HK1150793A - Rapid acting injectable insulin compositions - Google Patents

Description

Injectable rapid acting insulin composition

Cross Reference to Related Applications

This patent application claims priority from U.S. patent application No.11/869,693 filed by Roderike Pohl and Solomon s.steiner on 2007, 10, 9.

Technical Field

The present invention is generally in the field of injectable, rapid acting drug delivery insulin formulations.

Background

Overview of diabetes

Glucose is a monosaccharide that is utilized by all cells of the body to produce energy and sustain life. Whenever a person requires a minimum level of glucose in their blood to sustain life. The main way the body produces blood glucose is by digesting food. When a person cannot take this glucose from food digestion, the glucose is produced from stores within the tissue or released by the liver. The glucose level of the body is controlled by insulin. Insulin is a peptide hormone naturally secreted by the pancreas. Insulin helps glucose enter the cells of the body to provide an important source of energy.

When a healthy individual begins to eat, the pancreas secretes a natural peak of insulin, referred to as first-phase insulin secretion. In addition to providing sufficient insulin to process glucose entering the blood from food digestion, first-phase insulin secretion signals the liver to stop preparing glucose while food digestion is taking place. Because the liver does not produce glucose and there is sufficient insulin to process glucose from digesta, the blood glucose levels of healthy individuals remain relatively constant and their blood glucose levels do not become too high.

Diabetes is a disease characterized by abnormally high blood glucose levels and inadequate levels of insulin. There are two major types of diabetes-type 1 and type 2. In type 1 diabetes, the body does not produce insulin. In the early stages of type 2 diabetes, the pancreas produces insulin, but either the body does not produce insulin at the right time or the body cells ignore insulin, a condition known as insulin resistance.

One of the first effects of type 2 diabetes, even before any other symptoms are the loss of the first phase of insulin secretion caused by food. In the absence of first phase insulin secretion, the liver cannot receive a signal to stop producing glucose. As a result, the liver continues to produce glucose as the body begins to produce new glucose by food digestion. As a result, diabetic patients have postprandial blood glucose levels that become too high, a condition known as hyperglycemia. Hyperglycemia causes glucose to attach abnormally to certain proteins in the blood, interfering with the normal function of proteins in maintaining the integrity of small blood vessels. With hyperglycemia occurring after each meal, it eventually leads to rupture and leakage of the fine blood vessels. Long-term adverse effects of hyperglycemia include blindness, renal failure, nerve damage and loss of sensation, and poor peripheral blood circulation, and may also require amputation of limbs.

Between 2 and 3 hours after a meal, the blood glucose of untreated diabetics rises to such an extent that the pancreas receives signals to secrete unusually large amounts of insulin. In early type 2 diabetic patients, the pancreas can still respond to and secrete large amounts of insulin. However, these phenomena occur when digestion is nearly over and blood glucose levels should begin to drop. This unusually large amount of insulin causes two adverse effects. First, it places undue extreme demands on the pancreas that has been damaged, which can lead to its more rapid deterioration, ultimately rendering the pancreas incapable of producing insulin. Second, after digestion, too much insulin results in weight gain, which can further exacerbate the condition.

Current treatment of diabetes and its limitations

Because type 1 diabetics do not produce insulin, the primary treatment for type 1 diabetes is intensive daily insulin therapy. Treatment of type 2 diabetes usually begins with diet control and exercise. While helpful in the short term, treatment with diet and exercise alone is not an effective long-term treatment regimen for the vast majority of type 2 diabetics. When diet and exercise were no longer effective, various non-insulin oral medications were initiated. These oral drugs act by increasing the amount of insulin produced by the pancreas, increasing the sensitivity of insulin-sensitive cells, decreasing glucose production by the liver, or some combination of these mechanisms. These treatments are limited in their ability to effectively control the disease and often have significant side effects such as weight gain and hypertension. Over time, the condition of many type 2 diabetic patients worsens due to the limitations of non-insulin therapy, and eventually insulin therapy is required to support their metabolism.

Insulin therapy has been used to treat diabetes for over 80 years. The treatment method typically involves the administration of several injections of insulin daily. These injections include 1 or 2 daily administrations of long-acting basal injections, as well as fast-acting insulin injections at meals. While this treatment regimen is considered effective, it has limitations. First, patients often dislike injecting themselves with insulin because of the inconvenience and needle pain. As a result, patients tend to fail to adequately comply with prescribed treatment regimens and are often improperly dosed.

