HK1030747B - Stable insulin formulations - Google Patents

Description

Stable insulin formulations

The present invention is in the field of human medicine, in particular the treatment of diabetes and hyperglycaemia by administering monomeric insulin analogues. More particularly, the present invention relates to monomeric insulin analog formulations having superior long term physical stability when exposed to high mechanical energy inputs and high temperatures.

Stable formulations of therapeutic agents are particularly desirable for use in delivery devices that expose these agents to high temperatures and/or mechanical stress. For example, stable insulin formulations are needed for use in continuous infusion systems and pen (pen) delivery devices. Current formulations provide only limited stability in these types of delivery devices.

In continuous infusion systems, the fluid containing the therapeutic agent is typically pumped from a reservoir to a subcutaneous, intravenous, or intraperitoneal depot. The reservoir, which must be periodically replenished, is attached to or implanted in the patient's body. In either case, the body heat and body motion of the patient plus turbulence in the tube and pump impart relatively high thermodynamic energy to the formulation. Formulations with relatively high concentrations of therapeutic agents are highly advantageous in reducing the frequency with which the depot is replenished and the importance of reducing the volume of the depot.

Massey and Sheliga, u.s. patent No. 4,839,341 (Eli Lilly and company, 1989) discusses problems related to providing a stable continuous infusion of insulin formulations and presents a comprehensive review of the art throughout the year 1984. This problem is currently even greater because insulin preparations with a stabilization period of 1 to 3 months are now required.

Pen-type injection devices (injectors pen) have also been developed to assist diabetics in measuring and administering accurate, controlled doses of insulin. Typically, these pen type injection devices are mounted on a cartridge having a specific amount of liquid drug sealed therein. The cartridge comprises a plunger in the cartridge for dispersing the medicament in such a way and a mechanism for pushing the plunger. Pen type injection devices are either reusable or disposable. In a re-used pen-type injection device, the user may replace the spent cartridge and readjust the push screw at the rear of the pen-type injection device to its initial position. In disposable pen injection devices, the cartridge is permanently trapped in the disposable pen injection device after the contents of the cartridge have been consumed. The insulin formulations used in these pen-type injection devices are exposed to physical stress and limited stability is often observed.

With the introduction of new monomeric insulin analogues for the treatment of diabetes, there remains a need to use these compounds in therapeutic regimens that compromise the intrinsic stability of the formulation. Fast acting insulins, termed monomeric insulin analogs, are well known in the art and published in 1996, 5/7, Chance et al, U.S. patent No. 5,514,646, Brems et al, Protein Engineering, 6: 527-533(1992), EPO publication No. 214,826 (published 3/18 1987) by Brange et al, and CurrentOpition in Structural Biology 1: 934 and 940 (1991). Monomeric insulin analogs absorb much more rapidly than insulin and are ideally suited for controlling blood glucose levels after meals in patients in need thereof. They are also particularly suitable for administration by continuous infusion for dietary and basal blood glucose level control, due to their rapid absorption from the site of administration.

Unfortunately, monomeric insulin analog formulations have a tendency to aggregate and become unstable when exposed to thermodynamic stress [ U.S. patent No. 5,474,978 to Bakaysa et al, issued 12.12.1995 ]. Aggregation may even manifest as precipitation of higher (higher-order) insulin species. In this manner, the aggregation can prevent reproducible delivery of therapeutically effective doses of monomeric insulin analogs, and can also elicit a stimulatory or more systemic immune response at the site of administration. Insulin analog formulations stabilized against aggregation are desirable.

Formulations of monomeric insulin analogs for use in continuous infusion systems must remain soluble and substantially non-aggregating, even subject to body heat and movement of the patient from days to months. Instability is promoted by the higher protein concentration required for continuous infusion systems and by the thermodynamic stress to which the formulation is exposed in continuous infusion systems. Therefore, improvements in the physical and chemical stability of concentrated insulin analogue formulations are urgently needed to make them successfully used in continuous infusion systems. In addition to use in continuous infusion, improvements in the stability of monomeric insulin formulations are also beneficial.

Stable formulations of fast acting monomeric insulin analogues are known. Bakaysa et al, U.S. patent No. 5,474,978, disclose and claim a human insulin analogue complex comprising a six molecule human insulin analogue (hexameric complex), two zinc atoms and at least three molecules of a phenolic preservative, a formulation comprising the hexameric complex and a method of treating diabetes by administering the formulation. Bakaysa et al also claim formulations of hexameric complexes further comprising isotonic drugs and physiologically tolerated buffers.

