KR20190090061A - Formulations for the treatment of diabetes - Google Patents

Abstract

There is disclosed a parenteral dosage form comprising an insulin and aprotic polar solvent comprising a pH memory from 1 to 4 or 6 to 8, wherein the insulin is solubilized within the aprotic polar solvent and the solubilized insulin is a stable insulin Or a combination thereof, wherein the water content of the formulation is equal to or less than 15% w / v.

Description

[0001] FORMULATIONS FOR THE TREATMENT OF DIABETES [0002]

The present invention claims the benefit of U.S. Provisional Application No. 61 / 609,123 (filed on March 9, 2012) and U.S. Provisional Application No. 61 / 553,388 (filed on August 31, 2011), the contents of which are incorporated herein by reference Integrated.

A. Field of the Invention

The present invention relates to insulin formulations for parenteral administration. Such formulations may comprise a stabilized monomeric or dimeric form of insulin, thereby accelerating the rate of insulin absorption within the bloodstream of the subject.

B. Describing related technology

* Type 1 diabetics rarely produce insulin, so the primary treatment for type 1 diabetes is exogenous insulin therapy. In addition, due to the limitations of non-insulin therapy, many patients with type 2 diabetes eventually require insulin therapy. Historically, insulin has been used for over 90 years to treat diabetes. It involves administering multiple injections of insulin daily with a common diet: sustained action basal insulin is once or twice a day, rapid acting insulin is meal time. Although these therapies are recognized as effective, they have limitations. First, patients are generally reluctant to inject insulin themselves because of the pain and discomfort of needles. As a result, patients tend not to follow prescribed therapy properly. More importantly, even when properly administered, insulin products injectable at meal times do not adequately mimic the natural physiology of human insulin. In particular, the first-stage response within a person who is not diabetic is due to insulin spikes as well as increased blood insulin levels within a few minutes of blood glucose inflow from the meal. Blood insulin levels will be between 30 and 60 minutes after initiation of the reaction. In contrast, when insulin is slowly injected into the blood, the maximum observed concentration (Cmax) occurs 90 minutes or more after the injection of normal human insulin.

Various types of therapeutic insulin and insulin analogs have been developed to achieve other pharmacokinetic (PK) profiles, such as balancing the time for the beginning of the reaction and the peak of plasma insulin during the reaction. A key improvement in insulin therapy was the introduction of a rapid-acting insulin genome including Humalog®, Novolog® and Apidra®. But to talk about these analogs, a pick of insulin levels typically occurs in about 60 minutes after injection. Failure of the insulin product, which properly mimics the first step of insulin secretion currently marketed, results in insufficient insulin levels at the beginning of the meal and excess insulin levels in the diet, which may be due to hypoglycemia early in the meal and physiological effects of hypoglycemia Lt; / RTI > Both of these situations present significant problems with the promise of a closed circuit of artificial pancreatic technology, a complex algorithm that is required to manage both latencies.

In the treatment of diabetic patients through insulin, the basic route of the injection of exogenous insulin is subcutaneous, and the basic parameter of the PK profile depends on the subcutaneous absorption. A number of variables (e.g., blood flow, rate of diffusion, and connection status) affect the absorption of insulin injected subcutaneously. When blood flow is appropriate, the rate-limiting factor for the absorption of soluble insulin is (i) the transport between capillaries by diffusion and (ii) the restriction of transmission on capillary membranes, both dominated by the size of the molecule (For example, the connection state of insulin).

Typically, the insulin formulations are aqueous-based. One of the reasons for this is that most of the human body is composed of water containing plasma, which is an aqueous environment. It is therefore a natural tendency to inject drug formulations compatible with the environment in which the drug is intended to arrive. Insulin is generally present as a pharmaceutical composition in a stabilized form of a zinc-bound complex, while the monomer and dimer forms are more readily absorbed into the blood stream because of their smaller size compared to the insulin form of the insulin . The monomeric insulin in the aqueous solution is unstable, forms amyloid fibrils, and degrades through it as a mediator. While the hexahedral structure promotes stability within the solution (pH 5-8), it also interferes with diffusion and subsequent absorption. In addition, the volume of the syringe reservoir will also be affected by the diffusion, which results in a larger volume and a slower diffusion rate. The combination of factors is primarily responsible for delaying the onset of plasma insulin levels and response.

Insulin analogs have also been developed where amino acid sequences are altered to reduce the tendency to self-connect while receptor-binding affinity is maintained, in order to promote subcutaneous absorption and prevent insulin degradation and fibrosis in aqueous solutions. These types of insulins are often referred to as "dimeric" insulins, but they are in fact very weakly linked to the body. The absorption of these substances will still be retarded because it depends on the diffusion and subsequent reduction within the subcutaneous concentrations required to break down the hexaamer and dimer / monomer. Equilibrium insulin analogues that support the monomeric state (such as the insulin analog lispro) show a more rapid absorption and short reaction times. However, these analog molecules tend to be less stable and more irreversibly aggregated under thermal and mechanical stress compared to mixed insulin. In addition, such aggregation not only reduces the dosage of available insulin, but can also lead to a patient ' s immune response or stimulation. In addition, experimental and epidemiological studies on the induction of prolonged signaling and tumor proliferation of receptor tissue by several new insulin analogues have also been made. Despite these shortcomings, insulin analogues are expensive - about twice the normal human insulin.

The present invention provides a solution to the current problem faced by insulin formulations. The present invention resides in a dry insulin in a buffer for making a dried form of insulin which is dissolved in an aprotic polar solvent and reconstituted and then maintained at a desired pH. The resulting formulations include dissolved and stabilized monomeric and dimeric forms of insulin. In particular, the formulation may have a relatively low amount of water (20, 15, 10, 5, 4, 3, 2, 1% or less), or may be non- By reducing the volume of insulin contained in the formulation administered to the subject. In addition, the present invention allows both non-denatured or native and denatured or analog forms of insulin to be used. In other words, insulin analogs can be used in the present invention, while non-denatured / natural insulin can be used, and insulin is all held in stabilized monomeric and dimeric forms.

 In one aspect of the invention, there is disclosed a formulation comprising an insulin and an aprotic polar solvent in which the pH memory is 1 to 4 (or 1 to 3 or about 2) or 6 to 8 (or 6.5 to 7.5 or about 7) The insulin may be dissolved in the apical polar solvent and the dissolved insulin may be a stabilized monomer or dimer form of insulin or a mixture thereof and the water content of the formulation is 20, 15, 10, 5, 4 , 3, 2, 1% w / v or w / w or less (e.g., anhydrous). The formulations may be used for parenteral administration. In certain embodiments, the non-polar magnetic polar solvent is selected from the group consisting of dimethylsulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, dimethylformamide DMF), dimethylacetamide (DMA), propylene carbonate, or a mixture thereof. In certain embodiments, the aprotic polar solvent may be dimethylsulfoxide (DMSO). In one embodiment, the formulation comprises an insulin of 3 mg / ml to 50 mg / ml, 3 mg / ml to 10 mg / ml or 10 mg / ml to 50 mg / ml. In another aspect, it is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound is administered at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 70, 75, 80. 90, 100 mg / mL or more, or as needed or any range therein. In one embodiment, the majority of the insulin in the formulation is in monomeric or dimeric form or a combination of monomeric and dimeric forms. The formulation may further comprise a component capable of reducing the monomeric or dimeric form of aggregate of insulin. Non-limiting examples of such components include urea, guanidinium chloride, amino acids, sugars, polyols, polymers, acids, surfactants, or mixtures thereof. In certain embodiments, the acid may be acetic acid, ascorbic acid, citric acid, glutamic acid, aspartic acid, succinic acid, fumaric acid, maleic acid, adipic acid or a mixture thereof. The formulation may contain a cosolvent. In one non-limiting embodiment, the co-solvent is water. In one embodiment, the formulation does not include zinc, and includes zinc, which is a chelating agent to reduce / reduce the likelihood of, or to contain, low amounts of zinc. In certain embodiments, the insulin may be pre-dried in a non-volatile buffer and the buffer mentioned may have a pH range of 1 to 4 or 1 to 3 or about 2 or 6 to 8 or 6.5 to 7.5 or about 7 have. Examples of non-volatile buffers may be glycine buffers, citrate buffers, phosphate buffers, or mixtures thereof. In one embodiment, the buffer may comprise a chelating agent. Non-limiting examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), tartaric acid, glycerin, or citric acid, or any combination thereof. The formulations may also contain ingredients capable of lowering the freezing point of the aprotic polar solvent to about 0 ° C, and non-limiting examples of such ingredients include water, sugars, sugar alcohols, or mixtures thereof. In some cases, the insulin may be unmodified or natural human insulin. In another embodiment, the composition may further comprise an insulin adjuvant such as an amylin analog. The amylin analog may be dissolved in the formulation. A non-limiting example of an amylin analog is pramlintide. The pramlineide can be processed to have a pH memory of 1 to 5 or 2, 3 or 4 or about 2. In certain embodiments of the co-formulations, the pH memory of the insulin may be about 2, and the pH memory of the flavin tide may be about 2. In certain embodiments, the step of treating the pramlintide may comprise drying the indicated pramlintide in a non-volatile buffer, wherein the buffer referred to comprises from 1 to 5 or 2, 3, or 4 or about 2 Lt; / RTI > The moisture content in formulations comprising insulin and pramlintide may be 5-20% w / v or w / w or 5-15% w / v or w / w or 7-12% w / v or w / w or 8 to 10% w / v or w / w or about 9% w / v or w / w. The formulation may be in liquid form. The formulation may be a solution. In certain embodiments, at least 50, 60, 70, 80, or 90% or more of the insulin in the formulation can be chemically and physically stabilized and maintained when the formulation is stored at room temperature for one month . In one embodiment, the formulation may be configured within the container. The container may be a syringe, a pen injection device, an auto-injector device, a pump, or a perfusion bag. In certain embodiments, the aprotic polar solvent may be a continuous phase of the formulation. The formulation may comprise at least 75, 80, 85, 90, 95, 96, 97, 98, 99 or% w / v or w / w of the aprotic polar solvent. The insulin in the formulation may be metastable.

