Improved methods and kits for treating the long-term complication of diabetes that reduce the risk of the patient developing hypoglycemia during C-peptide therapy. The use of such methods and kits, can also maintain good glycemic control, and avoid excessive weight gain that may otherwise be associated with excessive insulin administration or caloric intake during C-peptide therapy.

Description of the Invention

Overview of Methods and Kits for Reducing the Risk of Hypoglycemia

The present invention relates to the development of improved methods and kits for treating the long-term complication of diabetes that reduce the risk of the patient developing hypoglycemia during or when starting C-peptide therapy. Significantly, such improved dosing regimens also maintain good glycemic control, and avoid excessive weight gain that may otherwise be associated with excessive insulin administration or caloric intake during C-peptide therapy. In one aspect, these methods may be applied to patients with neuropathy who exhibit altered sensitivity to hypoglycemia and reduced insulin usage during C-peptide therapy.

Thus in one aspect, the present invention includes a method for treating an insulin-dependent human patient, comprising the steps of; a) administering insulin to said patient, wherein said patient has neuropathy; b) administering subcutaneously to said patient a therapeutic dose of C-peptide in a different site as that used for said patient's insulin administration; c) adjusting the dosage amount, type, or frequency of insulin administered based on monitoring said patient's altered insulin requirements resulting from said therapeutic dose of C-peptide, wherein said adjusted dose of insulin reduces the risk, incidence, or severity of hypoglycemia, wherein said adjusted dose of insulin is at least 10% less than said patient's insulin dose prior to starting C-peptide treatment.

In one aspect of this method, the hypoglycemia is severe hypoglycemia. In another aspect of this method, the hypoglycemia is asymptomatic hypoglycemia.

In another aspect, the present invention includes a method of reducing insulin usage in an insulin-dependent human patient, comprising the steps of; a) administering insulin to said patient, wherein said patient has neuropathy; b) administering subcutaneously to said patient a therapeutic dose of C-peptide in a different site as that used for said patient's insulin administration; c) adjusting the dosage amount, type, or frequency of insulin administered based on monitoring said patient's altered insulin requirements resulting from said therapeutic dose of C-peptide, wherein said adjusted dose of insulin does not induce hyperglycemia, wherein said adjusted dose of insulin is at least 10% less than said patient's insulin dose prior to starting C-peptide treatment.

In another aspect, the present invention comprises a method for treating an insulin-dependent patient without substantially increasing the risk, incidence, or severity of hypoglycemia in comprising the steps of; a) administering insulin to the patient; b) administering subcutaneously to the patient a therapeutic dose of C-peptide in a different site as that used for the patient's insulin administration; and c) adjusting the dosage amount, type, or frequency of insulin administered with the therapeutic dose of C-peptide based on the patient's altered insulin requirements.

In another embodiment, the present invention includes a method of reducing insulin usage in an insulin-dependent patient without inducing hyperglycemia comprising the steps of; a) administering insulin to the patient; b) administering subcutaneously to the patient a therapeutic dose of C-peptide in a different site as that used for the patient's insulin administration; and c) adjusting the dosage amount, type, or frequency of insulin administered with the therapeutic dose of C-peptide based on said patient's altered insulin requirements.

In another embodiment, the present invention includes a method for reducing weight gain in an insulin-dependent patient comprising the steps of; a) administering insulin to the patient; b) administering subcutaneously to the patient a therapeutic dose of C-peptide in a different site as that used for the patient's insulin administration; and c) adjusting the dosage amount, type, or frequency of insulin administered with the therapeutic dose of C-peptide based on the patient's altered insulin requirements.

In a further embodiment, the invention includes a method for identifying patients that respond to C-peptide by a change in insulin requirements by monitoring the patient to determine the patient's basal C-peptide levels prior to starting C-peptide therapy.

In an additional embodiment, the invention includes a method for identifying patients that respond to C-peptide by a change in insulin requirements by monitoring the patient to determine the patient's change in nerve conduction velocity during C-peptide treatment.

In an additional further embodiment, the invention includes a method for identifying patients that respond to C-peptide by a change in insulin requirements by monitoring the patient to determine the patient's change in autonomic nerve function during C-peptide treatment.

In one further embodiment, the present invention includes a method for the treatment of a patient with a long-term complication of diabetes by C-peptide which minimizes the risk of the patient developing hypoglycemia, comprising the steps of; a) monitoring one or more clinical parameters, biomarkers, or analytes from the patient and/or monitoring the patient's insulin usage over a baseline period prior to starting C-peptide therapy; b) administering a therapeutically effective dose of C-peptide for an evaluation period; c) re-monitoring one or more of the clinical parameters, biomarkers, or analytes and/or re-monitoring the patient's insulin usage over the evaluation period to determine a new reference range of parameters; and d) reducing the dose, frequency, or type of insulin administration based on a comparison of the baseline reference range and new reference range of the parameters.

In another embodiment, the present invention includes a method for the treatment for a long-term complication of type 1 diabetes via treatment with C-peptide, which reduces the risk of the patient developing hypoglycemia during the therapy, comprising the steps of; a) monitoring one or more clinical parameters, biomarkers, or analytes and/or monitoring the patient's insulin usage over an evaluation period of C-peptide therapy to determine a reference range of insulin sensitivity for the patient when treated with C-peptide; and b) reducing the dose, frequency, or type of insulin administration based on a comparison of the patient's clinical parameters, biomarkers, or analytes obtained during the evaluation period compared to an index reference range of parameters for that patient.

In a further embodiment, the present invention includes a method for the treatment of a long-term complication of type 1 diabetes via treatment with C-peptide which reduces the risk of the patient developing hypoglycemia during the therapy, of comprising the steps of; a) monitoring at least the incidence of hypoglycemic events experienced by the patient during an evaluation period of C-peptide therapy; and b) reducing the average daily requirement of insulin administered to the patient calculated prior to starting C-peptide therapy by about 5% to about 50% if the incidence of hypoglycemic events observed during the evaluation period of C-peptide therapy is increased compared to the incidence of hypoglycemic events observed prior to starting C-peptide therapy.

In another embodiment, the present invention includes a method for reducing the risk of a patient with insulin-dependent diabetes developing hypoglycemia when treated with C-peptide comprising the steps of; a) assessing the patient's C-peptide levels prior to starting C-peptide therapy; and b) reducing the average daily dose of insulin administered to the patient by about 10% to about 35%.

In another embodiment, the present invention includes a method for reducing weight gain in a patient with insulin-dependent diabetes when treated with C-peptide comprising the steps of; a) assessing the patient's C-peptide levels prior to starting C-peptide therapy; and b) reducing the average daily dose of insulin administered to the patient by about 10% to about 35%.

In another embodiment, the present invention includes a method for the treatment of a patient with insulin-dependent diabetes who is starting a therapy of a long-term complication of type 1 diabetes via treatment with C-peptide which reduces the risk of the patient developing hypoglycemia, comprising the steps of; a) assessing the patient's average daily requirement of insulin including number of international units of short-, intermediate-, and long-acting insulin that the patient administers prior to starting C-peptide therapy; b) administering subcutaneously to the patient a therapeutically effective dose of C-peptide for a lead-in period; and c) independently reducing the average daily requirement of either short-, intermediate-, or long-acting insulin administered to the patient by about 5% to about 50% compared to the amount administered prior to starting C-peptide therapy.

Insulin-Dependent Diabetes

In any of the methods and kits disclosed herein, the terms "insulin-dependent patient" or "insulin-dependent diabetes" encompasses all forms of diabetics/diabetes who/that require insulin administration to adequately maintain normal glucose levels.

In broad terms, the term "diabetes" refers to the situation where the body either fails to properly respond to its own insulin, does not make enough insulin, or both. The primary result of impaired insulin production is the accumulation of glucose in the blood, and a C-peptide deficiency leading to various short- and long-term complications. Three principal forms of diabetes exist:

Type 1: Results from the body's failure to produce insulin and C-peptide. It is estimated that 5-10% of Americans who are diagnosed with diabetes have type 1 diabetes. Presently almost all persons with type 1 diabetes must take insulin injections. The term "type 1 diabetes" has replaced several former terms, including childhood-onset diabetes, juvenile diabetes, and insulin-dependent diabetes mellitus (IDDM). For patients with type 1 diabetes, basal levels of C-peptide are typically less than about 0.20 nM (Ludvigsson et al.: New Engl. J. Med. 359: 1909-1920, (2008)).

Type 2: Results from tissue insulin resistance, a condition in which cells fail to respond properly to insulin, sometimes combined with relative insulin deficiency. The term "type 2 diabetes" has replaced several former terms, including adult-onset diabetes, obesity-related diabetes, and non-insulin-dependent diabetes mellitus (NIDDM). For type 2 patients in the basal state, C-peptide levels of about 0.8 nM (range 0.64 to 1.56 nM), and glucose stimulated levels of about 5.7 nM (range 3.7 to 7.7 nM) have been reported. (Retnakaran R et al.: Diabetes Obes. Metab. (2009) DOI 10.11 111/j.1463-1326.2009.01129.x; Zander et al.: Lancet 359: 824-830, (2002)).

Gestational diabetes: Pregnant women who have never had diabetes before but who have high blood sugar (glucose) levels during pregnancy are said to have gestational diabetes. Gestational diabetes affects about 4% of all pregnant women. It may precede development of type 2 (or rarely type 1).

Several other forms of diabetes mellitus are categorized separately from these. Examples include congenital diabetes due to genetic defects of insulin secretion, cystic fibrosis-related diabetes, steroid diabetes induced by high doses of glucocorticoids, and several forms of monogenic diabetes.

Acute complications of diabetes include hypoglycemia, diabetic ketoacidosis, or nonketotic hyperosmolar coma that may occur if the disease is not adequately controlled. Serious long-term complications can also occur, and are discussed in more detail below.

Long-Term Complications of Diabetes

In any of these methods and kits, the terms "long-term complication of type 1 diabetes", or "long-term complications of diabetes" refers to the long-term complications of impaired glycemic control, and C-peptide deficiency associated with insulin-dependent diabetes. Accordingly in any of these methods and kits, the patient may be being treated for a long-term complication of diabetes. Typically long-term complications of type 1 diabetes are associated with type 1 diabetics. However the term can also refer to long-term complications of diabetes that arise in type 1.5 and type 2 diabetic patients who develop a C-peptide deficiency as a consequence of losing pancreatic islet .beta.-cells and therefore also become insulin dependent. In broad terms, many such complications arise from the primary damage of blood vessels (angiopathy), resulting in subsequent problems that can be grouped under "microvascular disease" (due to damage to small blood vessels) and "macrovascular disease" (due to damage to the arteries).

Specific diseases and disorders included within the term long-term complications of diabetes include, without limitation; retinopathy including early stage retinopathy with microaneurysms, proliferative retinopathy, and macular edema; peripheral neuropathy including sensorimotor polyneuropathy, painful sensory neuropathy, acute motor neuropathy, cranial focal and multifocal polyneuropathies, thoracolumbar radiculoneuropathies, proximal diabetic neuropathies, and focal limb neuropathies including entrapment and compression neuropathies; autonomic neuropathy involving the cardiovascular system, the gastrointestinal tract, the respiratory system, the urigenital system, sudomotor function, and papillary function; and nephropathy including disorders with microalbuminuria, overt proteinuria, and end-stage renal disease.

Impaired microcirculatory perfusion appears to be crucial to the pathogenesis of both neuropathy and retinopathy in diabetics. This in turn reflects a hyperglycemia-mediated perturbation of vascular endothelial function that results in: over-activation of protein kinase C, reduced availability of nitric oxide (NO), increased production of superoxide and endothelin-1 (ET-1), impaired insulin function, diminished synthesis of prostacyclin/PGE1, and increased activation and endothelial adherence of leukocytes. This is ultimately a catastrophic group of clinical events.

Diabetic retinopathy is an ocular manifestation of the systemic damage to small blood vessels leading to microangiopathy. In retinopathy, growth of friable and poor-quality new blood vessels in the retina as well as macular edema (swelling of the macula) can lead to severe vision loss or blindness. As new blood vessels form at the back of the eye as a part of proliferative diabetic retinopathy (PDR), they can bleed (hemorrhage) and blur vision. It affects up to 80% of all patients who have had diabetes for 10 years or more.

The symptoms of diabetic retinopathy are often slow to develop and subtle and include blurred version and progressive loss of sight. Macular edema, which may cause vision loss more rapidly, may not have any warning signs for some time. In general, however, a person with macular edema is likely to have blurred vision, making it hard to do things like read or drive. In some cases, the vision will get better or worse during the day.

Accordingly in any of these methods and kits, a patient who is in need of treatment for a long-term complication of diabetes can include a patient with one of more of the symptoms of diabetic retinopathy.

Diabetic neuropathies are neuropathic disorders that are associated with diabetic microvascular injury involving small blood vessels that supply nerves (vasa nervorum). Relatively common conditions which may be associated with diabetic neuropathy include third nerve palsy; mononeuropathy; mononeuropathy multiplex; diabetic amyotrophy; a painful polyneuropathy; peripheral neuropathy; autonomic neuropathy; and thoracoabdominal neuropathy.

