Classifications

• • 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
  • A61K9/2009—Inorganic compounds
• • 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
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
  • 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
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
  • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin

Definitions

• This invention relates to the oral delivery of insulin in a therapeutically effective amount to the bloodstream as part of a therapeutic regimen for the treatment of diabetes.
• This invention also relates to oral administration of compositions of insulin and a delivery agent that facilitates insulin transport in a therapeutically effective amount to the bloodstream for the treatment of diabetes.
• the present invention is also directed to therapies and protocols for administration of oral pharmaceutical dosage forms of insulin on a chronic basis to pre-diabetics, including those with impaired glucose tolerance and/or insulin resistance, to early stage diabetics, and to late stage diabetics.
• the present invention further relates to methods for reducing adverse effects and the incidence of diseases that are associated with systemic hyperinsulinemia and hyperglycemia, especially to the 3-cells ofthe pancreas.
• biological macromolecules namely biological polymers such as proteins and polypeptides
• proteins are increasingly being use in many diverse areas of science and technology.
• proteins are employed as active agents in the fields of pharmaceuticals, vaccines and veterinary products.
• biological macromolecules as active agents in pharmaceutical compositions is 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.
• 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.
• delivery vehicles can be used to facilitate absorption through the gastro-intestinal tract. These delivery vehicles must ⁇ c aoie iu release acuve molecules at a rate that is consistent with the needs ofthe particular patient or the disease process.
• insulin contributes to the normal regulation of blood glucose levels through its release by the pancreas, more specifically by the 3-cells of a major type of pancreatic tissue (the islets of Langerhans), so that the glucose can be used as a source of energy.
• Insulin secretion is a regulated process that, in normal subjects, provides stable concentrations of glucose in blood during both fasting and feeding. In healthy humans, insulin is secreted from the pancreas into the portal vein, which carries the insulin to the liver.
• the liver utilizes and/or metabolizes a large portion ofthe insulin that it receives from the portal circulation, hi very basic terms, the liver plays a key role in the metabolism of glucose as follows: in the presence of excess insulin, excess glucose, or both, the liver modulates the production of glucose released into the blood; and, in the absence of insulin or when the blood glucose concentration falls very low, the liver manufactures glucose from glycogen and releases it into the blood.
• the liver acts as a key blood glucose buffer mechanism by keeping blood glucose concentrations from rising too high or from falling too low.
• Blood glucose concentration is the principal stimulus to insulin secretion in healthy humans.
• the exact mechanism by which insulin release from the pancreas is stimulated by increased glucose levels is not fully understood, but the entry of glucose into the j3-cells ofthe pancreas is required.
• Glucose enters the pancreatic /3-cells 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 /3-cells.
• the capacity of sugars to undergo phosphorylation and subsequent glycolysis correlates closely with their ability to stimulate insulin release. It is noted that not all tissues are dependent on insulin for glucose uptake. For example, the brain, kidneys and red blood cells are insulin independent tissues, while the liver, adipose and muscle are insulin dependent tissues.
• insulin secretion When evoked by the presence of glucose (e.g., after a solid meal is ingested) in a non- diabetic individual, insulin secretion is biphasic: shortly after ingesting food, the pancreas releases the stored insulin in a burst, called a first phase insulin response, and then approximately 15-20 minutes later outputs further insulin to control the glycemic level from the food.
• the first phase insulin response reaches a peak after 1 to 2 minutes and is short-lived, whereas a second phase of secretion has a delayed onset but a longer duration.
• secretion of insulin rises rapidly in normal human subjects as the concentration of blood glucose rises above base levels (e.g., 100 mg/lOOml of blood), and the turn-off of insulin secretion is also rapid, occurring within minutes after reduction in blood glucose concentrations back to the fasting level.
• base levels e.g. 100 mg/lOOml of blood
• Insulin facilitates (and increases the rate of) glucose transport through the membranes of many cells ofthe body, particularly skeletal muscle and adipose tissue. Insulin has three basic effects: the enhanced rate of glucose metabolism, the promotion of increased glycogen stores in muscle and adipose tissue, and decreased circulating blood glucose concentration.
• Diabetes Mellitus (“diabetes”) is a disease state in which the pancreas does not release insulin at levels capable of controlling blood glucose and/or in which muscle, fat and liver cells respond poorly to normal insulin levels because of insulin resistance. Diabetes thus can result from a dual defect of insulin resistance and "burn out" ofthe /3-cells ofthe pancreas. Diabetes Mellitus is classified into two types: Type 1 and Type 2. Approximately 5 to 10% of diagnosed cases of diabetes are attributed to Type 1 diabetes, and approximately 90% to 95% are attributed to Type 2 diabetes.
• Type 1 diabetes is diabetes that is insulin dependent and usually first appears in young people.
• the islet cells ofthe pancreas stop producing insulin mainly due to autoimmune destruction, and the patient must self-inject the missing hormone.
• insulin therapy is essential and is intended to replace the absent endogenous insulin with an exogenous insulin supply.
• Type 2 diabetes is commonly referred to as adult-onset diabetes or non-insulin dependent diabetes and may be caused by a combination of insulin resistance (or decreased insulin sensitivity) and, in later stages, insufficient insulin secretion. This is the most common type of diabetes in the Western world. Close to 6% ofthe adult population of various countries around the world, including the United States, have Type 2 diabetes, and about 30% of these patients will need exogenous insulin at some point during their lifespans due to secondary pancreatic exhaustion and the eventual cessation of insulin production. For type 2 diabetics, therapy has consisted first of oral antidiabetic agents, which increase insulin sensitivity and/or insulin secretion, and only then insulin if, and when, the oral agents fail.
• Diabetes is the sixth leading cause of death in the United States and accounted for more than 193,000 deaths in 1997. However, this figure is an underestimate because complications resulting from diabetes are a major cause of morbidity in the population. Diabetes is associated with considerable morbidity and mortality in the form of cardiovascular disease, stroke, digestive diseases, infection, metabolic complications, ophthalmic disorders, neuropathy, kidney disease and failure, peripheral vascular disease, ulcerations and amputations, oral complications, and depression. Thus, diabetes contributes to many deaths that are ultimately ascribed to other causes.
• the main cause of mortality with Diabetes Mellitus is long term micro- and macro- vascular disease.
• Cardiovascular disease is responsible for up to 80% ofthe deaths of type 2 diabetic patients, and 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.
• heart disease, high blood pressure, heart attacks and strokes occur two to four times more frequently in adult diabetics than in adult non-diabetics.
• 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.
• These vascular complications lead to neuropathies, retinopathies and peripheral vascular disease.
• Diabetic retinopathy (lesions in the small blood vessels and capillaries supplying the retina ofthe eye, i.e., the breakdown ofthe lining at the back ofthe eye) is the leading cause of blindness in adults aged 20 through 74 years, and diabetic kidney disease, e.g., nephropathy (lesions in the small blood vessels and capillaries supplying the kidney, which may lead to kidney disease, and the inability ofthe kidney to properly filter body toxins), accounts for 40% of all new cases of end-stage renal disease (kidney failure). Furthermore, diabetes is also the leading cause for amputation of limbs in the United States. Diabetes causes special problems during pregnancy, and the rate of congenital malformations can be five times higher in the children of women with diabetes.
• Insulin resistance is also prevalent in the population, especially in overweight individuals, in those with risk of diabetes (i.e., pre-diabetic, wherein blood glucose levels are higher than normal but not yet high enough to be diagnosed as diabetes) and in individuals with type 2 diabetes who produce enough insulin but whose tissues have a diminished ability to adequately respond to the action of insulin.
• pre-diabetic wherein blood glucose levels are higher than normal but not yet high enough to be diagnosed as diabetes
• type 2 diabetes who produce enough insulin but whose tissues have a diminished ability to adequately respond to the action of insulin.
• the liver becomes insulin-resistant, the mechanism by which insulin affects the liver to suppress its glucose production breaks down, and the liver continues to produce glucose, even under hyperinsulinemic conditions (elevated plasma insulin levels). This lack of suppression can lead to a hyperglycemia (elevated blood glucose levels), even in a fasting state.
• pancreatic /3 ⁇ cells In order to compensate and to overcome the insulin resistance, the pancreatic /3 ⁇ cells initially increase their insulin production such that insulin resistant individuals often have high plasma insulin levels. This insulin is released into the portal vein and presented to the liver constantly or almost constantly. It is believed that the liver's constant exposure to high levels of insulin plays a role in increased insulin resistance and impaired glucose tolerance. After a period of high demand placed on the pancreatic /3-cells, the cells start to decompensate and exhaust, and insulin secretion, or insulin secretory capacity, is reduced at later stages of diabetes. It is estimated that, by the time an individual is diagnosed with type 2 diabetes, roughly 50% ofthe / 3-cells have already died due to increased demand for insulin production.
• Insulin resistance plays an important role in the pathogenesis of hyperglycemia in type 2 diabetes, eventually inducing the development of diabetic complications. Furthermore, insulin resistance ostensibly plays a role in the pathogenesis of macrovascular disease, cardiovascular diseases and microvascular disease. See, for example, Shinohara K. et al., Insulin Resistance as an Independent Predictor of Cardiovascular Mortality in Patients With End-Stage Renal Disease, J. Am. Soc. Nephrol, Vol. 13, No. 7, July 2002, pp. 1894-1900. Research currently shows that insulin resistance reaches a maximum and then plateaus. Once the insulin resistance plateaus, it is believed to not get appreciably worse, but can improve.
• Diabetes or insulin resistance can be diagnosed in many ways, as is known to those in the art.
• the initial diagnose may be made from a glucose tolerance test (GTT), where a patient is given a bolus of glucose, usually orally, and then the patient's blood glucose levels are measured at regular time intervals for approximately 2 hours, or as many as 6 hours in the case of an extended GTT.
• GTT glucose tolerance test
• Another method of testing for diabetes or insulin resistance is a test ofthe patients fasting or postprandial glucose.
• Other tests, such as Glycosolated Hemoglobin, often reported as Hemoglobin A ⁇ c (HbA ⁇ c ) can be used to assess blood glucose over 2-3 months.
• HOMA-JR fasting plasma glucose
• F ⁇ RI fasting immunoreactive insulin
• Several oral hypoglycemic agents have been developed for specifically improving a patient's insulin resistance, such as thiazolidinediones, which make the patient more sensitive to insulin, and biguanides, which decrease the amount of glucose made by the liver, and these are currently available clinically for patients with diabetes and insulin resistance.
• thiazolidinediones which make the patient more sensitive to insulin
• biguanides which decrease the amount of glucose made by the liver
• sulfonylureas stimulate the pancreas to make more insulin
• alpha-glucosidase inhibitors slow the absorption ofthe starches eaten by an individual
• meglitinides stimulate the pancreas to make more insulin
• D-phenylalanine derivatives help the pancreas make more insulin quickly.
• the aim of insulin treatment of diabetics is typically to administer enough insulin such that the patient will have normal carbohydrate metabolism throughout the day. Because the pancreas of a diabetic individual does not secrete sufficient insulin throughout the day, in order to effectively control diabetes through insulin therapy, a long-lasting insulin treatment, known as basal insulin, must be administered to provide the slow and steady release of insulin that is needed to control blood glucose concentrations and to keep cells supplied with energy when no food is being digested. Basal insulin is necessary to suppress glucose production between meals and overnight, and preferably mimics the patient's normal pancreatic basal insulin secretion over a 24-hour period. Thus, a diabetic patient may administer a single dose of a long-acting insulin each day subcutaneously, with an action lasting about 24 hours.
• bolus insulin which has generally been administered subcutaneously, provides a rise in plasma insulin levels at approximately 1 hour after administration, thereby limiting hyperglycemia after meals.
• these additional quantities of regular insulin with a duration of action of, e.g., 5-6 hours, may be subcutaneously administered at those times ofthe day when the patient's blood glucose level tends to rise too high, such as at meal times.
• basal insulin in combination with bolus insulin, repeated and regular lower doses of bolus insulin maybe administered in place ofthe long-acting basal insulin, and bolus insulin may be administered postprandially as needed.
• hyperinsulinemia can also occur, such as by the administration of insulin in a location (and manner) that is not consistent with the normal physiological route of delivery. Insulin circulates in blood as the free monomer, and its volume of distribution approximates the volume of extracellular fluid. Under fasting conditions, the concentration of insulin in portal blood is, e.g., about 2-4 ng/mL, whereas 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. In human diabetics who receive insulin via subcutaneous injection, the portal vein to periphery ratio is changed to about 0.75: 1.
• the liver does not receive the necessary concentrations of insulin to adequately control blood glucose, while the peripheral circulation is subjected to higher concentrations of insulin than are found in healthy subjects. Elevated systemic levels of insulin may lead to increased glucose uptake, glycogen synthesis, glycolysis, fatty acid synthesis, cortisol synthesis and triacylglycerol synthesis, leading to the expression of key genes that result in greater utilization of glucose.
• One aspect ofthe physiological response to the presence of insulin is the stimulation of glucose transport into muscle and adipose tissue. It has been reported that hyperglycemia and/or hyperinsulinemia is related to vascular diseases associated with diabetes. Impairment to the vascular system is believed to be the reason behind conditions such as microvascular complications or diseases, such as retinopathy, neuropathy (impairment ofthe function ofthe autonomic nerves, leading to abnormalities in the function ofthe gastrointestinal tract and bladder and loss of feeling in lower extremities) and nephropathy, or macrovascular complications or diseases, such as cardiovascular disease, etc.
• microvascular complications or diseases such as retinopathy, neuropathy (impairment ofthe function ofthe autonomic nerves, leading to abnormalities in the function ofthe gastrointestinal tract and bladder and loss of feeling in lower extremities) and nephropathy, or macrovascular complications or diseases, such as cardiovascular disease, etc.
• Oral delivery of insulin is a particularly desirable route of administration, for safety and convenience considerations, because it can minimize or eliminate the discomfort that often attends repeated hypodermic injections. It has been a significant unmet goal in the art to imitate normal insulin levels in the portal and systemic circulation via oral administration of insulin. [0028] Oral delivery of insulin may have advantages beyond convenience, acceptance and compliance issues.
• Insulin absorbed in the gastrointestinal tract is thought to mimic the physiologic route of insulin secreted by the pancreas because both are released into the portal vein and carried directly to the liver before being delivered into the peripheral circulation. Absorption into the portal vein maintains a peripheral-portal insulin gradient that regulates insulin secretion. In its first passage through the liver, roughly 60% ofthe insulin is retained and metabolized, thereby reducing the incidence of peripheral hyperinsulinemia, a factor linked to complications in diabetes.
• insulin exemplifies the problems confronted in the art in designing an effective oral drug delivery system for biological macromolecules. Insulin absorption in the gastrointestinal tract is prevented presumably by its molecular size and its susceptibility for enzymatic degradation. The physicochemical properties of insulin and its susceptibility to enzymatic digestion have precluded the design of a commercially viable oral or alternate delivery system.
• Emisphere Technologies, Inc. has developed compositions of insulin that are orally administrable, e.g., absorbed from the gastrointestinal tract in adequate concentrations, such that the insulin is bioavailable and bioactive following oral administration and provide sufficient absorption and pharmacokinetic/pharmacodynamic properties to provide the desired therapeutic effect, i.e., cause a reduction of blood glucose, as disclosed in U.S. Patent Applications Nos.
• the novel drug delivery technology of Emisphere Technologies, hie. is based upon the design and synthesis of low molecular weight compounds called "delivery agents.”
• the delivery agent which is in a preferred embodiment sodium N-[4-(4- chloro-2 hydroxybenzoyl)amino]butyrate (4-CNAB)
• 4-CNAB sodium N-[4-(4- chloro-2 hydroxybenzoyl)amino]butyrate
• 4-CNAB sodium N-[4-(4- chloro-2 hydroxybenzoyl)amino]butyrate
• 4-CNAB is not intended to possess any inherent pharmacological activity and serves only to increase the oral bioavailability of insulin by facilitating the transport of insulin across the gastrointestinal wall.
• the pharmacology of insulin is the desired therapeutic effect and is well characterized.
• Insulin/4-CNAB capsules were evaluated by Emisphere Technologies, Inc. in a nonclinical program that included pharmacological screening, pharmacokinetic and metabolic profiles, and toxicity assessments in rats and monkeys. These studies in rats and monkeys showed that 4-CNAB is absorbed rapidly following oral administration and that, over the range tested, insulin absorption increased with increasing doses of 4-CNAB. Similarly, for a fixed oral dose of 4-CNAB, insulin absorption increased with increasing doses of insulin.