More importantly, even when administered correctly, insulin injections do not replicate the natural time-effect characteristics of insulin. In particular, the natural peak of first-phase insulin secretion in people without diabetes results in elevated blood insulin levels within minutes after glucose from food enters the blood. In contrast, injected insulin enters the blood slowly, with peak insulin levels occurring within 80 to 100 minutes after injection of regular human insulin.

A viable solution is to inject insulin directly into the vein of a diabetic patient prior to a meal. In the insulin intravenous study, patients showed better control of their blood glucose 3 to 6 hours after a meal. However, the method of intravenous injection of insulin before each meal is not a practical treatment method for various medical reasons.

One of the important improvements in insulin therapy is the introduction of rapid acting insulin analogs, such as Humalog, in the nineties of the twentieth century、NovoLogAnd Apidra. However, even with rapid-acting insulin analogs, peak insulin levels typically occur within 50 to 70 minutes after injection. Since rapid-acting insulin analogs do not adequately mimic first-phase insulin secretion, diabetic patients using insulin therapy still have inadequate insulin levels at the beginning of a meal, with insulin remaining between mealsIn excess of insulin. This lag in insulin delivery can lead to hyperglycemia early after meals. In addition, too much insulin between meals may result in abnormally low blood glucose levels, which is referred to as hypoglycemia. Hypoglycemia can result in loss of mental acuity, confusion, increased heart rate, hunger, sweating, and dizziness. At very low glucose levels, hypoglycemia can result in loss of consciousness, coma, and even death. Diabetes patients taking insulin have an average of 1.2 severe hypoglycemic events per year as recorded by the american diabetes association or ADA, with most events requiring the patient to visit a hospital emergency room.

Since the time course of insulin delivery to the blood plays such an important role in overall control of blood glucose, there is a clear need for injectable insulin that reaches the blood more rapidly than rapid acting insulin analogs.

It is therefore an object of the present invention to provide an injectable rapid acting insulin composition with improved stability and rapid onset of action.

Summary of The Invention

Injectable insulin formulations having improved stability and rapid onset of action are described herein. The formulation may be administered subcutaneously, intradermally, or intramuscularly, and in a preferred embodiment, the formulation is administered by subcutaneous injection. The formulation contains insulin in combination with a chelating agent, and a dissolution agent, and optionally an additional excipient. In a preferred embodiment, the formulation contains human insulin, a zinc chelator (e.g., EDTA) and a dissolution agent (citric acid). When these formulations are administered by subcutaneous injection, they are rapidly absorbed into the blood stream.

In a preferred embodiment, the insulin is provided in the form of a dry powder within a sterile vial. Prior to or at the time of administration, it is mixed with a diluent containing a pharmaceutically acceptable carrier (e.g., water), a zinc chelator (e.g., EDTA), and a dissolution agent (citric acid). In another embodiment, the insulin is stored as a frozen mixture and ready for use after thawing.

Brief description of the drawings

FIG. 1 is a three-dimensional schematic of insulin showing the exposed surface charge and shielding of the charge by covering with appropriately sized molecules ("lytic and chelating agents").

Fig. 2 is a schematic diagram of a transwell device 10 for measuring insulin absorption from a feeding chamber 12 into a receiving chamber 18 through 4-5 layers of immortalized oral epithelial cells 14 on a 0.1 micron filter 16.

Figures 3a and 3b are graphs comparing in vitro insulin transport (accumulated insulin in micro units) through oral epithelial cells in the transwell system shown in figure 2 with and without 0.45mg EDTA/ml, depending on the change in acid selected as the lytic agent. EDTA was constant at 0.45mg/mL and the acid concentration varied as follows: FIG. 3a, aspartic acid (0.47mg/mL), glutamic acid (0.74mg/mL), succinic acid (0.41mg/mL), adipic acid (0.73mg/mL), and citric acid (0.29mg/mL and 0.56mg/mL), with a pH range of 3.2-3.8. FIG. 3b, maleic acid (0.32mg/mL), fumaric acid (1.28mg/mL), and oxalic acid (0.32mg/mL), pH range 2-3. Two time points (10 min and 30 min) were selected for comparative analysis. The results are the mean of 4 determinations plus or minus the standard error.

