteriopathies associated with diabetes.
  • a method for examining the mRNA levels of genes associated with various forms of vascular injury is described.
  • Vascular diseases are a complex set of processes that involve numerous changes in mRNA levels. While the mRNA markers of vascular injury presented here were seen in this specific study, several others are likely to exist. These include early response genes (i.e. c-myb and c-fos), cytokines (i.e. interleukins, and chemokines), growth factors and their receptors (i.e. fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor beta), adhesion molecules (i.e. selectins, and integrins), extracellular matrix proteins (i.e. collagen and actin), matrix metalloproteinases and their inhibitors, cell cycle proteins (i.e.
  • cyclins and cyclin dependent kinases i.e. mitogen activated protein kinases, and protein kinase C
  • protein kinases i.e. mitogen activated protein kinases, and protein kinase C
  • FIGS. 20 A and 20 B show early response gene mRNA expression compared to sham dosing. In our studies, and as shown in FIGS. 20A and 20B, a differential expression in the levels of Egr-1, c-myc, Jun B and Ets-1 was observed.
  • Egr-1 is associated with several elements of the vascular response to injury. Its expression is very low in uninjured vessels but increases with mechanical or oxidative injury. Egr-1 has been demonstrated to drive increases in mRNA levels of cytokines, adhesion molecules, growth factors and members of the coagulation cascade. In our study and as shown in FIGS. 20A and 20B, Egr-1 is immediately up regulated by subcutaneous insulin administration to a level 4.7-fold higher than with oral. Oral administration does not induce an early increase in Egr-1 mRNA levels. Instead, levels are maintained at near sham levels until at 180 minutes when they are elevated to slightly below that of the subcutaneously animals.
  • the switch in the cell cycle state from the dormant G 0 to the proliferative G 1 is accompanied by increased levels of c-myc.
  • Vascular damage induces expression of c-myc, and inhibition of c-myc through antisense oligonucleotides prevents intimal hyperplasia following balloon injury in the rat and porcine. It is therefore a critical marker of vascular injury.
  • FIGS. 20A and 20B subcutaneous dosing and not oral dosing lead to a significant increase in the mRNA levels of c-myc at 180 minutes.
  • the orally dosed samples remained at near sham levels; however no significant difference between the two dosing routes was seen when compared directly.
  • Ets-1 mRNA levels for subcutaneous and oral dosing are shown in FIG. 20A.
  • IGF I and II Insulin-like growth factor (IGF) I and II are a single chain polypeptides sharing homology with proinsulin. They play an important role in systemic glucose metabolism but have also been shown to effect cell cycle progression, mitogenesis, cell migration and apoptosis. Much of IGF's function is regulated by IGF-binding proteins (IGFBPs). In general, IGFBPs bind to IGF, preventing its binding to the IGF receptor. IGFBP-3 is the most prevalent in this respect, binding >90% of the IGF in adult serum. Though their primary function is the regulation of IGF, IGFBPs have been shown to have a biological effect at sites of vascular injury. IGFBP-1 stimulates the migration of vascular smooth muscle cells (VSMC) independent of IGF-1. IGFBP-1 to -5 have been shown to be expressed in restenotic tissue suggesting a role in the arterial response to vascular injury.
  • IGFBPs IGF-binding proteins
  • FIG. 21 shows IGFBP mRNA expression compared to sham dosing.
  • IGFBP-1 was reduced 39-fold as opposed to 3-fold
  • IGFBP-2 was reduced 15-fold compared to 6-fold at two hours compared to the sham dosing.
  • IGFBP expression is decreased, opposite to the effect seen in vascular injury. The mechanism driving this change may be beyond that of a direct effect of insulin on the VSMC's. Nonetheless, it is clear that the level of IGFBP-1 and -2 expression is higher in the subcutaneously dosed animals and this correlates with an increased response to injury. Little change was observed in IGFBP-3.
  • ICAM-1 intercellular adhesion molecule-1
  • VCAM-1 vascular cellular adhesion molecule-1
  • selectins the selectins and the integrins.
  • FIG. 22 shows intercellular adhesion molecule-1 mRNA expression compared to sham dosing. As shown in FIG. 22, no significant effect was seen in either dosing group except in the case of ICAM-1. ICAM-1 was increased at 180 minutes in the subcutaneously dosed animals. Increased expression of ICAM-1 is seen in several different forms of vascular injury, and is associated with the recruitment of inflammatory cells to the site of injury. This difference is seen both in the comparison to sham and in the direct comparison of the arrays from the two dosing groups.
  • ICAM-1 intercellular adhesion molecule-1
  • VCAM-1 vascular cellular adhesion molecule-1
  • FIGS. 23A and 23B show cytokine mRNA expression compared to sham dosing. In our studies and as shown in FIGS.
  • gp130 was seen to be equally increased in both subcutaneously and orally dosed animals.
  • a significant increase in IL-6 mRNA was seen only in the subcutaneously dosed group, as shown in FIGS. 23A and 23B. This is a critical difference as it shows the aorta of the both groups getting “primed” for a response to injury, but only the subcutaneous dosing actually drives significant production of the expected signal.
  • Cytokine data are represented graphically also for the cytokines Eotaxin, MCP-1, IL-12 and EL-13 in FIG. 23A.
  • FIG. 24 shows lipid peroxidation mRNA expression compared with sham dosing.
  • FIG. 24 shows lipid peroxidation mRNA expression compared with sham dosing.
  • this up-regulation is reversed at 120 and 180 minutes with 3.4- and 2.1-fold reductions in mRNA levels compared to sham.
  • the mRNA levels remain high until 180 minutes, at which time a 6.4-fold decrease is observed. It is important to note that the values at 120 minutes represent greater than 14-fold higher levels of this mRNA in the subcutaneously dosed samples.