• Toxicology studies were also conducted in rats and monkeys to assess the potential toxicity of 4-CNAB, alone or in combination with insulin. Based on the 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, i the 90-day oral repeated dose toxicity studies, NOAELs of 250 mg/kg and 600 mg/kg were observed in rats and monkeys, respectively. Four genotoxicity studies have also been conducted with 4-CNAB, with no positive findings. Developmental and reproductive toxicology studies have not yet been conducted.
• NOAEL No-Adverse Effect Level
• Oral insulin/4-CNAB capsules were also evaluated by Emisphere Technologies, Inc. in clinical human studies for safety, pharmacokinetics, pharmacodynamics, and the effect of food on the absorption of insulin/4-CNAB.
• 4-CNAB was shown to enhance the gastrointestinal absorption of insulin following oral administration in diabetic patients and healthy subjects.
• Oral administration of Insulin/4-CNAB capsules resulted in rapid absorption (t max ⁇ 20- 30 minutes) of both insulin and 4-CNAB, and the insulin absorbed orally in combination with 4-CNAB was pharmacologically active, as demonstrated by a reduction of blood glucose in healthy and diabetic subjects and by a blunting of postprandial glucose excursion in diabetic patients.
• the oral dosing method developed by Emisphere Technologies, Inc. is thought to mimic natural physiology, namely, the ratio of concentration of insulin in the portal circulation to that in the systemic circulation approaches the normal physiological ratio, e.g., from about 2:1 to about 6: 1.
• 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.
• the oral insulin formulations of Emisphere Technologies, Inc. provide an advantage over subcutaneously administered insulin that is currently the state ofthe art, beyond the benefit of ease of administration, pain-free administration, and the potential for improved patient compliance. Because subcutaneously administered insulin is delivered peripheral to the Gl tract and portal vein, and absorption of large biomolecules from the subcutaneous space is generally more prolonged, the first-phase insulin response is not well-replicated by subcutaneous insulin administration.
• the oral insulin formulations ofthe present invention the plasma levels of insulin that occur upon the first (acute) phase of insulin secretion by the pancreas can be simulated by rapid, direct absorption from the Gl tract.
• first-phase insulin secretion takes place 5 to 20 minutes after the start of a meal, and this effect has a well-known impact on prandial glucose homeostasis.
• the first phase of insulin secretion while of short duration, has an important role in priming the liver to the metabolic events ahead (meal).
• the loss of first-phase insulin secretion is a characteristic feature of Type 2 diabetic patients in the early stages ofthe disease, and it is also observed in prediabetic states, namely individuals with impaired glucose tolerance.
• the stimulatory effect of glucagon on gluconeogenesis is not suppressed and may contribute to the development of prandial hyperglycemia.
• compositions of insulin that can be administered closer to as meal than previously known and to provide a protocol for insulin treatment for patients with impaired glucose tolerance or with early stage or late stage diabetes, which treatment can be administered orally multiple times daily, such as at or shortly prior to mealtime and or at or shortly prior to bedtime, has a short duration of action, and has positive and long lasting effects on the patient's glucose tolerance, glycemic control, insulin secretory capacity and insulin sensitivity, but does not increase the risk of hypoglycemia, hyperinsulinemia and weight gain that are normally associated with insulin therapy treatments.
• compositions for oral administration comprising insulin and a delivery agent that facilitates insulin transport in a therapeutically effective amount to the bloodstream, which compositions are therapeutically and quickly effective.
• the invention provides a method for treating a mammal with impaired glucose tolerance or with early or late stage diabetes, and of achieving glucose homeostasis in mammals, comprising orally administering to a mammal a therapeutically effective dose of a pharmaceutical formulation comprising insulin such that the mammal achieves improved glucose tolerance and glycemic control as compared with baseline levels prior to treatment.
• the invention also provides a method for treating a mammal with impaired glucose tolerance or with early or late stage diabetes, comprising orally administering to a mammal a therapeutically effective dose of a pharmaceutical formulation comprising insulin such that the mammal achieves improved glucose tolerance and glycemic control as compared with baseline levels prior to treatment without any statistically significant weight gain by the mammal over the treatment period.
• the invention also provides a method for treating a mammal with impaired glucose tolerance or with early or late stage diabetes, comprising orally administering to a mammal a therapeutically effective dose of a pharmaceutical formulation comprising insulin such that the mammal achieves improved glucose tolerance and glycemic control as compared with baseline levels prior to treatment without any statistically significant risk of hypoglycemia in the mammal over the treatment period.
• the invention also provides a method for treating a mammal with impaired glucose tolerance or with early or late stage diabetes, comprising orally administering to a mammal a therapeutically effective dose of a pharmaceutical formulation comprising insulin such that the mammal achieves improved glucose tolerance and glycemic control as compared with baseline levels prior to treatment without any statistically significant risk of hyperinsulinemia in the mammal over the treatment period.
• the improved glucose tolerance is demonstrated by better endogenous capacity ofthe mammal to handle sugar load as measured by blood glucose concentration, following a sugar load, that is reduced by a statistically significant amount as compared with baseline blood glucose concentration, following a glucose load, prior to treatment.
• the statistically significant amount is a mean of about 10-20%, preferably about 15%.
• the improved glucose tolerance is demonstrated by better endogenous capacity ofthe mammal to handle sugar load as measured by an AUC of blood glucose excursion, following a glucose load, that is reduced by a statistically significant amount as compared with AUC of blood glucose excursion, following a glucose load, prior to treatment.
• the statistically significant amount is a mean of about 10-30%, preferably about 20%.
• the improved glycemic control is demonstrated by decreased fasting blood glucose levels as measured by fasting blood glucose concentration that is reduced by a statistically significant amount as compared with baseline fasting blood glucose concentration prior to treatment.
• the statistically significant amount is a mean of about 10-30%, preferably about 19%.
• the improved glycemic control is demonstrated by decreased serum fructosamine levels, as measured by serum fructosamine assay, that is reduced by a statistically significant amount as compared with baseline serum fructosamine levels prior to treatment.
• the statistically significant amount is a mean of about 5-20%, preferably about 9%.
• the improved glycemic control is demonstrated by improved HbAlc levels after treatment compared with baseline levels prior to treatment.
• the improved HbAlc levels are measured by a statistically significant decline in HbAlc levels.
• administration ofthe pharmaceutical formulation ofthe present invention can preferably be made to a mammal with impaired glucose tolerance or with early or late stage diabetes having an HbA ⁇ c level ranging from normal to elevated prior to treatment.
• the mammal may have an HbA] C level preferably of less than about 8.0 prior to treatment.
• the improved glucose tolerance and glycemic control are achieved without the need for monitoring the mammal's blood glucose concentrations or HbAi c levels over the treatment period.
• the mammal achieves improved insulin utilization and insulin sensitivity after the treatment as compared with baseline levels prior to treatment.
• the improved insulin utilization and insulin sensitivity are measured by a statistically significant decline in HOMA (Homeostasis Model Assessment).
• the mammal achieves improved insulin secretion capacity after the treatment as compared with baseline levels prior to treatment.
• the improved insulin secretion capacity is measured by a statistically significant decline in Stumvoll first-phase insulin secretion capacity index.
• the invention is also directed in part to an oral solid dosage form comprising a dose of insulin that achieves a therapeutically effective reduction in blood glucose after oral administration to a human diabetic patient, and which maintains a physiological (portal/peripheral) gradient, and in certain embodiments provides a ratio of portal vein insulin concentration 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 therapeutically effective amount of insulin, said dosage form upon pre-prandial oral administration to diabetic patients causing the post prandial blood glucose concentration in said patients to be reduced for the first hour after oral administration relative to a post-prandial blood glucose concentration without treatment or following subcutaneous insulin administration or other standard treatment regimen.
• the invention is further directed in part to an oral dosage form comprising a therapeutically effective amount of 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 administration ofthe oral insulin formulation ofthe present invention achieves a reduction in blood glucose concentration in human diabetic patients comparable to a subcutaneous insulin injection in those patients, while providing a lower (e.g., 20% or greater) total exposure of insulin to the peripheral blood circulation under acute, sub-acute and chronic conditions as compared to the peripheral blood insulin exposure achieved via subcutaneous injection.
• the present invention provides methods of treating mammals with impaired glucose tolerance, early stage diabetes and late stage diabetes; for achieving glucose homeostasis; for reducing the incidence and/or severity of systemic hyperinsulinemia associated with chronic dosing of insulin. It is believed that the present invention also provides methods for reducing the incidence and/or severity of one or more disease states associated with chronic dosing of insulin; for prophylactically sparing /3-cell function or for preventing /3-cell death or dysfunction, in a mammal which has impaired glucose tolerance or early stage diabetes mellitus; and for long-term protection from developing overt or insulin dependent diabetes, or for delaying the onset of overt or insulin dependent diabetes, in a mammal which has impaired glucose tolerance or early stage diabetes.
• such methods comprise orally administering a therapeutically effective dose of a pharmaceutical formulation comprising insulin and a delivery agent that facilitates the absorption ofthe insulin from the gastrointestinal tract, to provide a therapeutically effective reduction in blood glucose and a plasma insulin concentration, to provide a therapeutically effective reduction and/or control in blood glucose concentration and a plasma insulin concentration that is reduced relative to the plasma insulin concentration provided by a therapeutically equivalent dose of subcutaneously injected insulin.
• a pharmaceutical formulation comprising insulin and a delivery agent that facilitates the absorption ofthe insulin from the gastrointestinal tract, to provide a therapeutically effective reduction in blood glucose and a plasma insulin concentration, to provide a therapeutically effective reduction and/or control in blood glucose concentration and a plasma insulin concentration that is reduced relative to the plasma insulin concentration provided by a therapeutically equivalent dose of subcutaneously injected insulin.
• administration ofthe pharmaceutical formulation takes place multiple times daily, preferably at bedtime and preprandially during the day time, e.g., preprandially for breakfast, lunch and dinner. More preferably, administration ofthe pharmaceutical formulation is at or shortly prior to bedtime and concurrently with or shortly prior to ingestion of a meal, i.e., within about 15 minutes or less of ingestion ofthe meal.
• the oral pharmaceutical formulation will be administered about once daily to about four times daily, preprandially and/or at bedtime, depending upon the extent ofthe patient's impaired glucose tolerance and need for exogenous glycemic control. If the patient has a need for fasting glycemic control, the oral pharmaceutical formulation will be administered only at or shortly prior to bedtime. If the patient has a need for post-prandial glycemic control, the oral pharmaceutical formulation will be administered preprandially for all meals. If the patient has a need for comprehensive glycemic control, the oral pharmaceutical formulation will be administered preprandially for all meals and at or shortly prior to bedtime.
• the dosage form ofthe present invention will be administered for at least one day, more preferably on a chronic basis, and can be administered for the life ofthe patient. Most preferably, the dosage form ofthe present invention will be administered on a chronic basis, e.g., for at least about two weeks.
• the therapeutic insulin treatment ofthe present invention will be administered to patients having some form of impaired glucose tolerance. This can range from insulin resistance seen in pre-diabetics and early stage Type 2 diabetics to failure of insulin production by the pancreas seen in Type 1 diabetes and late stage Type 2 Diabetes.
• the resulting improved insulin utilization or insulin sensitivity ofthe patient's body is measured by HOMA (Homeostasis Model Assessment).
• the resulting improved insulin secretion capacity ofthe patient's body is measured by Stumvoll first-phase insulin secretion capacity index.
• the therapeutic insulin treatment ofthe present invention can be administered to a mammal with an HbAic ranging from normal to elevated levels. More particularly, the treatment can be administered to anyone in the range of normal glycemic control to impaired glycemic control to late stage type 2 diabetes or type 1 diabetes.
• the resulting improved glycemic control in the patient's body is measured by a reduced serum fructosamine concentration. Preferably the average decline will be about 8.8% after at least two weeks of treatment with the present invention.
• 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 one or more dosage forms 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 450 Units (15.3 mg) insulin, more preferably from about 200 Units (7.66 mg) to about 350 Units (13.4 mg), and still more preferably about 300 Units (11.5 mg), based on the accepted conversion of factor of 26.11 Units per mg.
• the dosage forms begin delivering insulin into the systemic circulation via the portal vein (via absorption through the mucosa ofthe gastrointestinal tract) to achieve peak levels within about 30 minutes or less.
• the dosage forms ofthe invention provide a t max for insulin at from about 5 minutes to about 30 minutes, and more preferably at from about 10 minutes to about 25 minutes after oral administration to diabetic patients, hi certain preferred embodiments ofthe invention, the dosage forms begin delivering insulin into the systemic circulation to produce a peak plasma insulin concentration at about 10 to about 20 minutes post oral administration and in further preferred embodiments, a peak plasma insulin concentration at about 10 minutes to about 15 minutes post oral administration to patients who ingested the dosage at about 0 or about 10 minutes prior to ingestion of a meal.
• the invention is also directed in part to an oral dosage form comprising a dose of unmodified insulin that achieves a therapeutically effective control of post prandial blood glucose after oral administration to human diabetic patients in tablet form at or shortly before mealtime, the oral solid dosage form providing an insulin t max at a time point from about 10 minutes to about 15 minutes after oral administration to said patients, at least about 30% ofthe blood glucose concentration reduction caused by said dose of insulin occurring within about less than 1 hour after oral administration of said dosage form.
• the oral dosage form is a tablet.
• the composition provides a t max for maximum control of glucose excursion at about 0.25 to about 1.5 hours, more preferably at about 0.75 to about 1.25 hours, after oral administration.
• the t max for post-prandial glucose control occurs preferably at less than about 120 minutes, more preferably at less than about 80 minutes, and still more preferably at about 45 minutes to about 60 minutes, after oral administration ofthe composition.
• the pharmaceutical composition contained in one or more dosage forms comprises from about 5 mg to about 800 mg of delivery agent, preferably about 20 mg to about 600 mg, more preferably from about 30 mg to about 400 mg, even more preferably from about 40 mg to about 200 mg, most preferably about 40 mg, 80 mg or 160 mg.
• the composition provides a peak plasma delivery agent concentration C max from about 3,000 to about 15,000 ng/mL, and a t max at about 10 minutes to about 35 minutes. More preferably, the composition provides a peak plasma delivery agent concentration within about 15 minutes to about 35 minutes after oral adminisfration and more preferably within about 20 minutes after oral administration to fed diabetic patients.
• 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”.
• Patient ⁇ refers to any mammal in whom there is determined to be.
• Diabetic patient refers to a mammal with a form of pre-diabetes or diabetes, either diagnosed or undiagnosed, and/or with a condition that would respond to an anti-diabetic and/or insulin treatment.
• Mammal includes but is not limited to rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and preferably humans.
• Diabetes or Diabetes Mellitus is deemed to encompass type 1 and type 2 diabetes mellitus, unless specifically specified otherwise.
• Overt Diabetes is deemed to encompass type 1 and type 2 diabetes mellitus that is insulin dependent.
• Early stage diabetes ⁇ refers to a condition of impaired glycemic control, absent freatment, wherein the function ofthe islet cells ofthe pancreas still exist, although in an impaired state, also including impaired glucose tolerance (IGT) and impaired fasting blood glucose (IFG), e.g., the patient's endogenous insulin production is insufficient to provide a first phase insulin response following ingestion of a meal but is sufficient to provide a second phase insulin response following ingestion of a meal.
• ITT impaired glucose tolerance
• IGF impaired fasting blood glucose
• Late stage diabetes refers to a condition of impaired glycemic confrol, absent treatment, wherein the islet cells ofthe pancreas are approaching or have reached total failure, e.g., the patient's endogenous insulin production is insufficient to provide a first or a second phase insulin response following ingestion of a meal.
• luuiuzj treatment ⁇ when used herein with respect to diabetes is deemed to include prevention of diabetes, delay ofthe onset of diabetes, delay of worsening of diabetic conditions and delay of progression from an earlier stage of diabetes to a later stage of diabetes, unless specifically specified otherwise.
• Delivery agent ⁇ refers to carrier compounds or carrier molecules that are effective in the oral delivery of therapeutic agents, and may be used interchangeably with "carrier”.
• Therapeutically effective amount of insulin ⁇ refers to an amount of insulin included in the dosage forms ofthe invention which is sufficient to achieve a clinically relevant confrol 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 refers to an amount ofthe delivery agent that has been shown to deliver the drug following oral administration by measurement of pharmacokinetic and/or pharmacodynamic endpoints.
• Organic solvents ⁇ refers to 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 refers to 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 ⁇ refers to 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 sub-units of proteins, such as peptides. The molecular weight range for proteins includes peptides of 1000 Daltons to glycoproteins of 600 to 1000 kiloDaltons.
• Reconstitution refers to dissolution of compositions or compositions in an appropriate buffer or pharmaceutical composition.