FIGS. 4a and 4b are graphs of insulin transport (cumulative insulin in units of micro units) through oral epithelial cells in vitro and in vivo in the transwell system shown in FIG. 2 with and without 0.56mg EDTA/mL for comparison of different lytic agents, where the acid is in the following equimolar (1.50X 10)^-3Mol) concentration: aspartic acid (0.20mg/mL), glutamic acid (0.22mg/mL), and citric acid (0.29mg/mL) (FIG. 4a) and citric acid (1.80mg/mL) (FIG. 4 b). Two time points (10 min and 30 min) were selected for comparative analysis.

FIG. 5 is insulin transport through oral epithelial cells in vitro and in vivo in the transwell system shown in FIG. 2 to compare different chelatorsGraph of efficacy. Measurement of insulin transport (accumulation of insulin, micromolar) through oral epithelial cells from a solution containing glutamic acid, citric acid or HCl and added at equimolar concentration (4.84X 10)^-3Moles) of different chelating agents. The chelators were no chelator (control), EDTA, EGTA, DMSA, CDTA and TSC.

FIG. 6 shows the relationship with HUMALOG(12U) and HUMULIN R(12U) graph of the in vivo pharmacodynamic effects of insulin prepared with citric acid and EDTA (12U) in human subjects, measured as mean GIR/kg, compared to each other.

FIG. 7 shows a graph of HUMULIN RIn contrast, a graph of the in vivo pharmacokinetic effect of insulin prepared with citric acid and EDTA on insulin concentration (micro units/ml) in human subjects as a function of time (minutes). The mean value of insulin doses (+ SEM, n ═ 10) was 12U/subject.

FIG. 8 shows a graph of the relationship with HUMULIN RAnd HUMALOGIn contrast, insulin prepared with citric acid and EDTA is a plot of pharmacodynamics in vivo as blood glucose (mg/dl) as a function of time (minutes) in 16 type 2 diabetic patients. The dose used for the patient trial is specific to the individual patient, which is adjusted for each patient based on their current insulin therapy regimen.

Detailed Description

An insulin formulation of injectable human insulin as described herein is administered either before or immediately after a meal. In a preferred embodiment, the formulation is used in combination with recombinant human insulin and specific components that are generally recognized as safe by the FDA. The formulations are designed to be absorbed into the blood more rapidly than currently marketed fast-acting insulin analogues. One of the important features of the insulin preparation is that it dissociates or separates the six molecular or hexameric forms of insulin, thereby forming a single molecular or monomeric form of insulin, and preventing reassociation to the hexameric form. It is believed that by preferentially forming the monomeric form, the formulation allows insulin to be more rapidly delivered to the blood as the single molecule form of insulin required by the human body before the formulation can be absorbed into the body to produce its desired biological effect. Most commercially available human insulin for injection is in the hexameric form. This makes it more difficult to absorb into the body, since the insulin hexamer must first dissociate to form a dimer and then a monomer.

I. Definition of

As used herein, unless otherwise indicated, "insulin" refers to recombinant, purified, or synthetic insulin or insulin analogs, human or non-human.

As used herein, "human insulin" is a human peptide hormone secreted by the pancreas, either isolated from a natural source or prepared from a genetically engineered microorganism. As used herein, "non-human insulin" is insulin identical to human insulin, except that it is derived from an animal source, such as porcine or bovine.

An insulin analogue as used herein is a modified insulin, which, although different from insulin secreted by the pancreas, can still produce the same effect on the body as native insulin. Through DNA-based genetic engineering, the amino acid sequence of insulin can be altered, thereby altering its ADME (uptake, partitioning, metabolism, and excretion) characteristics. Examples thereof include insulin lispro, insulin glargine, insulin aspart, insulin glulisine, insulin detemir. These insulins can also be engineered chemically, for example by acetylation. A human insulin analogue as used herein is an improved human insulin capable of performing the same effect as human insulin.

As used herein, "chelating agent" refers to a compound capable of forming a single bond or multiple bonds with a zinc ion. The bond is typically an ionic or coordinate bond. The chelating agent may be an inorganic or organic compound. A chelate complex is a complex in which a metal ion is bonded to two or more atoms of a chelating agent.