The specification of U.S. patent No. 5,474,978 discloses that zinc complexes of monomeric insulin analogues can be formulated in the presence of "physiologically tolerable buffers". The buffers mentioned for use in the formulation are sodium phosphate, sodium acetate, sodium citrate and TRIS. The examples in U.S. patent No. 5,474,978 only describe formulations in which the buffer is sodium phosphate, and only sodium phosphate buffer is required in the claims (claim 5). None of the examples in U.S. patent No. 5,474,978 specifically disclose the use of TRIS buffer in zinc-monomer insulin analogue complex formulations.

Monomeric insulin analog formulations containing protamine with a moderate duration of action at the time of use have also been developed. Monomeric insulin analogue-protamine formulations are described in U.S. patent No. 5,461,031. Methods of crystallizing a monomeric insulin analog with a basic peptide protamine to obtain a neutral protamine suspension are known in the art. In addition, a two-phase mixture containing a solution of the monomeric insulin analogue and a suspension of the monomeric insulin analogue-protamine can be prepared. These mixtures have the optimum time-action properties and basic activity of the analogs. Mixtures of monomeric insulin analogues are also described in U.S. patent No. 5,461,031.

The monomeric insulin analogue-protamine suspension formulation and the two-phase mixture are suitable for use in a cartridge container. However, as these devices require frequent handling by the patient, the stress on the formulation is increased. The protamine salt formulations in particular have limited stability when exposed to thermodynamic stress. Therefore, there is a need to develop stable, intermediate-acting monomeric insulin analog-protamine formulations as well as two-phase mixture formulations.

We have now found that when certain physiologically tolerable buffers other than phosphate are used in a biphasic mixture of a zinc-monomeric insulin analogue complex formulation, a protamine salt formulation or a monomeric insulin analogue, the physical stability of these formulations is unexpectedly greater compared to when phosphate buffers are used. The greatest advantage is that our finding that the soluble formulations provided by the present invention are sufficiently stable for safe use in long-term insulin infusion given that the physical stability of soluble formulations of zinc-monomeric insulin analogue complexes with phosphate buffers as exemplified in detail in U.S. patent No. 5,474,978 is not sufficient for long-term administration using a continuous infusion pump system. We have also found that the addition of arginine to a protamine salt formulation of a monomeric insulin analog results in an unexpected improvement in both chemical and physical stability of the formulation.

Accordingly, the present invention provides a solution formulation comprising a physiologically tolerable buffer selected from TRIS and arginine, a monomeric insulin analog, zinc and a phenolic preservative.

The invention also includes insulin analog formulations containing monomeric insulin analog, zinc, a phenolic preservative, protamine, and a buffer selected from TRIS and arginine.

The invention further provides a method of using the insulin analogue formulation in the treatment of diabetes and hyperglycaemia in a patient in need thereof, which method comprises administering to the patient the stable formulation of the invention.

For the purposes of the present invention, the following terms and abbreviations have the following meanings, as disclosed and claimed herein.

The term "administering" means introducing a formulation of the invention into a patient in need thereof to treat a disease or disorder.

The verb "aggregate" refers to the process by which a single molecule or complex is combined to form an aggregate. An assemblage is a polymeric population of molecules or complexes comprising monomeric insulin analogs. For the purposes of the present invention, the monomeric insulin analog hexamer is not an aggregate but a complex. Monomeric insulin analogs and their hexameric complexes have a tendency to aggregate when exposed to thermodynamic stress. Aggregation can proceed to the point where a visible precipitate forms.

The term "arginine" refers to an amino acid and includes the D-and L-enantiomers and mixtures thereof. The term also includes any pharmacologically acceptable salt thereof. Arginine is also known in the art as 1-amino-4-guanidinopentanoic acid. Arginine tends to form salts such as hydrochloride.

The term "complex" means a compound in which a transition metal is coordinated to at least one ligand. Ligands include nitrogen-containing molecules such as proteins, peptides, amino acids, and TRIS in many other compounds. The monomeric insulin analog can be a ligand for a divalent zinc ion.