• Unit-Dose Forms refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. It is contemplated for purposes ofthe present invention that dosage forms ofthe present invention comprising therapeutically effective amounts of insulin may include one or more unit doses (e.g., tablets, capsules, powders, semisolids (e.g. gelcaps or films), liquids for oral administration, ampoules or vials for injection, loaded syringes) to achieve the therapeutic effect. It is further contemplated for the purposes ofthe present invention that a preferred embodiment ofthe dosage form is an oral dosage form.
• multiple dose means that the patient has received at least two doses ofthe drug composition in accordance with the dosing interval for that composition.
• single dose means that the patient has received a single dose ofthe drug composition or that the repeated single doses have been administered with washout periods in between.
• Unmodified insulin refers to 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. Patent No. 6,309,633 and/or which not has been subjected to amphiphilic modification such as that described in U.S. Patent Nos. 5,359,030; 5,438,040; and/or 5,681,811, which patents are hereby inco ⁇ orated by reference in their entireties.
• a maximal reduction of blood glucose concenfration achieved by a first method of insulin administration is not more than 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.
• the phrase may also mean the dose required to approximate normoglycemia by any method of adminisfration, normoglycemia being defined as variability from a subject's baseline blood glucose of not more than 20%, preferably 10%, more preferably 5%, in the fasted state.
• mean means a standard, ADA and/or a mixed meal.
• mean when preceding a pharmacokinetic value (e.g., mean t max ), represents the arithmetic mean value ofthe pharmacokinetic value unless otherwise specified.
• mean baseline level means the measurement, calculation or level of a certain value that is used as a basis for comparison, which is the mean value over a statistically significant number of subjects, e.g., across a single clinical study or a combination of more than one clinical study.
• C max is the highest plasma concentration ofthe drug or delivery agent observed within the sampling interval.
• t max is the time post-dose at which C max is observed.
• AUC area under the plasma concenfration-time curve, as calculated by the trapezoidal rule over the complete sample collection interval.
• AU o-iast means the area under the plasma concentration-time curve using linear frapezoidal summation from time zero (dosing) to the time ofthe last quantifiable concentration post-dose.
• AUC ( o- t) means the area under the plasma concentration-time curve using linear frapezoidal summation from time zero (dosing) to time t post-dose, where t is any quantifiable time point.
• AUC ( o- mf) as used herein means an estimate ofthe area under the plasma concenfration-time curve from time zero (dosing) to infinity.
• CL/F means the apparent total body clearance calculated as Dose/AUC(o-inf uncorrected for absolute bioavailability.
• VJF means the apparent volume of distribution calculated as (CL/F)/K e i, uncorrected for absolute bioavailability.
• E b means the maximum observed effect (baseline subtracted) prior to intervention for hypoglycemia.
• E max means the maximum observed effect (baseline subtracted).
• K e i is the terminal elimination rate constant calculated by linear regression ofthe terminal linear portion ofthe log concentration vs. time curve [00130]
• t /2 as used herein means the terminal half-life calculated as ln(2)/Kei.
• BMI body mass index
• Bioavailability means the degree or ratio (%) to which a drug or agent is absorbed or otherwise available to the treatment site in the body relative to a parenteral route. This is calculated by the formula
• Biopotency means the degree or ratio (%) to which a drug or agent is effective relative to a parenteral route. This is calculated by the formula
• nighttime or “bedtime” as used herein means a time before the patient goes to sleep and is not limited to clock time or cycles of light and dark, and alternately refers to a time during a day or night of longest fast, a period without external glucose source.
• the phrase administered "at nighttime” or “at or shortly before (prior to) bedtime” means administered less than about 3 hours, preferably less than about 2 hours and more preferably less than about 1 hour prior to a prolonged period of sleep, or relative physical and/or mental inactivity, and fast, e.g., overnight. Whereas overnight typically means from the late night (p.m.) hours to the early morning (a.m.) hours, it could mean any period of a sleep-wake cycle during which a person obtains his/her necessary period of sleep.
• administration should also occur at least about one hour, preferably at least about 1.5 hours, more preferably at least about 2 hours and still more preferably at least about 2 to about 3 hours after the last meal ofthe day.
• the phrase administered "at mealtime” or “at or shortly before (prior to) ingestion of a meal” means administered within about 30 minutes prior to the meal.
• the adminisfration is preferably within about 25 minutes, more preferably within about 20 minutes, even more preferably within about 15 minutes, still more preferably within about 10 minutes, further more preferably within about 5 minutes of ingestion ofthe meal, and most preferably administered concurrently with ingestion ofthe meal (within about 0 minutes).
• Figure 1 shows a plot ofthe arithmetic means of postprandial blood glucose excursions for all subjects.
• Figure 2 shows a plot of 4-CNAB plasma concentrations (ng/mL) vs. time (arithmetic means).
• Figure 5 shows a plot of insulin plasma concentrations (pmol/1) vs. time (arithmetic means).
• Figure 6 shows a plot of C-peptide plasma concentrations (nmol/l) vs. time (arithmetic means).
• Figure 9 is a bar graph showing the effect of nighttime dosing of insulin and 4-CNAB on blood glucose concentration.
• Figure 10 is a bar graph showing the effect of nighttime dosing of insulin and 4-CNAB on blood C-peptide concentration.
• Figure 11 is a bar graph showing the effect of nighttime dosing of insulin and 4-CNAB on blood insulin concentration.
• Figure 12 is a plot of Preliminary Mean +/- SD % Change in Baseline Blood Glucose (SuperGL) Following Oral Adminisfration of h sulin/4-CNAB Tablets to Fed of Fasted Type 2 Diabetic Patients.
• Figure 13 is a plot of Preliminary Mean +/- SD % Change in Blood Glucose (SuperGL) Following Oral Administration of Insulin/ 4-CNAB Tablets to Type 2 Diabetic Patients with a Standard Meal.
• Figure 14 is a plot of Preliminary Mean +/- SD % Change in Blood Glucose (SuperGL) following Oral Adminisfration of Insulin/ 4-CNAB Tablets to Type 2 Diabetic Patients with or without a Meal.
• Figures 15-22 are plots of Preliminary Percent Change in Blood (SuperGL) Glucose for Subjects 101-108, respectively.
• Figure 23 is a plot of Preliminary Mean +/- SD Plasma Glucose Change (%) Following Oral Tablet Adminisfration of Insulin/ 4-CNAB to Type 2 Diabetic Patients with or without a Meal.
• Figure 24 is a plot of Preliminary Mean +/- SD Plasma Glucose concentration Following Oral Administration of Insulin/4-CNAB Tablets to Type 2 Diabetic Patients with or without a Meal.
• Figure 25 is a plot of Preliminary Mean +/- SD Blood (SuperGL) Glucose concentration following Oral Administration of Insulin/ 4-CNAB Tablets to type 2 diabetic patients with or without a Meal.
• Figures 26-33 are plots of Preliminary Blood (SuperGL) Glucose concentrations for Subjects 101-108, respectively.
• Figure 34 is a plot of Mean +/- SD Serum Insulin Concentration Following a Single Oral Administration of Insulin/ 4-CNAB Tablets to Fasted or Fed Type 2 Diabetic Patients.
• Figure 35 is a plot of Mean +/- SD Serum Insulin Concentration Following a Single Oral Administration of Insulin/ 4-CNAB Tablets to Fasted or Fed Type 2 Diabetic Patients.
• Figures 36-43 are plots of Serum Insulin Concenfration Following a Single Oral Administration of Insulin 4-CNAB Tablets to Fasted Type 2 Diabetic Patients Subjects 101-108, respectively.
• Figure 44 is a plot of Mean +/- SD Plasma 4-CNAB Concentration Following a Single Oral Tablet Administration of Insulin/4-CNAB to Fed or Fasted Type 2 Diabetic Patients.
• Figure 45 is a plot of Mean +/- SD Plasma C-Peptide Concentration Following a Single Oral Administration of Insulin/4-CNAB Tablets to Fed or Fasted Type 2 Diabetic Patients.
• Figure 46 is a prior art graph showing mean change in plasma glucose concentration from baseline for administration of placebo, glipizide, nateglinide, and glipizide plus nateglinide.
• Figures From 1022 ⁇ - Study 175A-C-10 (Profil) Multidose [00165]
• Figures 48A and 48B are curves showing PRELIMINARY Mean and Individual Fasting Blood Glucose Prior to Oral Glucose Tolerance Test at Screening and on Day 15 Following 14 Days of Daily QID Adminisfration of 300U Insulin/ 160mg 4-CNAB to Type 2 Diabetic Subjects (48A) and 200mg 4-CNAB Alone to Type 2 Diabetic Subjects (48B).
• Figures 49A and 49B are curves showing PRELIMINARY Mean and Individual Blood Glucose AUC0-240min Following Oral Glucose Tolerance Test At Screening and on Day 15 Following 14 Days of Daily QID Administration of 300U Insulin 160mg 4-CNAB to Type 2 Diabetic Subjects (49A) and 200mg 4-CNAB Alone to Type 2 Diabetic Subjects (49B).
• Figures 50A and 50B are curves showing PRELIMINARY Mean and Individual Blood Glucose 2 Hours Following Oral Glucose Tolerance Test at Screening and on Day 15 Following 14 Days of Daily QID Administration of 300U Insulin/160mg 4-CNAB to Type 2 Diabetic Subjects (50A) and 200mg 4-CNAB Alone to Type 2 Diabetic Subjects (50B).
• Figures 52A and 52B are curves showing PRELIMINARY Mean and Individual Plasma Insulin AUCo -24 omi n Following Oral Glucose Tolerance Test At Screening and on Day 15 Following 14 Days of Daily QID Adminisfration of 300U Insulin/160mg 4-CNAB to Type 2 Diabetic Subjects (52A) and 200mg 4-CNAB Alone to Type 2 Diabetic Subjects (52B).
• Figures 53A and 53B are bar graphs showing PRELIMINARY Mean and Fasting Blood Glucose and Plasma Insulin Prior to Oral Glucose Tolerance Test At Screening and on Day 15 Following 14 Days of Daily QID Administration of 300U fr ⁇ sulin/160mg 4-CNAB to Type 2 Diabetic Subjects (53) and 200mg 4-CNAB Alone to Type 2 Diabetic Subjects (53B).
• Figures 54A and 54B are bar graphs showing PRELIMINARY Mean Blood Glucose and Plasma Insulin 2 Hours Following Oral Glucose Tolerance Test at Screening and on Day 15 Following 14 Days of Daily QID Administration of 300U Insulin/160mg 4-CNAB to Type 2 Diabetic Subjects (54A) and 200mg 4-CNAB Alone to Type 2 Diabetic Subjects (54B).
• Figures 59A and 59B are curves showing PRELIMINARY Serum Fructosamine on Days 0 and 15 Following 14 Days of Daily QID Administration of 300U fr ⁇ sulin/160mg 4-CNAB to Type 2 Diabetic Subjects (59A) and 200mg 4-CNAB alone (confrol) to Type 2 Diabetic Subjects (59B).
• the oral dosage forms ofthe invention facilitate the oral delivery of insulin, and after insulin is absorbed into the bloodsfream, the composition produces a maximal decrease in blood glucose in treated type 2 diabetic patients from about 5 to about 60 minutes after oral administration, hi another embodiment ofthe present invention, the pharmaceutical composition produces a maximal decrease in blood glucose in treated type 2 diabetic patients from about 10 to about 50 minutes post oral administration. More particularly, the pharmaceutical composition produces a maximal decrease in blood glucose in treated type 2 diabetic patients within about 20 to about 40 minutes after oral adminisfration.
• the magnitude ofthe decrease in blood glucose produced by insulin absorbed into the bloodsfream following entry into the gastrointestinal tract varies with the dose of insulin.
• type 2 diabetic diabetic patients show a maximal decrease in blood glucose by at least 10% within one hour post oral administration
• type 2 diabetic 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 adminisfration.
• 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 115 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 concenfration from about 95 to about 110 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 confrol very high levels of blood glucose from being reached and/or 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 190 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 postprandial blood glucose concentration from about 150 to about 180 mg/dl, more preferably, the subject manifests fasting blood glucose concentrations at less than about 175 mg/dl.
• the present invention provides pharmaceutical compositions for oral adminisfration 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 90 to about 125 mg/dl.
• the present invention provides pha ⁇ naceutical 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 115 mg/dl.
• the present invention provides pharmaceutical compositions for oral adminisfration which include insulin as the active agent and a delivery agent in an amount effective to achieve blood glucose concentrations within the normal range during the evening period from about 70 to about 120 mg/dl.
• the present invention provides pharmaceutical compositions for oral adminisfration 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 about 4 hours after bed time from about 80 to about 120 mg/dl.
• the present invention provides a method of administering insulin and pharmaceutical compositions useful for administering insulin such that the insulin is bioavailable and biopotent.
• the delivery agent enables insulin to be orally absorbable through the mucosa ofthe stomach and facilitates the abso ⁇ tion 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).
• the delivery agent passes though the mucosal barriers ofthe gastrointestinal tract and is absorbed into the blood stream where it can be detected in the plasma and/or blood of subjects.
• the level of delivery agent in the bloodsfream as measured in the plasma and/or blood is dose-dependent.
• the ratio of portal (unmodified) insulin concentration to systemic (unmodified) insulin concentration approaches in human diabetic patients approaches that which is obtained in normal healthy humans.
• the chronic administration of oral dosage forms of the present invention result in a higher portal insulin concenfration and lower systemic insulin concentration over time than that obtained with an equi-effective dose of insulin administered subcutaneously (i.e., which provide similar confrol of blood glucose levels).
• Transient peaks in insulin levels that may occur by virtue ofthe oral adminisfration of insulin in accordance with the present invention is not believed to be associated with vascular diseases.
• the present invention provides a method for reducing the incidence and/or severity of systemic hyperinsulinemia associated with chronic dosing of insulin, and it is believed that the present invention also provides a method for reducing the incidence and/or severity of one or more disease states associated with chronic dosing of insulin.
• the patient achieves improved glucose tolerance and glycemic confrol as compared with baseline levels prior to treatment, even without any statistically significant increase in weight, risk of hypoglycemia or risk of hyperinsulinemia over the treatment period. Further, by virtue ofthe chronic administration of oral dosage forms ofthe present invention, the patient achieves improved insulin utilization, insulin sensitivity insulin secretion capacity and HbAic levels as compared with baseline levels prior to freatment.
• the prefe ⁇ ed pharmaceutical compositions ofthe 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 ofthe 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 oral insulin formulations ofthe invention may be administered to a patient at meal time, and preferably slightly before (e.g., about 10-30 minutes before) ingestion of a meal, such that the peak plasma insulin concenfrations are attained at or about the time of peak blood glucose concenfrations resulting from the meal.
• the adminisfration of a relatively short-acting insulin (e.g., such as the insulin used to prepare the capsules administered in the clinical studies reported in the appended examples) will further result in plasma insulin levels returning to baseline levels within about 4 hours (and preferably within about 3 hours or less) after oral administration ofthe insulin formulations ofthe present invention.
• insulin refers to insulin from a variety of sources. Naturally occurring insulin and structurally similar bioactive equivalents (insulin analogues including short acting and analogues with protracted action) can be used. Insulin useful in the invention can be may be obtained by isolating it from natural source, such as different species of mammal. For example, animal insulin preparations extracted from bovine or porcine pancreas can be used. Insulin analogues, fragments, mimetics or polyethylene glycol (PEG)-modified derivatives of these compounds, derivatives and bioequivalents thereof can also be used with the invention.
• PEG polyethylene glycol
• the insulin used in the present invention may be obtained by chemically synthesizing it using protein chemistry techniques such as peptide synthesis, or by using the techniques of molecular biology to produce recombinant insulin in bacteria or eukaryotic cells.
• the physical form of insulin may include crystalline and/or amo ⁇ hous solid forms, h 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. Patents Nos. 4,421,685, 5,474,978, and 5,534,488, the disclosure of each of which is hereby inco ⁇ orated by reference in its entirety.
• the most prefe ⁇ ed insulin useful in the pha ⁇ naceutical compositions and methods ofthe present invention is human recombinant insulin optionally having counter ions including zinc, sodium, calcium and ammonium or any combination thereof.
• 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 eukaryotic 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.
• insulin Zinc, human recombinant
• Calbiochem San Diego, CA
• human recombinant Zinc-Insulin Crystals Proinsulin Derived (Recombinant DNA Origin) USP Quality can be obtained from Eli Lilly and Company (Indianapolis, IN).
• insulin including insulin analogues (including but not limited to Insulin Lispro, Insulin Aspart, Insulin Glargine, and Insulin Detemir) are deemed for the pu ⁇ oses of this specification and the appended claims are considered to be encompassed by the term "insulin.”