As used herein, a "solubilizer" is a compound that increases the solubility of a substance in a solvent, such as insulin, in an aqueous solution. Examples of solubilizing agents include surfactants (TWEENS)) (ii) a Solvents such as ethanol and the like; micelle (micell) forming compounds such as oxyethylene monostearate and the like; and a pH adjusting agent.

As used herein, a "lytic agent" is an acid that, when added to insulin and EDTA, enhances insulin transport and absorption relative to HCl and EDTA under the same pH conditions, as determined using the epithelial transwell plate assay described in the examples below. HCl is not a dissolving agent but may be a solubilizing agent. Citric acid is a dissolving agent when determined by this assay.

As used herein, an "excipient" is an inert substance other than a chelating or solubilizing agent, which may be used as an insulin carrier or to aid in the product manufacturing process. In this case, excipients are used to dissolve or mix the active substance.

Preparation II

The formulation comprises insulin, a chelator and a dissolution agent, optionally together with one or more other excipients. In a preferred embodiment, the formulation is suitable for subcutaneous administration and is rapidly absorbed into the adipose subcutaneous tissue. The selection of the lytic and chelating agents, the concentration of both the lytic and chelating agents, and the adjusted pH of the formulation all have profound effects on the efficacy of the system. When multiple combinations are functional, preferred embodiments are selected for a variety of reasons, including safety, stability, general characteristics, and performance.

In a preferred embodiment, at least one of the formulation ingredients is selected to shield any charge on the active agent. This may facilitate transmembrane transport of insulin, thereby accelerating the onset and bioavailability of insulin. The ingredients are also selected to form a composition that dissolves rapidly in an aqueous medium. Preferably, insulin is rapidly absorbed and transported into the plasma, thereby having a rapid onset of action (preferably, onset occurs within about 5 minutes after administration and peaks at about 15-30 minutes after administration).

Chelating agents such as EDTA chelate the zinc in the insulin, thereby removing the zinc from the insulin solution. This allows insulin to take its dimeric and monomeric forms and prevents re-association to form hexamer morphology. Since both forms exist in a concentration-driven equilibrium state, more monomer is produced as it is absorbed. Thus, as the monomer is absorbed through the subcutaneous tissue, additional dimers depolymerize to form more monomer. The molecular weight of the monomeric form is less than one sixth of the molecular weight of the hexameric form, thus significantly increasing the rate and amount of insulin absorption. In the case of chelators (e.g., EDTA) and/or lytic agents (e.g., citric acid) that hydrogen bond with insulin, it is believed that they shield the charge on insulin, thereby facilitating its transport across the membrane, thereby increasing the onset and bioavailability of insulin.

Insulin

The insulin may be recombinant or purified from natural sources. The insulin may be human or non-human insulin. Preferably human insulin. In a most preferred embodiment, the insulin is a human recombinant insulin. Recombinant human insulin is obtained from a number of sources. The insulin may also be an insulin analogue, which may be based on the amino acid sequence of human insulin but with one or more different amino acids, or may be a chemically modified insulin or insulin analogue.

The dosage of insulin depends on its bioavailability and the patient to be treated. The dosage form comprises insulin generally in a dose of 1.5-100 IU/human, preferably 3-50 IU/human.

Dissolving agent

As shown in fig. 1, certain acids appear to shield the charge on insulin, enhancing uptake and transport. In contrast to hydrochloric acid, acids including acetic acid, ascorbic acid, citric acid, glutamic acid, aspartic acid, succinic acid, fumaric acid, maleic acid, and adipic acid were effective as solubilizing agents, as determined by the transwell plate assay described in the examples below. For example, if the active agent is insulin, the preferred dissolution agent is citric acid. In combination with any formulation, hydrochloric acid can be used to adjust the pH, but it is not a dissolution agent.

The range of solubilizers corresponding to an effective amount of citric acid in combination with insulin and EDTA is 9.37X 10^-4M citric acid to 9.37X 10^-2M is citric acid.

Chelating agents

In a preferred embodiment, the zinc chelator is mixed with the active agent. The chelating agent may be ionic or non-ionic. Suitable chelating agents include ethylenediaminetetraacetic acid (EDTA), EGTA, alginic acid, alpha-lipoic acid, dimercaptosuccinic acid (DMSA), CDTA (1, 2-cyclohexanediaminetetraacetic acid), trisodium citrate (TSC). Hydrochloric acid may be used in conjunction with the TSC to adjust the pH and in the process form citric acid as a dissolution agent.