Also disclosed is a method for reducing blood glucose levels comprising administering to a subject in need of any of said formulations of the invention an effective amount to reduce blood glucose levels within a subject. The subject may be a person (adult or child), an animal (e.g., chimpanzee, horse, cow, pig, rabbit, rat, mouse, etc.). In certain embodiments, the blood glucose level of the subject is reduced within 10, 20, 30, 60 or 90 minutes after administration. In one embodiment, the subject's initial 1/2 Tmax blood insulin level occurs within 10, 20, 30, 40, 50, or 60 minutes or 30 to 60 minutes after administration. The subject may already be diagnosed with Type-I or Type-II diabetes and may be sensitive to the development of Type-I or Type-II diabetes. In one embodiment, the formulation is administered within 30, 20, 15, 10, 5 or 1 minutes prior to ingestion of the food by the subject, or 1, 5, 10, 15, 20 or 30 minutes.

Also disclosed is a method of making the formulation of the present invention. The method may comprise a dry mixture comprising a non-volatile buffer for drying insulin and dried insulin, wherein the dried insulin is from 1 to 4 (or from 2 to 3 or about 2) or from 6 to 8 7.5, or about 7), and thereafter reconstituting the dried insulin within the apical polar solvent, wherein the insulin is soluble in the aprotic polar solvent and the dissolved insulin is insulin And the moisture content of the formulation may be in the range of 20, 15, 10, 5, 4, 3, 2, 1% w / v or w / w or less (E. G., Anhydrous). ≪ / RTI > The method may further comprise a dry mixture comprising an amylin analog and a dried amylin analog and a second nonvolatile buffer to obtain a dried amylin analogue, wherein the dried amylin analogue in the aprotic polar solvent And the dried amylin analog can be dissolved in a non-apolar polar solvent. As noted above, the amylin analog may be pramlintide and may be treated with a pH memory of a particular embodiment of 1 to 5 or 2, 3 or 4 or about 2. In certain embodiments of the formula, the pH memory of the insulin may be about 2, and the pH memory of the promintide may be about 2. The second non-volatile buffer may have a pH range of from 1 to 5, or about 2, 3 or 4 or especially about 2. Wherein the amount of the formulation is in the range of 5-20% w / v or w / w or 5-15% w / v or w / w or 7-12% w / v or w / w or 8-10% w / v or w / w, or about 9% w / v or w / w.

Another particular feature of the formulation of the present invention is that it is prepared immediately for parenteral administration, optionally contained in a container, and stored as needed without diluting or reconstituting the formulation. The container in which the formulation is to be stored may thus be a syringe, pen injection device, auto-injector device, pump or perfusion bag. Non-limiting examples of additional additives / pharmacological agents that are also contemplated for use in the formulation include: antioxidants (e.g., ascorbic acid, cysteine, methionine, Monothioglycerol, sodium thiosulfate, sulfites, BHT, BHA, ascorbyl palmitate, propyl gallate or vitamin E); Chelating agents (including, for example, EDTA, EGTA, tartaric acid, glycerin or citric acid); Or preservatives (including, for example, alkyl alcohols, benzyl alcohols, methyl parabens or propyl parabens or mixtures thereof). The formulations may be in liquid, semi-solid or gel form. As described below, the formulations may have a desired viscosity range (in one non-limiting example, this range may be from 0.5 to 15 cps).

It is contemplated that any embodiment referred to herein may be implemented with respect to any method or composition of the present invention, and vice versa. In addition, the compositions of the present invention may be used to achieve the methods of the present invention.

"Insulin" means human, non-human, recombinant, conformational, and / or synthetic (e.g., modified insulin or insulin analog) analogs. "Human insulin" means human peptide hormone insulin secreted from the pancreas - it can be isolated from a natural source that can be made from an organism manipulating organism, such as manufactured or purchased through synthetic chemistry. "Non-human insulin" is insulin derived from an animal (e. G., Pig, cattle, etc.).

"Modified insulin" or "insulin analogue" is a modified form of insulin that is different from natural (e.g., chemical alterations, other structures, other amino acid sequences) - It is applicable to perform functions such as modified insulin. For example, through genetic engineering of DNA coding, the amino acid sequence of the insulin may be altered to alter its ADME (adsorption, distribution, metabolism and / or excretion) properties . Modified insulin or insulin analogs include Lispro®, Aspart®, Glulisine®, Detemir®, Degludec®, and the like. Non-modified or rolled insulin includes natural or naturally occurring amidodic acid arrangements.

"Stabilized insulin" means an insulin that is not irreversibly aggregated in the formulation or lost activity if the formulation is not administered. Once insulin is absorbed into the blood, activity is maintained. Without wishing to be bound by theory, it is believed that insulin within the formulations of the invention is considered to be "quasi-stabilized" while the shape of the dissolved insulin can be altered, and once the insulin is absorbed or administered into the blood Go back to the natural form. In addition, structural changes in insulin within the formulation reduce the likelihood of monomer and dimer aggregation of adjuvants or other insulins such as amylin analogs in the formulation. The monomeric insulin form means insulin inside the monomeric form. The dimeric form of insulin means insulin in the form of a dimer (e. G., Two monomers linked together or linked together). Combined insulin forms refer to insulin in the form of a combinatorial form (eg, a serotyped form linked together or bound together).

"Zinc-free" or "low-zinc" means that the formulation contains about 0.6% or less (eg, 0.5, 0.4, 0.3, 0.2, 0.1, 0%) of zinc relative to the insulin content, or 6 insulin monomers Zinc ion or less (e.g., 2, 1, 0).

By "aprotic polar solvent" is meant a polar solvent that does not contain acidic hydrogen and does not act as a hydrogen bond donor. As noted above, non-limiting examples include dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone NMP), dimethylacetamide (DMA), and propylene carbonate.

"Parenteral administration" means the infusion of a formulation through or through one or more layers of skin or mucous membranes of an animal such as a human. Standard parenteral administration is provided within the subcutaneous or muscular region of an animal such as, for example, a human patient. This deep position is a goal because the tissue is more easily inflated, a relatively thin skin position, and to accommodate the injection volume to provide insulin formulations. The administration may be by a needle, a pump, an injection device, a catheter, or the like.

By "pharmaceutically acceptable carrier" is meant an acceptable solvent, which is a suspending reagent or solution for the pharmaceutical delivery of insulin to a mammal such as a human or animal.

May be suitable for use in humans and / or animals without side effects (such as toxicity, irritation, and allergic response) corresponding to a reasonable benefit / risk ratio among the "pharmaceutically acceptable" components, excipients or components.

"Biocompatibility" may be suitable for use in humans and / or animals without side effects (such as toxicity, irritation, and allergic response) corresponding to reasonable benefit / risk ratios.

"Bioavailability" means the extent to which insulin is absorbed from the formulation into the subject.

The "effect on the whole body" of the administration or delivery of insulin towards the subject means that the therapeutic agent can be detected at a significant biological level in the plasma of the subject.

"Patient", "subject" or "individual" means a mammal (eg, a human, a primate, a dog, a cat, a cow, a sheep, a pig, a horse, a rat, a speed, a hamster, a rabbit or a guinea pig) .

"Suppression" or "decrease, " or any variation of such term, includes any measurable reduction or complete inhibition to achieve the desired result when used in the claims and / or the detailed description.

&Quot; Effective "or" treatment "or" prevention, " or any variation of such term is sufficient to achieve the intended, expected, intended result when used in the claims and / or the detailed description.

The term " about "or" approximately "is defined as being close to that understood by those of ordinary skill in the art and includes, by way of example and not limitation, within 10%, preferably within 5%, more preferably within 1% And is preferably defined within 0.5%. In addition, "substantially water-insoluble" means that the volume or volume of water is less than 5%, 4%, 3%, 2%, 1% or less.

The use of the term " one "or" one "when used in the claims and / or the detailed description with the word" comprising " Matches "one or more than one".