Diabetic neuropathy affects all peripheral nerves: pain fibers, motor neurons, autonomic nerves. It therefore necessarily can affect all organs and systems since all are innervated. There are several distinct syndromes based on the organ systems and members affected, but these are by no means exclusive. A patient can have sensorimotor and autonomic neuropathy or any other combination. Symptoms vary depending on the nerve(s) affected and may include symptoms other than those listed. Symptoms usually develop gradually over years.

Symptoms of diabetic neuropathy may include: numbness and tingling of extremities, dysesthesia (decreased or loss of sensation to a body part), diarrhea, erectile dysfunction, urinary incontinence (loss of bladder control), impotence, facial, mouth and eyelid drooping, vision changes, dizziness, muscle weakness, difficulty swallowing, speech impairment, fasciculation (muscle contractions), anorgasmia, and burning or electric pain.

Additionally, different nerves are affected in different ways by neuropathy. Sensorimotor polyneuropathy, in which longer nerve fibers are affected to a greater degree than shorter ones, because nerve conduction velocity is slowed in proportion to a nerve's length. In this syndrome, decreased sensation and loss of reflexes occurs first in the toes on each foot, then extends upward. It is usually described as glove-stocking distribution of numbness, sensory loss, dysesthesia, and nighttime pain. The pain can feel like burning, pricking sensation, achy, or dull. Pins and needles sensation is common. Loss of proprioception, the sense of where a limb is in space, is affected early.

These patients cannot feel when they are stepping on a foreign body, like a splinter, or when they are developing a callous from an ill-fitting shoe. Consequently, they are at risk for developing ulcers and infections on the feet and legs, which can lead to amputation. Similarly, these patients can get multiple fractures of the knee, ankle, or foot, and develop a Charcot joint. Loss of motor function results in dorsiflexion, contractures of the toes, loss of the interosseous muscle function, and leads to contraction of the digits, so called hammer toes. These contractures occur not only in the foot, but also in the hand where the loss of the musculature makes the hand appear gaunt and skeletal. The loss of muscular function is progressive.

Autonomic neuropathy impacts the autonomic nervous system serving the heart, gastrointestinal system, and genitourinary system. The most commonly recognized autonomic dysfunction in diabetics is orthostatic hypotension, or fainting when standing up. In the case of diabetic autonomic neuropathy, it is due to the failure of the heart and arteries to appropriately adjust heart rate and vascular tone to keep blood continually and fully flowing to the brain. This symptom is usually accompanied by a loss of the usual change in heart rate seen with normal breathing. These two findings suggest autonomic neuropathy.

Gastrointestinal system symptoms include delayed gastric emptying, gastroparesis, nausea, bloating, and diarrhea. Because many diabetics take oral medication for their diabetes, absorption of these medicines is greatly affected by the delayed gastric emptying. This can lead to hypoglycemia when an oral diabetic agent is taken before a meal and does not get absorbed until hours, or sometimes days later, when there is normal or low blood sugar already. Sluggish movement of the small intestine can cause bacterial overgrowth, made worse by the presence of hyperglycemia. This leads to bloating, gas, and diarrhea.

Genitourinary system symptoms include urinary frequency, urgency, incontinence, and retention. Urinary retention can lead to bladder diverticula, stones, reflux nephropathy, and frequent urinary tract infections.

Cranial neuropathy occurs when cranial nerves are affected, and oculomotor (3rd) neuropathies are most commonly observed. The oculomotor nerve controls all of the muscles that move the eye with the exception of the lateral rectus and superior oblique muscles. It also serves to constrict the pupil and open the eyelid. The onset of a diabetic third nerve palsy is usually abrupt, beginning with frontal or periorbital pain and then diplopia. All of the oculomotor muscles innervated by the third nerve may be affected, except for those that control pupil size. This is because pupillary function within the oculomotor nerve (CNIII) is found on the periphery of the nerve (in terms of a cross sectional view), which makes it less susceptible to ischemic damage (as it is closer to the vascular supply).

The sixth nerve, the abducens nerve, which innervates the lateral rectus muscle of the eye (moves the eye laterally), is also commonly affected but the fourth nerve, the trochlear nerve (innervates the superior oblique muscle, which moves the eye downward), involvement is unusual. Mononeuropathies of the thoracic or lumbar spinal nerves can occur and lead to painful syndromes that mimic myocardial infarction, cholecystitis, or appendicitis. Diabetics have a higher incidence of entrapment neuropathies.

Accordingly in any of these methods and kits, the insulin-dependent patient in need of treatment for a long-term complication of diabetes includes a patient with one of more of the symptoms of diabetic neuropathy. In another aspect of the claimed methods, the patient has neuropathy, and in one aspect the patient has incipient neuropathy, and in one aspect the patient has established neuropathy.

Diabetic nephropathy is a progressive kidney disease caused by angiopathy of capillaries in the kidney glomeruli. It is characterized by nephrotic syndrome and diffuse glomerulosclerosis. It is due to long-standing diabetes mellitus, and is a prime cause for dialysis in many Western countries.

The symptoms of diabetic nephropathy can be seen in patients with chronic diabetes (15 years or more after onset). The disease is progressive and is more frequent in men. Diabetic nephropathy is the most common cause of chronic kidney failure and end-stage kidney disease in the United States. People with both type 1 and type 2 diabetes are at risk. The risk is higher if blood-glucose levels are poorly controlled. Further, once nephropathy develops, the greatest rate of progression is seen in patients with poor control of their blood pressure. Also people with high cholesterol level in their blood have much more risk than others.

The earliest detectable change in the course of diabetic nephropathy is an abnormality of the glomerular filtration barrier. At this stage, the kidney may start allowing more serum albumin than normal in the urine (albuminuria), and this can be detected by sensitive medical tests for albumin. This stage is called "microalbuminuria". As diabetic nephropathy progresses, increasing numbers of glomeruli are destroyed by nodular glomerulosclerosis. Now the amounts of albumin being excreted in the urine increases, and may be detected by ordinary urinalysis techniques. At this stage, a kidney biopsy clearly shows diabetic nephropathy.

Kidney failure provoked by glomerulosclerosis leads to fluid filtration deficits and other disorders of kidney function. There is an increase in blood pressure (hypertension) and fluid retention in the body plus a reduced plasma oncotic pressure causes edema. Other complications may be arteriosclerosis of the renal artery and proteinuria.

Throughout its early course, diabetic nephropathy has no symptoms. They develop in late stages and may be a result of excretion of high amounts of protein in the urine or due to renal failure. Symptoms include, edema: swelling, usually around the eyes in the mornings; later, general body swelling may result, such as swelling of the legs, foamy appearance or excessive frothing of the urine (caused by the proteinura), unintentional weight gain (from fluid accumulation), anorexia (poor appetite), nausea and vomiting, malaise (general ill feeling), fatigue, headache, frequent hiccups, and generalized itching.

Accordingly in any of these methods and kits, a patient who is in need of treatment for a long-term complication of diabetes can include a patient with one of more of the symptoms of diabetic nephropathy.

Diabetic cardiomyopathy (DCM), damage to the heart, leading to diastolic dysfunction and eventually heart failure. Aside from large vessel disease and accelerated atherosclerosis, which is very common in diabetes, DCM is a clinical condition diagnosed when ventricular dysfunction develops in patients with diabetes in the absence of coronary atherosclerosis and hypertension. DCM may be characterized functionally by ventricular dilation, myocyte hypertrophy, prominent interstitial fibrosis, and decreased or preserved systolic function in the presence of a diastolic dysfunction.

One particularity of DCM is the long latent phase, during which the disease progresses but is completely asymptomatic. In most cases, DCM is detected with concomitant hypertension or coronary artery disease. One of the earliest signs is mild left ventricular diastolic dysfunction with little effect on ventricular filling. Also, the diabetic patient may show subtle signs of DCM related to decreased left ventricular compliance or left ventricular hypertrophy or a combination of both. A prominent "a" wave can also be noted in the jugular venous pulse, and the cardiac apical impulse may be overactive or sustained throughout systole. After the development of systolic dysfunction, left ventricular dilation and symptomatic heart failure, the jugular venous pressure may become elevated and the apical impulse would be displaced downward and to the left. Systolic mitral murmur is not uncommon in these cases.

These changes are accompanied by a variety of electrocardiographic changes that may be associated with DCM in 60% of patients without structural heart disease, although usually not in the early asymptomatic phase. Later in the progression, a prolonged QT interval may be indicative of fibrosis. Given that DCM's definition excludes concomitant atherosclerosis or hypertension, there are no changes in perfusion or in atrial natriuretic peptide levels up until the very late stages of the disease, when the hypertrophy and fibrosis become very pronounced.

Macrovascular diseases of diabetes include coronary artery disease, leading to angina or myocardial infarction ("heart attack"), stroke (mainly the ischemic type), peripheral vascular disease, which contributes to intermittent claudication (exertion-related leg and foot pain), as well as diabetic foot and diabetic myonecrosis ("muscle wasting").

Hypoglycemia and Hypoglycemic Events

In any of these methods and kits, the term "hypoglycemia" or "hypoglycemic events" refers to all episodes of abnormally low plasma glucose concentration that exposes the patient to potential harm. The American Diabetes Association Workgroup has recommended that people with insulin-dependent diabetes become concerned about the possibility of developing hypoglycemia at a plasma glucose concentration of less than 70 mg/dL (3.9 mmoL/L). Accordingly in one aspect of any of the claimed methods and kits, the terms hypoglycemia or hypoglycemic event refers to the situation where the plasma glucose concentration of the patient drops to less than about 70 mg/dL (3.9 mmoL/L).

Hypoglycemia is a serious medical complication in the treatment of diabetes, and causes recurrent morbidity in most people with type 1 diabetes and many with advanced type 2 diabetes and is sometimes fatal. In addition, hypoglycemia compromises physiological and behavioral defenses against subsequent falling plasma glucose concentrations and thus causes a vicious cycle of recurrent hypoglycemia. Accordingly the prevention of hypoglycemia is of significant importance in the treatment of diabetes, as well as the treatment of the long-term complications of diabetes.

Unfortunately hypoglycemia is a fact of life for most people with type 1 diabetes (Cryer P E et al.: Diabetes 57: 3169-3176, (2008)). The average patient has untold numbers of episodes of asymptomatic hypoglycemia and suffers two episodes of symptomatic hypoglycemia per week, with thousands of such episodes over a lifetime of diabetes. He or she suffers one or more episodes of severe, temporarily disabling hypoglycemia often with seizure or coma, per year.

Overall, hypoglycemia is less frequent in type 2 diabetes; however, the risk of hypoglycemia becomes progressively more frequent and limiting to glycemic control later in the course of type 2 diabetes. The prospective, population-based data of Donnelly et al. (Diabetes Med. 22: 749-755, (2005)) indicate that the overall incidence of hypoglycemia in insulin-treated type 2 diabetes is approximately one third of that in type 1 diabetes. The incidence of any hypoglycemia and of severe hypoglycemia was 4,300 and 115 episodes per 100 patient years, respectively, in type 1 diabetes and 1600 and 35 episodes per 100 patient years, respectively, in insulin-treated type 2 diabetes.

Hypoglycemia may be classified based on the severity of the hypoglycemic event. For example, the American Diabetes Association Workgroup has suggested the following classification of hypoglycemia in diabetes: 1) severe hypoglycemia (i.e., hypoglycemic coma requiring assistance of another person); 2) documented symptomatic hypoglycemia (with symptoms and a plasma glucose concentration of less than 70 mg/dL); 3) asymptomatic hypoglycemia (with a plasma glucose concentration of less than 70 mg/dL without symptoms); 4) probable symptomatic hypoglycemia (with symptoms attributed to hypoglycemia, but without a plasma glucose measurement); and 5) relative hypoglycemia (with a plasma glucose concentration of greater than 70 mg/dL but falling towards that level).

Thus in another aspect of any of the methods and kits disclosed herein, the term "hypoglycemia" refers to severe hypoglycemia, and/or hypoglycemic coma. In another aspect of any of these methods and kits, the term "hypoglycemia" refers to symptomatic hypoglycemia. In another aspect of any of these methods and kits, the term "hypoglycemia" refers to probable symptomatic hypoglycemia. In another aspect of any of these methods and kits, the term "hypoglycemia" refers to asymptomatic hypoglycemia. In another aspect of any of these methods and kits, the term "hypoglycemia" refers to relative hypoglycemia.

Although hypoglycemic events are typically associated with symptoms, the signs of hypoglycemia are not specific. Thus, clinical hypoglycemia is most convincingly documented by a combination of: 1) symptoms, signs, or both consistent with hypoglycemia, 2) a low measured plasma glucose concentration, and 3) resolution of these symptoms and signs after the plasma glucose concentration is raised.

Symptoms of hypoglycemia are categorized as neuroglycopenic (those that are the direct result of brain glucose deprivation per se) and neurogenic (or autonomic), and those that are largely the result of the perception of physiological changes caused by the sympathoadrenal (largely the sympathetic neural discharge triggered by hypoglycemia). Neuroglycopenic manifestations include cognitive impairments, behavioral changes, and psychomotor abnormalities, and, at lower plasma glucose concentrations, seizure and coma.