The term "continuous infusion system" refers to devices that continuously administer fluid to a patient parenterally over extended time intervals or intermittently administer fluid to a patient parenterally over extended time intervals without having to establish a new site of administration each time the fluid is administered. The fluid contains one or more therapeutic agents. The device includes a reservoir for storing fluid prior to its infusion, a pump, a catheter or other tube for connecting the reservoir to the administration site via the pump and controlling the device to adjust the pump. The device may be configured as an implant device, typically a subcutaneous device. In such cases, the insulin reservoir is typically adapted for transdermal re-supplementation. It will be apparent that when the device is implanted, the contents of the reservoir will be at body temperature and subject to physical movement by the patient.

An "isotonic drug" is a compound that is physiologically tolerated and imparts appropriate permeability to a formulation to prevent net flow of water across the cell membrane in contact with the formulation. Compounds such as glycerol are commonly used for such purposes at known concentrations. Other possible isotonic agents include salts such as sodium chloride, glucose and lactose.

The terms "monomeric human insulin analogue", "monomeric insulin analogue" and "human insulin analogue" are well known in the art and generally refer to rapid acting analogues of human insulin which include:

human insulin wherein Pro at position B28 is replaced by Asp, Lys, Leu, Val or Ala and wherein position B29 is Lys or substituted with Pro;

AlaB 26-human insulin

des (B28-B30) human insulin; and

des (B27) human insulin.

Such monomeric insulin analogues were issued in 1996, 5/7, Chance et al U.S. patent No. 5,514,646, Chance et al U.S. patent application serial No. 08/255,297, Brems et al Protein Engineering, 6: 527-533(1992), Brange et al EPO publication No. 214,826 (published 3/18 1987), and Brange et al Current Opinion in structural Biology 1: 934 and 940 (1991). The monomeric insulin analogue used in the present formulation is suitably cross-linked. Insulin analogues suitable for cross-linking contain three disulfide bridges: one bridge is between position 7 of the A chain and position 7 of the B chain, the second is between position 20 of the A chain and position 19 of the B chain, and the third is between position 6 and position 11 of the A chain.

The term "phenolic preservative" as used herein refers to chlorocresol, m-cresol, phenol, or mixtures thereof.

The term "stability" as used herein refers to the physical stability of the formulation of the monomeric insulin analogue. Physical instability of protein formulations can be caused by aggregation of protein molecules to form higher order polymers or even to form precipitates. A "stable" formulation is one in which the degree of aggregation of the protein is acceptably controlled and does not increase unacceptably over time. Monomeric insulin analog formulations have a tendency to aggregate when exposed to thermodynamic stress. Physical stability can be evaluated by methods well known in the art, including measuring the apparent attenuation (absorption or optical density) of light of a sample. Such a measure of light attenuation is related to the turbidity of the formulation. Aggregation or precipitation of the protein or complex in the formulation can produce turbidity. Other methods of assessing physical stability are well known in the art.

The term "treatment" refers to the treatment and care of patients with diabetes or hyperglycemia, or other disorders requiring the administration of insulin for the purpose of counteracting or alleviating the symptoms and complications of those disorders. Treatment includes administration of the formulations of the invention to prevent the onset of symptoms or complications, to alleviate symptoms or complications, or to eliminate a disease, disorder, or condition.

The term "TRIS" refers to 2-amino-2-hydroxymethyl-1, 3-propanediol and any pharmacologically acceptable salt thereof. The free base and hydrochloride forms are two common forms of TRIS. Tris is also known in the art as Tris, tromethamine and Tris (hydroxymethyl) aminomethane.

The present invention provides formulations of monomeric insulin analogs having greatly increased physical stability relative to those known in the art, as can be readily appreciated from the following data.

Comprising a monomeric insulin analogue, Lys prepared substantially as described herein in example 3^B28Pro^B29Formulations of human insulin analogues and TRIS accelerated stability tests were performed as described below. A sample of the prepared formulation was placed into a pre-cleaned 2ml capacity glass HPLC autosampler vial. Each tube contained three Teflon tubes having a diameter of about 3/16 inches^®A ball. The air was completely displaced from the tube with the formulation sample. After sealing, the vial was shaken continuously (20 × g, average linear acceleration) at 40Hz at 12mm peak-to-peak amplitude and provided a relatively high level of mechanical energy input into the formulation at 37 ℃ at temperatures conducive to aggregation and physical instability. The tubes were fixed to the shaker such that their length (i.e., from top to bottom) was oriented parallel to the direction of linear acceleration-i.e., they lay flat on the shaker surface. It has been shown for insulin preparations to increase under the accelerated conditions described aboveThe stability of (2) is greatly related to the practical stability.