It should be understood that the terms "comprising" and "comprising" as well as any form of having, such as "have" and "has", "includes" (including any form of including such as "include") and "containing" (including any form containing "contains" and "contain") do not exclude the inclusion or addition of constituent or method steps that are open and not mentioned .

The components and methods for their use may "comprise," " comprise, "or" constitute "any component or step disclosed herein. In one non-limiting embodiment, for a modified form of "essential composition ", the basic and novel features of the formulations and methods disclosed herein are the monomeric and / or dimeric forms of insulin in the formulations Stability and solubility. Therefore, ingredients that may affect the stability or solubility of the monomeric and / or dimeric forms of insulin in the formulation will be excluded from the formulations mentioned in the examples of the claims using modified forms of "essentially constitutional ".

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the description and examples are given by way of illustration only, while indicating specific embodiments of the invention. In addition, changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description.

The following drawings form part of the present invention and are included to further illustrate certain aspects of the present invention. The invention may be better understood with reference to one or more of these drawings in conjunction with the detailed description of specific embodiments presented below.
Figure 1 shows the FTIR spectra of DMSO / insulin and aqueous / insulin formulations.
Figure 2 shows the molecular weights of DMSO / insulin and aqueous / insulin formulations.
Figure 3 shows the hydrodynamic radius distribution when Ins-E is 50 mg / mL in DMSO. The horizontal axis is the algebraic spacing lattice of the hydrodynamic radius value (the other adjacent points by a factor of ~ 1.3). The analysis covers a radius range of ~ 0.01 nm to ~ 20 nm. Picks of 0.01 to 0.1 nm, which are artifacts arising from the backward pulse of the photodetector, are suppressed.
4 is a hydrodynamic radius distribution when Ins-E is 30 mg / mL in DMSO. (See the graph in FIG. 3)
5 is a hydrodynamic radius distribution when Ins-E is 10 mg / mL in DMSO. (See the graph in FIG. 3)
6 is a hydrodynamic radius distribution when Ins-E is 3 mg / mL in DMSO. (See the graph in FIG. 3)
7 is a hydrodynamic radius distribution when Ins-H ₂ O is 10 mg / mL in the buffer E. (See the graph in FIG. 3)
8 is a hydrodynamic radius distribution when Ins-H ₂ O is 10 mg / mL in the buffer F (see the graph of FIG. 3)

As described above, problems associated with formulations of monomeric or dimeric forms of insulin for parenteral administration are well documented. Current solutions to these problems are also well known and accepted as standard practice in the field of formulation. For example, insulin analogues / modified insulin have been prepared to reduce self-binding affinity to prevent the formation of derivatives of derivatives. Such analogs are generally administered or administered in an aqueous environment, which reduces their stability and causes irreversible aggregation to occur more readily as flocculation occurs. In addition, these analogs can be expensive and induce a patient ' s immune response or stimulation.

On the contrary, the present inventor has found a solution to the above-mentioned problem. The solution consists in preparing insulin with a specific pH memory and reconstituting and melting the insulin in aprotic polar solvents. The insulin thus obtained can have a small amount of water, or can not have water, and the formulation includes dissolved and stabilized monomeric and dimeric forms of insulin. In addition, the increased solubility of insulin in aprotic polar solvents results in low volume formulations with large amounts of monomeric and dimeric forms of insulin. In particular, the formulations can be used for modified or unmodified insulin. In the case of unmodified insulin, problems associated with the use of modified / analogous insulin molecules such as inflammation, immune response and cost can be avoided.

Embodiments of the present invention and other non-limiting embodiments are described below.

A. Insulin

Insulin helps the body store or use blood sugar from food. People with type 1 diabetes do not make insulin anymore. People with type 2 diabetes make insulin, but their body's response to it is not efficient or appropriate, and is often called insulin resistance.

Insulin itself is a well-known and characteristic peptide hormone. The monomeric form of human insulin is composed of a B chain coupled by a disulfide bond and a 51 amino acid chain having two peptide chains called the A chain. In most species, the A chain consists of 21 amino acids and the B chain consists of 30 amino acids. Although the amidoxyl sequence of insulin varies from species to species, certain segments of the molecule are very well conserved. This similarity in the amino acid sequence of insulin leads to a three-dimensional three-dimensional structure of insulin very similar to each species, and insulin from one animal can react with other species biologically. For example, pig insulin is widely used for human patients. Monomeric forms of insulin can be linked together to form dimers. The dimer may be linked together to form a typically occurring complex in the presence of zinc.

Both the monomeric and dimeric forms of insulin readily diffuse into the bloodstream. In contrast, hexagons diffuse poorly in many areas due to their considerably large size. As noted above, this is advantageous in the context of the present invention and is not limited to the use of commercially available modified insulin or insulin analogues (e.g., Lispro®, Aspart®, Glulisine®, Detemir®, Degludec®, and others). In addition, conventional unmodified insulins are also readily commercially available (e.g., Humulin ® R, Humulin ® N, Humulin ® 70/30, Novolin ®, Or the like) or may also be used in the context of the present invention. In certain embodiments, the normal / unmodified form of insulin may be used in place of the modified form to reduce the cost of the formulation, immunity or allergic costs. Insulin is currently produced from a variety of manufacturers, including pharmaceutical companies and subcontracted pharmaceutical companies. Pharmaceutical companies include Eli Lilly, Co., Novo Nordisk, and Sanofi. Subcontractors include Sigma-Aldrich, Lonza and Biocon. Insulin used as an example of the detailed description of the present invention was a recombinant unmodified human insulin purchased from Sigma-Aldrich (St. Louis, Mo.).

B. pH memory

The present inventors have also found a step of preparing such that the insulin dissolved in the formulation can be used more stabilized. This step involves mixing the non-volatile buffer in the aqueous solution with insulin followed by drying the mixture to obtain dried insulin. Prior to the drying step, the aqueous solution has a pH range of 1 to 4 or 6 to 8, which is the optimum pH for the stabilization of insulin in an aqueous environment. Thus, as soon as the mixture is dried, it produces dried insulin with a "pH memory" of 1 to 4 or 6 to 8, so that after the insulin dissolved in the aprotic polar solvent has dried, the pH memory is maintained do. In certain embodiments wherein the pramlintide is additionally included, the insulin pH memory can be about 2, and the promontinide pH memory can be about 2 days.

In particular, the "pH memory" of insulin is the charge profile (proton state) obtained after the step of drying the insulin from a buffered aqueous solution (for example, from a non-volatile buffer). The proton state, i.e. the solubility and stability of insulin in the aprotic polar solvent, is affected by the pH of the aqueous insulin mixture or solution prior to the drying step. When insulin is dried in a buffer species that is both basic and acid non-volatile, the pH memory of the dried insulin will approximate the pH of the aqueous insulin mixture or solution. See, for example, Enzymatic Reactions in Organic Media , Koskinen, AMP, and Klibanov, AM, eds., Springer (1996). In addition, the pH of the insulin-dried buffered aqueous solution (non-volatile buffer) is optimized for optimal stability, maximum solubility and minimal degradation of insulin when the reconstituted insulin is subsequently reconstituted in the apical polar solvent pH memory can be provided. Thus, when dried insulin in such a solvent is reconstituted, insulin in the reconstituted formulation will maintain the solubility and stability characteristics of the optimized pH memory.

The pH memory of insulin can be measured in a variety of ways. As one method, the pH memory may be determined by reconstituting the dried insulin within the unbuffered water and measuring the pH of the reconstituted insulin mixture or solution through a pH meter, such as a pH paper or a calibrated pH electrode . Alternatively, the pH memory may be measured by adding at least 20% water to the insulin / aprotic polar solvent formulation and measuring the pH of the formulation with a pH meter. See, for example, Baughman and Kreevoy, "Determination of Acidity in 80% Dimethyl Sulfoxide-20% Water," Journal a / Physical Chemistry, 78 (4): 421-23 (1974). The pH measurement inside the aprotic polar solvent-water solution may require minor corrections (for example, below 0.2 pH units per Baughman and Kreevoy).

In view of the above, useful nonvolatile buffers in the formulations described herein are those useful for setting the pH of maximum stability / minimum degradability, as well as those useful for removing moisture content or residual moisture of insulin. Non-volatile buffers include buffers that will not evaporate in a manner similar to dry / freeze-dried water. For example, suitable non-volatile buffers include glycine buffers, citrate buffers, phosphate buffers, and the like. In a particular embodiment, the non-volatile buffer is a glycine buffer or a citrate buffer.

The step of drying insulin, such as a non-volatile buffer, is carried out using spray-drying techniques, freeze-drying techniques, freeze-drying techniques, vacuum centrifugation techniques, and the like. Spray drying techniques are well known to those skilled in the art. Spray drying involves spraying a solution containing one or more solids (e.g., therapeutic) through a nozzle spinning disk or other device by evaporating the solvent from the droplets. The nature of the resulting powder is a function of various parameters including the initial solute concentration, the size distribution of the droplets produced, and the solute removal rate. The produced particles may comprise agglomerates of primary particles composed of crystalline and / or amorphous solids depending on the state and proportion of the solute removal rate. For example, reference is made to U.S. Patent No. 6,051,256 for spray-drying steps for the preparation of super-fine powders of pharmaceuticals. The freeze-drying step is well known in the art, see, for example, U.S. Patent No. 4,608,764 and U.S. Patent No. 4,848,094. For example, the spray-freeze-drying step is referred to U.S. Pat. No. 5,208,998. Other spray-drying techniques are described in U.S. Patent Nos. 6,253,463; 6,001,336; 5,260,306; And PCT International Patent Nos. WO 91/16882 and WO 96/09814.