Adrenergic neurogenic symptoms include palpitations, tremor, and anxiety/arousal. Cholingeric neurogenic symptoms include sweating, hunger, and paresthesias. Central, as well as peripheral mechanisms may be involved in the generation of some symptoms such as hunger. Awareness of hypoglycemia is largely the result of the perception of the neurogenic symptoms. Pallor and diaphoresis (the result of adrenergic cutaneous vasoconstriction and cholinergic stimulation of sweat glands, respectively) are common signs of hypoglycemia.

Accordingly in one aspect of any of the methods and kits disclosed herein, hypoglycemic events may be determined by monitoring one or more of the symptoms of hypoglycemia. In one aspect, or any of these methods and kits, the symptoms of hypoglycemia can be selected from palpitations, tremor, anxiety/arousal, sweating, hunger, and paresthesias. In a preferred aspect, hypoglycemia may be determined by measuring or monitoring the patient's blood glucose levels.

In another aspect of any of the claimed methods and kits, a patient undergoing or initiating C-peptide therapy may be evaluated for one or more risk factors for developing hypoglycemia.

Conventional risk factors for hypoglycemia in diabetes include:

Insulin or insulin secretagogue doses are excessive, ill-timed, or of a wrong type.

Exogenous glucose delivery is decreased (e.g., following missed meals and during the overnight fast).

Glucose utilization is increased (e.g., during and shortly after exercise).

Endogenous glucose production is decreased (e.g., following alcohol ingestion).

Sensitivity to insulin is increased (e.g., in the middle of the night and following weight loss, improved fitness, or improved glycemic control).

Insulin clearance is decreased (e.g., with renal failure).

Additionally, recent antecedent hypoglycemia, as well as prior exercise or sleep, causes both defective glucose counter regulation and hypoglycemia unawareness and therefore, a vicious cycle of recurrent iatrogenic hypoglycemia referred to as hypoglycemia-associated autonomic failure (HAAF).

Accordingly in one aspect of any of the claimed methods and kits, one or more risk factors for hypoglycemia may be monitored. In one aspect of these methods and kits, the risk factors for hypoglycemia may be monitored prior to initiating C-peptide therapy. In another aspect, the risk factors may be monitored during an evaluation period of C-peptide therapy.

The risk factors indicative of HAAF include the degree of endogenous insulin deficiency; a history of severe hypoglycemia, hypoglycemia unawareness, or both as well as recent antecedent hypoglycemia, prior exercise, or sleep, and lower mean glycemic associated with aggressive glycemic therapy per se (lower HbA1c levels, lower glycemic goals).

Accordingly in another aspect of any of the methods and kits disclosed herein the risk of hypoglycemia events may be assessed by monitoring one or more of the risk factors of hypoglycemia or HAAF.

Methods for Monitoring Patient Parameters and Determination of Risk Factors

An assessment of one or more biomarkers, analytes, and clinical parameters in any of the claimed methods and kits of the invention allows one of skill in the art to identify and assess those patients who respond to C-peptide therapy with an increase in insulin sensitivity, and who are therefore at risk for developing hypoglycemia, or for excessive weight gain.

Effective regulation of glucose levels and the prevention of hypoglycemia however are ultimately dependent on modifying human behavior. Consequently, people with diabetes face a life-long behaviorally controlled optimization problem to reduce hyperglycemic excursions and maintain strict glycemic control, without increasing their risk for hypoglycemia.

While intensive treatment with insulin to maintain nearly normal levels of glucose markedly reduces the incidence of long-term complications of diabetes, this approach may also increase the risk of severe hypoglycemia. Additionally, the present invention recognizes for the first time that co-treatment with C-peptide may present the patient with an additional risk of developing a hypoglycemia coma caused by a C-peptide-mediated reduction in insulin requirements in a subset of patients. Accordingly, the development of hypoglycemia can be considered a primary barrier to the successful treatment of the long-term complications of diabetes with C-peptide.

The warning symptoms and hormonal defenses against hypoglycemia are typically attenuated in type 1 diabetes, and may be particularly problematic in patients that have suffered from diabetes for several years and who are therefore at increased risk of, or have, one or more long-term complications of diabetes. Accordingly, the present invention provides methods for monitoring and assessing a patient's risk of hypoglycemia by providing one or more periods of evaluation of the patient either prior to, during, or as the patient starts C-peptide therapy.

In one aspect, the present invention is based on the discovery that patients who exhibit a reduced severity of peripheral neuropathy relative to their duration of disease, may respond more effectively to C-peptide therapy, and are therefore at increased risk of developing hypoglycemia. Accordingly in one aspect, disease severity may be monitored by measuring or assessing the patient's height-adjusted sensory or motor nerve conduction velocity. In one aspect of this method, initial nerve conduction velocity is assessed. In another embodiment, peak nerve conduction velocity is assessed. In another aspect of any of the claimed methods and kits, patients are identified to be at risk of developing hypoglycemia based on their baseline nerve conduction velocity, and taking into account their length of disease duration. In one aspect, identification is based on the patient exhibiting a peak nerve conduction velocity that is at least about 2 standard deviations from the mean peak nerve conduction velocity for a similar height-matched patient group comprising patients that have had insulin-dependent diabetes for a comparable time. In one aspect, the patients with a disease duration of about 30 years are selected with a peak nerve conduction velocity of greater than about 35 m/s. In one aspect of any of the claimed methods and kits, the patients are selected with a peak nerve conduction velocity of greater than about 40 m/s. In one aspect, the patients are selected with a peak nerve conduction velocity of greater than about 45 m/s. In one aspect, the patients are selected with a peak nerve conduction velocity of greater than about 50 m/s. In another aspect of any of the claimed methods and kits, the patients are selected based on their relative improvement in nerve conduction velocity.

In one aspect of any of the claimed methods and kits, patients are selected based on exhibiting an improvement in nerve conduction velocity of greater than about 1.5 m/s after starting C-peptide therapy. In another aspect of these methods and kits, patients are selected based on exhibiting an improvement in nerve conduction velocity of greater than about 2 m/s after starting C-peptide.

In embodiment of any of the claimed methods and kits, the patient is monitored over a baseline period prior to initiating C-peptide therapy. In one aspect, monitoring involves measuring, monitoring, or assessing a change in a biomarker, analyte, or clinical parameter in a patient starting C-peptide therapy. In one aspect, such monitoring is conducted by the patient. In another aspect, the results are downloaded to a computer or PC for data analysis at a remote site.

In one aspect of any of the claimed methods and kits, the analyte is selected from blood glucose concentration, glycosylated hemoglobin level, C-peptide, and insulin concentration and/or usage. In one aspect of these methods and kits, an assessment of insulin usage can include an assessment of the type of insulin administered, as well as the type, timing, and mode of administration.

In one aspect of any of these methods and kits, a computer is used to capture long-term trends towards increased risk for hypoglycemia. In one aspect of these methods and kits, a computer program may be used to identify periods of increased risk for hypoglycemia that may be associated with C-peptide therapy. These analyses may be based on specific algorithms that enable the comprehensive evaluation of glycemic control. (See generally, e.g., US Patent Application No. 20080154513 entitled, "Systems, Methods and Computer Program Codes for Recognition of Patterns of Hyperglycemia and Hypoglycemia, Increased Glucose Variability, and Ineffective Self-Monitoring in Diabetes".)

In one aspect of any of the claimed methods and kits, blood glucose concentrations are determined by the patient by self-monitoring of blood glucose (SMBG). Contemporary home blood glucose meters offer convenient means for frequent and accurate blood glucose determinations through self-monitoring of blood glucose. Most meters are capable of storing hundreds of SMBG readings, together with the date and time of each reading, and have interfaces to download these readings into a PC. The meters are usually accompanied by software that has capabilities for basic data analyses (e.g., calculation of mean blood glucose [BG], estimates of the average BG over the previous two weeks, percentages in target, hypoglycemic, and hyperglycemic zones, etc.), log of the data, and graphical representation (e.g., histograms, pie charts).

In another aspect of any of these methods and kits, glycosylated hemoglobin levels are determined. Glycosylated hemoglobin includes three components; namely, HbA1a, HbA1b, and HbA1c. It has been shown that a normal level of HbA1c in a diabetic patient's blood is a good indication that the treatment regime is effective and the risk of secondary complications of diabetes is low. The level of HbA1c in a healthy person's blood is between 4% and 6% of the total hemoglobin while in a diabetic person the level may be significantly higher (e.g., greater than 8%). It is generally sought to reduce the level of HbA1c in a diabetic patient's blood to between 6% and 7%. The HbA1c level reflects the idiosyncratic (i.e., patient-specific) effectiveness of blood glucose treatment over a period of several months preceding the HbA1c measurement. The HbA1c level is commonly measured by laboratory tests in order to provide information related to the long-term effectiveness of diabetes treatment.

In another aspect of the claimed methods and kits, blood glucose concentrations may be measured by a continuous glucose monitor that determines blood glucose levels on a continuous basis (every few minutes). A typical system comprises a disposable glucose sensor placed under the skin, a link from the sensor to a non-implanted transmitter which communicates to a radio receiver, and an electronic receiver worn like a pager that displays blood glucose levels in a continuous manner, as well as monitors rising and falling trends. In an additional aspect, blood glucose levels may be monitored by non-invasive technique not requiring access to blood. Such techniques include infra-red detection, ultrasound measurements, or dielectric spectroscopy. While HbA1c levels provide valuable information, HbA1c levels are measured infrequently for typical patients and give little direct information as to the variability associated with a patient's glycemic control or the propensity for hypoglycemia or hyperglycemia. For example, a patient may have an acceptable HbA1c level ranging between 4% and 7%, but may have frequent hypoglycemic and/or hyperglycemic episodes because such episodes are not reflected in an HbA1c level.

In one aspect of any of these methods and kits, the baseline period of evaluation extends from about one hour to any period of time prior to starting C-peptide therapy. In another aspect of any of these methods and kits, the baseline period may include historical logs of glucose, insulin, nerve conduction velocity, C-peptide levels, or HbA1c data collected over any period of time prior to starting C-peptide therapy. In one embodiment, the baseline period extends from about one day prior to starting C-peptide therapy to about 24 weeks prior to starting therapy. In another aspect, the baseline period extends from about one day to about 12 weeks. In another aspect, the baseline period extends from about one day to about 6 weeks. In another aspect, the baseline period extends from about one day to about 4 weeks. In another aspect, the baseline period extends from about one day to about 2 weeks. In another aspect, the baseline period extends from about one day to about one week.

In one aspect, the present invention is based on the discovery that patients who exhibit a reduced basal or meal-stimulated C-peptide level may respond more effectively to C-peptide therapy, and are therefore at increased risk of developing hypoglycemia. Accordingly in one aspect of the invention, C-peptide levels are monitored prior to initiating C-peptide therapy to assess the relative risk of the patient developing hypoglycemia. In one embodiment of any of the claimed methods and kits, the patient's C-peptide levels are measured and the patients are grouped according to their basal C-peptide level prior to starting C-peptide therapy. In one aspect, patients are grouped into group I (about 0.05 nM or less C-peptide); group II (about 0.05 to about 0.1 nM C-peptide); group III (about 0.1 to about 0.2 nM C-peptide), group IV (about 0.2 to about 0.3 nM C-peptide); group V (about 0.3 to about 0.4 nM C-peptide); group VI (about 0.4 to about 0.5 nM C-peptide). In one aspect of the invention, patients with altered C-peptide levels display an altered risk of developing hypoglycemia. In one aspect of the invention, an altered risk of developing hypoglycemia when initiating C-peptide therapy is found in patients in groups I to III compared to groups IV to VI. Accordingly in one aspect of any of the methods and kits described herein, the invention includes a method for assessing a patient's risk for developing hypoglycemia by measuring the patient's basal or meal-stimulated C-peptide levels prior to initiating C-peptide therapy, and reducing the insulin dosage of the patient at risk of hypoglycemia when the patient starts C-peptide therapy.

In another aspect of any of these methods and kits, the clinical parameter is selected from energy expenditure, weight gain, caloric intake, body mass index, and nerve conduction velocity, amplitude of the nerve signal, or vibration perception threshold. In one aspect, the clinical parameter is sensory nerve conduction velocity. In one aspect, the clinical parameter is motor nerve conduction velocity. In one aspect, sensory nerve conduction is measured using the sural, ulnar, or median nerves. In one aspect, motor nerve conduction is measured using the median, ulnar, or peroneal nerves.

In one aspect, energy expenditure may be estimated based on reference ranges of energy expenditure for various activities, including work-related activity, exercise regimen, and other daily activities and functions, and as refined by the patient's weight, gender, and age. Such data may be collected, or monitored directly by the patient. In another aspect of any of these methods and kits, the patient's weight gain when starting C-peptide therapy is used as a proxy of appropriate insulin usage when combined with C-peptide therapy, and is therefore used to assess the relative long-term risk of the patient developing hypoglycemia.