In a preferred embodiment, the chelating agent is EDTA. For example, when the active agent is insulin, the chelator is known to abstract zinc from the insulin, and therefore, the insulin is more advantageously maintained in a dimeric form relative to a hexamer form and promotes insulin absorption to the surrounding tissue (e.g., mucosal or adipose tissue). In addition, the chelating agent may hydrogen bond with the active agent, thereby facilitating charge shielding of the active agent and facilitating transmembrane delivery of the active agent.

The effective amount of chelating agent corresponding to EDTA in combination with insulin and citric acid ranges from 2.42X 10^-4M EDTA to 9.68X 10^-2M EDTA.

Excipient

Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. For example, Pharmaceutical formulations are discussed in Hoover, John E.S., Pharmaceutical Sciences, Remington, eds (Mack publishing company, Easton, Pa., USA (1975)), and in Pharmaceutical Dosage Forms, edited by Liberman, H.A., and Lachman, L.

In a preferred embodiment, the insulin preparation comprises one or more solubilizers to allow rapid dissolution in aqueous media. Suitable solubilizing agents include: wetting agents such as polysorbates, glycerols and poloxamers, non-ionic and ionic surfactants, food acidulants and food alkalizing agents (e.g. sodium bicarbonate), alcohols, and buffer salts for pH control.

Stabilizers are used to inhibit or delay drug decomposition reactions, such as oxidation reactions. A variety of stabilizers may be used. Suitable stabilizers include: polysaccharides such as cellulose and cellulose derivatives; simple alcohols such as glycerol; bacteriostatic agents such as phenol, m-cresol and methylparaben; isotonic agents such as sodium chloride, glycerol and glucose; lecithins such as natural lecithins (e.g. egg yolk lecithin or soybean lecithin) and synthetic or semi-synthetic lecithins (e.g. dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine or distearoyl phosphatidylcholine); phosphatidic acid; phosphatidylethanolamine; phosphatidylserines such as distearoylphosphatidylserine, dipalmitoylphosphatidylserine, and dineotetraenoylphosphatidylserine; phosphatidylglycerol; phosphatidylinositol; cardiolipin; sphingomyelin. In one example, the stabilizing agent may be a combination of glycerin, a bacteriostatic agent, and an isotonic agent.

Preparation method of preparation

The injectable formulation comprises insulin, a chelator and a dissolution agent. In a preferred embodiment, the injectable formulation comprises insulin, EDTA, citric acid, and saline, and/or glycerol.

In a preferred embodiment, the subcutaneously injectable formulation is prepared by mixing saline and/or glycerol, citric acid and EDTA to form a solution and sterilizing the solution (which will be referred to as "diluent"). The insulin is separately added to sterile water to form a solution, filtered, and the specified amounts are separately added to a number of sterile injection vials. The insulin solution is lyophilized to form a powder, which should be stored separately from the diluent in order to maintain its stability. The dilution is added to the insulin injection bottle prior to administration. After subcutaneous injection of a prescribed amount of insulin to the patient, the remaining insulin solution may be stored, preferably refrigerated.

In another embodiment, insulin may be combined with a diluent and, prior to use, sterile filtered to form a multiple use injection and frozen.

Methods of use of the formulations

The formulation may be administered subcutaneously or intramuscularly. The formulation is designed to be rapidly absorbed and transported into the plasma for systemic delivery.

A type 1 or type 2 diabetic patient may be administered a formulation comprising insulin as the active agent before or between meals. The composition can stop converting glycogen into glucose in the liver due to rapid absorption, thereby preventing hyperglycemia, which is a major cause of diabetic complications and is the first symptom of type 2 diabetes, from occurring. In order to stop producing glucose in the liver, the currently available standard subcutaneous injections of human insulin must be administered about half an hour to 1 hour prior to a meal, but produce less than the expected effect because insulin absorption is too slow to stop producing glucose in the liver. In addition, the subcutaneous insulin composition may be able to slow or stop the progression of type 2 diabetes if the injection is given early in the development of the condition.

The invention will be further understood by reference to the following non-limiting examples.