• insulin analogues including but not limited to Insulin Lispro, Insulin Aspart, Insulin Glargine, and Insulin Detemir
• the present invention also provides compositions of recombinant human zinc insulin and a delivery agent as a drug for oral adminisfration of insulin in humans.
• the insulin is a modified insulin, such as that conjugated with an oligomer such as that described in U.S. Patent No. 6,309,633 and/or which not has been subjected to amphiphilic modification such as that described in U.S. Patent Nos. 5,359,030; 5,438,040; and/or 5,681,811.
• the conjugated (modified) insulin may be inco ⁇ orated into the oral formulations ofthe present invention in addition to or in the absence of any ofthe types of insulin described above, as well as with other insulin analogues.
• the oral formulations include the modified insulin either with or without a pharmaceutically acceptable delivery agent that facilitates abso ⁇ tion of said insulin from the gastrointestinal tract.
• a pharmaceutically acceptable delivery agent that facilitates abso ⁇ tion of said insulin from the gastrointestinal tract.
• the total amount of insulin to be used can be determined by those skilled in the art. It is preferable that the oral dosage form comprise a therapeutically effective amount of insulin, i.e., a pharmacologically or biologically effective amount, or an amount effective to accomplish the pu ⁇ ose of insulin.
• the dose of insulin administered should preferably be in such an amount that, upon oral administration, it results in a measurable and statistically significant reduction in blood glucose levels in normal healthy human subjects.
• 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 tablet, 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, biologically or chemically active amounts of biologically or pharmacologically active agent.
• Prefe ⁇ ed insulin doses contained in one or more dosage forms when dosed in combination with the delivery agents described herein, are about 50 to about 600 insulin Units USP (from about 2 to about 23 mg), preferably from about 100 Units (3.8 mg) to about 450 Units (15.3 mg), more preferably from about 200 Units (7.66 mg) to about 350 Units (13.4 mg), and still more preferably about 300 Units (11.5 mg), based on the accepted conversion of factor of 26.11 Units per mg.
• Regular insulin has an onset of action of 30-60 minutes, peak time of effect of 1.5 to 2 hours, and a duration of activity of 5 to 12 hours.
• Rapid acting insulins such as aspart (Humalog ® )/lispro (Novolog ® ), have an onset of action of 10-30 minutes, peak time of effect of 30- 60 minutes, and a duration of activity of 3 to 5 hours.
• Intermediate-acting insulins such as NPH (neutral protamine Hagedorn) and Lente insulins (insulin zinc suspension), have an onset of action of 1-2 hours, peak time of effect of 4 to 8 hours, and a duration of activity of 10 to 20 hours.
• Ulfralente insulin has an onset of action of 2-4 hrs, peak time of effect of 8-20 hours, and a duration of activity of 16 to 24 hours
• Glargine insulin has an onset of action of 1 to 2 hours, a duration of action of 24 hours but no peak effect.
• soluble insulin There are over 180 individual insulin preparations available world-wide. Approximately 25% of these are soluble insulin (unmodified form); about 35% are basal insulins (mixed with NPH or Lente insulins, increased pi, or isoelectric point (insulin glargine), or acylation (insulin detemir); these forms have reduced solubility, slow subcutaneous abso ⁇ tion and long duration of action relative to soluble insulins); about 2% are rapid-acting insulins (e.g., which are engineered by amino-acid change, and have reduced self-association and increased subcutaneous abso ⁇ tion); and about 38% pre-mixed insulins (e.g., NPH/soluble/rapid-acting insulins; these preparations have the benefit, e.g., of reduced number of daily injections). In many cases, regimens that use insulin in the management of diabetes combine long-acting and short-acting insulin.
• basal insulins mixed with NPH or Lente insulins, increased pi, or isoelectric point
• the oral insulin formulations ofthe present invention which include insulin preferably together with a pharmaceutically acceptable delivery agent that facilitates abso ⁇ tion of said insulin from the gastrointestinal tract, may be utilized in combination therapy to include an insulin that has rapid action, intermediate action, and/or slow action, as described above, in order to provide effective basal insulin levels in the diabetic patient.
• the rate of action ofthe insulin may be caused by virtue of its solubility, and/or by virtue of its half-life, etc.
• the oral formulations ofthe present invention may be designed to provide the intermediate activity which is found with, e.g., a subcutaneously administered NPH insulin, or a slow action which is found with protamine zinc insulin.
• the oral formulations ofthe invention which preferably include a pharmaceutically acceptable delivery agent which facilitates abso ⁇ tion ofthe insulin (as described herein) provide effective control of blood glucose levels, albeit for different time periods and with different plasma glucose time curves.
• Intermediate-acting and long-acting insulin may be prepared using methodologies known to those skilled in the art to provide a continuous level of insulin, similar to the slow, steady (basal) secretion of insulin provided by the normal pancreas.
• Lantus ® from Aventis Pharmaceuticals Inc., is a recombinant human insulin analog that is a long-acting, parenteral blood- glucose-lowering agent whose longer duration of action (up to 24 hours) is directly related to its slower rate of abso ⁇ tion.
• Lantus ® is administered subcutaneously once a day, preferably at bedtime, and is said to provide a continuous level of insulin, similar to the slow, steady (basal) secretion of insulin provided by the normal pancreas.
• Such a long-acting insulin results in a relatively constant concentration/time profile over 24 hours with no pronounced peak, thus allowing it to be administered once a day as a patient's basal insulin.
• Such long-acting insulin has a long-acting effect by virtue of its chemical composition, rather than by virtue of an addition to insulin when administered.
• administration ofthe pharmaceutical formulation comprising long-acting insulin is once or twice a day.
• administration ofthe dosage form providing short-acting insulin effect can be once, twice, three times, four times or more than four times daily, and can be at nighttime, in the morning and/or preprandially.
• administration ofthe dosage form is preferably at nighttime or morning and three times preprandially, and more preferably is at nighttime and preprandially for breakfast, lunch and dinner.
• the insulin formulations are administered to such human patients on a chronic basis, e.g., for at least about 2 weeks.
• the oral formulations include an insulin conjugated with an oligomer such as that described in U.S. Patent No. 6,309,633 and/or which not has been subjected to amphiphilic modification such as that described in U.S. Patent Nos. 5,359,030; 5,438,040; and/or 5,681,811.
• the conjugated (modified) insulin may be inco ⁇ orated into the oral formulations ofthe present invention in addition to or in the absence of any ofthe types of insulin described above, as well as with other insulin analogues.
• the oral formulations preferably include the modified insulin together with a pharmaceutically acceptable delivery agent which facilitates abso ⁇ tion of said insulin from the gastrointestinal tract.
• Oral adminisfrable drugs cu ⁇ ently available for management of type 2 diabetes fall into two general categories: those that increase insulin supply (sulfonylureas, other secretagogues and insulin itself) and those that decrease insulin resistance or improve its effectiveness (biguanides, thiazolidinediones).
• Oral sulfonylurea secretagogues include the first and second generation insulin secretagogues which are believed to interact with ATP-sensitive potassium channels in the beta cell membrane to increase secretion of insulin.
• the more commonly used second-generation agents (glyburide, glipizide, and glimepiride), which are more potent than the first-generation drugs (acetohexamide, chlo ⁇ ropamide, tolbutamide, and tolazamide), are similar to each other in efficacy, but differ in dosage and duration of action.
• such secretagogues are useful for increasing insulin levels sufficiently to achieve desired basal insulin levels in patients with early stages of type II diabetes, who are still able to produce their own insulin.
• secretagogues would be useful for increasing insulin levels sufficiently to achieve desired basal insulin levels in patients with later stages of type II diabetes, who have very little pancreatic function left and produce very little insulin endogenously.
• the basal insulin levels are achieved, e.g., via the use of subcutaneous injections of insulin (such as a long-acting insulin, for example Lantus ).
• the oral insulin formulations include one or more ofthe various types of secretagogues mentioned above in addition to a type of insulin as described above.
• tolbutamide Orinase
• Tolazamide Tolinase ®
• Tolinase ® has an onset of action of 4-6 hours and a duration of action of 10-14 hours, and is usually given in a dose of 250 mg to 500 mg either once or in divided daily doses (1000 mg/day).
• Acetohexamide (Dymelor ® ) has an onset of action of one (1) hour and a duration of action of 10-14 hours, and is usually given in a dose of 500 mg to 750 mg either once or in divided daily doses (maximum daily dose 1500 mg/day).
• Chlo ⁇ ropamide (Diabinese ® ) has an onset of action of one hour and a duration of action of 72 hours, and is usually given in a dose of 250 mg to 375 mg once a day (maximum daily dose, 750 mg/day).
• glyburide (DiaBeta ® ); Micronase ® ; Glynase ® ) has an onset of action of 1.5 hours and a duration of action of 18-24 hours. It is usually given in a dose of 5 to 20 mg either once or in divided daily doses (maximum daily dose, 20 mg/day).
• Glipizide (Glucofrol ® ) has an onset of action of one hour and a duration of action of 10- 24 hours. It is usually given in a dose of 10 to 20 mg either once or in divided daily doses (maximum daily dose, 40 mg/day).
• Glimepiride (Amaryl ® ) has an onset of action of 2 hours and a duration of action of 18-28 hours. It is usually administered in a dose of 1 to 4 mg once a day (maximum daily dose, 8 mg/day). Lastly, gliclazide (Diamicron ® ) is usually administered in a dose of 40 to 80 mg per day (maximum daily dose, 320 mg).
• Oral non-sulfonylurea secretagogues such as repaglinide and nateglinide, although structurally different from the sulfonylureas, also bind to ATP-sensitive potassium channels on beta-cells and increase insulin release.
• repaglinide and nateglinide are rapidly absorbed, resulting in plasma levels of insulin that peak within 30 to 60 minutes and return to baseline before the next meal. These drugs must be taken before each meal; if a meal is missed, the drug should be omitted.
• Repaglinide and nateglinide are much more expensive than sulfonylureas, but repaglinide may be a useful alternative to a sulfonylurea in patients with renal impairment (because it is cleared primarily by hepatic metabolism) or in patients who eat sporadically. Hypoglycemia may be slightly less frequent with nateglinide and repaglinide than with sulfonylureas, but data are limited. Nateglinide (Starlix ® ) stimulates pancreatic insulin secretion within 20 minutes of oral adminisfration.
• nateglinide is rapidly absorbed with a mean peak plasma drug concentration (C max ) generally occu ⁇ ing within one hour (t max ) after dosing.
• C max mean peak plasma drug concentration
• t max mean peak plasma drug concentration
• Nateglinide is usually administered in a dose of 60 to 120 mg tliree times daily before meals (maximum daily dose, 360 mg/day).
• AUC extent of nateglinide abso ⁇ tion
• biguanides which decrease the amount of glucose made by the liver, and thiazolidinediones, which make the patient more sensitive to insulin, are oral hypoglycemic agents that are cu ⁇ ently used clinically for improving insulin resistance.
• Biguanides such as Metformin (Glucophage ® and Glucophage ® XR by Bristol-Myers Squibb Company of Princeton, NJ), which is the only biguanide available for therapeutic use, decreases hepatic glucose production (gluconeogenesis), decreases intestinal abso ⁇ tion of glucose and improves insulin sensitivity by increasing peripheral glucose uptake and utilization.
• Glucophage ® There is no fixed dosage of Glucophage ® for the management of hyperglycemia, and dosage must be individualized based upon effectiveness and tolerance, while not exceeding the maximum recommended daily dose of 2550 mg in adults and 2000 mg in pediatric patients, once or in divided doses. In general, clinically significant results are not seen at doses below 1500 mg per day. However, a lower recommended starting dose and gradually increased dosage is advised in order to minimize gastrointestinal symptoms.
• Thiazolidinediones improve sensitivity to insulin in muscle and adipose tissue and inhibit hepatic gluconeogenesis, and thus depend on the presence of insulin for their action.
• the two cu ⁇ ently approved thiazolidinedione compounds are pioglitazone (Actos ® by Takeda Pharmaceuticals America, Inc. of Lincolnshire, IL) and rosiglitazone (Avandia ® by GlaxoSmithKline of Research Triangle Park, NC). Actos ® also improves hepatic sensitivity to insulin and improves dysfunctional glucose homeostasis.
• Actos ® is first measurable in serum, following oral adminisfration in the fasting state, within 30 minutes, with peak concentrations observed within 2 hours. Food slightly delays the time to peak serum concenfration to 3 to 4 hours but does not alter the extent of abso ⁇ tion. Actos ® is usually given once daily without regard to meals, and dosage must be individualized based upon HbAi c for a period of time adequate to evaluate changes in HbA ⁇ c . Monotherapy dosage in patients not adequately controlled with diet and exercise maybe initiated at 15 mg or 30 mg and can be increased incrementally up to 45 mg (maximum dose 45 mg per day).
• Avandia reaches peak plasma concentrations within about 1 hour after dosing, and adminisfration with food results in no change in overall exposure (AUC) but results in a 28% decrease in maximum plasma concentrations and a delay ofthe time to reach peak plasma concentrations to about 1.75 hours after dosing.
• Dosage of Avandia ® must be individualized, and Avandia ® may be administered either at a starting dose of 4 mg as a single daily dose or divided and administered twice a day with or without food. For patients who respond inadequately, as dete ⁇ nined by reduction in fasting blood glucose, the dose may be increased to 8 mg daily (maximum dose 8 mg per day).
• the oral formulations ofthe invention provide two forms of insulin having different activity rates in order to simulate the biphasic release of insulin in non-diabetic humans.
• such oral formulations may include a rapid-acting form of insulin together with a slow acting form of insulin so as to provide a first peak of insulin which occurs rapidly and is short-lived, followed by a second peak of insulin which occurs at a later time, but which preferably has a longer duration.
• the oral formulations of the invention include a rapid-acting form of insulin together with a secretagogue that promotes the secretion of insulin from the beta-cells at a time and to an extent which mimics the second phase release of insulin in non-diabetic humans.
• the methods of insulin administration ofthe invention provide two separate forms of insulin having different activity rates in order for the regimen to simulate the biphasic release of insulin in non-diabetic humans.
• the oral formulations may include a rapid-acting form of insulin so as to provide a first peak of insulin which occurs rapidly and is short-lived.
• Such fast-acting effect may be provided by the delivery agent that facilitates the abso ⁇ tion of insulin from the gastrointestinal tract.
• the slow acting form of insulin provides a second peak of insulin that occurs at a later time but that preferably has a longer duration.
• Such slower acting insulin may be provided by a separate dosage form, which may be administered orally or subcutaneously.
• 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 a sulfonylurea, a biguanide (such as Metformin), an alpha-glucosidase, insulin delivered via a different pathway (e.g., parenteral insulin), and/or an insulin sensitizer such as thiazolidinedione.
• an additional drug such as a sulfonylurea, a biguanide (such as Metformin), an alpha-glucosidase, insulin delivered via a different pathway (e.g., parenteral insulin), and/or an insulin sensitizer such as thiazolidinedione.
• the oral dosage forms described herein reduce the likelihood of hypoglycemic events.
• Hypoglycemia usually results from a mismatch between insulin levels and degree of glycemia, e.g., when the administration of insulin and the ingestion ofthe meal are not timed such that the insulin peak occurs at peak glycemia, and administration of insulin shortly before a meal is more practical for a patient and is also safer, because glucose is ingested soon thereafter.
• the risk of hypoglycemia is lowered mainly due to the portal-physiologic route of adminisfration of oral insulin.
• the pancreas will not sequester additional glucose but rather will only cease producing endogenous insulin.
• the brief peak of insulin that results from the oral composition described herein shows that, even if insulin were to reach high peripheral levels, the peak quickly drops precipitously.
• further embodiments ofthe oral dosage forms described herein avoid the risk of hypoglycemic events that may occur in certain short acting insulin formulations, which may, between the time of administration of insulin and the time of ingestion ofthe meal, contribute to a lowering of blood glucose to a level that could range from undesirable to clinically hypoglycemic.
• dosing closer to a meal eliminated the dip in blood glucose levels, which was precarious by itself. The effect seems to have also translated to lowering ofthe subsequent glucose excursion
• 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 administration.
• Previous disclosures by Emisphere Technologies, hie. solved the problem of oral abso ⁇ tion of insulin by providing delivery agents that facilitate transport of insulin through the gut wall and into the bloodstream where the insulin can perform its biological function.
• effective oral drug delivery methods are provided to increase the oral bioavailability and abso ⁇ tion of insulin, which is cu ⁇ ently administered parenterally.