In a clinical setting, insulin administration is frequently associated with body weight gain secondary to increased caloric intake to prevent the development of hypoglycemia combined with the anabolic effects of insulin, resulting in increased lipogenesis in adipose tissue and skeletal muscle. In type 2 diabetes patients starting on insulin therapy, the weight gain may amount to 2-4 kg over a period of 4-8 months (Goudswaard et al., Cochrane Data Base Syst Rev, 4: CD 003418 (2004); Carver C, Diabetes Educ., 32:910-917, (2006)). Indeed it is well-established that insulin exerts potent effects on lipid metabolism. It stimulates catabolism of lipoproteins and triglycerides via activation of lipoprotein lipase in the vascular capillaries and enhances esterification of free fatty acids, resulting in storage of lipids in adipose tissue, skeletal muscle, and liver. Moreover, epidemiological data and experimental evidence indicate that hyperinsulinemia may be linked to the development of atherosclerotic vascular disease. Thus increased weight gain is indicative of increased caloric intake and/or increased insulin usage that can signify that the patient is at increased risk of developing hypoglycemia, or of developing a hypoglycemic coma when starting, or during, C-peptide therapy.

Accordingly in one aspect of any of the claimed methods and kits, the patient's body mass index (BMI), or a change in BMI upon starting, or during, C-peptide therapy, may be monitored. BMI is measured (kg/m.sup.2 [or lb/in.sup.2.times.704.5]). Alternatively, changes in any of the following parameters (alone or in any combination) may be used to estimate a change in BMI; weight, waist circumference (estimates fat distribution), waist-to-hip ratio (estimates fat distribution), skin fold thickness (if measured at several sites, estimates fat distribution), or bio-impedance (based on principle that lean mass conducts current better than fat mass [i.e., fat mass impedes current], estimates % fat). The parameters for normal, overweight, or obese individuals are as follows: Underweight: BMI less than 18.5; Normal: BMI 18.5 to 24.9; Overweight: BMI=25 to 29.9. Obese individuals are characterized as having a BMI of 30 to 34.9, being greater than 20% above "normal" weight for height. Individuals with severe or morbid obesity are characterized as having a BMI of greater than 35.

In one aspect of any of these methods and kits, caloric intake over the baseline, or evaluation periods of C-peptide therapy may be monitored. In one aspect of these methods and kits, caloric intake may be further differentiated based on the type of food ingested; e.g., based on the relative consumption of carbohydrates, proteins, and fat. Such estimates may be based on any art-recognized methods for determining such parameters.

In another embodiment of any of the claimed methods and kits, the parameters monitored over the baseline are compared to one or more biomarkers, analytes, or clinical parameters monitored after the patient has initiated C-peptide therapy. In one aspect of any of these methods and kits, the same biomarker, analyte, or clinical parameter is compared before and after therapy. In another aspect, distinct biomarker, analyte, or clinical parameters are used before and after starting C-peptide therapy to assess the risk of the patient developing hypoglycemia, or potential for the patient gaining weight.

In one aspect of any of these methods and kits, one or more biomarkers, analytes, or clinical parameters are monitored during a lead-in evaluation period of C-peptide therapy. In one aspect of any of these methods and kits, the evaluation period of C-peptide therapy extends from about one hour, to any period of time after starting C-peptide therapy. In another aspect of any of these methods and kits, the evaluation period may include historical logs of glucose, insulin, C-peptide, nerve conduction velocity, or HbA1c data collected over any period of time after starting C-peptide therapy. In one embodiment, the evaluation period extends from about one day after starting C-peptide therapy to about 24 weeks after starting therapy. In another aspect, the evaluation period extends from about one day to about 12 weeks. In another aspect, the evaluation period extends from about one day to about 6 weeks. In another aspect, the evaluation period extends from about one day to about 4 weeks. In another aspect, the evaluation period extends from about one day to about 2 weeks. In another aspect, the evaluation period extends from about one day to about one week.

In another aspect of any of these methods and kits, data from a biomarker, analyte, or clinical parameter may be compared to an index reference value that represents the expected normal value, or range, for that patient variable. For example, such index reference values may be based on historical data sets matched for the patient's age, gender, race, medical history, pre-existing medical conditions, length of diabetes duration, etc. In any of these methods and kits, data from the patient may be obtained prior, during, or when starting C-peptide therapy, and compared to an index reference value or range, e.g., by variance of greater than about 2 standard deviations from the mean.

Accordingly, in another aspect of any of the claimed methods and kits, risk prediction for the development of hypoglycemia can also encompass risk prediction algorithms and computed indices that assess and estimate a patient's absolute risk for developing hypoglycemia with reference to a historical cohort. Risk assessment using such predictive mathematical algorithms and computed indices has increasingly been incorporated into guidelines for diagnostic testing and treatment, and encompass indices obtained from and validated with, inter alia, multistage, stratified samples from a representative population.

Tests to measure biomarkers, analytes, and clinical parameters can be implemented on a wide variety of diagnostic test systems, including remote monitoring systems that can be connected via wireless communication systems and web-based patient monitoring and tracking systems. Such sensors typically provide continuous monitoring of a particular physiological function and an alarm output if a critical event arises. The alarm output can be transmitted utilizing conventional communication technology such as a wired hospital network, radio frequency, Bluetooth or magnetic coupling (B-field), or via a cell phone. Representative monitoring systems include, e.g., those described in US Patent Applications Nos. 20090231125, 20090182204, and 20090171169.

Insulin Types and Administration Forms

There are over 180 individual insulin preparations available worldwide which have been developed to provide different lengths of activity (activity profiles). Approximately 25% of these are soluble insulin (unmodified form); about 35% are long- or intermediate-acting basal insulins (mixed with NPH [neutral protamine Hagedorn] insulin or Lente insulin [insulin zinc suspension], or forms that are modified to have an increased isoelectric point [insulin glargine], or acylation [insulin detemir]; these forms have reduced solubility, slow subcutaneous absorption, 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 absorption); and about 38% are pre-mixed insulins (e.g., mixtures of short-, intermediate-, and long-acting insulins; these preparations have the benefit of a reduced number of daily injections).

Short-acting insulin preparations that are commercially available in the US include regular insulin and rapid-acting insulins. Regular insulin has an onset of action of 30-60 minutes, peak time of effect of 1.5 to 2 hours, and duration of activity of 5 to 12 hours. Rapid-acting insulins, such as Aspart (Novo Rapid), Lispro (Humalog), and Glulisine (Apidra), have an onset of action of 10-30 minutes, peak time of effect of around 30 minutes, and a duration of activity of 3 to 5 hours.

Intermediate-acting insulins, such as NPH and Lente insulins, have an onset of action of 1 to 2 hours, peak time of effect of 4 to 8 hours, and a duration of activity of 10 to 20 hours.

Long-acting insulins, such as Ultralente insulin, have an onset of action of 2 to 4 hours, peak time of effect of 8 to 20 hours, and a duration of activity of 16 to 24 hours. Other examples of long-acting insulins include Glargine and Determir. Glargine insulin has an onset of action of 1 to 2 hours, and a duration of action of 24 hours, but with no peak effect.

In many cases, regimens that use insulin in the management of diabetes combine long-acting and short-acting insulin. For example, 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 absorption. 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. The activity of 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.

More recently automated wireless controlled systems for continuous infusion of insulin, such as the system sold under the trademark OMNIPOD.TM. Insulin Management System (Insulet Corporation, Bedford, Mass.) have been developed. These systems provide continuous subcutaneous insulin delivery with blood glucose monitoring technology in a discreet two-part system. This system eliminates the need for daily insulin injections, and does not require a conventional insulin pump which is connected via tubing.

OMNIPOD.TM. is a small lightweight device that is worn on the skin like an infusion set. It delivers insulin according to pre-programmed instructions transmitted wirelessly from the Personal Diabetes Manager (PDM). The PDM is a wireless, hand-held device that is used to program the OMNIPOD.TM. Insulin Management System with customized insulin delivery instructions, monitor the operation of the system, and check blood glucose levels using blood glucose test strips sold under the trademark FREESTYLE.TM.. There is no tubing connecting the device to the PDM. OMNIPOD.TM. Insulin Management System is worn beneath the clothing, and the PDM can be carried separately in a backpack, briefcase, or purse. Similar to currently available insulin pumps, the OMNIPOD.TM. Insulin Management System features fully programmable continuous subcutaneous insulin delivery with multiple basal rates and bolus options, suggested bolus calculations, safety checks, and alarm features.

The aim of insulin treatment of diabetics is typically to administer enough insulin such that the patient will have blood glucose levels within the physiological range and 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.

Furthermore, in order to effectively control diabetes through insulin therapy by dealing with postprandial rises in glucose levels, a bolus, fast-acting treatment must also be administered. The bolus insulin, which is generally administered subcutaneously, provides a rise in plasma insulin levels at approximately 1 hour after administration, thereby limiting hyperglycemia after meals. Thus, these additional quantities of regular insulin, with a duration of action of, e.g., 5 to 6 hours, may be subcutaneously administered at those times of the day when the patient's blood glucose level tends to rise too high, such as at meal times. As an alternative to administering basal insulin in combination with bolus insulin, repeated and regular lower doses of bolus insulin may be administered in place of the long-acting basal insulin, and bolus insulin may be administered postprandially as needed.

Currently, regular subcutaneously injected insulin is recommended to be dosed at 30 to 45 minutes prior to mealtime. As a result, diabetic patients and other insulin users must engage in considerable planning of their meals and of their insulin administrations relative to their meals. Unfortunately, intervening events that may take place between administration of insulin and ingestion of the meal may affect the anticipated glucose excursions.

Furthermore, there is also the potential for hypoglycemia if the administered insulin provides a therapeutic effect over too great a time, e.g., after the rise in glucose levels that occur as a result of ingestion of the meal has already been lowered. As outlined in the Examples, this risk of hypoglycemia is increased in patients who have been treated with C-peptide due to a reduced requirement for insulin.

Accordingly, in one aspect of any of the methods and kits disclosed herein, the present invention includes a method for reducing the risk of the patient developing hypoglycemia by reducing the average daily dose of insulin administered to the patient by about 5% to about 50% after starting C-peptide therapy. In another aspect, the dose of insulin administered is reduced by about 5% to about 45% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 5% to about 40% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 5% to about 35% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 5% to about 30% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 5% to about 25% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 5% to about 20% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 5% to about 15% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 5% to about 10% compared to the patient's insulin dose prior to starting C-peptide treatment.

In another aspect, the dose of insulin administered is reduced by about 2% to about 10% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 2% to about 15% compared to the patient's insulin dose prior to starting C-peptide treatment.

In another aspect, the dose of insulin administered is reduced by about 2% to about 20% compared to the patient's insulin dose prior to starting C-peptide treatment.

In another aspect, the dose of insulin administered is reduced by about 10% to about 50% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 10% to about 45% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 10% to about 40% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 10% to about 35% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 10% to about 30% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 10% to about 25% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by about 10% to about 20% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect, the dose of insulin administered is reduced by at least 10% compared to the patient's insulin dose prior to starting C-peptide treatment.

In one aspect of any of these methods and kits, the dose of short-acting insulin administered is selectively reduced by any of the prescribed ranges listed above. In another aspect of any of these methods and kits, the dose of intermediate-acting insulin administered is selectively reduced by any of the prescribed ranges. In one aspect of any of these methods and kits, the dose of long-acting insulin administered is selectively reduced by any of the prescribed ranges listed above.

In another aspect of any of these methods and kits, the dose of intermediate- and long-acting insulin administered is independently reduced by any of the prescribed ranges listed above, while the dose of short-acting insulin remains substantially unchanged.

In one aspect of these methods and kits, the dose of short-acting insulin administered is reduced by about 5% to about 50% compared to the patient's insulin dose prior to starting C-peptide treatment. In another embodiment, the dose of short-acting insulin administered is reduced by about 5% to about 35% compared to the patient's insulin dose prior to starting C-peptide treatment. In another embodiment, the dose of short-acting insulin administered is reduced by about 10% to about 20% compared to the patient's insulin dose prior to starting C-peptide treatment. In one aspect of these methods and kits, the dose of short-acting insulin administered preprandially for a meal is reduced. In another aspect of these methods and kits, the dose of short-acting insulin administered in the morning or at nighttime is reduced. In another aspect of any of these methods and kits, the dose of short-acting insulin administered is reduced while the dose of long-acting and/or intermediate-acting insulin administered to the patient is substantially unchanged.

In another aspect of any of the methods and kits disclosed herein, the present invention includes a method for reducing the risk of the patient developing hypoglycemia by reducing the average daily dose of intermediate-acting insulin administered to the patient by about 5% to about 35% after starting C-peptide therapy. In one aspect of these methods and kits, the dose of intermediate-acting insulin administered is reduced by about 5% to about 50% compared to the patient's insulin dose prior to starting C-peptide treatment. In another embodiment, the dose of intermediate-acting insulin administration is reduced by about 5% to about 35% compared to the patient's insulin dose prior to starting C-peptide treatment. In another embodiment, the dose of intermediate-acting insulin administered is reduced by about 10% to about 20% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect of these methods and kits, the dose of intermediate-acting insulin administered in the morning or at nighttime is reduced. In another aspect of any of these methods and kits, the dose of intermediate-acting insulin administered is reduced while the dose of short-acting insulin administered to the patient is substantially unchanged.