Example 1: in vitro comparison of insulin uptake and transport according to changes in lytic agents using epithelial cell Transwell assay

Materials and methods

As shown in fig. 2, oral epithelial cells were grown on a transwell chamber (transwell insert) for 2 weeks until a multi-layered (4-5-layered) cell layer was formed. The transport test was performed by adding the appropriate solution to the supply well (donor well) and removing the sample from the receiving well after 10 minutes. The solution consisted of water, +/-EDTA (0.45mg/ml), NaCl (0.85% w/v), 1mg/ml insulin and an amount of acid sufficient to maintain a pH of 3.8. The amount of insulin in the receiving wells was analyzed by ELISA.

Results

The results shown in figures 3a and 3b demonstrate that some acids are more effective in enhancing the uptake and transport of insulin through epithelial cells. These acids can be easily tested and compared to the results obtained using HCl, providing a standard for any acid that can be tested and determining whether it is a lytic agent (e.g., improved uptake and transport relative to HCl).

The results obtained using acids with a pH range of 3.2-3.8 are shown in FIG. 3 a. The stronger acid (pH < 3) is shown in FIG. 3 b.

This result demonstrates that acid selection has a substantial effect on insulin transport through cell culture when using chelating agents at the same concentration as the acid. The preferred acid is citric acid.

Example 2: in vitro comparison of insulin uptake and transport according to changes in lytic agent concentration using epithelial cell Transwell assay

Materials and methods

The materials and methods of example 1 were used except for the concentration of the reagents. In this test, equimolar concentrations of acid and chelating agent were added. The solution consisted of water, +/-EDTA (0.56mg/mL), NaCl (0.85% w/v), 1mg/mL of insulin, and an acid which was: aspartic acid (0.20mg/mL), glutamic acid (0.22mg/mL), or citric acid (0.29 mg/mL). Citric acid was tested at a higher concentration of 1.8mg/mL, in the presence or absence of chelating agents. The data show data for two time points, 10 minutes and 30 minutes after cell administration to the chamber.

Results

The results obtained using aspartic acid (0.20mg/mL), glutamic acid (0.22mg/mL) or citric acid (0.29mg/mL) are shown in FIG. 4 a. In this case, no significant difference was found in the addition of the chelating agent.

In contrast, in the experiment using a higher concentration of 1.80mg/mL of citric acid, a significant improvement was shown after adding the chelating agent to the solution (t-test comparison, single sided). Please refer to fig. 4 b. This result demonstrates that the concentration of both components is important for optimal uptake and transport.

Example 3: in vitro comparison of insulin uptake and transport based on changes in chelator, using epithelial Transwell assay

Materials and methods

Oral epithelial cells were grown on a transwell chamber for 2 weeks until a multi-layered (4-5 layers) cell layer was formed. Transport tests were performed by adding the appropriate solution to the supply wells and removing the samples from the receiving wells after 10, 20 and 30 minutes.

Immediately prior to the transwell test, the solution was prepared as follows: 1.8mg/ml citric acid was dissolved in 0.85% w/v saline and then to this solution was added a chelating agent at the concentration shown below: 1.80mg/ml EDTA, 1.84mg/ml EGTA, 0.88mg/ml DMSA and 1.42mg/ml TSC. Since liquid CDTA was used, citric acid was added directly to the CDTA. In each case, the concentration of chelating agent was always 4.84X 10^-3And (3) mol.

Subsequently, 1mg/ml of insulin was added and the pH was readjusted to 3.8 if necessary. Control samples using only pH-corrected HCl were included for comparison. Alginic acid solidifies at pH3.8, so this example is not to be compared. Transwell tests were performed by adding 0.2ml of each solution to the supply wells.

The amount of insulin in the receiving wells was analyzed by ELISA.

Results

The data curve 30 minutes after insulin administration is shown in figure 5. When citric acid or glutamic acid was used, more insulin was delivered by the cells except when compared to the results obtained with TSC (sodium citrate). In the case of TSC, HCl was used for pH correction. The pH correction causes TSC to produce citric acid, which may explain the above results.

These results demonstrate that the increase in uptake and transport depends on the choice of chelator.

Example 4: preclinical evaluation of chelators in citric acid-based insulin formulations in pigs

Materials and methods

The pharmacokinetics of fast acting insulin analogues, insulin aspart, were studied in rats, dogs and pigs, as well as the pharmacodynamics of insulin aspart in pigs, as disclosed in a. In Drug meta. 155-60(2000), elimination half-life is a useful determinant in determining insulin absorption, since elimination delay means slow absorption from the injection site. Therefore, in order to detect PK and PD parameters, in particular, elimination half-life, a non-compartmental analysis study of pigs was performed.