• the invention is thus directed to an methods involving oral administration of a dosage form comprising insulin together with a pharmaceutically acceptable delivery agent that serves to render the insulin orally absorbable through the gastrointestinal mucosa, the delivery agent being present in an amount effective to facilitate the abso ⁇ tion of said insulin, such that a therapeutically effective amount of said dose of insulin is absorbed from the gastrointestinal tract of human diabetic patients. .
• a pharmaceutically acceptable delivery agent that serves to render the insulin orally absorbable through the gastrointestinal mucosa
• the delivery agent being present in an amount effective to facilitate the abso ⁇ tion 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 oral dosage forms ofthe present invention comprise a mixture of insulin and a delivery agent, e.g., monosodium N-(4-chlorosalicyloyl)-4-aminobutyrate (4-CNAB), a novel compound discovered by Emisphere Technologies, hie, or separately containing insulin and the delivery agent.
• a delivery agent e.g., monosodium N-(4-chlorosalicyloyl)-4-aminobutyrate (4-CNAB), a novel compound discovered by Emisphere Technologies, hie, or separately containing insulin and the delivery agent.
• 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 ⁇ -C 3 alkylene, substituted or unsubstituted C ⁇ -C 3 alkenylene.
• the delivery agents ofthe invention preferably have the following structure:
• X is halogen
• R is substituted or unsubstituted C ⁇ -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 oral dosage forms ofthe present invention comprise a mixture of insulin and a delivery agent, e.g., monosodium N-(4- chlorosalicyloyl)-4-aminobutyrate (4-CNAB), a novel compound discovered by Emisphere Technologies, Inc., or separately containing insulin and the delivery agent.
• a delivery agent e.g., monosodium N-(4- chlorosalicyloyl)-4-aminobutyrate (4-CNAB), a novel compound discovered by Emisphere Technologies, Inc., or separately containing insulin and the delivery agent.
• the delivery agents may be in the form ofthe 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- vaient 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 United States Patents 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 inco ⁇ orated herein by reference. Additional suitable delivery agents are also described in International Publications Nos.
• WO 01/34114 WO 01/21073, WO 01/41985, WO 01/32130, WO 01/32596, WO 01/44199, WO 01/51454, WO 01/25704, WO 01/25679, WO 00/50386, WO 02/02509, WO 00/47188, WO 00/07979, WO 00/06534, WO 98/25589, WO 02/19969, WO 00/59863, WO 95/28838, WO 02/19969, WO 02/20466, WO 02/069937 and WO 02/070438, the disclosure of each of which is inco ⁇ orated herein by reference.
• Salts ofthe delivery agent compounds ofthe 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 United States Patents Nos. 5,643,957 and 5,650,386, the disclosure of each of which is inco ⁇ orated 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 Publications Nos. WO 02/02509 and WO 03/057170, the disclosure of each of which is inco ⁇ orated herein by reference.
• the delivery agents may also be prepared by alkylation ofthe appropriate salicylamide according to the methods of International Publication No. WO 00/46182, the disclosure of which is inco ⁇ orated herein by reference.
• the salicylamide may be prepared from salicylic acid via the ester by reaction with sulfuric acid and ammonia.
• polyamino 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 fo ⁇ ned 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 ofthe Gl tract and is absorbed into the blood stream where it can be detected in the plasma of subjects.
• the delivery agent facilitates the abso ⁇ tion ofthe 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).
• the mechanism by which 4-CNAB facilitates the gastrointestinal abso ⁇ tion of insulin has not yet been fully elucidated.
• the amount of delivery agent in the present composition is a delivery effective amount and can be determined for any particular delivery agent/insulin combination by methods known to those skilled in the art.
• the amount of delivery agent necessary to adequately deliver the therapeutic amount of insulin into the blood stream of a subject needing the therapeutic effect of insulin may vary depending on one or more ofthe following; the chemical nature of insulin; the chemical structure ofthe particular delivery agent; the nature and extent of interaction between insulin and delivery agent; the nature ofthe unit dose, i.e., solid, liquid, tablet, capsule or suspension; the concenfration of delivery agent in the Gl tract; the feeding state ofthe subject; the diet ofthe subject; the health ofthe subject and the ratio of delivery agent to insulin, hi certain prefe ⁇ ed embodiments ofthe invention, the amount ofthe delivery agent prefe ⁇ ed for the pharmaceutical composition and contained in one or more dosage forms is from about 1 mg to about 2,000 mg, more preferably from about 5 mg to about 800 mg, more preferably about 20 mg to about 600 mg, even more preferably from about 30 mg to
• the time it takes for the delivery agent to reach a peak in the bloodsfream may depend on many factors such as the following: the nature ofthe unit dose, i.e., solid, liquid, tablet, capsule, suspension; the concenfration of delivery agent in the Gl tract; the feeding state ofthe subject; the diet ofthe subject; the health ofthe 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, preferably in tablet form, and preferably provide a peak plasma delivery agent concenfration within about 5 minutes to about 40 minutes after oral adminisfration, and preferably at about 10 minutes to about 35 minutes after oral adminisfration.
• the composition provides a peak plasma delivery agent concenfration within about 25 minutes to about 35 minutes after oral administration to fasting diabetic patients and within about 15 minutes to about 25 minutes after oral adminisfration to fed diabetic patients.
• a peak plasma concenfration (C max ) of the delivery agent achieved after oral adminisfration is preferably from about 10 to about 250,000 ng/ml, after oral administration, preferably from about 100 to about 125,000 ng/ml, and preferably the peak plasma concenfration ofthe delivery agent is from about 1,000 to about 50,000 ng/ml, after oral administration. More preferably, the peak plasma concenfration ofthe delivery agents of the present invention is from about 3,000 to about 15,000 ng/ml after oral administration.
• the composition provides a peak plasma 4-CNAB 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.
• the ratio of active agent to delivery agent may vary for different active agent/delivery agent combinations, hi certain prefe ⁇ ed embodiments ofthe invention where the oral pharmaceutical composition includes insulin as the active agent and the delivery agent is the compound 4-CNAB, the amount ofthe delivery agent included in the pha ⁇ naceutical composition may be from about 20 mg to about 600 mg of said delivery agent.
• the pha ⁇ naceutical 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].
• Prefe ⁇ ed 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.
• Abso ⁇ tion of insulin can be detected in subjects treated with the pharmaceutical compositions ofthe 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 ofthe unit dose, i.e., solid, liquid, tablet, capsule, suspension; the concentration of active agent and delivery agent in the Gl tract; the feeding state of the subject; the diet ofthe subject; the health ofthe subject and the ratio of active agent to the delivery agent.
• the composition provides a peak plasma insulin concenfration from about 0.1 to about 1 hour after oral administration. In another embodiment, the composition provides a peak plasma insulin concenfration from about 0.2 to about 0.6 hours after oral administration, hi a prefe ⁇ ed 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 concenfration within about 1 hour after oral administration.
• the pharmaceutical composition comprises insulin and the compound 4-CNAB as a delivery agent to facilitate the oral delivery of insulin, and after insulin is absorbed into the bloodsfream, the plasma insulin levels in freated patients peak at about 20 minutes post oral adminisfration with a second peak at about 105 minutes.
• the pharmaceutical composition comprises insulin 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 freated patients from about 80 and about 120 minutes post oral adminisfration. More particularly, the composition produces a maximal decrease in C-peptide concenfration in treated patients from about 90 and about 110 minutes post oral adminisfration.
• Emisphere Technologies, Inc. disclosed structures of various delivery agents, comparisons of their effectiveness of abso ⁇ tion and effectiveness of delivery, the preparation ofthe prefe ⁇ ed delivery agent 4-CNAB, its preparation for human studies, and data regarding previous non-clinical and clinical studies involving the delivery agent 4-CNAB.
• the delivery agent may be used directly by mixing with the unmodified insulin prior to administration, either in dry powder form or wet granulated together. To this mixture, other pharmaceutically acceptable excipients may be added. The mixture may be then tableted or placed into gelatin capsules containing a unit dose ofthe active agent and the delivery agent. Alternatively, the delivery agent/insulin mixture may be prepared as an oral solution or suspension. The delivery agent and insulin do not need to be mixed together prior to administration, such that, in certain embodiments, the unit dose of insulin (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 insulin.
• the oral dosage forms ofthe present invention are solid.
• the insulin in dry powder form is stable, and in certain prefe ⁇ ed 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 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 dosage forms ofthe present invention may be produced by first dissolving insulin and the delivery agent 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 co ⁇ ect amount of either insulin 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 adminisfration.
• the administration mixtures may be prepared, e.g., by mixing an aqueous solution ofthe delivery agent with an aqueous solution of insulin just prior to adminisfration.
• the delivery agent and insulin 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.
• the oral dosage form is solid, and is preferably provided inco ⁇ orated within a gelatin capsule or is contained in a tablet.
• Stabilizing additives may be inco ⁇ orated into the delivery agent solution. With some drugs, the presence of such additives promotes the stability and dispersibility ofthe agent in solution.
• the stabilizing additives maybe employed at a concenfration 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 prefe ⁇ ed stabilizing additives are gum acacia, gelatin and methyl cellulose.
• the oral dosage forms ofthe present invention containing a mixture ofthe 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, dichlorotefrafluoroethane, 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, trolamin ⁇ ); Anticaking agents (see glidant); Anti
• Some ofthe factors that are believed to contribute to insulin stability in this formulation are: reduced surface area exposure to atmospheric conditions (only the outside surface ofthe tablet is exposed, while the inner tablet core is not); formulation ofthe tablet to provide an "insulin-friendly" local pH, perhaps in part due to the presence of dicalcium phosphate; and low moisture content (anhydrous excipients were used whenever possible, and 4-CNAB is not hygroscopic(residual moisture content ⁇ 0.5%) below 75% RH and has moisture content below 0.5% w/w).
• HPLC stability- indicating assay This method determines the amount of intact insulin molecules present in a sample, but does not determine whether these molecules are in a bioactive conformation, which is necessary in order to have an effective product.
• Other methods are measurement of related substances (impurities) by HPLC and assessing the bioactivity ofthe product, which could be an in vivo assay or an in vitro predictor of in vivo performance.
• the insulin present in the dosage unit form is absorbed into the circulation.
• the circulating levels ofthe insulin itself can be measured directly.
• levels of 4-CNAB delivery agent in the blood can be measured.
• the bioavailability ofthe insulin is readily assessed by measuring a known pharmacological activity in blood, e.g., decreased blood glucose. Further physiologic effects ofthe insulin can be measured using tests, for example, measurement of plasma C-peptide concentration as a measure of endogenous insulin production.
• a fructosamine assay can be performed to determine the measure ofthe diabetic patient's glycemic control over the previous period of two to three weeks.
• Fructosamine is formed by a non-enzymatic reaction between glucose and amino acid residues of proteins, and serum fructosamine levels are elevated in diabetic patients with elevated blood glucose concentration.
• blood glucose concenfration is a short-term indicator of diabetes confrol
• fructosamine is a short- to medium-term indicator of diabetes confrol that co ⁇ elates well with both fasting and mean blood glucose over a 2-week period.
• the methods for treating a mammal with impaired glucose tolerance or with early or late stage diabetes comprise orally administering to the mammal a pha ⁇ naceutical formulation that includes a therapeutically effective amount of insulin or an insulin analog and a delivery agent in an amount effective to facilitate the abso ⁇ tion ofthe insulin from the gastrointestinal tract. It is prefe ⁇ ed that the administration be on a chronic basis, e.g., for at least two weeks, and be preprandially and at bedtime such that, after two weeks of freatment, the mammal achieves improved glucose tolerance and glycemic control, as well as improved insulin utilization, insulin sensitivity, insulin secretion capacity and HbAic levels, as compared with baseline levels prior to treatment.
• Improved glucose tolerance can be demonstrated by better endogenous capacity ofthe mammal to handle sugar load as measured by blood glucose concentration, following a sugar load, that is reduced by a statistically significant amount as compared with baseline blood glucose concentration, following a glucose load, prior to freatment.
• the statistically significant reduction in blood glucose concentration is a mean of about 10-20%, preferably about 15%.
• Improved glucose tolerance and better endogenous capacity ofthe mammal to handle sugar load can also be measured by an AUC of blood glucose excursion, following a glucose load, that is reduced by a statistically significant amount as compared with AUC of blood glucose excursion, following a glucose load, prior to treatment.
• the statistically significant reduction in AUC of blood glucose excursion is a mean of about 10-30%, preferably about 20%.
• Improved glycemic confrol can be demonstrated by decreased fasting blood glucose levels as measured by fasting blood glucose concentration that is reduced by a statistically significant amount as compared with baseline fasting blood glucose concentration prior to treatment.
• the statistically significant reduction in fasting blood glucose concenfration is a mean of about 10-30%), preferably about 19%.
• Improved glycemic control can also be demonstrated by decreased serum fructosamine concentrations, as measured by serum fructosamine assay, that is reduced by a statistically significant amount as compared with baseline serum fructosamine concentrations prior to treatment.
• the statistically significant reduction in serum fructosamine concentrations is a mean of about 5-20%, preferably about 9%.
• Improved glycemic confrol can also be demonstrated by improved HbAlc levels after treatment compared with baseline levels prior to freatment.
• the improved HbAlc levels are measured by a statistically significant decline in HbAlc levels.
• adminisfration ofthe pharmaceutical formulation ofthe present invention can preferably be made to a mammal having an HbA ⁇ c level ranging from normal to elevated prior to freatment.
• the mammal may have an HbAi c level preferably of less than about 8.0 prior to freatment.
• Improved insulin utilization and insulin sensitivity ofthe patient's body can be measured by a statistically significant decline in HOMA (Homeostasis Model Assessment), and the improved insulin secretion capacity ofthe patient's body is measured by Stumvoll first-phase insulin secretion capacity index.
• HOMA Homeostasis Model Assessment
• the patient achieves improved glucose tolerance and glycemic confrol as compared with baseline levels prior to freatment even without any statistically significant increase in weight, any statistically significant increase in risk of hypoglycemia or any statistically significant increase in risk of hyperinsulinemia in the mammal over the treatment period, and without the need for monitoring the mammal's blood glucose concentrations or HbAic levels.
• the chronic administration of oral dosage fo ⁇ ns ofthe present invention the patient achieves improved insulin utilization, insulin sensitivity insulin secretion capacity and HbAic levels as compared with baseline levels prior to freatment.
• the administration ofthe oral pharmaceutical formulation will be about once daily to about four or more times daily, preprandially and/or at bedtime.
• administration ofthe pharmaceutical fo ⁇ nulation takes place once daily, either at bedtime or preprandially for one meal during the day time, e.g., for breakfast, lunch or dinner.
• administration ofthe pharmaceutical fo ⁇ nulation takes place multiple times daily, preferably at bedtime and preprandially for one meal during the day time, e.g., for breakfast, lunch or dinner.
• adminisfration ofthe pharmaceutical formulation takes place multiple times daily, preferably at bedtime and preprandially for more than one meal during the day time.
• Administration ofthe pharmaceutical formulation can also be is at or shortly prior to bedtime and concu ⁇ ently with or shortly prior to ingestion of each meal, i.e., within about 15 minutes or less of ingestion of each meal.
• the insulin formulations are administered to such human patients on a chronic basis, e.g., for at least about two weeks.
• the dosage form ofthe present invention can be administered for at least one day, for one week, for two weeks, for longer periods, for alternating on-off time periods, or for the life ofthe patient.
• the frequency of adminisfration ofthe oral pharmaceutical fo ⁇ nulation on a daily basis (i.e., how often during one day-night period) and on a chronic basis (i.e., for how many days), will depend upon the patient's position along a "diabetes continuum", i.e., the extent of the patient's impaired glucose tolerance, the patient's stage of diabetes and the patient's need for exogenous glycemic control.
• This continuum ranges from no ⁇ nal glycemic confrol, to simple impaired glucose tolerance and insulin resistance seen in pre-diabetics or early stage type 2 diabetics, to failure of insulin production by the pancreas seen in type 1 diabetics and late stage type 2 diabetics. This can also be measured by the patient's HbAic concenfration, ranging from normal to elevated levels.
• the oral pharmaceutical fo ⁇ nulation should preferably be administered only at or shortly prior to bedtime. If the patient has some need for post-prandial glycemic control, the oral pharmaceutical formulation should preferably be administered preprandially for some meals. If the patient has a need for total postprandial glycemic control, the oral pharmaceutical formulation should preferably be administered preprandially for all meals. If the patient has a need for comprehensive glycemic control, the oral pharmaceutical formulation should preferably be administered preprandially for all meals and at or shortly prior to bedtime.
• the oral insulin formulations ofthe present invention may be utilized in combination therapy, and may also include an additional treatment, either oral or subcutaneously administered, such as an anti-diabetic drug or insulin that has rapid action, intermediate action and/or slow action.