In another aspect of any of the methods and kits disclosed herein, the present invention includes a method for reducing the risk of the patient developing hypoglycemia by reducing the average daily dose of long-acting insulin administered to the patient by about 5% to about 50% after starting C-peptide therapy. In one embodiment, the dose of long-acting insulin administered is reduced by about 5% to about 35% compared to the patient's insulin dose prior to starting C-peptide treatment. In another embodiment, the dose of long-acting insulin administered is reduced by about 10% to about 20% compared to the patient's insulin dose prior to starting C-peptide treatment. In another aspect of these methods and kits, the dose of long-acting insulin administered in the morning or at nighttime is reduced. In another aspect of any of these methods and kits, the dose of long-acting insulin administered is reduced while the dose of short-acting insulin administered to the patient is substantially unchanged.

In certain preferred embodiments, the patient achieves improved insulin utilization and insulin sensitivity while experiencing a reduced risk of developing hypoglycemia after treatment with C-peptide as compared with baseline levels prior to treatment. Preferably, the improved insulin utilization and insulin sensitivity are measured by a statistically significant decline in HOMA (Homeostasis Model Assessment) (Turner et al.: Metabolism 28(11): 1086-1096, (1979)).

Therapeutic forms of C-peptide

The terms "C-peptide" or "proinsulin C-peptide" as used herein includes all naturally-occurring and synthetic forms of C-peptide that retain C-peptide activity. Such C-peptides include the human peptide, as well as peptides derived from other animal species and genera, preferably mammals. Preferably, "C-peptide" refers to human C-peptide having the amino acid sequence

EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (Seq ID No. 1 in Table D1 (see Original Patent)).

C-peptides from a number of different species have been sequenced, and are known in the art to be at least partially functionally interchangeable. It would thus be a routine matter to select a variant being a C-peptide from a species or genus other than human. Several such variants of C-peptide (i.e., representative C-peptides from other species) are shown in Table D1 (see Seq ID Nos. 1-29) (see Original Patent).

Thus all such homologues, orthologs, and naturally-occurring isoforms of C-peptide from human as well as other species (Seq ID Nos. 1-29) are included in any of the methods and kits of the invention, as long as they retain detectable C-peptide activity.

The C-peptides may be in their native form, i.e., as different variants as they appear in nature in different species which may be viewed as functionally equivalent variants of human C-peptide, or they may be functionally equivalent natural derivatives thereof, which may differ in their amino acid sequence, e.g., by truncation (e.g., from the N- or C-terminus or both) or other amino acid deletions, additions, insertions, substitutions, or post-translational modifications. Naturally-occurring chemical derivatives, including post-translational modifications and degradation products of C-peptide, are also specifically included in any of the methods and kits of the invention including, e.g., pyroglutamyl, iso-aspartyl, proteolytic, phosphorylated, glycosylated, oxidatized, isomerized, and deaminated variants of C-peptide.

It is known in the art to synthetically modify the sequences of proteins or peptides, while retaining their useful activity, and this may be achieved using techniques which are standard in the art and widely described in the literature, e.g., random or site-directed mutagenesis, cleavage, and ligation of nucleic acids, or via the chemical synthesis or modification of amino acids or polypeptide chains. Similarly it is within the skill in the art to address and/or mitigate immunogenicity concerns if they arise using C-peptide variants, e.g., by the use of automated computer recognition programs to identify potential T cell epitopes, and directed evolution approaches to identify less immunogenic forms.

Any such modifications, or combinations thereof, may be made and used in any of the methods and kits of the invention, as long as activity is retained. The C-terminal end of the molecule is known to be important for activity. Preferably, therefore, the C-terminal end of the C-peptide should be preserved in any such C-peptide variants or derivatives, more preferably the C-terminal pentapeptide of C-peptide (EGSLQ) (Seq ID No. 31) should be preserved or sufficient (see Henriksson M et al.: Cell Mol. Life. Sci. 62: 1772-1778, (2005)). As mentioned above, modification of an amino acid sequence may be by amino acid substitution, e.g., an amino acid may be replaced by another that preserves the physicochemical character of the peptide (e.g., A may be replaced by G or vice versa, V by A or L; E by D or vice versa; and Q by N). Generally, the substituting amino acid has similar properties, e.g., hydrophobicity, hydrophilicity, electronegativity, bulky side chains, etc., to the amino acid being replaced.

Modifications to the mid-part of the C-peptide sequence (e.g., to residues 13 to 25 of human C-peptide) allow the production of functional derivatives or variants of C-peptide. Thus, C-peptides which may be used in any of the methods or kits of the invention may have amino acid sequences which are substantially homologous, or substantially similar to the native C-peptide amino acid sequences, e.g., to the human C-peptide sequence of Seq ID No. 1 or any of the other native C-peptide sequences shown in Table D1 (see Original Patent). Alternatively, the C-peptide may have an amino acid sequence having at least 30% preferably at least 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identity with the amino acid sequence of any one of Seq ID Nos. 1-29 as shown in Table D1, preferably with the native human sequence of Seq ID No. 1. In a preferred embodiment, the C-peptide for use in any of the methods or kits of the present invention is at least 80% identical to a sequence selected from Table D1. In another aspect, the C-peptide for use in any of the methods or kits of the invention is at least 80% identical to human C-peptide (Seq ID No. 1). Although any amino acid of C-peptide may be altered as described above, it is preferred that one or more of the glutamic acid residues at positions 3, 11, and 27 of human C-peptide (Seq ID No. 1) or corresponding or equivalent positions in C-peptide of other species, are conserved. Preferably, all of the glutamic acid residues at positions 3, 11, and 27 (or corresponding Glu residues) of Seq ID No. 1 are conserved. Alternatively, it is preferred that Glu27 of human C-peptide (or a corresponding Glu residue of a non-human C-peptide) is conserved. An exemplary functional equivalent form of C-peptide which may be used in any of the methods or kits of the invention includes the amino acid sequences:

EXEXXQXXXXELXXXXXXXXXXXXALBXXXQ (Seq ID No. 30).

GXEXXQXXXXELXXXXXXXXXXXXALBXXXQ (Seq ID No. 33).

As used herein, X is any amino acid. The N-terminal residue may be either Glu or Gly (Seq ID No. 30 or Seq ID No. 33 respectively). Functionally equivalent derivatives or variants of native C-peptide sequences may readily be prepared according to techniques well-known in the art, and include peptide sequences having a functional, e.g., a biological activity of a native C-peptide.

Fragments of native or synthetic C-peptide sequences may also have the desirable functional properties of the peptide from which they were derived and may be used in any of the methods or kits of the invention. The term "fragment" as used herein thus includes fragments of a C-peptide provided that the fragment retains the biological or therapeutically beneficial activity of the whole molecule. The fragment may also include a C-terminal fragment of C-peptide. Preferred fragments comprise residues 15-31 of native C-peptide, more especially residues 20-31. Peptides comprising the pentapeptide EGSLQ (Seq ID No. 31) (residues 27-31 of native human C-peptide) are also preferred. The fragment may thus vary in size from, e.g., 4 to 30 amino acids or 5 to 20 residues. Suitable fragments are disclosed in WO 98/13384 the contents of which are incorporated herein by reference.

The fragment may also include an N-terminal fragment of C-peptide, typically having the sequence EAEDLQVGQVEL (Seq ID No. 32), or a fragment thereof which comprises 2 acidic amino acid residues, capable of adopting a conformation where said two acidic amino acid residues are spatially separated by a distance of 9-14 A between the alpha-carbons thereof. Also included are fragments having N- and/or C-terminal extensions or flanking sequences. The length of such extended peptides may vary, but typically are not more than 50, 30, 25, or 20 amino acids in length. Representative suitable fragments are described in U.S. Pat. No. 6,610,649, which is hereby incorporated by reference in its entirety.

In such a case it will be appreciated that the extension or flanking sequence will be a sequence of amino acids which is not native to a naturally-occurring or native C-peptide, and in particular a C-peptide from which the fragment is derived. Such a N- and/or C-terminal extension or flanking sequence may comprise, e.g., from 1 to 10, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 amino acids.

The term "derivative" as used herein thus refers to C-peptide sequences or fragments thereof, which have modifications as compared to the native sequence. Such modifications may be one or more amino acid deletions, additions, insertions, and/or substitutions. These may be contiguous or non-contiguous. Representative variants may include those having 1 to 6, or more preferably 1 to 4, 1 to 3, or 1 or 2 amino acid substitutions, insertions, and/or deletions as compared to any of Seq ID Nos. 1-33. The substituted amino acid may be any amino acid, particularly one of the well-known 20 conventional amino acids (Ala (A); Cys (C); Asp (D); Glu (E); Phe (F); Gly (G); His (H); Ile (I); Lys (K); Leu (L); Met (M); Asn (N); Pro (P); G in (O); Arg (R); Ser (S); Thr (T); Val (V); Trp (W); and Tyr (Y)). Any such variant or derivative of C-peptide may be used in any of the methods or kits of the invention.

Fusion proteins of C-peptide to other proteins are also included, and these fusion proteins may enhance C-peptide's biological activity, targeting, biological life, or pharmacokinetic properties. Examples of fusion proteins that improve pharmacokinetic properties include without limitation, fusions to human albumin (Osborn et al.: Eur. J. Pharmacol. 456(1-3): 149-158, (2002)), antibody fc domains, poly Glu or poly Asp sequences, and transferrin. Additionally, fusion with conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and Ser (`PASylation`) or hydroxyethyl starch (sold under the trademark HESYLATION.RTM.) provides a simple way to increase the hydrodynamic volume of the C-peptide. This additional extension adopts a bulky random structure, which significantly increases the size of the resulting fusion protein. By this means the typically rapid clearance of the C-peptide via kidney filtration is retarded by several orders of magnitude.

An additional fusion protein approach contemplated for use within the present invention includes the fusion of C-peptide to a multimerization domain. Representative multimerization domains include without limitation coiled-coil dimerization domains such as leucine zipper domains which are found in certain DNA-binding polypeptides, the dimerization domain of an immunoglobulin Fab constant domain, such as an immunoglobulin heavy chain CH.sub.1 constant region or an immunoglobulin light chain constant region. In a preferred embodiment, the multimerisation domain is derived from tetranectin, and more specifically comprises the tetranectin trimerising structural element, which is described in detail in WO 98/56906.

It will be appreciated that a flexible molecular linker (or spacer) optionally may be interposed between, and covalently join, the C-peptide and any of the fusion proteins disclosed herein. Any such fusion protein many be used in any of the methods or kits of the present invention.

Chemical modifications of the native C-peptide structure, which retain or stabilize C-peptide activity or biological half-life, may also be used with any of the methods or kits described herein. Such chemical modification strategies include, without limitation, pegylation, glycosylation, and acylation (Clark et al.: J. Biol. Chem. 271(36): 21969-21977, (1996); Roberts et al.: Adv. Drug. Deliv. Rev. 54(4): 459-476, (2002); Felix et al.: Int. J. Pept. Protein. Res. 46(3-4): 253-264, (1995); Garber A J: Diabetes Obes. Metab. 7(6): 666-74 (2005)). C- and N-terminal protecting groups and peptomimetic units may also be included.

A wide variety of PEG derivatives are both available and suitable for use in the preparation of PEG-conjugates. For example, NOF Corp.'s SUNBRIGHT.RTM. Series provides numerous PEG derivatives, including methoxypolyethylene glycols and activated PEG derivatives such as methoxy-PEG amines, maleimides, and carboxylic acids, for coupling by various methods to drugs, enzymes, phospholipids, and other biomaterials and Nektar Therapeutics' Advanced PEGylation also offers diverse PEG-coupling technologies to improve the safety and efficacy of therapeutics.

A search of patents, published patent applications, and related publications will also provide those skilled in the art reading this disclosure with significant possible PEG-coupling technologies and PEG-derivatives. For example, U.S. Pat. Nos. 6,436,386; 5,932,462; 5,900,461; 5,824,784; and 4,904,584; the contents of which are incorporated by reference in their entirety, describe such technologies and derivatives, and methods for their manufacture. Thus, one skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could couple PEG, a PEG-derivative, or some other polymer to C-peptide for its extended release.

PEG is a well-known polymer having the properties of solubility in water and in many organic solvents, lack of toxicity, lack of immunogenicity, and also clear, colorless, odorless, and stable. One use of PEG is to covalently attach the polymer to insoluble molecules to make the resulting PEG-molecule conjugate soluble. For these reasons and others, PEG has been selected as the preferred polymer for attachment, but it has been employed solely for purposes of illustration and not limitation. Similar products may be obtained with other water-soluble polymers, including without limitation; polyvinyl alcohol, other poly(alkylene oxides) such as poly(propylene glycol) and the like, poly(oxyethylated polyols) such as poly(oxyethylated glycerol) and the like, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl purrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride, and polyaminoacids. One skilled in the art will be able to select the desired polymer based on the desired dosage, circulation time, resistance to proteolysis, and other considerations.