Freeze-drying techniques are well known in the art. Freeze-drying is a dehydration technique that occurs while the product is frozen and dried through a mild heat treatment under vacuum (ice sublimation under vacuum). These conditions stabilize the product and minimize oxidation and other degradation processes. The freeze-drying conditions allow the process to proceed at low temperatures, and thus thermally unstable products can be protected. The freeze-drying step includes a pre-treatment, a freezing, a main drying and a sub-drying step. The pretreatment includes any method of pretreating the product prior to refrigeration. This may include flocculating the product, modifying the formulation (e.g., adding stability or increasing the composition for step enhancement), decreasing the step of increasing the vapor pressure, or increasing the surface area. Methods of pretreatment include frozen agglomeration, solution phase agglomeration and specific formulations to protect the appearance of the product or to provide lyoprotection of the reactive product, see, for example, U.S. Patent 6,199,297. "Standard" lyophilization conditions are described, for example, in U.S. Patent Application 5,031,336 and "Freeze Drying of Pharmaceuticals" (DeLuca, Patrick P., J. Vac. Sci. Technol., Vol. 14, No. 1, January / February 1977 ) And "The Lyophilization of Pharmaceuticals: A Literature Review" (Williams, NA, and GP Polli, Journal of Parenteral Science and Technology, Vol.38, No. 2, March / April 1984).

In certain embodiments, the lyophilization cycle can be performed in part on the glass transition temperature (Tg) of insulin, which leads to the collapse of mass to form a dense cake containing residual moisture. In another embodiment, the lyophilization cycle was performed below the glass transition temperature of insulin to prevent collapse to achieve complete drying of the insulin particles.

C. non-magnetic polar solvent

After the dried insulin with the selected pH memory is obtained, the dried insulin can be dissolved and reconstituted in the aprotic polar solvent. The aprotic polar solvent includes a solvent which is insufficient in the hydrogen (Acidic Hydrogen) of the acid. This feature is useful for maintaining the pH memory of dried insulin. Non-limiting examples of aprotic polar solvents include dimethylsulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, n-methyl pyrrolidone (NMP) ), Dimethylacetamide (DMA), propylene carbonate, or mixtures thereof. Each of these solvents is well known and is commercially available from a variety of sources.

As shown in the examples, dissolved insulin causes stabilized monomeric or dimeric forms of insulin and may result in super-fast or rapid-response insulin products. In addition, as noted above, it is not desired to be limited by theory, and insulin dissolved in aprotic polar solvents is considered to be "quasi-stabilized ". This quasi-stability is believed to be derived from the combination of the solubility of insulin in the apolar polar solvent and the pH memory of the insulin.

D. Reduced aggregation of insulin

Additional components that further reduce the likelihood of aggregation of monomeric and / or dimeric forms of insulin may be added to the formulation. These components may be used to reduce aggregation within the formulation before administration (e.g., storage) or after administration (e.g., prior to administration or prior to injection into the bloodstream of the subject). These components may be used including urea, guanidinium chloride, amino acids, sugars, polyols, polymers, acids, surfactants, or mixtures thereof. These ingredients are commercially available from a variety of sources.

E. Moisture content of the formulation

The formulations of the present invention may have a low moisture or moisture content as the aprotic polar solvent is used in a relatively high amount. This can provide additional stability so that the monomer or dimer form of the insulin can be present in the formulation by reducing the possibility of aggregation of the monomer or dimer. For example, the formulations of the present invention may comprise 20%, 19%, 18%, 17%, 16%, 15%, 10%, 9%, 8%, 7%, 6% Moisture content or moisture of 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, 0.05%, 0.025%, 0.01% to 0%. However, in one embodiment, water may be used as a co-solvent, such as when the formulations of the present invention comprise insulin or pramine tide.

F. Insulin / Porphyrin Tie Co-Formulations

Amylin, a beta-cell hormone commonly secreted in insulin by glucose uptake, is also deficient in type 1 diabetics. Amylin shows some glucose-regulating hormone effects that complement insulin for postprandial glucose control. The natural human amylin is unsuitable for therapeutic or pharmaceutical use due to some physicochemical properties, poor solubility, self-aggregation and the formation of amyloid fibers and amyloid plaques.

Prandintide is an analog of human amylin developed by the selective substitution of proline for ara-25 (Ala-25), cell-28 (Ser-28) and cell 29 (Ser-29). It is the physicochemical properties of the human amylin lane while maintaining important metabolic activities. Pramlintide is widely available from several commercial sources (e.g., Symlin ' s from Amylin Pharmaceuticals).

Pramlintide is typically administered via separate subcutaneous injection in addition to insulin. This method is acceptable in some populations of patients, but injecting additional injections is a great burden on patients who have already been injected multiple times daily. In addition, some patients, either accidentally or intentionally, may mix adrenaline and insulin into the same syringe prior to injection, resulting in adverse reactions or undesirable events.

One of the reasons for the separate administration of pramine tide and insulin is that these drugs collide with their buffering system, making the compatibility of the mixed formulations difficult. For example, some insulin and insulin analogues have an isoelectric point of from 5 to 6 and are therefore formulated at a pH of about 7. [ Pharmintide has an isoelectric point greater than 10.5, is optimally stable at low pH, and is typically formulated at a pH of about 4. The interaction of pramineide and insulin formulations under different pH and other buffering capacities often leads to precipitation of soluble insulin components or solubilization of crystalline insulin components. In vitro studies of promynidine and short- and long-acting insulin formulations have found significant changes in insulin solubility when various amounts of insulin are mixed with fixed amounts of pramlintide.

The problems of such co-formulation issues are addressed in the present invention. For example, pramlintide with a pH memory of 1 to 5 or 2, 3 or 4 or more particularly about 2 can be dried in a buffer system. Insulin having a pH memory of 1 to 4, 1 to 3 or about 2 or 6 to 8 or about 7 can be dried in the same or a separate buffer system. The dried promontide and insulin can then be dissolved or reconstituted in the same non-polar polar solvent and their respective solubility and stability characteristics can be maintained in the same formulation. Thus, only a single formulation requires the administration of both pramlintide and insulin to the subject. These co-formulations will lower the resistance of the subject by treating them more closely mimicking the natural physiological response of postprandial blood glucose elevation. In some specific embodiments of the co-formulations, the pH memory of the insulin may be about 2, and the pH memory of the promintide may be about 2.

In addition to pramlintide, other amylin agonists may be used in accordance with the context of the present invention. These agents can be purified or recombined by natural sources. The amylin agonists may be human or non-human. The amylin agonists may also be amylin analogs based on amino acid sequences of human amylin, but with one or more amino acid differences, or chemically modified amylin or amylin analogs. Quantification of the amylin agonist will depend on its bioavailability and the patient being treated. "Human amylin" is either secreted by the pancreas, isolated from natural sources, produced by synthetic peptide chemistry, genetically modified by microorganisms, or includes human peptide hormones. "Amylin analogue" is different from amylin secreted from the pancreas by modified amylin, but it is still valid to accept the same response as the natural amylin in the human body.

G. Quantification

Any suitable amount of insulin, pramine tiez, or a mixture of both may be administered by using the formulations of the present invention. Of course, the dosage administered will depend extensively on the following known factors: the pharmacokinetic properties of the particular drug, salt or mixture thereof; Age, health or weight of the subject; Degree and nature of symptoms; Types of simultaneous treatment, metabolism characteristics of patients and therapeutic agents; Frequency of treatment; Or desired effect. Generally, insulin may occur in formulations in an amount ranging from about 0.5 mg / mL to about 1 mg / mL. In one embodiment, insulin is administered to a subject in a dosage amount ranging from about 3 mg / mL to 100 mg / mL, from 3 mg / mL to about 100 mg / mL, from 10 mg / mL to about 50 mg / mL, or from about 50 mg / mL to about 100 mg / Lt; / RTI > In certain embodiments, the range of amounts of insulin with the formulation is from about 3 mg / mL to about 10 mg / mL, which can result in a significant portion of the insulin that occurs in the monomer form (see data in the examples). In yet another embodiment, the range of amounts of insulin with the formulation is from about 10 mg / mL to about 50 mg / mL and can cause most of the insulin that occurs in dimeric form (see data in the examples) . In one embodiment, pramlintide is present in the formulation in an amount ranging from 0.1 to 10 mg / mL or 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 8 or 9 mg / mL or as needed. It will also be apparent to those skilled in the art that the dosage of the drug can vary depending on the drug used and the disease, disorder or condition to be treated, and the degree of concentration of the drug in the dosage form will vary depending on the solubility, will be.