The optical densities of the sample and control formulations at 450nm were measured in stages using a Shimadzu1201 spectrophotometer. The control formulation was prepared in the same way as the sample formulation, but stored without stirring at 4 ℃. The net optical density of the sample was calculated by subtracting the optical density of the control from the optical density of the sample. The values in table 1 are the mean net optical density and the standard deviation of the number of samples (n) given. Samples and control formulations (pH 7.4 ± 0.1) containing phosphate as buffer were prepared essentially as described in example 4.

TABLE 1 buffer and time of exposure to high mechanical energy input at 37 ℃ versus Lys^B28Pro^B29Effect of turbidity (optical Density at 450 nm) of human insulin analogues

	Optical density at 450nm
					16 hours	70 hours	100 hours	500 hours
	Example 3(TRIS) example 4 (phosphate)	0.02±0.01n＝50.81±0.71n＝5	0.03±0.02n＝5N.D.	0.01±0.01n＝5N.D.					0.04±0.01n＝4N.D.

Not determined (N.D.)

Under the conditions described above, the turbidity reached a very high unacceptable level at 16 hours in the formulation with phosphate buffer compared to the control formulation containing phosphate stored without stirring at 4 ℃ (table 1, example 4). On the other hand, the optical density of the formulation containing TRIS as a buffer remained virtually the same as that of the control formulation containing TRIS (example 3) for 500 hours. The data in Table 1 clearly demonstrate Lys^B28Pro^B29The optical density of human pancreatic hours remained the same. The data in Table 1 clearly demonstrate Lys^B28Pro^B29Replacement of phosphate buffer with TRIS buffer in human insulin analogue formulations significantly increases the stability of the formulations. Because energy input in accelerated testing is greater than during the expected use, it is believed that the surprising and significant stability of the monomeric insulin analog formulation in TRIS buffer in accelerated testing would translate into a practical stability that is much greater than 500 hours, based on observations made for other insulin formulations.

Preparation of insulin analogues containing monomers, Lys as described in examples 3, 4 and 5, respectively^B28Pro^B29Formulations of human insulin analogues and TRIS, phosphate or L-arginine as buffer. Lys of three lots^B28pro^B29Human insulin analogues are used for the preparation of the formulations. For each mixture of analog lot and buffer, six samples were subjected to the stability test described above. Four different shaker platforms were used to impart mechanical energy to the vials. Each shaker has at least one sample and buffer mixture for each lot. The stability of these formulations was evaluated in stages by measuring the optical density of the samples and controls as described above. The results are shown in Table 2. The values in table 2 are the average net optical density and standard deviation for six samples for each lot number and buffer.

TABLE 2 Exposure at 37 deg.CBuffer, analog lot number and time to Lys at high mechanical energy input^B28Pro^B29Effect of turbidity (optical Density at 450 nm) of human insulin analogue preparation

Buffering agent	Analog batch number	Optical density at 450nm
						23 hours	47 hours	87 hours	139 hours
		TRIS	Batch number 1 batch number 2 batch number 3	0.02±0.020.00±0.010.02±0.02	0.06±0.020.04±0.020.05±0.03					0.05±0.020.03±0.010.04±0.03	0.00±0.020.00±0.020.01±0.02
Arginine	Batch number 1 batch number 2 batch number 3					0.01±0.020.01±0.020.00±0.02	0.04±0.020.04±0.020.03±0.02	0.04±0.020.06±0.081.84±0.66	2.12±1.031.80±0.60N.D.
		Phosphate salts	Batch number 1 batch number 2 batch number 3	0.13±0.060.21±0.240.29±0.23	2.68±0.172.14±0.752.75±0.14					2.61±0.112.75±0.142.79±0.11	N.D.N.D.N.D.

Under the conditions described above, the turbidity of the formulations containing phosphate buffer reached very high unacceptable levels at 23 hours, ignoring the lot number of insulin analogue used (table 2). In contrast, the turbidity of the formulation containing TRIS as buffer remained essentially unchanged for 139 hours, ignoring the lot number of insulin used. Formulations containing L-arginine buffer demonstrated better physical stability compared to formulations containing phosphate, and the duration of their stability was somewhat dependent on the lot number of insulin analogue used. The data in Table 2 clearly demonstrate that Lys with TRIS buffer or L-arginine buffer at pH 7.4 compared to formulations with phosphate buffer^B28Pro^B29Formulations of human insulin analogues are able to maintain a stable anti-aggregation effect for a significantly longer time. Furthermore, because the energy input in accelerated testing is greater than the desired use, it is believed that the surprising and significant stability of the formulation of monomeric insulin analogs in TRIS and L-arginine buffer will translate into greater practical stability than observed in accelerated testing.