  • Heme Oxygenase-1 (HO-1) is induced by mildly oxidized LDL. It serves a protective antioxidant function through elimination of heme and the further antioxidant capabilities of its reaction products. As shown in FIG. 24, the mRNA levels of this gene are seen to be 6-fold higher in the subcutaneously dosed animals when compared to the orally dosed animals at 60 minutes. Though the function of this enzyme is protective, its up regulation represents a response to injury and may well be in response to the increased levels of 12-LO or its stimulation of LDL oxidation.
  • FIG. 25 shows plasminogen activator inhibitors mRNA expression compared to sham dosing.
  • PAI-1 and ⁇ 2 plasminogen activator inhibitors
  • FIG. 25 shows plasminogen activator inhibitors mRNA expression compared to sham dosing.
  • PAI-1 levels were elevated in the subcutaneous samples only. A 2.6-fold increase over sham and a 2.8-fold increase over oral were seen at 120 minutes. A 2.6-fold decrease in these levels was seen in the oral samples at 60 minutes, which returned to sham levels at 120 and 180 minutes.
  • PAI-2 expression was similar in both sets of dosing. At 60 minutes, the orally dosed samples exhibited a significantly greater (4.8-fold) level of PAI-2. This elevation is not present at 120 and 180 minutes. There was no significant difference between the two dosing routes for this mRNA.
  • FIG. 26 compares the effects of subcutaneous delivery of insulin and oral delivery of insulin on the mRNA expression of NPY, TGF-beta, ICAM-1 and 12-LO.
  • FIG. 28 shows a graph of blood glucose (mg/mL) over time following a single administration with subcutaneous and oral delivery for a streptozotocin diabetic model. Two different oral dosages of insulin are demonstrated.
  • FIG. 29 shows the serum insulin levels (mU/mL) over time using the administrations of FIG. 28.
  • the examples discussed above demonstrate the ability of an oral composition of insulin to alleviate the undesirable effects on the vasculature of the traditional subcutaneous dosing at the level of messenger RNA regulation, and document changes in glucose metabolism caused by altering the dosing route.
  • the pharmacodynamic data demonstrates the ability of the orally dosed composition to achieve a glucose depression similar to that of the traditional dosing method. Though the pharmacodynamic data is similar, the pharmacokinetic data shows a greatly lower serum insulin level in the orally dosed composition compared with that of the traditional dosing method. . The difference in serum insulin level must be a result of direct administration of the insulin to the liver. The liver reacts to the bolus of insulin in two ways.
  • first-pass metabolism decreases the level of insulin reaching the systemic circulation. The result is a rapid decrease in blood glucose and a decrease in the level of insulin to which the systemic circulation is exposed. In achieving a similar glucose control as subcutaneous dosing while lowering the exposure of the peripheral circulation to insulin, the undesirable effects of insulin on non-target tissues can be prevented.
  • VSMCs Although not considered a major site of glucose metabolism, VSMCs do possess glucose regulatory capacity and therefore yield insight into differences in the peripheral response to changing the dosing route. What is surprising is that despite drastically lower levels of circulating insulin, little difference in the mRNA levels of key enzymes involved in glucose regulation is observed. In fact, the levels seen for hexokinase II and glycogen synthase suggest a stronger response to the oral composition.
  • natural regulation of glucose involves the liver controlling peripheral glucose metabolism and utilization through a messenger other than insulin. The fact that higher circulating levels of insulin can compensate for the loss of this natural process may simply be due to the fact that the two proteins bind the same or similar receptors.
  • the IGF system is a prime candidate for such secondary signaling and is known to exhibit glucose regulatory activity.
  • IGFBP-1 and -2 were down regulated in both sets of data. This is contrary to published data on vascular injury and may not be associated with a vascular injury response so much as playing a role in glucose control.
  • the data previously reported on the liver's response to changes in dosing route of insulin does not demonstrate a differential response in either the IGF's or their binding proteins. This does not rule out this pathway, as no data is available on the proteases responsible for degradation of the IGFBP's. It is quite possible that the liver responds to elevated insulin levels by releasing IGFBP proteases that then degrade IGFBP's freeing IGF to drive a reduction in glucose. Further study is required to determine if this is the case, but the liver and aorta gene array data supports this hypothesis. If correct, this hypothesis supports the use of an oral composition of insulin simply based on its ability to mimic the natural glucose control pathway.
  • the first step is to assess any effects from administration of the carrier alone. It is believed that, upon entering the systemic circulation, the carrier and insulin no longer interact due to the dilution effect.
  • An exhaustive analysis of the array data from animals receiving the carrier without insulin was performed including adding a second three-hour experiment to try to further identify any response. The analysis yielded no mRNA's whose levels appear to be affected by administration of the carrier. To clarify, any response seen in the carrier alone samples was also seen in the animals receiving insulin orally without carrier suggesting that the effect was due to an experimental parameter not accounted for in the sham dosing. This study is not designed to identify specific genes regulated by the carrier, as such a study would require multiple animals at each time point. Nonetheless, this data supports the view that the carrier has a minimal effect on the vasculature.
  • the vascular response to injury is a complex set of processes that occur over an extended period of time. Some of these, such as atherosclerotic plaque formation, occur over years or even decades, while the more rapid examples, such as Restenosis, occur on the order of months. Thus, the time scale for studying vascular damage in animal models is on the order of days or weeks and not hours. In this study, we aimed to identify any signs of vascular injury induced by a single dose of insulin and to document any effect changing the route of administration had on these markers.

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WO2003057170A8 (fr) 2004-01-15
US7429564B2 (en) 2008-09-30
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CA2471769C (fr) 2011-03-22
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