H. Additional Ingredients / Pharmaceutical Additives

The formulations of the present invention may include additional component / pharmaceutical additives to further protect insulin or pramlintide or to develop additional formulations to have the desired tactile properties, viscosity range. For example, the formulations may be in the form of any one, any combination or all of the antioxidants (non-limiting examples of which are ascorbic acid, cysteine, methionine, monothioglycerol, Sodium thiosulfate, sulfites, BHT, BHA, ascorbyl palmitate, propyl gallate or vitamin E or any combination thereof); Chelating reagents (non-limiting examples include EDTA, EGTA, tartaric acid, glycerin, citric acid and their salts); And / or preservatives (including alkyl alcohols, benzyl alcohols, methyl parabens or propyl parabens or mixtures thereof). In addition, the formulations of the present invention may also include non-aqueous pro- tic solvents (non-limiting examples include polyethylene glycol (PEG), propylene glycol (PG), polyvinylpyrrolidone (PVP) , Methoxypropylene glycol (MPEG), glycerol, glycofurol, and mixtures thereof).

I. Kit / Container

Kits are also contemplated for use in certain embodiments of the present invention. For example, the formulations of the invention may be included in a kit. The kit may include a container. For example, in one embodiment, the formulation may be configured within a container that is ready for parenteral administration to the subject without diluting or reconstituting the formulation. That is, the formulations to be administered can be stored in a container and used immediately as needed. The storage container may be a syringe, a pen injection device, an auto-injector device, or a pump. Suitable pen / autosamplers include, but are not limited to, pen / autosampler manufactured by Becton-Dickenson, Swedish Healthcare Limited (SHL Group), YpsoMed Ag, But is not limited thereto. Suitable pumps include, but are not limited to, pumps manufactured in Tandem Diabetes Care, Inc., Delsys Pharmaceuticals, and the like.

Alternatively, the kit of the present invention may comprise a plurality of sections in a plurality of containers or containers. Each container or plurality of sections may be used, for example, to store a biocompatible non-aqueous solvent and a separate small molecule drug. If necessary, the solvent and the drug may be mixed together and administered together or may be stored for later, if necessary.

J. Preparation of Formulation

The formulations of the present invention can be made using the following steps. The following steps were used to make the formulation as one embodiment of the specification.

1. Insulin powder (for example, recombinant human insulin, Sigma-Aldrich, St. Louis, MO) was placed in a desired aqueous buffer (specific buffer species, pH and concentration; citric acid, pH 2.0 included) at an insulin concentration of 10 mg / St. Louis, Mo.). ≪ / RTI >

 2. At a concentration of 2 mg / ml, pramlintide (for example, AmbioPharm, Inc., Beech Island, SC and CS, except for pramlintide, which can be dissolved in an aqueous buffer, CS Bio, Inc., Menlo Park, CA were used as one embodiment of the specification) can be similarly prepared.

3a. The insulin or pramine tide solution was dispensed into a clean HPLC or lyophilized bottle and lyophilized similar to or following the lyophilization cycle of Table 1.

step Temperature condition Speed / duration Vacuum (mTorr) Shelf load 5 ℃ 1 hr N / A frozen -50 ℃ 1 ° C / min N / A Frozen dipping -50 ℃ 2 hrs N / A Lamp for annealing -15 ℃ 1 ° C / min N / A Annealing -15 ℃ 1 hrs N / A Foundation drying -15 ℃ 24 hrs 100 The second lamp 25 ℃ 1 ° C / min 100 Auxiliary drying 25 ℃ 8 hrs 100 Stopper ring 25 ℃ 100

3b. In addition, aqueous insulin or pumulinide solution was dispensed into microcentrifuge tubes and dried by light heat treatment (25 - 30 ° C) and centrifugation in a vacuum atmosphere.

4. In the selected pH memory, the dried insulin or pramine tie powder was dissolved in DMSO with the appropriate concentration or the appropriate pipette at the concentration allowed by the specific buffer system and pH.

5. The resulting solution was visually evaluated for clarity and / or scattered light was analyzed using a visible spectrophotometer at 630 nM and various downstream applications were used.

Example

The present invention will more specifically describe the method of the specific embodiment. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. Various non-limiting embodiments which can be easily modified or modified to yield essentially the same result will be readily apparent to those skilled in the art.

This example provides information on how an insulin / DMSO formulation is prepared, wherein the insulin has a pH memory of about 2. Insulin / H ₂ O formulations for comparison would also be provided and Fourier-Transform Infrared Spectroscopic (FTIR) and Dynamic Light Scattering (DLS) analyzers were used (see Examples 2 and 3) Described later in each embodiment). The insulin used in the examples of this specification is the recombinant non-modified human insulin purchased from Sigma-Aldrich (Saint Louis, Mo.).

Recombination of insulin / buffer A / DMSO: human insulin (Sigma-Aldrich, St. Louis, Mo.) was dissolved at a concentration of 10 mg / ml in buffer A (for example 10 mM citrate + 1 mM EDTA, pH 2.0) , 0.25 mL-aliquots were dispensed in HPLC bottles and lyophilized according to the procedure outlined in steps 1-5 above in the section "Formulation Preparation" section above. The lyophilized insulin inside each bottle had a pH memory of 2.0 and was reconstituted with 100 μL of DMSO at a concentration of 25 mg / mL (insulin was dissolved in DMSO by visual inspection). The stocks of this stock were then further diluted in buffer A as desired to produce a citrate-buffered insulin / DMSO / H ₂ O solution (for example, 12.5 and 5 mg / mL of DMSO and insulin in buffer A ). These formulations are referred to as "Ins-A / DMSO" or represent dilutions thereof.

Insulin / Buffer A / H ₂ O : Insulin was dissolved at a concentration of 10 mg / mL in distilled water, deionized water, and 0.25 mL aliquots were lyophilized. The source of insulin and the lyophilization process are the same as those mentioned above. The bottles were reconstituted with 250 μL of buffer A (eg, H ₂ O + 10 mM citrate + 1 mM EDTA, pH 2.0) and insulin was prepared in a 10 mg / mL buffer A solution. The aliquots of this stock were diluted in buffer A as desired (for example, 5 mg / mL of insulin in buffer A) to make an insulin / Buffer A solution. Insulin was dissolved in buffer A by visual inspection. These formulations are referred to as "Ins-A / H ₂ O ".

Insulin / Buffer E / DMSO : Insulin was dissolved at a concentration of 10 mg / mL in buffer E (for example, H ₂ O + 10 mM citrate + 1 mM EDTA + 10 mM MaCl, pH 2.0) and 0.5 mL aliquots were lyophilized . The source of insulin and the lyophilization process are the same as those mentioned above. The lyophilized insulin inside each bottle has a pH memory of 2.0 and is reconstituted with 100 μL of DMSO at a concentration of 50 mg / mL. Insulin was dissolved in DMSO by visual inspection. The stocks of this stock were diluted in DMSO as desired to make an insulin / DMSO solution (e.g., 30, 25, 10, 5 and 3 mg / mL of insulin in DMSO). These formulations are referred to as "Ins-E / DMSO ".

Insulin / Buffer E and F / H ₂ O : Insulin was dissolved at a concentration of 10 mg / mL in distilled water, deionized water, and 0.5 mL aliquots were lyophilized. The source of insulin and the lyophilization process are the same as those mentioned above. One bottle was reconstituted with 500 μL of buffer E, and insulin was prepared in 10 mg / mL buffer E solution. The other bottle was reconstituted with 500 μL of buffer F (for example, H ₂ O + 10 mM phosphate-citrate + 1 mM EDTA + 10 mM NaCl, pH 7.0) and resuspended in 10 mg / mL buffer F solution Gt; insulin < / RTI > Insulin was dissolved in buffers E and F by visual inspection. These samples are referred to as "Ins-E / H ₂ O" and "Ins-F / H ₂ O", respectively.

This example provides FTIR data showing the effect of DMSO on insulin form. BioTools Inc. (Jupiter, Florida USA) performed FTIR analysis and provided the data (see below).

MATERIALS AND METHODS FOR FTIR ANALYSIS: The following formulations were prepared for FTIR analysis.

Formulation 1 (F1): Ins-A / DMSO diluted in 1 part of buffer A to 12.5 mg / mL

Formulation 2 (F2): Ins-A / H ₂ O diluted to 5 mg / mL in buffer A

Formulation 3 (F3): Ins-A / H ₂ O reconstituted to 10 mg / mL in buffer A

Formulation 4 (F4): 25 mg / mL Ins-A / DMSO

Formulation 5 (F5): Ins-A / DMSO diluted in 4 portions of buffer A to 5 mg / mL

FTIR spectra were collected with a PROTA FTIR spectrometer (BioTools) installed with a DTGS detector with a 20 min, 4 cm-1 resolution for each sample and buffer. Samples were dissolved as described and placed inside a 6 um biocell with 75 microns for DMSO-based samples and CaF ₂ windows for aqueous samples (windows). All spectral analyzes (buffer shrinkage and structural description) were performed using the PROTA software suite.