Isomers of the native L-amino acids, e.g., D-amino acids may be incorporated in any of the above forms of C-peptide, and used in any of the methods or kits of the invention. Additional variants may include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acids. Longer peptides may comprise multiple copies of one or more of the C-peptide sequences, such as any of Seq ID Nos. 1-33. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced at a site in the protein. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Variants may include, e.g., different allelic variants as they appear in nature, e.g., in other species or due to geographical variation. All such variants, derivatives, fusion proteins, or fragments of C-peptide are included, may be used in any of the methods claims or kits disclosed herein, and are subsumed under the term "C-peptide".

The variants, derivatives, and fragments are functionally equivalent in that they have detectable C-peptide activity. More particularly, they exhibit at least 40%, preferably at least 60%, more preferably at least 80% of the activity of proinsulin C-peptide, particularly human C-peptide. Thus they are capable of functioning as proinsulin C-peptide, i.e., can substitute for C-peptide itself. Such activity means any activity exhibited by a native C-peptide, whether a physiological response exhibited in an in vivo or in vitro test system, or any biological activity or reaction mediated by a native C-peptide, e.g., in an enzyme assay or in binding to test tissues, membranes, or metal ions. Thus, it is known that C-peptide increases the intracellular concentration of calcium. An assay for C-peptide activity can thus be made by assaying for changes in intracellular calcium concentrations upon addition or administration of the peptide (e.g., fragment or derivative) in question. Such an assay is described in, e.g., Ohtomo Y et al. (Diabetologia 39: 199-205, (1996)), Kunt T et al. (Diabetologia 42(4): 465-471, (1999)), Shafqat J et al. (Cell Mol. Life. Sci. 59: 1185-1189, (2002)). Further, C-peptide has been found to induce phosphorylation of the MAP-kinases ERK 1 and 2 of a mouse embryonic fibroblast cell line (Swiss 3T3), and measurement of such phosphorylation and MAPK activation may be used to assess, or assay for C-peptide activity, as described, e.g., by Kitamura T et al. (Biochem. J. 355: 123-129, (2001)). C-peptide also has a well-known effect in stimulating Na.sup.+K.sup.+-ATPase activity and this also may form the basis of an assay for C-peptide activity, e.g., as described in WO 98/13384 or in Ohtomo Y et al. (supra) or Ohtomo Y et al. (Diabetologia 41: 287-291, (1998)). An assay for C-peptide activity based on endothelial nitric oxide synthase (eNOS) activity is also described in Kunt T et al. (supra) using bovine aortic cells and a reporter cell assay. Binding to particular cells may also be used to assess or assay for C-peptide activity, e.g., to cell membranes from human renal tubular cells, skin fibroblasts, and saphenous vein endothelial cells using fluorescence correlation spectroscopy, as described, e.g., in Rigler R et al. (PNAS USA 96: 13318-13323, (1999)), Henriksson M et al. (Cell Mol. Life. Sci. 57: 337-342, (2000)) and Pramanik A et al. (Biochem Biophys. Res. Commun. 284: 94-98, (2001)).

C-Peptide Therapeutic Dose Forms

Human C-peptide may be produced by recombinant technology, e.g., as a by-product in the production of human insulin from human proinsulin, or using genetically modified E. coli (see WO 1999007735) or synthetically using standard solid-phase peptide synthesis.

Administration of the C-peptide may be by any suitable method known in the medicinal arts, including oral, parenteral, topical, or subcutaneous administration, inhalation, or the implantation of a sustained delivery device or composition. In one aspect, administration is by subcutaneous administration. The C-peptide may be administered at any time during the day. For humans, the daily dosage used may range from about 0.1 to 10 mg/24 hours of C-peptide, e.g., from about 0.1 to 0.3 mg, about 0.3 to 1.5 mg, about 1.5 to 2.25 mg, about 2.25 to 3.0 mg, about 3.0 to 6.0 mg, and about 6.0 to 10 mg/24 hours. Preferably the total daily dose used is about 0.45 to 0.9 mg, about 0.6 to 1.2 mg, about 1.2 to 2.4 mg, or about 2.5 to 3.0 mg/24 hours. The total daily dose may be about 0.3 mg, about 0.45 mg, about 0.6 mg, about 0.9 mg, about 1.2 mg, about 1.5 mg, about 1.8 mg, about 2.1 mg, about 2.4 mg, about 2.7 mg, about 3.0 mg, about 3.3 mg, about 3.6 mg, about 3.9 mg, about 4.2 mg, or about 4.5 mg/24 hours. (It will be appreciated that masses of C-peptide referred to above are dependent on the bioavailability of the delivery system and based on the use of C-peptide with a molecular mass of approximately 3,020 Da).

In another aspect of any of these methods and kits, the therapeutic dose of C-peptide comprises a daily dose ranging from about 1.5 to about 4.5 mg per 24 hours. In another aspect of any of these methods and kits, the therapeutic dose of C-peptide comprises a daily dose ranging from about 0.3 mg to about 1.5 mg per 24 hours. In another aspect of any of these methods and kits, the therapeutic dose of C-peptide comprises a daily dose ranging from about 3.0 mg to about 6 mg per 24 hours. In another aspect of any of these methods and kits, the therapeutic dose of C-peptide maintains the average steady state concentration of C-peptide (C.sub.ss-ave) in the patient's plasma of between about 0.2 nM and about 6 nM.

In another aspect of any of these methods and kits, the therapeutic dose of C-peptide is provided to the patient so as to maintain the average steady state concentration of C-peptide in the patient's plasma between about 0.2 nM and about 6 nM for at least 24 hours. In another aspect of any of these methods and kits, the therapeutic dose of C-peptide is provided to the patient so as to maintain the average steady state concentration of C-peptide in the patient's plasma between about 0.2 nM and about 6 nM for at least 48 hours. In another aspect of any of these methods and kits, the therapeutic dose of C-peptide is provided to the patient so as to maintain the average steady state concentration of C-peptide in the patient's plasma between about 0.2 nM and about 6 nM for at least 72 hours. In another aspect of any of these methods and kits, the therapeutic dose of C-peptide is provided to the patient so as to maintain the average steady state concentration of C-peptide in the patient's plasma between about 0.2 nM and about 6 nM for at least one week. In any of these methods and kits, the therapeutic dose is administered by daily subcutaneous injections. In another aspect of any of these methods and kits, the therapeutic dose is administered by a sustained release formulation or device.

It will be further appreciated that for sustained delivery devices and compositions the total dose of C-peptide contained in such delivery system will be correspondingly larger depending upon the release profile of the sustained release system. Thus, a sustained release composition or device that is intended to deliver C-peptide over a period of 5 days will typically comprise at least about 5 to 10 times the daily dose of C-peptide; a sustained release composition or device that is intended to deliver C-peptide over a period of 365 days will typically comprise at least about 400 to 600 times the daily dose of C-peptide (depending upon the stability and bioavailability of C-peptide when administered using the sustained release system). Typically such devices and systems will maintain an average steady state concentration of C-peptide in the patient's plasma of between about 0.2 nM and about 6 nM.

In one aspect of any of these modes of administration, the total daily dose of C-peptide may be administered in multiple, single doses throughout the day to maintain the steady-state level of C-peptide above the minimum effective therapuetic level. The size of the single dose as administered will vary depending on the frequency of administration and bioavailability, but may typically be in the region of about 0.15 to 6.0 mg, about 0.15 to 4.5 mg, about 0.15 to 3.0 mg, about 0.15 to 2.4 mg, about 0.15 to 1.8 mg, or about 0.15 to 1.2 mg. Other ranges include about 0.1 to 4.5 mg, about 0.3 to 0.6 mg, about 0.3 to 1.5 mg, or about 0.5 to 3.0 mg. Representative single doses include about 5.0 mg, about 4.5 mg, about 4.0 mg, about 3.5 mg, about 3.0 mg, about 2.5 mg, about 2.0 mg, about 1.5 mg, about 1.0 mg, or about 0.5 mg. In one aspect, the dosing interval of such multiple administration regimens will be about 3 hours between doses, or about 4 hours between doses, or about 6 hours between doses.

In one aspect of any of these methods and kits, the dose and dosing interval of C-peptide administered may vary depending on the time of administration. For example, a total daily dose of 1.8 mg/24 hours may be divided into 4 doses; 0.45 mg in the morning (06:00-10:00); at lunch (11:00-14:00); at dinner (16:00-19:00); and 0.9 mg at bedtime (20:00-24:00). Typically such dosing schedules maintain the average steady-state C-peptide level in the blood above the minimum effective therapeutic level for at least 50% of the time for any one 24 hour dosing period. In a preferred aspect, the dosing schedule maintains the C-peptide level in the blood above the minimum effective therapeutic level for at least 75% of the time for any one 24 hour dosing period. In a more preferred aspect, the dosing schedule maintains the C-peptide level in the blood above the minimum effective therapeutic level for at least 85% of the time for any one 24 hour dosing period. In another aspect of any of these modes of administration, the total daily dose of C-peptide may be administered continuously throughout the day to coordinate C-peptide levels with insulin levels, meals, or periods of exercise, sleep, or any other patient-specific clinical parameter or biomarker.

The dose may or may not be in solution. If the dose is administered in solution, it will be appreciated that the volume of the dose may vary, but will typically be 10 .mu.L-2 mL. Preferably the dose for S.C. administration will be given in a volume of 1000 uL, 900 .mu.L, 800 .mu.L, 700 .mu.L, 600 .mu.L, 500 .mu.L, 400 .mu.L, 300 .mu.L, 200 .mu.L, 100 .mu.L, 50 .mu.L, or 20 .mu.L. Sustained release compositions and depot formulations may include doses in volumes of about 2 mL to about 50 uL.

C-peptide doses in solution can also comprise a preservative and/or a buffer. For example, the preservative m-cresol can be used. Typical concentrations of preservatives include 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, or 5 mg/mL. Thus, a range of concentration of preservative may include 0.2 to 10 mg/mL, particularly 0.5 to 6 mg/mL, or 0.5 to 5 mg/mL. Examples of buffers that can be used include histidine (pH 6.0), sodium phosphate buffer (pH 7.3), or sodium bicarbonate buffer (pH 7.3). It will be appreciated that the C-peptide dose may comprise one or more of a native or intact C-peptide, fragments, derivatives, or other functionally equivalent variants of C-peptide.

A dose of C-peptide may comprise full-length human C-peptide (Seq ID No. 1) and the C-terminal C-peptide fragment EGSLQ (Seq ID No. 31) and/or a C-peptide homolog or C-peptide derivative. Further, the dose may if desired only contain a fragment of C-peptide, e.g., EGSLQ. Thus, the term "C-peptide" may encompass a single C-peptide entity or a mixture of different "C-peptides".

Pharmaceutical compositions for use in the present invention may be formulated according to techniques and procedures well-known in the art and widely discussed in the literature and may comprise any of the known carriers, diluents, or excipients. In one aspect, the compositions may be in the form of sterile aqueous solutions and/or suspensions of the pharmaceutically active ingredients, aerosols, ointments, and the like. Formulations which are aqueous solutions are most preferred. Such formulations typically contain the C-peptide itself, water, and one or more buffers which act as stabilizers (e.g., phosphate-containing buffers) and optionally one or more preservatives. Such formulations containing, e.g., about 0.3 to 12.0 mg, about 0.3 to 10.0 mg, about 0.3 to 8.0 mg, about 0.3 to 6.0 mg, about 0.3 to 4.0 mg, about 0.3 to 3.0 mg, or any of the ranges mentioned above, e.g., about 12 mg, about 10 mg, about 8 mg, about 6 mg, about 5 mg, about 4 mg, about 3 mg, about 2 mg, or about 1 mg of the C-peptide and constitute a further aspect of the invention.

Pharmaceutical compositions may include pharmaceutically acceptable salts of C-peptide. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Suitable base salts are formed from bases which form non-toxic salts. Representative examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases may also be formed, e.g., hemisulphate and hemicalcium salts. In one embodiment, C-peptide may be prepared as a gel with a pharmaceutically acceptable positively charged ion. In one aspect, the positively charged ion may be a divalent metal ion. In one aspect, the metal ion is selected from calcium, magnesium, and zinc.

Compositions to be used in the invention suitable for parenteral administration may comprise sterile aqueous solutions and/or suspensions of the pharmaceutically active ingredients preferably made isotonic with the blood of the recipient, generally using sodium chloride, glycerin, glucose, mannitol, sorbitol, and the like.

Compositions of the invention suitable for oral administration may, e.g., comprise peptides in sterile purified stock powder form preferably covered by an envelope or envelopes (enterocapsules) protecting from degradation of the peptides in the stomach and thereby enabling absorption of these substances from the gingiva or in the small intestines. The total amount of active ingredient in the composition may vary from 99.99 to 0.01 percent of weight.

Methods for Administration of C-Peptide

Pharmaceutical compositions suitable for the delivery of C-peptide and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, e.g., in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

Pharmaceutical compositions of C-peptide may be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques. Subcutaneous administration of C-peptide is preferred. Subcutaneous administration of C-peptide will typically not be into the same site as that most recently used for insulin administration. In one aspect of any of the claimed methods and kits, C-peptide is administered to the opposite side of the abdomen to the site most recently used for insulin administration. In another aspect of any of the claimed methods and kits, C-peptide is administered to the upper arm. In another aspect of any of the claimed methods and kits, C-peptide is administered to the abdomen. In another aspect of any of the claimed methods and kits, C-peptide is administered to the upper area of the buttock. In another aspect of any of the claimed methods and kits, C-peptide is administered to the front of the thigh.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates, and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, e.g., by lyophilization, may readily be accomplished using standard pharmaceutical techniques well-known to those skilled in the art.