• the oral dosage forms described herein can be orally administered as described herein in combination with an additional yet separate therapy to treat diabetes or impaired glucose tolerance or to achieve glucose homeostasis, such as an additional drug such as sulfonylurea, a biguanide, an alpha-glucosidase, insulin delivered via a different pathway (e.g., parenteral insulin), an insulin sensitizer such as thiazolidinedione, and/or an insulin secretagogue.
• an additional drug such as sulfonylurea, a biguanide, an alpha-glucosidase, insulin delivered via a different pathway (e.g., parenteral insulin), an insulin sensitizer such as thiazolidinedione, and/or an insulin secretagogue.
• the additional freatment may comprise a second form of insulin, so as to provide the patient with two separate forms of insulin having different activity rates in order for the regimen to simulate the biphasic release of insulin in non-diabetic humans.
• the oral formulations may include a rapid-acting form of insulin so as to provide a first insulin peak that occurs rapidly and is short-lived, and the fast-acting effect may be provided by the delivery agent that facilitates the abso ⁇ tion of insulin from the gastrointestinal tract.
• the slow acting form of insulin provides a second insulin peak that occurs later but has a longer duration.
• Such slower acting insulin may be provided by the same oral formulation as the rapid-acting insulin or by a separate dosage form that may be administered orally or subcutaneously.
• the particular combination therapy and its frequency of administration will depend upon the patient's position along the "diabetes continuum". For example, if the patient has a need for fasting glycemic control, the oral pharmaceutical formulation should be administered only at or shortly prior to bedtime. If the patient has some need for post-prandial glycemic control, the oral pharmaceutical formulation should be administered preprandially for meals. If the patient has a need for basal insulin, as in late stage type 2 diabetes or type 1 diabetes, the supplemental slow-acting insulin or anti-diabetic drug should be administered daily.
• the oral pharmaceutical fo ⁇ nulation should preferably be administered preprandially for all meals and at or shortly prior to bedtime in combination with the slow-acting insulin or anti-diabetic drug.
• the invention provides a method of achieving glucose homeostasis in mammals, comprising orally administering to a mammal a pharmaceutical formulation comprising a therapeutically effective amount of insulin or an insulin analog and a delivery agent in an amount effective to facilitate the abso ⁇ tion ofthe insulin from the gastrointestinal tract.
• the administration be on a chronic basis, e.g., for at least two weeks, and be preprandially and at bedtime such that, after two weeks of treatment, the mammal achieves improved glucose tolerance and glycemic control as compared with baseline levels prior to treatment.
• the chronic adminisfration ofthe oral dosage forms ofthe present invention will reduce the incidence and/or severity of systemic hyperinsulinemia associated with chronic dosing of insulin or of one or more disease states associated with chronic dosing of insulin in a mammal that has impaired glucose tolerance or early stage diabetes.
• the chronic adminisfration of oral dosage fo ⁇ ns ofthe present invention result in a higher portal insulin concentration and lower systemic insulin concentration over time than that obtained with an equi-effective dose of insulin administered subcutaneously (i.e., which provide similar confrol of blood glucose levels).
• Transient peaks in insulin levels that may occur by virtue ofthe oral adminisfration of insulin in accordance with the present invention are not believed to be associated with vascular diseases.
• the chronic administration of oral dosage forms of the present invention instead of equi-effective subcutaneous doses of insulin, lower levels of hyperinsulinemia are obtained, e.g., systemic insulin concentrations are at least about 20% lower when compared to a comparably effective subcutaneous dose of insulin.
• the present invention thus provides methods for reducing the incidence and/or severity of systemic hyperinsulinemia associated with chronic dosing of insulin, and it is believed that the present invention also provides a method for reducing the incidence and/or severity of one or more disease states associated with chronic dosing of insulin.
• Such methods also comprise orally administering a therapeutically effective dose of a pharmaceutical fo ⁇ nulation comprising insulin and a delivery agent that facilitates the abso ⁇ tion of the insulin from the gastrointestinal tract, to provide a therapeutically effective reduction and/or confrol in blood glucose concenfration and a plasma insulin concentration that is reduced relative to the plasma insulin concenfration provided by a therapeutically equivalent dose of subcutaneously injected insulin.
• Such methods also achieve a reduction in blood glucose concentration in human diabetic patients comparable to a subcutaneous insulin injection in those patients, while providing a lower (e.g., 20% or greater) total exposure of insulin to the peripheral blood circulation under acute, sub-acute and chronic conditions as compared to the peripheral blood insulin exposure achieved via subcutaneous injection.
• the determinations of blood or insulin concentration obtained in patients who have been administered subcutaneous insulin are well known to those skilled in the art.
• pancreas In a non-diabetic individual, during times of fasting, such as during sleeping hours or between meals, the pancreas is able to store insulin for future use and is given a rest from secretion. In a diabetic or insulin resistant patients, the pancreas continues to secrete insulin without allowing a proper insulin store to be achieved. It is believed that one ofthe first defects ofthe pancreas in insulin resistance and type 2 diabetes is this defect in insulin storage.
• Cu ⁇ ent literature shows that patients in the United States with type 2 diabetes are being diagnosed 8-10 years after the diabetic process has begun.
• the cunent American Diabetes Association guideline for diagnosing diabetes is two consecutive fasting blood glucose levels above 110 mg/dL. It is believed that, by the time of diagnosis, a diabetic patient has already lost function of about 50% of his islet cells.
• therapy can be initiated at an early stage to prophylactically spare / 3-cell function and aid in preventing / 3-cell death and the progression to overt diabetes.
• Many factors may be taken into account when therapy becomes necessary or desirable including, but not limited to: defects in GTT indicating signs of insulin resistance, reactive hypoglycemia, or early /3-cell dysfunction, elevated fasting or postprandial blood glucose levels, family history for diabetes, obesity, HbA ⁇ c above approximately 6.5 or an elevation of HbA ⁇ c of more than about 10% over patient's past values, even if still within normal ranges.
• a mammal at this early stage can be treated, prophylactically sparing /3-cell function, aiding in preventing /3-cell death and/or the progression to overt diabetes, by administering one time daily an effective dose of a pharmaceutical formulation, preferably an oral formulation, comprising insulin (as described herein) at nighttime, in the morning or preprandially, preferably at nighttime or in the morning.
• a pharmaceutical formulation preferably an oral formulation, comprising insulin (as described herein) at nighttime, in the morning or preprandially, preferably at nighttime or in the morning.
• the insulin formulation is administered to such human patients on a chronic basis, e.g., for at least about two weeks.
• a mammal at this early stage of impaired glucose tolerance or early stage diabetes mellitus can be treated, prophylactically sparing remaining 3-cell function, aiding in preventing /3-cell death and/or the progression to overt diabetes and treating the cu ⁇ ent level of glycemic confrol dysfunction, by administering an effective dose of a pharmaceutical formulation, preferably an oral formulation, twice daily comprising insulin (as described herein) at nighttime, in the morning and/or preprandially, preferably at nighttime or morning and preprandially, more preferably at nighttime and preprandial for breakfast.
• a pharmaceutical formulation preferably an oral formulation, twice daily comprising insulin (as described herein) at nighttime, in the morning and/or preprandially, preferably at nighttime or morning and preprandially, more preferably at nighttime and preprandial for breakfast.
• the insulin formulation is administered to such human patients on a chronic basis, e.g., for at least about two weeks.
• the patient can be treated, prophylactically sparing remaining /3-cell function, aiding in preventing /3-cell death and/or the progression to overt diabetes and treating the cu ⁇ ent level of glycemic control dysfunction, by administering an effective dose of a pharmaceutical formulation, preferably an oral formulation, three times daily comprising insulin (as described herein) preprandially or postprandially.
• a pharmaceutical formulation preferably an oral formulation
• insulin preprandially or postprandially.
• This treatment regime can be carried through to later stages o the diabetes.
• the insulin formulation is administered to such human patients on a chronic basis, e.g., for at least about two weeks.
• a mammal at this stage of impaired glucose tolerance or diabetes mellitus can be treated, prophylactically sparing remaining / 3-cell function, aiding in preventing ⁇ - cell death and/or the progression to overt diabetes and treating the cu ⁇ ent level of glycemic control dysfunction, by administering an effective dose of a pharmaceutical formulation, preferably an oral formulation, three times daily comprising insulin (as described herein) at nighttime, in the morning and/or preprandially, preferably at nighttime or morning and twice preprandially, more preferably at nighttime and preprandial for breakfast and lunch.
• the insulin formulation is administered to such human patients on a chronic basis, e.g., for at least about two weeks.
• a mammal at this stage of impaired glucose tolerance or diabetes mellitus can be freated, prophylactically sparing remaining /3-cell function, and/or aiding in preventing /3-cell death and treating the cu ⁇ ent level of glycemic confrol dysfunction, by administering an effective dose of a pharmaceutical formulation, preferably an oral formulation four times daily comprising insulin (as described herein) at nighttime, in the morning and/or preprandially, preferably at nighttime or morning and three times preprandially, more preferably at nighttime and preprandially for breakfast, lunch and dinner.
• a pharmaceutical formulation preferably an oral formulation four times daily comprising insulin (as described herein) at nighttime, in the morning and/or preprandially, preferably at nighttime or morning and three times preprandially, more preferably at nighttime and preprandially for breakfast, lunch and dinner.
• the insulin formulation is administered to such human patients on a chronic basis, e.g., for at least about two weeks.
• a mammal at this stage of diabetes mellitus can be treated, prophylactically sparing any remaining /3-cell function, and/or aiding in preventing (3-cell death and treating the cu ⁇ ent level of glycemic confrol dysfunction, by administering an effective dose of a pharmaceutical fo ⁇ nulation, preferably oral, comprising long- acting insulin; and an effective dose of a pharmaceutical fo ⁇ nulation, preferably an oral formulation, four times daily comprising insulin (as described herein) at nighttime, in the morning and/or preprandially, preferably at nighttime or morning and three times preprandially, more preferably at nighttime and preprandially for breakfast, lunch and dinner.
• the insulin formulations are administered to such human patients on a chronic basis
• a mammal at this stage of diabetes mellitus can be treated by administering an effective dose of a pharmaceutical formulation, preferably oral, comprising long- acting insulin; and an effective dose of a pharmaceutical formulation, preferably an oral formulation, three or four times daily comprising insulin (as described herein) at nighttime, in the morning and/or preprandially, preferably nighttime, or in the morning and three preprandially, preferably at nighttime and three times preprandially.
• an effective dose of a pharmaceutical formulation of should be dosed, preferably preprandially.
• the insulin fo ⁇ nulations are administered to such human patients on a chronic basis, e.g., for at least about two weeks.
• a continuum of development of diabetes comprising a pre-diabetic stage, an early stage diabetes and late stage diabetes, and the invention comprises identifying a patient's stage along the continuum of development of diabetes.
• a prefe ⁇ ed embodiment ofthe invention comprises a method for treating a patient in accordance with his/her stage of development of diabetes comprising: identifying a patient's stage along the continuum of development of diabetes, devising a course of treatment for that patient in accordance with his stage along the continuum of development of diabetes and administering the treatment to the patient.
• 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 (SC) injected short acting insulin [Humalog 18 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.
• Visits 7 and 8 were combined (i.e., final examination was performed at Visit 7, immediately after finishing experimental procedures).
• the SC insulin dose of 12 U was selected to fall within a range typical for type 2 diabetic patients.
• the oral dose of 300 U insulin (in combination with 400 mg 4-CNBA) had been shown to be effective in Example 5 above.
• the oral dose of 150 U insulin (in combination with 200 mg 4- CNBA) was chosen to investigate whether or not an effect on hepatic glucose production could be achieved also by a lower insulin dose.
• the time point of study drug administration (SC injection: 15 minutes prior to meal intake; oral administration: 30 minutes prior to meal intake) was selected in order to match the PK and PD properties ofthe administered insulin formulations with the postprandial rise of blood glucose.
• the wash-out period between the first three treatment sessions was 1-20 days.
• the duration of each session was approximately 8-9 hours, and all experiments were performed after an overnight fast of approx. 12 hours.
• Visit 1 the patients came to the clinical research unit in a fasted state (i.e., not having had any caloric intake for at least 12 hours).
• each patient was randomized to one of six treatment sequences shown in Table 2 below and received either one ofthe two active treatments (300 U oral Insulin/400 mg 4- CNAB or 12 U short-acting SC insulin) or no supplemental insulin (placebo).
• the patients ate a standardized breakfast, and postprandial blood glucose concenfrations were monitored for six hours.
• Serial blood samples were also collected in regular intervals for measurement of plasma insulin, 4-CNAB, and C-peptide concentrations. The study patients were released from the institute at the end ofthe freatment session.
• Visits 3 and 4 the study patients returned to the clinical unit to receive the alternative treatments in conjunction with the test meal according to their treatment sequence. All experimental procedures and measurements were identical with those ofthe preceding freatment days. A final examination (Visit 5) was perfo ⁇ ned after Visit 4, preferably immediately after the experimental procedures were completed, but no longer than fourteen days after Visit 4. [00300] The patients were invited to attend a fourth treatment session (Visit 7) with a single oral adminisfration of 150 U Insulin/200 mg 4-CNAB thirty minutes prior to a test meal. All experimental procedures and measurements were the same as on the preceding treatment days.
• Visit 6 Patients attended a screening (Visit 6), no more than twenty days prior to the additional session, as well as a final examination (Visit 8), preferably immediately after the experimental procedures of Visit 7 were completed, but no longer than fourteen days thereafter. Visits 7 and 8 were generally combined (i.e., for all patients final examination was performed at Visit 7, immediately after completion of experimental procedures).
• each patient received on the first three treatment sessions (Visits 2-4), in addition to his scheduled treatment administration (oral or SC), the alternative adminisfration (SC or oral) as placebo preparation.
• oral or SC his scheduled treatment administration
• SC or oral the alternative adminisfration
• both treatments oral and SC
• placebo preparations On the last treatment session (Visit 7), all patients received in an open fashion one oral dose of 150 U Insulin/200 mg 4-CNAB.
• 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, IN).
• the h ⁇ sulin 4-CNAB capsules contained 150 Insulin Units USP and 200 mg 4-CNAB.
• the insulin/4-CNAB capsules were prepared by AAI Pharma Inc., Wilmington NC.
• 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.
• exogenous insulin was administered by oral insulin adminisfration or by subcutaneous injection at two ofthe three experimental days.
• the oral treatments (Insulin/4-CNAB capsules and placebo capsules) were administered 30 minutes, and the injections (short-acting insulin and placebo solution) 15 minutes, before start of meal intake.
• the pharmacodynamic response elicited was studied by measurements of blood glucose concentrations in 5 minute intervals for another six hours, and no food intake was allowed during this period, although water was consumed as desired.
• Blood samples for blood glucose determination (0.25 mL per sample) were taken at -1 min (baseline), 5 minutes after start of meal intake and thereafter in 5 minute intervals until 120 minutes, 10 minute intervals until 240 minutes, and 15 minute intervals until 360 minutes after start of meal intake (45 samples per session). Blood glucose concentrations were measured immediately after sample collection using an automated GOD method (Super GL Ambulance Glucose Analyzer, Ruhrtal Labortechnik, Delecke-M ⁇ hnesee, Germany).
• Plasma samples for determination of 4-CNAB plasma concentrations (2 mL in sodium heparin tube) were drawn 10, 20, 30, 40, 60, 90, 120, 240 and 360 minutes after start of meal intake (9 samples per session).
• Blood samples for determination of insulin and C-peptide plasma concenfrations (5 mL in sodium heparin tube) were drawn at -60 and -30 minutes, at time 0 (start of meal intake), and after 10, 20, 30, 40, 50, 60, 75, 90, 105, 120, 150, 180, 210, 240, 300, and 360 minutes (19 samples per session).
• Plasma concenfrations of insulin were determined by a GLP- validated microparticle enzyme immunoassay (MELA).
• Plasma samples for the determination of plasma concentrations of insulin, 4-CNAB and C-peptide were collected at defined intervals, as discussed above. Plasma samples were stored at approximately -20°C (4-CNAB at -70°C) until determination by immunoassay is performed. After the end ofthe sampling period, the study subjects were released from the clinic.
• BG max Maximal blood glucose excursion
• I BG max Area under the blood glucose excursion curve in defined time-intervals
• BGabs max maximal absolute blood glucose concenfrations
• tBGabs max time to BGabs max
• Plasma insulin concentrations were subjected to appropriate pharmacokinetic analyses. Parameters determined include C max , t max , and the area under the plasma concenfration versus time curve from the time of dosing until a return to the baseline measurement (AUCo-?), where t' is the time that the level of plasma insulin concentration returns to the baseline.