Results: FIG. 1 shows the FTIR spectrum of the amide 1 region corresponding to the form. This data confirms that insulin is irreversibly unfolded in DMSO when the insulin profiles of tested Formulations 1 to 5 remain relatively constant. In particular, Formulation 3 shows a typical insulin spectrum of mixed a-helix, beta-sheet protein. Formulation 4 shows shifting to a higher frequency while maintaining its profile. This may be the result of strong hydrogen bonding characteristics or morphological changes of the DMSO solvent. Formulation 5 is essentially the same as the aqueous spectrum of insulin. The data in FIG. 1 confirms that insulin can not irreversibly spread in DMSO.

This example provides a DLS assay (e. G., Monomeric, dimeric, and hexahedral forms) with comparisons controlling the samples to confirm the connectivity of insulin in DMSO. Alliance Protein Laboratories (Thousand Oaks, California USA) performed a DLS analysis and provided corresponding data (see below). Buffer E and Buffer F systems were used because of the presence of NaCl needed to perform DLS analysis.

Materials and methods for DLS analysis: The following formulations are prepared for DLS analysis:

Formulation 6 (F6): 50 mg / ml Ins-E / DMSO

Formulation 6 (F6): 30 mg / ml Ins-E / DMSO

Formulation 6 (F6): 10 mg / ml Ins-E / DMSO

Formulation 6 (F6): 3 mg / ml Ins-E / DMSO

Formulation 6 (F6): 10 mg / ml Ins-E / H ₂ O

Formulation 6 (F6): 10 mg / ml Ins-F / H ₂ O

Measure the variation over time of scattered light in DLS (also known as quasi-elastic light scattering or photon correlation spectroscopy). This variation is related to the Brownian motion of the molecule and can therefore be used to determine the diffusion coefficient. These diffusion coefficients are generally converted to the hydrodynamic (stoke) radius, R _h , through the Stoke-Einstein relationship:

는 볼츠만 상수,

는 절대온도,

는 용매 속도 및

는 확산 계수이다.

Is the Boltzmann constant,

The absolute temperature,

Lt; / RTI >

Is the diffusion coefficient.

Data were collected using a Protein solution (now Wyatt Technology) DaynaPro MS / X instrument using 12 μL quartz scattering cells at a nominal 25 ° C temperature. Samples were centrifuged for 10 minutes in a centrifuge (Fisher Model 235A) to remove dust and large particles prior to loading in the assay cuvette. Typically, 25 seconds of data accumulation were recorded and averaged for signal / noise improvement. The resulting data was analyzed with the Dynamic Version 6.12.0.3 software provided by the manufacturer. The mean (z-average) size was based on the cumulants method. The size distribution was calculated using the Dynals analysis method. The instrument was perfect and was based on units of time and distance (the distance was measured by the wavelength of the light source). However, instrumentation is verified each year using calibrated latex spherical size criteria (diameter 21 + 1.5 nm, 35266 from Thermo Scientific, many products 3020A). The viscosity index of DMSO was assigned to 1.991 cp and 1.4768.

Overall Results : The aggregation state of insulin in DMSO was tested using dynamic scattering (DLS). Monomeric insulin has a true MW of approximately 6 kDa. Thus, dimer insulin will have a true MW of 12 kDa and mixed insulin will have a true MW of 36 kDa. Figure 2 summarizes the molecular weights (MW) of the insulin measured in prepared DMSO and aqueous solutions (i.e. Formulations 6 to 10). The indicated MW of insulin in aqueous formulations at a concentration of pH 7.0 and 10 mg / ml is 53 kDa (Form 10). At these high concentrations, the solution is not nearly ideal and the intermolecular effect causes an apparent MW that is greater than true MW. Regardless, the apparent MW measured represents insulin in a mixed state.

For the insulin / DMSO formulation, the apparent MW at 10 mg / ml (Formulation 7) is 16 kDa, which is one third of the MW of aqueous insulin (Formulation 10) at approximately 10 mg / ml, Indicating that insulin is close to the dimer. This may also be the case for concentrations above 50 mg / ml (Formulation 8-9), since the approximate linear difference within the apparent MW observed with increasing concentration is similar to that of artifacts typical of the excluded volume effect typical for this technique . However, a decrease in apparent MW of 13 kDa (Form 6) at 3 mg / ml deviates from this tendency, indicating that monomer remains at this concentration and is reversibly separated.

This DLS study suggests that the largest multimeric state of INS-2E in DMSO is a dimer over a range of suitable concentrations of use and that there is a monomer-dimer equilibrium at the lower end of the concentration range. These findings are in contrast to aqueous insulin formulations, where the complex is dominant - even in the absence of zinc - monomers are unstable and rapidly fibrous. Based on the current kinetic model of buprenorphine association and absorption, insulin formulations other than the cointegrate insulin are currently used for the rapid-response insulin (e.g., Lispro®, Aspart®, etc.) containing a significant number of insulin- ≪ RTI ID = 0.0 > kinetics. ≪ / RTI > In conclusion, these physicochemical studies show that the approach using non-aqueous solvents is almost more successful than the approach to aqueous to develop super-fast response insulin formulations. In aqueous solutions containing rapid-reaction formulations, monomeric insulin is unstable and predominantly formed in the body, whereas insulin monomer / dimers absorbed more rapidly in DMSO are thermodynamically favored and even relatively high Concentration. Additionally, this data (including DLS and FTIR data) shows that any structural change that appears to be induced by DMSO is reversibly reconstituted within the aqueous medium.

Specific results of formulation 6 (50 mg / mL of Ins-E / DMSO) : The size distribution of Ins-E at 50 mg / mL in DMSO (histogram of the scattering profile vs hydrodynamic radius) is shown in FIG. The main pick (by weight) is the first pick having an average radius of 2.12 nm and representing 34.7% of the total scattered light. The radius corresponds to a molar mass of about 20 kDa based on the aqueous spherical protein standard. At the average radius of 110 nm, 2.29 μm and 9.85 μm, an additional large peak was detected in addition to the main pick. These different sensitivities contribute about two-thirds of the total scattering intensity, and as expected in Table 2 below, they actually represent a very small percentage of the weight basis by weight. Unfortunately, there was no possibility of producing a meaningful fraction by predicting the weight of species larger than 1 μm because (1) scattering from these large particles is highly dependent on the detailed shape of the particles (due to internal reflection), (2) Is emitted forward only at a very small fraction at the 90 ° angle observed here. Some or all of these other species may be due to contaminants or minute dissolved buffer elements rather than insulin aggregation.

(Summary of 50 mg / ml Ins-E in DMSO) Pick # Average radius (nm) Estimated molar mass Percentage of intensity Percentage of weight (%) One 2.12 20 kDA 34.7 99.970 2 110 200 MDa 54.5 0.030 3 2,290 250 GDa 5.6 ** 4 9,850 7.4 TDa 5.2 **

* z-average radius 24.5 nm; Average intensity 182 kcnt / s.

** The weight fraction of this large species can not be predicted with certainty, and these picks are excluded from the calculation .

Specific results of Formulation 7 (30 mg / mL Ins-E / DMSO) : The size distribution obtained at 30 mg / mL Ins-E in DMSO is shown in FIG. At this concentration, the main peak shifted to a slightly lower radius of 2.02 nm (expected mass 17 kDa). In particular, species of 2.29 and 9.85 [mu] m were no longer detected and strongly predicted that these were dissolved buffer components. The relative intensities of species near 100 nm are also significantly lower. At this concentration, a new paper of 17.7 nm was detected. Species represented only 1.9% of the total scattered light, but it was possible that these species occurred at the same level in the 50 mg / mL sample, but because they were lost by flash (strong scatter from 10 nm and larger species) Was not detected. The raw data from the DLS (autocorrelation function) has a limit in the dynamic range, which means that the species occurring at less than ~ 1% of the total scattered light often fall below the threshold value. A summary of this data is provided by Table 3 in Formulation 7.

(Ins-E summary of 30 mg / ml in DMSO) Pick # Average radius (nm) Estimated molar mass Percentage of intensity Percentage of weight (%) One 2.02 17 kDa 76.7 99.9932 2 17.7 2.8 MDa 1.9 0.0037 3 102 170 MDa 21.4 0.0031

* z-average radius 2.12 nm; Average intensity 77.9 kcnt / s.

Specific results of Formulation 8 (10 mg / mL Ins-E / DMSO) : The size distribution obtained at 10 mg / mL Ins-E in DMSO is shown in FIG. The dilution has an additional major peak shifted below 1.94 nm (expected mass 16 kDa). No peak at 17.7 nm was detected at this concentration, and the relative intensities of species near 100 nm were further reduced. Traces of large particles of 5.45 μm were also present (but removal of these species by centrifugation is sometimes not perfect). Table 4 provides a summary of data for Formulation 8.

(Ins-E summary of 10 mg / ml in DMSO) Pick # Average radius (nm) Estimated molar mass Percentage of intensity Percentage of weight (%) One 1.94 16 kDa 87.1 99.9975 2 115 220 kDa 12.2 0.0025 3 5450 1.9 TDa 0.7 **

* z-average radius 1.07 nm; Average intensity 43.1 kcnt / s.