Formulations for parenteral administration may be formulated to be immediate and/or sustained release. Sustained release compositions include delayed, modified, pulsed, controlled, targeted and programmed release. Thus C-peptide may be formulated as a suspension or as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing sustained release of C-peptide. Examples of such formulations include without limitation, drug-coated stents and semi-solids and suspensions comprising drug-loaded poly(DL-lactic-co-glycolic)acid (PGLA), poly(DL-lactide-co-glycolide) (PLG) or poly(lactide) (PLA) lamellar vesicles or microparticles, hydrogels (Hoffman A S: Ann. N.Y. Acad. Sci. 944: 62-73 (2001)), poly-amino acid nanoparticles systems, such as the Medusa system developed by Flamel Technologies Inc., non aequous gel systems such as Atrigel developed by Atrix, Inc., and SABER (Sucrose Acetate Isobutyrate Extended Release) developed by Durect Corporation, and lipid-based systems such as DepoFoam developed by SkyePharma.

Sustained release devices capable of delivering desired doses of C-peptide over extended periods of time are known in the art. For example, U.S. Pat. Nos. 5,034,229; 5,557,318; 5,110,596; 5,728,396; 5,985,305; 6,113,938; 6,156,331; 6,375,978; and 6,395,292; teach osmotically-driven devices capable of delivering an active agent formulation, such as a solution or a suspension, at a desired rate over an extended period of time (i.e., a period ranging from more than one week up to one year or more). Other exemplary sustained release devices include regulator-type pumps that provide constant flow, adjustable flow, or programmable flow of beneficial agent formulations, which are available from, e.g., Insulet Corporation, Codman of Raynham, Mass., (sold under the trademark OMNIPODT.TM. Insulin Management System), Medtronic of Minneapolis, Minn., Intarcia Therapeutics of Hayward, Calif., and Tricumed Medinzintechnik GmbH of Germany. Further examples of devices are described in U.S. Pat. Nos. 6,283,949; 5,976,109; 5,836,935; and 5,511,355.

Generally, in an osmotic pump system, a core is encased by a semi-permeable membrane having at least one orifice. The semi-permeable membrane is permeable to water, but impermeable to the active agent. When the system is exposed to body fluids, water penetrates through the semi-permeable membrane into the core containing osmotic excipients and the active agent. Osmotic pressure increases within the core and the agent is displaced through the orifice at a controlled, predetermined rate.

In many osmotic pumps, the core contains more than one internal compartment. For example, a first compartment may contain the active agent. A second compartment contains an osmotic agent and/or "driving member." See, e.g., U.S. Pat. No. 5,573,776, the contents of which are incorporated herein by reference. This compartment may have a high osmolality, which causes water to flux into the pump through the semi permeable membrane. The influx of water compresses the first compartment. This can be accomplished, e.g., by using a polymer in the second compartment, which swells on contact with the fluid. Accordingly, the agent is displaced at a predetermined rate.

In another embodiment, the osmotic pump may comprise more than one active agent-containing compartment, with each compartment containing the same agent or a different agent. The concentrations of the agent in each compartment, as well as the rate of release, may also be the same or different.

The rate of delivery is generally controlled by the water permeability of the semi-permeable membrane. Thus, the delivery profile of the pump is independent of the agent dispensed, and the molecular weight of an agent, or its physical and chemical properties, generally have no bearing on its rate of delivery. Further discussion regarding the principle of operation, the design criteria, and the delivery rate for osmotic pumps is provided in Theeuwes and Yum (Ann. of Biomed. Eng. 4(4): 343-353, (1976)) and Urquhart J et al. (Ann. Rev. Pharmacol. Toxicol. 24:199-236, (1984)), the contents of which are incorporated by reference.

Sustained release devices based on osmotic pumps are well-known in the art and readily available to one of ordinary skill in the art from companies experienced in providing osmotic pumps for extended release drug delivery. For example, the technology sold under the trademark DUROS.RTM. which was originally developed by ALZA is an implantable, nonbiodegradable, osmotically-driven system that enables delivery of small drugs, peptides, proteins, DNA, and other bioactive macromolecules for up to one year; ALZA's technology sold under the trademark OROS.RTM. embodies tablets that employ osmosis to provide precise, controlled drug delivery for up to 24 hours; Osmotica Pharmaceutical's system sold under the trademark OSMODEX.RTM. includes a tablet, which may have more than one layer of the drug(s) with the same or different release profiles; Shire Laboratories' system sold under the trademark ENSOTROl.RTM. solubilizes drugs within the core and delivers the solubilized drug through a laser-drilled hole by osmosis; and osmotic pumps sold under the trademark ALZET.RTM. are miniature, implantable pumps used for research in mice, rats, and other laboratory animals.

A search of patents, published patent applications, and related publications will also provide those skilled in the art reading this disclosure with significant possible osmotic pump technologies. For example, U.S. Pat. Nos. 6,890,918; 6,838,093; 6,814,979; 6,713,086; 6,534,090; 6,514,532; 6,361,796; 6,352,721; 6,294,201; 6,284,276; 6,110,498; 5,573,776; 4,200,0984; and 4,088,864; the contents of which are incorporated herein by reference, describe osmotic pumps and methods for their manufacture. One skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents, could produce an osmotic pump for the sustained release of C-peptide.

Typical materials for the semi-permeable membrane include semi-permeable polymers known to the art as osmosis and reverse osmosis membranes, such as cellulose acylate, cellulose diacylate, cellulose triacylate, cellulose acetate, cellulose diacetate, cellulose triacetate, agar acetate, amylase triacetate, beta glucan acetate, acetaldehyde dimethyl acetate, cellulose acetate ethyl carbamate, polyamides, plyurethanes, sulfonated polystyrenes, cellulose acetate phthalate, cellulose acetate methyl carbamate, cellulose acetate succinate, cellulose acetate dimethyl aminoacetate, cellulose acetate ethyl carbamate, cellulose acetate chloracetate, cellulose dipalmitate, cellulose dioctanoate, cellulose dicaprylate, cellulose dipentanlate, cellulose acetate valerate, cellulose acetate succinate, cellulose propionate, succinate, methyl cellulose, cellulose acetate p-toluene sulfonate, cellulose acetate butyrate, cross-linked selectively semi-permeable polymers formed by the coprecipitation of a polyanion and a polycation, semi-permeable polymers, lightly cross-linked polystyrene derivatives, cross-linked poly(sodium styrene sulfonate), poly(vinylbenzyltrimethyl ammonium chloride), cellulose acetate having a degree of substitution up to 1 and an acetyl content up to 50%, cellulose diacetate having a degree of substitution of 1 to 2 and an acetyl content of 21 to 35%, cellulose triacetate having a degree of substitution of 2 to 3 and an acetyl content of 35 to 44.8%, as disclosed in U.S. Pat. No. 6,713,086, the contents of which are incorporated herein by reference.

The osmotic agent(s) present in the pump may comprise any osmotically effective compound(s) that exhibit an osmotic pressure gradient across the semi-permeable wall against the exterior fluid. Effective agents include, without limitation, magnesium sulfate, calcium sulfate, magnesium chloride, sodium chloride, lithium chloride, potassium sulfate, sodium carbonate, sodium sulfite, lithium sulfate, potassium chloride, sodium sulfate, d-mannitol, urea, sorbitol, inositol, raffinose, sucrose, flucose, hydrophilic polymers such as cellulose polymers, mixtures thereof, and the like, as disclosed in U.S. Pat. No. 6,713,086, the contents of which are incorporated herein by reference.

The "driving member" is typically a hydrophilic polymer that interacts with biological fluids and swells or expands. The polymer exhibits the ability to swell in water and retain a significant portion of the imbibed water within the polymer structure. The polymers swell or expand to a very high degree, usually exhibiting a 2- to 50-fold volume increase. The polymers can be non-cross-linked or cross-linked. Hydrophilic polymers suitable for the present purpose are well-known in the art.

The orifice may comprise any means and methods suitable for releasing the active agent from the system. The osmotic pump may include one or more apertures or orifices that have been bored through the semi-permeable membrane by mechanical procedures known in the art, including, but not limited to, the use of lasers as disclosed in U.S. Pat. No 4,088,864. Alternatively, it may be formed by incorporating an erodible element, such as a gelatin plug, in the semi-permeable membrane.

Because they can be designed to deliver a desired active agent at therapeutic levels over an extended period of time, implantable delivery systems can advantageously provide long-term therapeutic dosing of a desired active agent without requiring frequent visits to a healthcare provider or repetitive self-medication. Therefore, implantable delivery devices can work to provide increased patient compliance, reduced irritation at the site of administration, fewer occupational hazards for healthcare providers, reduced waste hazards, and increased therapeutic efficacy through enhanced dosing control.

Among other challenges, two problems must be addressed when seeking to deliver biomolecular material over an extended period of time from an implanted delivery device. First, the biomolecular material must be contained within a formulation that substantially maintains the stability of the material at elevated temperatures (i.e., 37.degree. C. and above) over the operational life of the device. Second, the biomolecular material must be formulated in a way that allows delivery of the biomolecular material from an implanted device into a desired environment of operation over an extended period time. This second challenge has proven particularly difficult where the biomolecular material is included in a flowable composition that is delivered from a device over an extended period of time at low flow rates (i.e., 100 .mu.L/day).

Peptide drugs such as C-peptide may degrade via one or more of several different mechanisms, including deamidation, oxidation, hydrolysis, and racemization. Significantly, water is a reactant in many of the relevant degradation pathways. Moreover, water acts as a plasticizer and facilitates the unfolding and irreversible aggregation of biomolecular materials. To work around the stability problems created by aqueous formulations of biomolecular materials, dry powder formulations of biomolecular materials have been created using known particle formation processes, such as by known lyophilization, spray-drying, or desiccation techniques. Though dry powder formulations of biomolecular material have been shown to provide suitable stability characteristics, it would be desirable to provide a formulation that is not only stable over extended periods of time, but is also flowable and readily deliverable from an implantable delivery device.

Accordingly in one aspect of any of the claimed methods and kits, the C-peptide is provided in a nonaqueous drug formulation, and is delivered from a sustained release implantable device, wherein the C-peptide is stable for at least two months of time at 37.degree. C.

Representative nonaqueous formulations for C-peptide include those disclosed in International Publication Number WO00/45790 that describes nonaqueous vehicle formulations that are formulated using at least two of a polymer, a solvent, and a surfactant.

WO98/27962 discloses an injectable depot gel composition containing a polymer, a solvent that can dissolve the polymer and thereby form a viscous gel, a beneficial agent, and an emulsifying agent in the form of a dispersed droplet phase in the viscous gel.

WO04089335 discloses nonaqueous vehicles that are formed using a combination of polymer and solvent that results in a vehicle that is miscible in water. As it is used herein, the term "miscible in water" refers to a vehicle that, at a temperature range representative of a chosen operational environment, can be mixed with water at all proportions without resulting in a phase separation of the polymer from the solvent such that a highly viscous polymer phase is formed. For the purposes of the present invention, a "highly viscous polymer phase" refers to a polymer containing composition that exhibits a viscosity that is greater than the viscosity of the vehicle before the vehicle is mixed with water.

Accordingly in another aspect of any of the claimed methods and kits, C-peptide is provided in a sustained release device comprising: a reservoir having at least one drug delivery orifice, and a stable nonaqueous drug formulation. In one aspect of these methods and kits, the formulation comprises: at least C-peptide; and a nonaqueous, single-phase vehicle comprising at least one polymer and at least one solvent, the vehicle being miscible in water, wherein the drug is insoluble in one or more vehicle components and the C-peptide formulation is stable at 37.degree. C. for at least two months. In one aspect, the solvent is selected from the group consisting of glycofurol, benzyl alcohol, tetraglycol, n-methylpyrrolidone, glycerol formal, propylene glycol, and combinations thereof.

In particular, a nonaqueous formulation is considered chemically stable if no more than about 35% of the C-peptide is degraded by chemical pathways, such as by oxidation, deamidation, and hydrolysis, after maintenance of the formulation at 37.degree. C. for a period of two months, and a formulation is considered physically stable if, under the same conditions, no more than about 15% of the C-peptide contained in the formulation is degraded through aggregation. A drug formulation is stable according to the present invention if at least about 65% of the C-peptide remains physically and chemically stable after about two months at 37.degree. C.

C-peptide for use in the present invention may also be administered topically, (intra)dermally, or transdermally to the skin or mucosa. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages, and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Penetration enhancers may be incorporated--see, e.g., Finnin and Morgan: J. Pharm. Sci. 88(10): 955-958, (1999). Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis, and microneedle or needle-free injection (e.g., the systems sold under the trademarks POWDERJECT.TM., BIOJECTT.TM.).

Formulations for topical administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.