• pharmacokinetic parameters such as / heading elimination rate constant ( ⁇ z ) and partial AUC values, were calculated, if considered appropriate, for each individual subject enrolled within the study.
• FIG. 1 shows a plot ofthe arithmetic means of postprandial blood glucose excursions (mg/dL) vs. time for all subjects.
• mean blood glucose excursions ofthe different treatments reach their maxima between 1 and 2 hours after start of meal intake and then return towards baseline.
• the time to maximal glucose excursion (median) was 1.3 hours for SC 12 U short-acting insulin, 1.7 hours for placebo, 1.8 hours for oral 150 U Insulin/200mg 4-CNAB, and 2.2 hours for oral 300 U Insulin/400 mg 4-CNAB.
• the lowest overall excursions were achieved with the 12 U SC short-acting insulin injection. Compared to both oral insulin treatments and placebo, blood glucose excursions after SC injection are markedly lower during the period from 45 to 360 minutes and, after crossing the baseline at about 180 minutes, values become increasingly negative until 360 minutes after meal intake.
• Subject 116 was chosen because he was a Type II diabetic in an early stage ofthe disease, i.e., was able to produce his own insulin, and the glucose curve for subject 116 shown in Figure 3 paralleled that of healthy (normal) non-diabetic humans.
• subject 117 was a type ⁇ diabetic in an advanced stage ofthe disease, i.e., having very little pancreatic function left and producing very little endogenous insulin. Accordingly, as shown in the glucose curve of Figure 4 for subject 117, it took much longer to lower the glucose level for this subject back to a level found in healthy (normal) non-diabetic humans.
• test results can be summarized as follows: When C max and AUCs for 3 hours and more are considered, no statistically significant differences ofthe oral treatments compared to no treatment (placebo) could be established. On the other hand, both oral treatments differ significantly from SC insulin injection, with oral treatments leading to higher mean values.
• Table 41 below presents the mean time data (with standard deviation) ofthe plasma 4- CNAB concentrations for the two treatments involving 4-CNAB.
• FIG. 2 shows profiles of 4-CNAB plasma concentrations (ng/mL) vs. time (arithmetic means) for the two treatments involving 4-CNAB.
• plasma 4-CNAB concentrations show a rapid decline within the first two hours after start of meal intake. After 2 hours, concentrations are less than 10% ofthe levels seen after 10 minutes. The results indicate that markedly higher concentrations might have been reached in the time between intake ofthe Insulin/4-CNAB capsules and the first measurement 10 minutes after start of meal intake. Concentrations after intake of 400 mg 4-CNAB are approximately twice as high as after intake of 200 mg.
• Table 42 below presents the mean time data (with standard deviation) ofthe plasma insulin concentrations per treatment.
• Figure 5 shows profiles of insulin plasma concentrations (pmol/1) vs. time (arithmetic means). As shown in Figure 5 and in Table B above, highest mean insulin plasma concentrations are reached after the 150 U oral dose, followed by 300 U oral, placebo, and 12 U SC injection.
• the curve of oral 300 U Insulin/400 mg 4-CNAB shows two maxima, the first at 0 min and the second at 120 min.
• the peak at 0 min is due to one particular patient who contributed with a value of 1803 pmol/L the most to this marked shift of mean insulin concentration. Almost all patients showed a more or less marked isolated increase of insulin concentrations at time 0 but not to such an extent as that patient.
• the rise of insulin concentrations under placebo is explained by the patients' endogenous insulin production, induced by the meal intake.
• Figures 7 and 8 show the insulin plasma concentration vs. time curves for subjects 116 and 117, respectively.
• subject 116 who was an early stage Type ⁇ diabetic who produced his own insulin
• the insulin plasma concentration vs. time curve shown in Figure 7 mimicked that of healthy (normal) non-diabetic humans, i.e., it had the same biphasic secretion time curve shape, although the insulin peaks occurred slightly earlier than normal.
• the insulin plasma concentration vs. time curve shown in Figure 8 shows levels of insulin after the initial peak that are lower that those for normal, non-diabetic humans and shows that no second peak of plasma insulin concentration occurred. This is an indication that this subject would also need to be administered basal long lasting insulin in order to maintain normal insulin plasma concentration and blood glucose levels.
• Table 43 below presents the mean time data (with standard deviation) ofthe plasma C-peptide concentrations per treatment.
• Figure 6 shows profiles of C-peptide plasma concentrations (nmol/l) vs. time (arithmetic means). Mean plasma concentrations of C-peptide, the indicator of endogenous insulin production, increased after all treatments. Decreasing, or more or less constant C-peptide concentrations, were seen only in a few patients and only after SC injection of short-acting insulin. This may reflect the fact that in most ofthe patients the ability to produce endogenous insulin was still maintained. As expected, the 150 U oral insulin dose and placebo show the most marked increase, whereas the increases after the 300 U oral dose and the 12 U SC injection are clearly lower.
• t* denotes time when baseline insulin level is reached again, or last data point (360 min)
• the primary objective of this study was to compare the effect of orally administered 300 U Insulin/400 mg 4-CNAB with that of 12 U subcutaneously injected short-acting insulin (Humalog ® ) on postprandial blood glucose excursions after a standardized breakfast.
• AUC 0-2h as main parameter for pharmacodynamic evaluation, the highest effect on blood glucose excursions was found for 12 U SC short-acting insulin, followed by oral 300 U Insulin 400 mg 4-CNAB, oral 150 U Insulin/200 mg 4-CNAB and placebo, and the effects ofthe two latter appeared more or less equal.
• these results were not consistent for all calculated AUCs.
• the insulin profiles showed the highest AUC after 150 U oral insulin, followed by placebo, 300 U oral insulin, and 12 U SC short-acting insulin.
• the marked increase of mean plasma insulin concentrations after placebo indicates that the patients' ability of endogenous insulin production, induced by meal intake, was still maintained.
• the high AUC for 150 U oral insulin probably reflects mainly endogenous insulin production, and also the curves ofthe other treatments may account for a certain amount of endogenous insulin.
• the C-peptide plasma concentration profiles confirm this view and also indicate the release of considerable amounts of endogenous insulin.
• the levels were highest after 150 U oral insulin, followed by placebo, 300 U oral insulin, and 12 U SC short-acting insulin.
• the 150 U oral dose and placebo led to the most marked increase, whereas the increase after the 300 U oral dose and the 12 U SC injection was clearly lower, and these findings correlate with the blood glucose lowering effect seen for the different treatments: the lower the effect ofthe external insulin dose, the higher were the amounts of C-peptide as indicator of endogenous insulin production.
• Example 6 of International Publication No. WO 03057170 The insulin concentration vs. time profiles seen for both oral doses in this study are considerably different from those obtained in Example 6 of International Publication No. WO 03057170, where mean insulin concentrations were back to baseline after approximately two hours and where maximum concentrations occurred after about half an hour. These differences might be due to the influence ofthe meal, stimulating endogenous insulin release and also possibly interfering with the resorption ofthe oral insulin preparations.
• Example 6 of WO 03057170 patients fasted during the entire experiment, and endogenous insulin production was suppressed by a constant low-dose insulin infusion. Therefore, the concentration vs. time curves of Example 6 of WO 03057170 represent more the pure pharmacokinetics ofthe administered exogenous insulin, whereas in the present study the effects of exogenous and endogenous insulin are overlapping.
• oral insulin capsule(s) described herein were orally administered to twenty human subjects with diabetes at night before going to sleep.
• the focus of this study is the assessment ofthe safety of insulin/4-CNAB, administered orally at bedtime, to type 2 diabetic subjects.
• the purpose ofthe study was to determine if the administration of oral insulin at bedtime could exert effects on overnight-fasting glucose homeostatsis and insulin secretion.
• the postulated mode of action e.g., suppressing the liver production of glucose, and thus preventing ⁇ -cell death or dysfunction of insulin producing was the basis for the design ofthe study.
• Group 1 twelve (12) type 2 diabetic subjects: (a) oral insulin/4-CNAB - fasted subjects, and (b) empty capsule - fasted subjects.
• Group 2 twelve (12) type 2 diabetic subjects: (a) standard meal with regular medication, and (b) standard meal with oral human insulin/4-CNAB.
• the subjects took one oral insulin dose that contained the following ingredients: 300 mg 4-CNAB and insulin according to the dose (200-400 U) that the subject received during the first phase of the trial. If the subject had received 200 U insulin in the first phase ofthe trail and there was no drop in blood glucose level ( ⁇ 15% reduction), he now received 300 U of insulin. If the subject had received 300 U insulin in the first phase ofthe trail and there was no drop in blood glucose level ( ⁇ 15% reduction), he now received 400 U of insulin. None ofthe subjects received more than 400 U of insulin.
• the capsules were prepared by AAI and have shown stability.
• a nurse was present at the home ofthe subjects when they took the oral insulin capsules and throughout the night. The nurse checked the blood glucose level with a glucometer before the subjects took the medication. In addition, blood was taken for further blood glucose levels, insulin and C-peptide. Orange juice was readily available for treatment in the unlikely event of hypoglycemia.
• a Glucowatch which is a monitor of blood glucose and measures and records blood glucose levels at regular intervals. The Glucowatch is equipped with an alarm triggered when blood glucose levels reach predetermined blood glucose levels (hypoglycemic levels) determined by the investigator or patient. The bedside private duty nurse was also present during the night to monitor the patient.
• the nurse checked their blood glucose level with the glucometer. Additional blood samples were taken for further blood glucose levels, insulin and C-peptide.
• the blood samples from the night before were stored in the refrigerator at home and in the morning the nurse brought the samples of blood (from the night and the morning) to the lab for analysis.
• This example describes the manufacturing procedure for Insulm/4-CNAB tablets.
• Each tablet contained about 75 units of insulin USP (equivalent to about 2.82 mg of recombinant human insulin with an as-is potency of about 26.6 U/mg) and about 100 mg of 4-CNAB monosodium salt.
• composition of formulation (theoretical, all numbers are approximate):
• KOLLIDON ® 90F (BASF Corporation, Mount Olive, NJ)
• Insulin obtained from Diosynth, Inc.
• 4-CNAB was granulated using the insulin suspension as granulation media.
• Granulation was completed with additional water, as required.
• Granules were dried in a vacuum oven at about 50°C. Partly dried granules (about 0-10% w/w, preferably about 2-3% w/w moisture) were milled through about 0.02 inch screen using hammer mill. Drying was continued to a final moisture content of less than about 0.6% w/w.
• the resulting tablet had a hardness of about 7.6 kP, a thickness of about 2.8 mm, a diameter of about 7.1 mm, a friability of 0.00% and a disintegration time of about 5 minutes.
• the dose for this preparation was about four tablets per patient, as described in Example 3 below.
• the insulin molecule appears to be stable (> 90%) in the 75 U Insulin/100 mg 4-CNAB insulin tablet fo ⁇ nulation when stored for two months at 25 degrees C and 60% Relative Humidity (25/60).
• This example describes the manufacturing procedure for Insulin/4-CNAB tablets.
• Each tablet contained about 150 units of insulin USP (equivalent to about 5.64 mg of recombinant human insulin with an as-is potency of about 26.6 U/mg) and about 80 mg of 4-CNAB monosodium salt.
• the insulin used in this study was obtained from Diosynth, Inc. and met the specifications for Human Insulin as described in the United States Pharmacopoeia.
• composition of formulation (theoretical, all numbers are approximate):
• KOLLIDON ® 90F was weighed. KOLLIDON ® 90F was dissolved in water. The amount of water used in this step was about 1-50%, preferably about 15% w/w ofthe amount of material used in the granulation. Insulin (obtained from Diosynth, Inc.) and 4-CNAB were geometrically blended and charged to the 5L bowl of a Key Instruments KG-5 high shear granulator. The insulin/4-CNAB blend was then granulated using the KOLLIDON ® solution. The granulation was finished with additional water as required. Granules were dried in a vacuum oven at about 20-80°C, preferably about 50°C.
• Partly dried granules (about 0-10% w/w, preferably about 2-3% w/w moisture) were milled through about 0.02 inch screen using hammer mill. Drying was continued to a final moisture content of less than about 0.6% w/w.
• Tablets were compressed on an EK-0 single station press with a hardness of about 5KP-10KP, preferably about 7KP.
• the resulting tablet had a hardness of about 7.8 kP, a thickness of about 2.8 mm, a diameter of about 6.5 mm, a friability of 0.02% and a disintegration time of about 6 minutes.
• the resulting tablets were studied to determine whether they would remain within specification when stored under recommended storage conditions in order to provide evidence on how the product quality varies with time under the influence of temperature and humidity.
• the stability tests were conducted in compliance with the U.S. Federal Drug Administration current Good Manufacturing Practice Standards, 21 C.F.R. ⁇ 210 and 211, and the International Conference on Harmonization (ICH) Guidance, ICH Q1A (R2), using qualified equipment, test methods and personnel.
• ICH International Conference on Harmonization
• the insulin molecule appears to be stable (> 90%) in the 150 U Insulin/80 mg 4-CNAB insulin tablet formulation when stored for six months at 25 degrees C and 60% Relative Humidity (25/60).
• a 150 Insulin/200 mg 4-CNAB capsule formulation yielded spurious HPLC data after two months, and a result below 90% after three months.
• the differences between the capsule and tablet formulations are that capsules allow higher surface area exposure to atmosphere, had a higher level of 4-CNAB, contained hydrous rather than anhydrous dicalcium phosphate, and contained sodium lauryl sulfate (a potential insulin denaturant).
• This example describes the manufacturing procedure for 4-CNAB tablets. Each tablet contains about 100 mg of 4-CNAB monosodium salt.
• composition of formulation (theoretical, all numbers are approximate):
• KOLLIDON ® 90F (BASF Corp., Mount Olive, NJ)
• the amount of water used in this step should be about 1 - 50%, preferably about 15% w/w ofthe amount of material used in the granulation.
• 4- CNAB was granulated using the KOLLIDON ® solution as granulation media. Granulation was completed with additional water, as required. Granules were dried in a vacuum oven at about 20- 80°C, preferably about 50°C.
• Partly dried granules (about 0-10% w/w, preferably about 2-3% w/w moisture) were milled through about 0.02 inch screen using hammer mill. Drying was continued to a final moisture content of less than about 1.0%, preferably less than about 0.6% w/w. Based on final moisture content, amounts of excipients (anhydrous EMCOMPRESS ® (dicalcium phosphate (JRS Pharma PL,Patterson, New York))and magnesium stearate) were calculated, weighed and screened. 4-CNAB granules and anhydrous EMCOMPRESS ® were blended in a V-blender for about 10-20 minutes, preferably about 15 minutes. Magnesium stearate was added and blended for about 1-5 minutes, preferably about 3 minutes. Tablets were compressed on an EK-0 single station press with a hardness of about 5KP-10KP, preferably about 7KP.
• a single-blind, crossover study was done in order to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of oral Insulin 4-CNAB tablets in fasted and pre-prandial type 2 diabetic patients.
• a crossover design was selected for this study so as to reduce the inter-subject variability and to allow maximum use ofthe limited sample size, and a blinded study design was selected in order to reduce the bias from the patient side.
• each study drug was administered with exactly 150 mL of water and followed by a 72 hour wash-out period in order to eliminate pharmacological treatment interactions.
• An indwelling catheter was inserted for pharmacokinetic, pharmacodynamic and clinical blood sample collection.
• the study drug regimen was as follows:
• the dose levels for this study were selected based upon previous human experience in both healthy and diabetic subjects, wherein up to 450 U Insulin/600 mg 4-CNAB has been safely tolerated in Diabetes Type 1 and Type 2 patients.
• the fo ⁇ nulation and dose ratio of insulin to 4-CNAB were chosen to investigate a drug carrier ratio-response relationship for formulation optimization.
• the 75 U Insulin/ 100 mg 4-CNAB tablets and the 150 U Insulin/80 mg 4-CNAB tablets were manufactured as described in Examples 1 and 2 above, respectively.
• Plasma or blood glucose 13 samples: 15 and 5 minutes pre-study dose; and 5, 10, 15, 20, 25, 30, 40, 50 and 60 minutes, and 2 and 4 hours post-study dose (blood glucose used the SuperGL equipment; plasma used an Elisa assay).
• the pharmacokinetic parameters determined or calculated from the plasma concentration time data for 4-CNAB were C max , t max , t-/din AUC 0 -iast, AUCM, AUC 0- t, and CL/F.