** The weight fraction of this large species can not be predicted with certainty, and these picks are excluded from the calculation .

Specific results of Formulation 9 (3 mg / mL Ins-E / DMSO) : The size distribution obtained at 3 mg / mL Ins-E in DMSO is shown in FIG. At this concentration, the main peak falls at 1.79 nm (expected mass 13 kDa). At this concentration, a new pick at 6.34 nm was detected and could show a small amount of insulin aggregation (possibly caused by freeze drying). The peak seen in the 60.8 nm sample is probably the same material measured at 100-110 nm at high concentrations-the shift look at this concentration may be due to either low signal / noise, or it may be the result of a new peak resolution at 6.34 nm have. Table 5 provides a summary of the data of Formulation 9.

(Ins-E summary of 3 mg / ml in DMSO) Pick # Average radius (nm) Estimated molar mass Percentage of intensity Percentage of weight (%) One 1.79 13 kDa 84.7 99.9393 2 6.34 250 kDa 2.2 0.060 3 60.8 50 MDa 13.1 0.0009

* z-average radius of 0.24 nm; Average intensity 31.4 kcnt / s.

Specific results of formulation 10 (Ins-E / H ₂ O): The size distribution of Ins-H ₂ O in buffer E (pH 2.0) is shown in FIG. The main pick occurs at a radius of 3.08 nm. The radius corresponds to an expected mass of 47 kDa, and at this low pH the sample still suggests that most of it is (or more). The concentration effect of a non-ideal ("molecular crowding") 10 mg / mL solution can cause some size distortion, but the distortion that occurs up and down is the dominance of blackout or excluded volume effects It depends on either one. Traces of large species at 27 nm and 165 nm were also detected, but it is unclear whether this represents aggregation of insulin or fine particle contaminants. Table 6 provides a summary of the data for Formulation 10.

(10 mg / ml Ins-H in Buffer E ₂ O summary) Pick # Average radius (nm) Estimated molar mass Percentage of intensity Percentage of weight (%) One 3.08 47 kDa 67.5 99.894 2 27.0 7.5 MDa 20.5 0.053 3 165 520 MDa 12.0 0.053

* z-average radius 4.06 nm; The average intensity is 375 kcnt / s.

Specific results of formulation 10 (Ins-F / H ₂ O): The size distribution of Ins-H ₂ O in buffer F (pH 7.0) is shown in FIG. The main pick occurs at a radius of 3.26 nm. Its radius corresponds to the expected mass of 53 kDa. Here again the effect of the 10 mg / mL non-ideal solution leads to some distortion of the size, but at neutral pH it may be similar to the volume dominance which excludes the low charge, and therefore the appearance of the size is slightly larger than the actual size. Traces of large species were also detected at 35 nm and 238 nm. Table 7 provides a summary of data for Formulation 11.

(10 mg / ml Ins-H in buffer F ₂ O summary) Pick # Average radius (nm) Estimated molar mass Percentage of intensity Percentage of weight (%) One 3.26 53 kDa 77.2 99.9688 2 35.0 14 MDa 18.2 0.023 3 238 1.2 GDa 4.6 0.0078

* z-average radius 3.80 nm; Average intensity 447 kcnt / s.

This example provides data relating to pramine tide and insulin / promastide co-formulations in the context of the formulation of the present invention.

Solubility of Pramine Tide in DMSO and DMSO-Water Solvents: A solution of pramlintide was prepared by mixing 10 mM citrate at pH 2.0 or 10 mM citrate at pH 4.0 with 2 mg / mL trehalose, respectively, or with 2 mg / mL. < / RTI > The solution was dried in a vacuum at 25-30 ° C for 3.5 hours in a centrifuge or lyophilized as described above.

The citrate-buffered phos- phorinide in a dried pH 2.0 (with or without trehalose) memory was completely dissolved for several minutes at 20 mg / mL in clean DMSO (intermittent soft pipette) (highest concentration experiment). As a result, the solution was visibly flowable and completely clean.

The citrate-buffered phorbolideide in a dried pH 4.0 memory had significant resistance to reconstitution to start at a concentration of 2 mg / mL in clean DMSO, and was particularly insoluble in water. Concentration of nominal pramlintide increases from 6% to 10% of water to pramlintide in DMSO at 2 to 5 mg / mL, resulting in a visually detectable or nearly complete peptide solubility.

Co-formulations of insulin and pramine tide : A co-formulation of insulin and pramine tide is prepared as follows: Recombinant human insulin is dissolved in a 1.0 mM EDTA buffer at 10 mM citrate / pH 2 at a concentration of 10 mg / mL. Pramlintide was dissolved with or without 2 mg / mL trehalose in 10 mM citrate, pH 2.0 at a concentration of 2 mg / mL. The solution was dried by centrifugation in a vacuum of 0.5-mL aliquots as described above. Insulin was reconstituted with 50 μL of DMSO at a concentration of 20 mg / mL. The same volume of peptide-DMSO solution was mixed with or without about 10 mg / mL of trehalose to obtain a mixed solution of 50 mg / ml insulin and 10 mg / ml pumulinide. The solution is fluid and visibly clean by visual inspection. The ratio of 5: 1 (w / w) of insulin: pramineide can be one representative treatment dose ratio, and the peptide remains stable within the monolithic, highly concentrated solution of the visual inspection over 6 hours. Through conventional formulation techniques, these peptides require a separate and incompatible buffer system, requiring re-administration through separate injections at the site of the body's separation - an important barrier to the implementation of this beneficial therapy.

This is a prophetic example for determining the bioactivity, pharmacological and pharmacodynamic capacity of the formulations of the present invention as compared to conventional fast-acting insulin products (Asparto, Glucosin, Lispro).

Bioactivity : The insulin response at the cellular level is regulated by binding to the insulin receptor (IR), phosphorylation of the receptor, IR-mediated phosphorylation of the insulin receptor material and subsequent activation of the PI3 kinase-Akt cascade . The receptor binding is partly determined by the association status of the insulin molecule, and thus the overall bioactivity of the insulin formulation as well as the multiple- (or excess-) form of the peptide can be determined. The bioactivity of the formulations of the present invention can be measured using an enzyme-linked immunosorbent assay (ELISA) kit from the R & D system using IR-B isoforms (mouse embryo fibroblasts IR binding can be measured and compared alone to determine glucose regulation and fatty acid uptake and lipolysis, and the ability of cell lysates to phosphorylate Akt (Marks, AG, et al. (2011), " Differential activation of insulin receptor substrates (IRS) -1 and 2 by IGF-activated < RTI ID = 0.0 > (2006), "Differential activation of insulin receptor isoforms by insulin-like growth factors is determined by the C domain," Endocrinology, 147: 1029-1036, all included by reference Which it can be similarly quantified are for reference) does not necessarily predict more adjacent downstream signaling the ultimate biological reactions.

Pharmacology: Pharmacological studies can be performed using a conscious pig model injected with octreotide in conjunction with a subcutaneous vascular access port (VAP). Conscious pig model Non-diabetic Yorkshire can be used on the basis of: (a) a human-like carbohydrate physiology; (b) a large vein suitable for IV catheter placement; (c) prolonged anesthesia , Difficult admission / intubation), and (d) a single pig that can be used for multiple studies. The studies were conducted at 0, 5, 10, 20, 30, 45, 60, 90, 120, 180, and 240 to test formulations of the invention with acceptable insulin doses (e.g., 0.2 mg / kg) Lt; RTI ID = 0.0 > minutes. ≪ / RTI >

Pharmacokinetics: Blood samples can be analyzed using a previously validated assay of native and analogous insulin of porcine serum in OHSU (Mercodia Iso-Insulin ELISA, Product number 10-1128-01, manufactured by Mercodia AB Uppsala, Sweden) ). The blood levels of human insulin as well as the insulin analogues (e.g., the formulations of the invention and the comparable aqueous formulations) can be quantified over time, and the area under the curve, Cmax, Tmax, The Tmax value and the terminal period 1/2 Tmax value can be compared. The primary endpoint may be an initial-or end-1/2 Tmax value that is substantially more sensitive to changes in PK than Tmax. Tmax can often be difficult to measure and can produce erroneous results, while long stasis occurs and the values of initial and terminal values are rapidly rising and falling and thus much more reliable.

* * * * * * * * * * * * * *

All components, compositions, or methods described and claimed in this specification can be made and executed without undue experimentation in light of the description of the invention. Although the components, compositions, or methods of the present invention are described in the specific examples, it will be appreciated that the reaction of the components in the order and step of the methods described herein and in the compositions and / It will be apparent to those skilled in the art that there may be variations that can be applied.