In another embodiment of a sustained release composition of C-peptide, the C-peptide is packaged in a liposome, which has demonstrated utility in delivering beneficial active agents in a controlled manner over prolonged periods of time. Liposomes are completely closed bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles possessing a single membrane bilayer or multilamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous layer. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) tails of the lipid orient toward the center of the bilayer while the hydrophilic (polar) heads orient towards the aqueous phase.

Generally, in a liposome-drug delivery system, the active agent is entrapped in the liposome and then administered to the patient to be treated. However, if the active agent is lipophilic, it may associate with the lipid bilayer. The immune system may recognize conventional liposomes as foreign bodies and destroy them before significant amounts of the active agent reaches the intended disease site. Thus in one embodiment, the liposome may be coated with a flexible water-soluble polymer that avoids uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen. Suitable hydrophilic polymers for surrounding the liposomes include, without limitation, polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxethylacrylate, hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol, polyaspartamide and hydrophilie peptide sequences as described in U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; 6,043,094; the contents of which are incorporated by reference in their entirety.

Liposomes may be comprised of any lipid or lipid combination known in the art. For example, the vesicle-forming lipids may be naturally-occurring or synthetic lipids, including phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phasphatidylglycerol, phosphatidylinositol, and sphingomyelin as disclosed in U.S. Pat. Nos. 6,056,973 and 5,874,104. The vesicle-forming lipids may also be glycolipids, cerebrosides, or cationic lipids, such as 1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-;I-(2,3-ditetradecyloxy)propyl; -N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N 2,3-dioleyloxy)propyl; N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE); N;I-(2,3-dioleyloxy)propyl N,N,N-trimethylammonium chloride (DOTMA); 3; N--(N',N'-dimethylaminoethane) carbamoly; cholesterol (DC-Choi); or dimethyldioctadecylamnionium (DDAB) also as disclosed in U.S. Pat. No. 6,056,973. Cholesterol may also be present in the proper range to impart stability to the vesicle as disclosed in U.S. Pat. Nos. 5,916,588 and 5,874,104.

The liposomes for use in any of the methods or kits of the invention can be manufactured by standard techniques known to those of skill in the art. For example, in one embodiment, as disclosed in U.S. Pat. No. 5,916,588, a buffered solution of the active agent is prepared. Then a suitable lipid, such as hydrogenated soy phosphatidylcholine, and cholesterol, both in powdered form, are dissolved in chloroform or the like and dried by rotoevaporation. The lipid film thus formed is resupsended in diethyl ether or the like and placed in a flask, and sonicated in a water bath during addition of the buffered solution of the active agent. Once the ether has evaporated, sonication is discontinued and a stream of nitrogen is applied until residual ether is removed. Other standard manufacturing procedures are described in U.S. Pat. Nos. 6,352,716; 6,294,191; 6,126,966; 6,056,973; 5,965,156; and 5,874,104. The liposomes of this invention can be produced by any method generally accepted in the art for making liposomes, including, without limitation, the methods of the above-cited documents (the contents of which are incorporated herein by reference).

Liposomes are also well-known in the art and readily available from companies experienced in providing liposomes for extended release drug delivery. For example, ALZA's (formerly Sequus Pharmaceutical's) liposomal technology for intravenous drug delivery sold under the trademark STEALTH.RTM. uses a polyethylene glycol coating on liposomes to evade recognition by the immune system; Gilead Sciences (formerly Nexstar's) liposomal technology sold under the trademark AMBISOME.RTM., and FDA approved treatment for fungal infections; and NOF Corp. offers a wide variety of Good Manufacturing Practice (GMP)-grade phospholipids, phospholipids derivatives, and PEG-phospholipids sold under the tradenames COATSOME.RTM. and SUNBRIGHT.RTM..

A search of patents, published patent applications, and related publications will also provide those skilled in the art reading this disclosure with significant possible liposomal technologies. U.S. Pat. Nos. 6,759,057; 6,406,713; 6,352,716; 6,316,024; 6,294,191; 6,126,966; 6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 5,215,680; and 4,684,479; the contents of which are incorporated herein by reference, describe liposomes and lipid-coated microbubbles, and methods for their manufacture. Thus, one skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could produce a liposome for the sustained release of C-peptide.

In another embodiment of the present invention, the sustained release of C-peptide into the blood comprises a sustained release composition comprising C-peptide that is packaged in a microsphere. Microspheres have demonstrated utility in delivering beneficial active agents to a target area in a controlled manner over prolonged periods of time. Microspheres are generally biodegradable and can be used for subcutaneous, intramuscular, and intravenous administration.

Generally, each microsphere is composed of an active agent and polymer molecules as disclosed in U.S. Pat. No. 6,268,053, the active agent may be centrally located within a membrane formed by the polymer molecules, or, alternatively, dispersed throughout the microsphere because the internal structure comprises a matrix of the active agent and a polymer excipient. Typically, the outer surface of the microsphere is permeable to water, which allows aqueous fluids to enter the microsphere, as well as solubilized active agent and polymer to exit the microsphere.

In one embodiment, the polymer membrane comprises cross-linked polymers as disclosed in U.S. Pat. No. 6,395,302. When the pore sizes of the cross-linked polymer are equal or smaller than the hydrodynamic diameter of the active agent, the active agent is essentially released when the polymer is degraded. On the other hand, if the pore sizes of the cross-linked polymers are larger than the size of the active agent, the active agent is at least partially released by diffusion.

Additional methods for making microsphere membranes are known and used in the art and can be used in the practice of the invention disclosed herein. Typical materials for the outer membrane include the following categories of polymers: (1) carbohydrate-based polymers, such as methylcellulose, carboxymethyl cellulose-based polymers, dextran, polydextrose, chitins, chitosan, and starch (including hetastarch), and derivatives thereof; (2) polyaliphatic alcohols such as polyethylene oxide and derivatives thereof including PEG, PEG-acrylates, polyethyleneimine, polyvinyl acetate, and derivatives thereof; (3) polyvinyl polymers such as polyvinyl alcohol, polyvinylpyrrolidone, poly(vinyl)phosphate, poly(vinyl)phosphonic acid, and derivatives thereof; (4) polyacrylic acids and derivatives thereof; (5) polyorganic acids, such as polymaleic acid, and derivatives thereof; (6) polyamino acids, such as polylysine, and poly-imino acids, such as polyimino tyrosine, and derivatives thereof; (7) co-polymers and block co-polymers, such as poloxamer 407 or Pluronic L-101; polymer, and derivatives thereof; (8) tert-polymers and derivatives thereof; (9) polyethers, such as poly(tetramethylene ether glycol), and derivatives thereof; (10) naturally-occurring polymers, such as zein, chitosan and pullulan, and derivatives thereof; (11) polyimids, such as poly n-tris(hydroxymethyl)methylmethacrylate, and derivatives thereof; (12) surfactants, such as polyoxyethylene sorbitan, and derivatives thereof; (13) polyesters such polyethylene glycol) (n) monomethyl ether mono(succinimidyl succinate)ester, and derivatives thereof; (14) branched and cyclo-polymers, such as branched PEG and cyclodextrins, and derivatives thereof; and (15) polyaldehydes, such as poly(perfluoropropylene oxide-b-perfluoroformaldehyde), and derivatives thereof as disclosed in U.S. Pat. No. 6,268,053, the contents of which are incorporated herein by reference. Other typical polymers known to those of ordinary skill in the art include poly(lactide-co-glycolide), polylactide homopolymer; polyglycolide homopolymer; polycaprolactone; polyhydroxybutyrate-polyhydroxyvalerate copolymer; poly(lactide-co-caprolactone); polyesteramides; polyorthoesters; poly 13-hydroxybutyric acid; and polyanhydrides as disclosed in U.S. Pat. No. 6,517,859, the contents of which are incorporated herein by reference.

In one embodiment, the microsphere of the present invention are attached to or coated with additional molecules. Such molecules can facilitate targeting, enhance receptor mediation, and provide escape from endocytosis or destruction. Typical molecules include phospholipids, receptors, antibodies, hormones, and polysaccharides. Additionally, one or more cleavable molecules may be attached to the outer surface of microspheres to target it to a predetermined site. Then, under appropriate biological conditions, the molecule is cleaved causing release of the microsphere from the target.

The microspheres for use in the sustained release compositions are manufactured by standard techniques. For example, in one embodiment, volume exclusion is performed by mixing the active agent in solution with a polymer or mixture of polymers in solution in the presence of an energy source for a sufficient amount of time to form particles as disclosed in U.S. Pat. No. 6,268,053. The pH of the solution is adjusted to a pH near the isoelectric point (pI) of the macromolecule. Next, the solution is exposed to an energy source, such as heat, radiation, or ionization, alone or in combination with sonication, vortexing, mixing or stirring, to form microparticles. The resulting microparticles are then separated from any unincorporated components present in the solution by physical separation methods well-known to those skilled in the art and may then be washed. Other standard manufacturing procedures are described in U.S. Pat. Nos. 6,669,961; 6,517,859; 6,458,387; 6,395,302; 6,303,148; 6,268,053; 6,090,925; 6,024,983; 5,942,252; 5,981,719; 5,578,709; 5,554,730; 5,407,609; 4,897,268; and 4,542,025; the contents of which are incorporated by reference in their entirety. Microspheres are well-known and readily available to one of ordinary skill in the art from companies experienced in providing such technologies for extended release drug delivery. For example, Epic Therapeutics, a subsidiary of Baxter Healthcare Corp., developed, a protein-matrix drug delivery system sold under the trademark PROMAXX.RTM. that produces bioerodible protein microspheres in a totally water-based process; OctoPlus developed, cross-linked dextran microspheres sold under the trademark OCTODEX.RTM. that release active ingredients based on bulk degradation of matrix rather than based on surface erosion.

A search of patents, published patent applications, and related publications will also provide those skilled in the art reading this disclosure with significant possible microsphere technologies for use in formulating sustained release compositions. For example, U.S. Pat. Nos. 6,669,961; 6,517,859; 6,458,387; 6,395,302; 6,303,148; 6,268,053; 6,090,925; 6,024,983; 5,942,252; 5,981,719; 5,578,709; 5,554,730; 5,407,609; 4,897,268; and 4,542,025; the contents of which are incorporated by reference in their entirety, describe microspheres and methods for their manufacture. One skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could make and use microspheres for the sustained release of C-peptide for use in any of the methods or kits claimed herein.

The C-peptide can be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, e.g., in a dry blend with lactose, or as a mixed component particle, e.g., mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler, as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electro hydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane, or as nasal drops. For intranasal use, the powder may comprise a bioadhesive agent, e.g., chitosan or cyclodextrin.

The pressurized container, pump, spray, atomizer, or nebulizer contains a solution or suspension of the compound(s) of the invention comprising, e.g., ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

Prior to use in a dry powder or suspension formulation, the drug product is micronized to a size suitable for delivery by inhalation (typically less than 5 .mu.m). This may be achieved by any appropriate method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

Capsules (made, e.g., from gelatin or hydroxypropylmethylcellulose), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose.

A suitable solution formulation for use in an atomizer using electro hydrodynamics to produce a fine mist may contain from 1 .mu.g to 20 mg of C-peptide per actuation and the actuation volume may vary from 1 .mu.L to 100 .mu.L. A typical formulation may comprise C-peptide propylene glycol, sterile water, ethanol, and sodium chloride. Alternative solvents that may be used instead of propylene glycol include glycerol and polyethylene glycol. Suitable flavors, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration. Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release using, e.g., PGLA. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve that delivers a metered amount. Units in accordance with the invention are typically arranged to administer a metered dose or "puff" containing from 0.1 mg to 10 mg of C-peptide. The overall daily dose will typically be in the range 0.1 mg to 20 mg that may be administered in a single dose or, more usually, as divided doses throughout the day.

Kits are also contemplated for this invention. A typical kit would comprise a container, preferably a vial, for the C-peptide formulation comprising C-peptide in a pharmaceutically acceptable formulation, and instructions, and/or a product insert or label. In one aspect, the instructions include a dosing regimen for administration of said C-peptide to an insulin-dependent patient to reduce the risk, incidence, or severity of hypoglycemia. In one aspect, the kit includes instructions to reduce the administration of insulin by about 5% to about 35% when starting C-peptide therapy. In another aspect, the instructions include directions for the patient to closely monitor their blood glucose levels when starting C-peptide therapy. In another aspect, the instructions include directions for the patient to avoid situations or circumstances that might predispose the patient to hypoglycemia when starting C-peptide therapy.
 

Claim 1 of 20 Claims

1. A method for treating an insulin-dependent human patient, comprising the steps of: a. administering insulin to said patient, wherein said patient has neuropathy; b. administering subcutaneously to said patient a therapeutic dose of C-peptide in a different site as that used for said patient's insulin administration; c. adjusting the dosage amount, type, or frequency of insulin administered based on monitoring said patient's altered insulin requirements resulting from said therapeutic dose of C-peptide, wherein said adjusted dose of insulin reduces the risk, incidence, or severity of hypoglycemia, wherein said adjusted dose of insulin is at least 10% less than said patient's insulin dose prior to starting C-peptide treatment.
 

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