• Table 48 sets forth the data for mean blood glucose change vs. time:
• Table 48 Mean Percent Change from Baseline Blood Glucose vs. Time
• Figure 12 shows graphs of mean (for all eight subjects) percent change in blood glucose concentration from baseline levels following oral administration ofthe various Insulin 4-CNAB tablet combinations and control described above to Type 2 diabetic patients, both with and without a meal.
• the mean Figure 12 is based upon Figures 15-22, which show percent change in blood glucose concentrations from baseline levels for subjects 101-108 following oral administration of the various Insulin 4-CNAB tablet combinations described above.
• the oral insulin/4-CNAB tablets appeared to be well-tolerated in Type 2 diabetes patients.
• Figure 13 also shows that the 300U insulin/160 mg 4-CNAB ratio appeared to have been more effective at delivering insulin than was the 300U insulin/400 mg 4-CNAB ratio, because the 300U insulin/160 mg 4-CNAB ratio produced a slightly greater decrease in blood glucose when administered 10 minutes prior to a meal than did the 300U insulin/400 mg 4-CNAB ratio. Therefore, at controlling post-prandial glucose excursion, the 300U insulin/160 mg 4-CNAB ratio appeared to be at least as effective as, if not more effective than, the 300U insulin/400 mg 4-CNAB ratio.
• Figure 23 shows graphs of mean (for all eight subjects) percent change in plasma glucose concenfration from baseline levels following oral administration ofthe various Insulin/ 4-CNAB tablet combinations described above to type 2 diabetic patients, both with and without a meal.
• r gure 24 snows a comparison ofthe mean (for all eight subjects) percent change in plasma glucose concentration for only the 300U Insulin/160 mg 4-CNAB tablets and the 4-CNAB alone tablets, in both cases 10 minutes before a meal.
• These figures are similar to Figures 12 and 14, except that plasma glucose concenfration, instead of blood glucose concenfration, was measured.
• Figure 25 shows graphs of mean (for all eight subjects) blood glucose concenfration following oral adminisfration ofthe various Insulin/4-CNAB tablet combinations described above to type 2 diabetic patients, both with and without a meal.
• the mean Figure 25 is based upon Figures 26-33, which show blood glucose concenfration vs. time curves for subjects 101-108 following oral adminisfration ofthe various Insulin/4-CNAB tablet combinations described above.
• Table 50 sets forth the data for mean serum insulin concenfration vs. time:
• Table 50 Mean Serum Insulin Concentration vs. Time
• Figure 34 shows curves of mean (for all eight subjects) serum insulin concenfration following oral adminisfration ofthe various Insulin/4-CNAB tablet combinations described above to type 2 diabetic patients, both with and without a meal.
• the mean Figure 34 is based upon Figures 36-43, which show graphs of serum insulin concentration vs. time following oral adminisfration ofthe various fr ⁇ sulin/4-CNAB tablet combinations described above for subjects 101-108, both with and without a meal.
• Figure 35 shows a comparison of graphs of mean (for all eight subjects) serum insulin concenfration vs. time for only the 300U Insulin/160 mg 4-CNAB tablets (both fasted afterwards and at 10 minutes before a meal) and the confrol (4-CNAB alone) 10 minutes before a meal.
• the C m a x was somewhat higher, and the t m ⁇ was somewhat later, for the fasted state than it was for the prandial state (adminisfration 0 or 10 minutes prior to a meal) in both the 300U insulin/160 mg 4-CNAB ratio group and the 300U insulin/400 mg 4-CNAB ratio group.
• mean fasted C max was about 170 ⁇ U/mL and mean fasted t m ⁇ was at about 20 minutes post adminisfration
• mean C ma ⁇ was about 65 ⁇ U/mL and mean t max was at about 15 minutes post administration when administered 10 minutes prior to a meal.
• mean fasted C ma was about 90 ⁇ U/mL and mean fasted t max was at about 20 minutes post administration, and mean C max was about 75 ⁇ U/mL and mean t max was at about 15 minutes post adminisfration when administered 10 minutes prior to a meal.
• Table 51 sets forth the data for mean plasma 4-CNAB concenfration vs. time:
• Table 51 Mean Plasma 4-CNAB Concentration vs. Time
• Figure 33 shows graphs of mean (for all eight subjects) plasma 4-CNAB concenfration vs. time following oral adminisfration ofthe various Insulin/4-CNAB tablet combinations described above to type 2 diabetic patients, both with and without a meal.
• mean fasted t max was at about 20-25 minutes post adminisfration, and mean t ma ⁇ was at about 15 minutes post adminisfration when administered 10 minutes prior to a meal.
• mean fasted t max was at about 30 minutes post adminisfration, and mean t ma ⁇ was at about 20 minutes post adminisfration when administered 10 minutes prior to a meal.
• Table 52 below sets forth the data for mean plasma C-peptide concentration vs. time:
• Figure 45 shows graphs of mean (for all eight subjects) plasma C-Peptide concentration vs. time following oral adminisfration ofthe various Insulin/4-CNAB tablet combinations described above to type 2 diabetic patients, both with and without a meal.
• the oral insulin formulation disclosed herein has the same effect on glucose suppression after a standard meal as observed with the secretagogues. This can be seen by comparing glucose excursion profiles shown in Figures 14 and 46.
• Figure 3 the change in blood glucose concenfration from baseline of 300U insulin/160 mg 4-CNAB administered 10 minutes before a meal is compared with that for 4-CNAB alone administered 10 minutes before a meal (confrol).
• the oral insulin dose resulted in about a 30% reduction in the post-prandial glucose excursion for about 60 minutes.
• Figure 46 which is taken from M.F.
• the aim of this study was to evaluate the safety, tolerability and pharmacokinetics of oral Insulin/4-CNAB following two weeks of preprandial and bedtime adminisfration and to investigate the effect on glycemic confrol, insulin secretion capacity, insulin sensitivity and glucose tolerance in diet-treated type 2 diabetic subjects in good to moderate metabolic control.
• this study was to evaluate whether repeated dosing of oral insulin multiple times daily can exert a sustained therapeutic effect in patients with type 2 diabetes.
• the information gained in this study is to provide further insight into the beneficial effects of a preprandial treatment with oral insulin/4-CNAB in early phase type 2 diabetic patients.
• Patients were randomized to one of two treatment groups to receive an active dose of Insulin/4-CNAB or a confrol dose of 4-CNAB alone for a two-week period four times daily, 10 minutes before breakfast, lunch and dinner and at bedtime).
• the seven patients in the active group were to be freated with two tablets totaling 300 U insulin/160 mg 4-CNAB, and the six patients in the confrol group were treated with two tablets totaling 200 mg 4-CNAB.
• Those chosen for active treatment had HbAic between 6.1% and 7.7%, with a mean HbAic of 6.5%, meaning that they were early stage type 2 diabetics.
• Those chosen for active treatment also had a body weight between 71.3 kg and 101.4 kg, with a median of 96.9 kg and a mean of 92.4 kg.
• Blood samples for the determination of blood glucose concentrations, insulin, pro-insulin, C-peptide and 4-CNAB were drawn at regular intervals over the first 24 hours beginning before the first dose of study dose, i.e., immediately before breakfast is served, and throughout lunch and dinner, at bedtime, and the following morning.
• a fasting blood sample was drawn for the evaluation of fructosamine, and an oral glucose tolerance test was performed as discussed above with ten blood samples being drawn over a 4 hour period for fasting glucose, C-peptide, insulin and pro-insulin.
• Plasma concenfrations of insulin were determined from approximately 0.5 ml of plasma at Huntingdon Life Sciences by means of a GLP validated RIA assay. Blood glucose concenfrations were measured immediately after sample collection using a laboratory method (Super GL Ambulance glucose analyzer, Ruhrtal Labortechnik, Delecke-M ⁇ hnesee, Germany) based on the glucooxidase-reaction.
• Plasma C-peptide was measured from 0.5 ml serum, obtained from a 3 ml blood sample, which was kept at -70 °C. An evaluated RIA- Assay with double determinations was used for the measurements. Urinary C-peptide was measured by liquid chromatography-tandem mass specfrometry with a stable isotopically-labeled internal standard. Plasma proinsulin was measured from 1 ml serum obtained from the same sample as C-peptide, and an evaluated RIA-Assay with double determinations was used for the measurements. [00425] Serum fructosamine was measured from 0.5 mL serum obtained from a 1 mL blood sample collection, which was kept at - 70 °C until assayed. Plasma 4-CNAB was measured from 0.5 mL plasma obtained from a 1 mL blood sample collected into a sodium heparin tube and stored frozen at - 70 °C until shipment to Huntingdon Life Science Laboratories for analysis by a validated
• AUC Areas under the curve
• AUCs were calculated for insulin (AUCms), proinsulin (AUC P R O ), and C-peptide (AUC CP ) but only for the first two hours, six hours and the total duration ofthe experiments (AUCo- ⁇ 2 o, AUCo -3 6o > and AUCo- ⁇ 44 o) (see Figures 52A and 52B for insulin AUC). All AUCs were calculated as incremental AUCs, i.e., area under the curve ofthe absolute values minus the baseline values, with the frapezoidal rule. In case of many negative incremental AUCs (which occur when baseline values are higher than subsequent values), absolute AUCs were calculated additionally. All AUCs were compared between the freatment arms.
• BG/AUC(Ins) area under the blood glucose (BG) or insulin (INS) concentration curve.
• BG/h s(0) blood glucose (BG) or insulin (INS) concenfration at time point 0, i.e., immediately before ingestion ofthe oral glucose load.
• BG blood glucose
• INS insulin
• BG/Ins(OGTT) mean blood glucose (BG) or insulin (INS) concenfrations during the oral glucose tolerance test.
• ISC insulin secretion capacity
• ISC Insulinogenic
• the HOMA and the FIRI indices will be calculated not only from the OGTT experiments, but also from the fasting samples drawn at the ambulatory and the follow-up visits.
• the primary efficacy parameters assess the effect of a two week freatment with an oral insulin/4-CNAB tablet formulation on insulin secretion capacity, insulin sensitivity, and glucose tolerance in diet-treated patients with type 2 diabetes.
• the Stumvoll indices of insulin secretion and first phase insulin secretion were determined.
• the secondary efficacy parameters assess the effect of a two week treatment with an oral insulin formulation on glycemic control in diet-treated patients with type 2 diabetes. Glycemic control was assessed by the measurement of fructosamine and 24-hour blood glucose profiles. To assess glycemic confrol, the absolute concentrations at the scheduled time-points as well as the maximal concentrations, time of maximal concenfrations, and the area under the curves for various time-intervals were determined. All other parameters of insulin sensitivity and insulin secretion, and the AUCs and maximal concenfrations of insulin, C-peptide and proinsulin are regarded as secondary outcome parameters.
• the safety parameters to be assessed are: physical examination, electrocardiograms, vital signs, clinical labs (chemistry, hematology, urinalysis), and continuous glucose monitoring.
• LUU437J As shown in Figure 47, patients receiving oral insulin tablets for two weeks showed clear improvements versus baseline levels on reduced fasting blood glucose by lowered levels of glucose excursion after an oral glucose tolerance test. As shown in Figure 48A, there were clinically relevant decreases in fasting blood glucose concentrations versus baseline levels after an oral glucose tolerance test at Day 15 (mean 19%). Thus, after two weeks of freatment, the patients achieved improved glycemic control compared with baseline levels prior to treatment.
• FIG. 49A which shows blood glucose AUC of Figure 48 A after an oral glucose tolerance test
• patients receiving oral insulin tablets for two weeks showed significantly lower exposure to glucose versus baseline levels based upon reduced average blood glucose concentration, as evidenced by a decrease in the AUC (mean 21%) following an oral glucose tolerance test at Day 15.
• patients receiving oral insulin tablets for two weeks showed decreased two-hour, post-load blood glucose concentration versus baseline levels (mean 16%) following an oral glucose tolerance test at Day 15.
• the patients achieved improved glucose tolerance and a better capacity to handle a sugar load compared with baseline levels prior to freatment.
• a patient's two-hour post-load glucose is a standard clinical marker for assessing a patient's diabetic disease state
• lowering of this marker is perhaps an indication of a reversal ofthe patients' diabetic disease states.
• this study showed improved sensitivity ofthe patients' livers to insulin or the improved ability ofthe patients' pancreas to produce insulin and to thereby confrol overnight glucose concenfrations, as seen from the significant decrease in fasting and two-hour, post-load blood glucose concenfration versus baseline levels.
• the improved insulin secretion capacity and insulin sensitivity based on at least two widely used indices (the Stumvoll first-phase insulin secretion capacity index and the Homeostasis Model Assessment, or HOMA, indices).
• the Stumvoll first-phase insulin secretion capacity index As a benchmark for insulin sensitivity, it was determined that patients receiving oral insulin tablets for two weeks showed a significant increase in insulin sensitivity from baseline (mean 0.010) to day 14 (mean 0.018) ofthe freatment.
• the Strumvoll first phase index as a benchmark for insulin secretion capacity, it was again determined that patients receiving oral insulin tablets for two weeks showed a significant increase in insulin secretion capacity from baseline (mean -37594) to day 14 (mean -30264).
• the HOMA index as a benchmark for insulin secretion capacity however, showed a slight decrease in insulin secretion capacity from baseline (mean 2.328) to day 14 (mean 2.134).
• Figure 57 shows a comparison post-prandial glucose excursion from day 1 ofthe freatment to day 14 ofthe freatment, in order to demonstrate the continuing and cumulative impact ofthe two-week treatment on post-prandial glucose excursion, i.e., that the lowered post-prandial glucose excursion on day 14 was not due to that day's dosage but rather was even lower than the post-prandial glucose excursion of day 1 , evidencing a cumulative effect ofthe two-week freatment on lowering post-prandial glucose excursions.
• post-prandial insulin abso ⁇ tion did not reach higher levels than baseline levels, indicating that no hyperinsulinemia resulted from the two-week freatment.
• post-prandial insulin abso ⁇ tion was lower, as evidenced by the decline in post-prandial (breakfast) insulin C ma ⁇ from a baseline level of 129.1 ⁇ U/mL to 123.7 ⁇ U/mL after one day of freatment and a further decline to 105.4 ⁇ U/mL after two weeks of freatment.
• the two weeks of oral insulin resulted in improved post-prandial glycemic confrol, evidenced by significantly lower C max values on Day 14 as compared to baseline values (an 11% decrease in mean maximum blood glucose), as well as significantly lower glucose AUC values on Day 14 as compared to baseline (12% decrease in mean glucose AUC).
• the two weeks of oral insulin resulted in no hyperinsulinemia, as evidenced by no significant differences in insulin C raa ⁇ and no significant differences in insulin AUC (there was, in fact, slightly lower mean AUC values from Day 0 to Day 14).
• none ofthe patients receiving oral insulin tablets for two weeks had episodes of hypoglycemia, and no subject required rescue throughout the study.
• the freatment also provided the patients with demonsfrably improved glycemic confrol over the previous period of two weeks, as evidenced by serum fructosamine levels.
• FIG 59 based upon fructosamine assay, patients receiving oral insulin tablets for two weeks (mean baseline HbAic of 6.5%) showed a mean 8.8% decrease in fructosamine levels versus baseline levels, meaning that the patients had a better average glycemic confrol over approximately the two week study period.
• confrol group also experienced some improvements in certain key parameters. For example, patients receiving tablets containing only delivery agent showed somewhat lowered levels of glucose excursion, decreased fasting blood glucose concenfrations versus baseline levels (mean 13%), lower exposure to glucose to glucose (AUC) versus baseline levels (mean 11%) and decreased two-hour post-load glucose concenfrations versus baseline levels (mean 5%) following a glucose tolerance test at Day 15.
• the oral insulin tablets proved to be safe and tolerable to the patients receiving them, based upon no hypoglycemic events even in patients with tight glycemic confrol (HbAlc -6.5%), as well as no serious adverse events and a low incidence of mild to moderate adverse events (of which two were deemed potentially related to the study drug).
• the invention may be characterized by any aspect of any ofthe in- vivo clinical (human) data set forth herein, as well as any combination thereof.
• the invention is deemed to encompass (i) patentable aspects ofthe efficacy values, onset and duration obtained for the tested formulations with respect to direct measurements of insulin levels, glucose levels and C-peptide levels (including but not limited to t max , C m a ⁇ 5 shape ofthe plasma concenfration curve (e.g., plasma insulin levels); (ii) any combination ofthe various direct measurements ofthe freatment efficacy characteristics set forth in the above specification and/or as demonstrated by the appended examples; (iii) any combination of any ofthe above-mentioned characteristics ofthe invention together with aspects ofthe contemplated formulations themselves, including but not limited to the method of manufacture ofthe formulation, the drug load, the delivery agent load, the drug and form ofthe drug used (e.g., unmodified insulin), the delivery agent used, the ratio of

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