Claims (50)

(a) insulin comprising a pH memory from 1 to 4 or from 6 to 8; And
(b) a formulation for parenteral administration comprising an aprotic polar solvent,
Wherein the insulin is dissolved in a non-apoptotic polar solvent and the dissolved insulin comprises insulin or a mixture thereof in the form of a stabilized monomer or dimer,
Wherein the moisture content of the formulation is equal to or less than 15% w / v,
Formulations for parenteral administration. The method according to claim 1,
Wherein the pH memory of the insulin is 1 to 4 or 1 to 3 or about 2,
Formulations for parenteral administration. The method according to claim 1,
Wherein the pH memory of the insulin is 6 to 8 or about 7,
Formulations for parenteral administration. 4. The method according to any one of claims 1 to 3,
The aprotic polar solvent may be selected from the group consisting of dimethylsulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, dimethylformamide (DMF), dimethylacetamide dimethylacetamide (DMA), propylene carbonate, or mixtures thereof.
Formulations for parenteral administration. 5. The method of claim 4,
Wherein the aprotic polar solvent is dimethylsulfoxide (DMSO)
Formulations for parenteral administration. 6. The method according to any one of claims 1 to 5,
Wherein the formulation comprises an insulin of 3 mg / ml to 50 mg / ml, 3 mg / ml to 10 mg / ml, or 10 mg / ml to 50 mg / ml.
Formulations for parenteral administration. 7. The method according to any one of claims 1 to 6,
Most of the dissolved insulin is in monomeric form,
Formulations for parenteral administration. 7. The method according to any one of claims 1 to 6,
Most of the dissolved insulin is in the dimeric form,
Formulations for parenteral administration. 9. The method according to any one of claims 1 to 8,
0.0 > 1, < / RTI > further comprising a component capable of reducing the aggregation of insulin in monomeric or dimeric form,
Formulations for parenteral administration. 10. The method of claim 9,
Such a component capable of reducing the aggregation of insulin in monomeric or dimeric form is one or more compounds selected from the group consisting of urea, guanidinium chloride, amino acids, sugars, polyols, polymers, acids, surfactants,
Formulations for parenteral administration. 11. The method of claim 10,
The acid may be acetic acid, ascorbic acid, citric acid, glutamic acid, aspartic acid, succinic acid, fumaric acid, maleic acid, adipic acid,
Formulations for parenteral administration. 11. The method according to any one of claims 1 to 10,
And further comprising a co-solvent,
Formulations for parenteral administration. 13. The method of claim 12,
Wherein the co-
Formulations for parenteral administration. 14. The method according to any one of claims 1 to 13,
Wherein the formulation does not comprise zinc, or wherein the zinc in the formulation is bound to a chelating agent.
Formulations for parenteral administration. 15. The method according to any one of claims 1 to 14,
Said insulin is already dried in a non-volatile buffer, said buffer having a pH range of 1 to 4 or 6 to 8,
Formulations for parenteral administration. 16. The method of claim 15,
The non-volatile buffer may be a glycine buffer, a citrate buffer, a phosphate buffer, or a mixture thereof.
Formulations for parenteral administration. 17. The method according to claim 15 or 16,
Wherein said buffer comprises a chelating agent.
Formulations for parenteral administration. 18. The method according to claim 14 or 17,
The chelating agent may be ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), tartaric acid, glycerin or citric acid,
Formulations for parenteral administration. 19. The method according to any one of claims 1 to 18,
Further comprising a component capable of lowering the freezing point of the aprotic polar solvent to about < RTI ID = 0.0 > 0 C. <
Formulations for parenteral administration. 20. The method of claim 19,
The component capable of lowering the freezing point of the polar aprotic solvent to about 0 캜 is water, sugar, sugar alcohol,
Formulations for parenteral administration. 21. The method according to any one of claims 1 to 20,
Wherein said insulin is a non-modified human insulin,
Formulations for parenteral administration. 22. The method according to any one of claims 1 to 21,
≪ / RTI > further comprising an amylin analog dissolved in said formulation.
Formulations for parenteral administration. 23. The method of claim 22,
The amylin analog is pramlintide,
Formulations for parenteral administration. 24. The method of claim 23,
The pramine tide has a pH memory of about 2,
Wherein the pramine tide has a pH memory of about 2 and the insulin has a pH memory of about 2,
Formulations for parenteral administration. 25. The method of claim 24,
The pramlintide was previously dried from a non-volatile buffer, and the buffer had a pH of about 2,
Formulations for parenteral administration. 26. The method according to any one of claims 22 to 25,
Wherein the moisture content of the formulation is 5 to 15% w / v, 7 to 12% w / v, 8 to 10% w / v or 9%
Formulations for parenteral administration. 32. The method according to any one of claims 1 to 30,
The formulation may be in the form of a liquid,
Formulations for parenteral administration. 28. The method of claim 27,
The formulation is a solution,
Formulations for parenteral administration. 29. The method according to any one of claims 1 to 28,
When the formulation is stored at room temperature for one month, at least 90% of the insulin in the formulation is chemically and physically stable,
Formulations for parenteral administration. 30. The method according to any one of claims 1 to 29,
Wherein the formulation is contained within a container,
Formulations for parenteral administration. 32. The method of claim 30, wherein the container is a syringe, a pen injection device, an auto-injector device, a pump or a perfusion bag,
Formulations for parenteral administration. 32. The method according to any one of claims 1 to 31,
Wherein said aprotic polar solvent is a contiuous phase of said formulation,
Formulations for parenteral administration. 33. The method according to any one of claims 1 to 32,
Wherein said formulation comprises at least 75, 80, 85, 90, or 95% w / v of an oily aprotic polar solvent.
Formulations for parenteral administration. 34. The method according to any one of claims 1 to 33,
The dissolved insulin is metastable,
Formulations for parenteral administration. Comprising administering to a subject in need thereof an effective amount of one of the formulations of claims 1 to 34 for reducing the subject's blood glucose level.
A method for reducing blood glucose levels. 36. The method of claim 35,
Wherein the blood glucose level of the subject is reduced within 30 minutes, 60 minutes, or 90 minutes after administration,
A method for reducing blood glucose levels. 37. The method according to any one of claims 35 to 36,
The initial < RTI ID = 0.0 > 1/2 < / RTI > _Tmax blood insulin level of the subject, which occurs within 30-60 minutes after administration,
A method for reducing blood glucose levels. 37. The method according to any one of claims 35 to 37,
The subject was diagnosed with Type-I or Type-II diabetes,
A method for reducing blood glucose levels. 39. The method according to any one of claims 35 to 38,
Wherein said formulation is administered within 10 minutes, 5 minutes or 1 minute prior to ingestion of the food by said subject, or within 1 minute, 5 minutes or 10 minutes after ingestion of food by said subject,
A method for reducing blood glucose levels. (a) drying a mixture comprising insulin and a nonvolatile buffer to obtain dried insulin having a pH memory from 1 to 4 or 6 to 8; And
(b) reconstituting said dried insulin in a non-apoptotic polar solvent, said method comprising the steps of:
Wherein the insulin is dissolved in the apical polar solvent, wherein the dissolved insulin comprises a stable monomeric or dimeric form of insulin or a combination thereof,
The moisture content of the formulation is 15% w / v or less,
A method for preparing a formulation for parenteral administration. 41. The method of claim 40,
Wherein the pH memory of the insulin is 1 to 4 or 1 to 3 or about 2,
A method for preparing a formulation for parenteral administration. 40. The method of claim 39,
Wherein the pH memory of the insulin is 6 to 8 or about 7,
A method for preparing a formulation for parenteral administration. 42. The method according to any one of claims 40 to 41,
The aprotic polar solvent may be selected from the group consisting of dimethylsulfoxide (DMSO), n-methyl pyrrolidone (NMP), ethyl acetate, dimethylformamide (DMF), dimethylacetamide dimethylacetamide (DMA), propylene carbonate, or mixtures thereof.
A method for preparing a formulation for parenteral administration. 44. The method of claim 43,
Wherein the aprotic polar solvent is dimethylsulfoxide (DMSO)
A method for preparing a formulation for parenteral administration. 45. The method according to any one of claims 40 to 44,
Wherein the formulation comprises an insulin of 3 mg / ml to 50 mg / ml, 3 mg / ml to 10 mg / ml, 10 mg / ml to 50 mg / ml or insulin of 50 mg / ml to 100 mg / ml ,
A method for preparing a formulation for parenteral administration. 46. The method according to any one of claims 40 to 45,
Wherein most of said dissolved insulin is in monomeric or dimeric form,
A method for preparing a formulation for parenteral administration. 46. The method according to any one of claims 40 to 46,
(c) drying the mixture comprising an amylin analog and a second nonvolatile buffer to obtain the same nonvolatile buffer or dried amylin analog as in step (a); And
(d) reconstituting the dried amylin analog in the aprotic polar solvent with the dried insulin, wherein the dried amylin analog is an amylin analog that is dissolved in the aprotic polar solvent,
A method for preparing a formulation for parenteral administration. 49. The method of claim 47,
Wherein the amylin analog is pramlintide, the pramlintide has a pH memory of about 2 or the pramlintide has a pH memory of about 2 and the insulin has a pH memory of about 2,
A method for preparing a formulation for parenteral administration. 49. The method of claim 47 or 48,
The non-volatile buffer has a pH range of about 2,
A method for preparing a formulation for parenteral administration. 49. A method according to any one of claims 46 to 49,
Further comprising adding 5 to 15% w / v water of said formulation as a cosolvent,
A method for preparing a formulation for parenteral administration.

Images