One of four insulin preparations was injected subcutaneously into diabetic pigs. Three formulations contained a chelator (EDTA, EGTA or TSC) and a fourth control formulation contained only conventional human insulin Humulin RWithout the inclusion of a chelating agent. In all formulations containing chelating agents, citric acid was used as acid and in all cases NaCl and m-cresol were added for isotonicity and sterility of the formulation. The concentrations of the chelating agents are all 4.84X 10^-3And (3) mol.

Pigs were fasted overnight and injected subcutaneously with 0.125U/kg human insulin containing EDTA (n ═ 3) or 0.08U/kg human insulin containing EGTA or TSC (n ═ 2). Because blood glucose is very low when high doses are used, the dose is reduced. Blood glucose and insulin levels were measured at all time points within 8 hours after dosing.

Pharmacokinetic models were analyzed using WinNonlin using a uniformly weighted non-compartmental model. In table 1, the elimination half-lives are compared.

Table 1: comparison of blood glucose in pigs as a function of chelating agent

Insulin	Half-life "lamda z" (min. +/-sd) terminal half-life
		Humulin/chelator free	120
insulin/EDTA	39.1+/-15.8
		insulin/EGTA	37.5+/-8.0
insulin/TSC	30.1+/-9.0

In preliminary experiments, the elimination half-life (120 min) of conventional human insulin in pigs was consistent with data published in the literature and used as test index for validating the data. This elimination half-life greatly exceeds the time after intravenous injection, thus demonstrating that slow absorption from the injection site still occurs after injection. The formulation of citric acid with a chelator significantly reduced this parameter, demonstrating that these three chelators were effective in increasing the uptake and transport of conventional human insulin, albeit to a different extent.

Example 5: comparison of EDTA-citric acid insulin formulations with regular human insulin in human clinical trials

Materials and methods

The objective of this study was to evaluate the Pharmacodynamic (PD) properties of the test formulation "CE" containing insulin in combination with citric acid and EDTA. For 10 fasting healthy volunteers (mean age 40(20-62 years range); BMI 22.5 (19.2-2)4.9)kg/m²) 5 normal glucose clamp tests were performed. An orthogonal design with a fixed treatment order was used, with 12IU of regular insulin and 12IU of CE insulin preparation injected subcutaneously in the abdominal region.

Results

The results are shown in FIGS. 6 and 7. The time-effect profile of CE by subcutaneous injection was: the glucose consumption was more significantly accelerated than when conventional human insulin was used (fig. 6). The mean pharmacokinetic data confirmed the PD results (fig. 7).

This experiment shows that the time to reach maximum concentration is faster (figure 7) and the onset is faster (figure 6) for conventional human insulin with citric acid and EDTA added compared to the formulation with conventional human insulin alone, which demonstrates that it improves the rate of absorption of insulin.

Example 6: pharmacokinetics and pharmacodynamics of CE, lispro and regular human insulin when administered subcutaneously to type 1 diabetics immediately before meals

Background and objects

The purpose of this test is to determine the VIAject after a standard meal for type 1 diabetic patients^TM(very fast acting formulation of Regular Human Insulin (RHI) in combination with citric acid and EDTA, denoted CE above), the effect of RHI and insulin Lispro (Lispro) on postprandial Blood Glucose (BG) excursions

Materials and methods

9 patients (5 male and 4 female; age 40 + -10 years, body mass index ("BMI") 24.0 + -2.0 kg/m) were stabilized by glucose clamp (glucose clamp) prior to meals²) Blood glucose ("BG") (target BG 120 mg/dl). Infusion of glucose was stopped prior to the standard meal and administration of insulin. Using an orthogonal design with a fixed treatment order, the same patient was given a subcutaneous injection of a specific dose of VIAject immediately before meal^TM(CE), Lispro or RHI. Followed byBlood glucose was monitored continuously for 8 hours and glucose infusion was resumed if BG < 60 mg/dl. The insulin level in the plasma was determined throughout the experiment.

Results

After a meal was given to type 2 diabetic patients, regular human insulin, citric acid and EDTA-supplemented insulin (CE) and insulin lispro were injected subcutaneously and the insulin Tmax was compared as mean ± standard deviation, the results of which are shown in table 2. The results of comparing blood glucose of the same test subjects are shown in table 3.

Table 2: comparison of insulin Tmax (minutes)

Pharmacokinetics	RHI	Lispro	VIAjectTM(CE)
				Insulin peak time	143±29*	62±37	43±36*

^*p <. 001, paired t-test

Table 3: insulin pharmacokinetic blood glucose comparison

Pharmacokinetics (0-180 minutes)	RHI	Lispro	VIAjectTM(CE)
				Blood sugar peak time (minutes)	93±56*	47±28	41±26*
Highest blood sugar (mg/dL)	185±44	158±33	157±27
				Minimum blood glucose value (mg/dL)	103±21	73±31	87±24
Blood glucose maximum-blood glucose minimum (mg/dL)	82±30	84±11*	70±18*

^*p < 0.05, paired t-test

The total number of hypoglycemic events occurring 3 to 8 hours after injection (time required for glucose infusion) is: 13 pieces were used for RHI, 11 pieces were used for Lispro, and 4 pieces were used for CE. To prevent hypoglycemia, the average total amount of glucose is infused over the above-mentioned time period, using RHI more than using VIAject^TM6 times higher for (CE), and more for Lispro than for VIAject^TM(CE) 2 times. The sum of the areas above and below the normoglycemic target zone (BG Area Under Curve (AUC) above 140mg/dL, below 80mg/dL) calculated for all patients per group was: 81,895mg/dL min with RHI, 57,423mg/dL min with Lispro, and VIAject^TM38,740mg/dL min. The mean blood glucose levels are shown in figure 8.

To summarize, VIAject^TM(CE) twist the standard postprandial rise in blood glucose is the fastest acting. By VIAject^TMThe treated patient experienced a decrease in postprandial blood glucose excursions. In contrast, RHI is used with the highest degree of blood glucose excursion, which corresponds to its lowest rate of absorption. VIAject was used compared to Lispro^TMThe change in glucose level (average difference between the highest and lowest values) was significantly smaller, confirming VIAject^TMBetter control of blood glucose in these type 1 diabetic patients.

It will be apparent to those skilled in the art from this disclosure that modifications and variations of the present invention are within the scope of the appended claims.

Claims (13)

1. An injectable insulin composition comprising insulin and an effective amount of a dissolution agent and a zinc chelator to increase uptake and transport of the insulin through epithelial cells as compared to insulin in combination with a zinc chelator and HCl.

2. The composition of claim 1, wherein the agent is selected from the group consisting of human insulin and insulin analogs.

3. The composition of claim 2, wherein the agent is human insulin.

4. The composition of claim 1, wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), EGTA, trisodium citrate (TSC), alginic acid, alpha-lipoic acid, dimercaptosuccinic acid (DMSA), CDTA (1, 2-cyclohexanediaminetetraacetic acid).

5. The composition of claim 4, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

6. The composition of claim 1, wherein the dissolution agent is an acid selected from the group consisting of acetic acid, ascorbic acid, citric acid, glutamic acid, succinic acid, aspartic acid, maleic acid, fumaric acid, and adipic acid.

7. The composition of claim 6, wherein the dissolution agent is citric acid.

8. The composition of claim 1, wherein the chelating agent is present in a concentration range corresponding to 2.42 x 10^-4M to 9.68X 10^-2M EDTA.

9. The composition of claim 1, wherein the concentration of the dissolution agent is in a range corresponding to 9.37 x 10^-4M to 9.37X 10^-2M citric acid.

10. The composition of claim 1, wherein the zinc chelator is EDTA, the dissolution agent is citric acid, and the chelator is present in a concentration range corresponding to 2.42 x 10^-4M to 9.68X 10^-2M EDTA, in a concentration range corresponding to 9.37X 10^-4M to 9.37X 10^-2M citric acid.

11. The composition of claim 1, wherein the insulin is disposed in a first container in the form of a dry powder and the at least one of a chelator and dissolution agent is disposed in a second container comprising a diluent.

12. The composition of claim 1, provided in the form of a frozen pharmaceutically acceptable medicament for the treatment of diabetes.

13. A method of treating a diabetic individual, the method comprising injecting an effective amount of an injectable insulin composition comprising insulin and an effective amount of a dissolution agent and a zinc chelator to increase uptake and transport of the insulin through epithelial cells as compared to insulin in combination with a zinc chelator and HCl.