Evaluation of Lys by Physical Stability Stress Test (PSST)^B28Pro^B29The susceptibility of suspension formulations to changes in morphology and appearance. In this thermodynamic process, a catalyst is preparedThe agent is enclosed in a headspace-free fixed volume container with glass beads. The vessel was placed in a chamber at elevated temperature (about 37 degrees celsius), rotated at a fixed speed (about 30rpm) for a period of time (about 4 hours), and then left to stand for 24 hours. The container was evaluated for changes when it was determined that aggregation (caking) had occurred and removed from the test. No failure for longer periods of time during the test and a greater number of containers remaining during the test are indicated as increased physical stability.

Has been tested to contain Lys^B28Pro^B29And Lys^B28Pro^B29Two different mixtures of protamine crystals. Lys for low (low) mixtures^B28Pro^B29And Lys^B28Pro^B29Protamine in a ratio of 25: 75, and for high (high) mixtures in a ratio of 75: 25. These mixtures were prepared as described in examples 6 and 7. When the PSST method was used to test the low mixture, only the formulation containing arginine had the remaining container after 18 days. After 44 days both experimental samples had the remaining containers. The PSST test in high mix showed similar results to the formulation containing arginine with approximately 50% of the containers remaining after 36 days of the test, whereas the control formulation containing phosphate buffer had 0 to 5% of the containers remaining after 36 days.

A preferred monomeric insulin analogue for use in the formulations of the invention is Lys^B28Pro^B29Human insulin, Asp^B28Human insulin and Ala^B26Human insulin.

The concentration of the monomeric insulin analogue in the present formulation ranges from 1.2mg/mL to 50 mg/mL. The preferred range of analog concentration is from about 3.0mg/mL to about 35 mg/mL. More preferred concentrations are about 3.5mg/mL, about 7mg/mL, about 14mg/mL, about 17.5mg/mL, and about 35mg/mL, which correspond to formulations having insulin activity of about 100 units, about 200 units, about 400 units, about 500 units, and about 1000 units per mL, respectively.

The concentration of zinc in these formulations ranges from about 4.5mg/mL to about 370mg/mL, and there must be at least two zinc atoms in the complex of six insulin molecules in each hexamer. The ratio of total zinc (complexed zinc plus uncomplexed zinc) to insulin analogue hexamer should be between 2 and 4. The ratio of total zinc per insulin analog hexamer complex is preferably about 3 to about 4 atoms.

A minimum concentration of phenolic preservative is required to form the monomeric insulin analog hexamer in the present formulation. For some purposes, to meet the requirement of the compendial preservative effectiveness of a multiple use formulation, the concentration of phenolic preservative in the formulation of the invention may be increased above the concentration required to form the hexamer in the formulation to the amount necessary to maintain preservative effectiveness. The necessary preservative concentration for effective preservation depends on the preservative used, the pH of the formulation, and whether a preservative-binding or chelating substance is present. Generally, the necessary amounts can be found, for example, in WALLHAUSER, k.dh., develop.bio.standard.24, pages 9-28 (Basel, s.krager, 1974). When formulated, insulin analog hexamer complexes used in the present formulations can bind up to seven phenols, although typically only six phenols are bound to the hexamer. A minimum of about three phenols is required for hexamer formation. When antimicrobial effectiveness of the preservative is desired, a preferred phenol concentration is about 23mM to 35 mM. M-cresol and phenol are preferred preservatives, either alone or in admixture.

The formulation may optionally contain an isotonic agent. The formulation preferably contains an isotonic agent and glycerol is the most preferred isotonic agent. When used, the concentration of glycerol is within the range known in the art for insulin formulations, preferably about 16 mg/mL.

The pH of the formulation is controlled by a buffer such as TRIS or L-arginine. The concentration of the buffer should not be considered critical in achieving the objectives of the present invention, and should be such as to provide sufficient pH buffering to maintain the pH between pH ± 0.1pH units during storage. The preferred pH is between about 7 and about 8 when measured at about 22 ℃.

Other additives, e.g. pharmaceutically acceptable cosolvents like tween 20^®(polyoxyethylene (20) sorbitan monolaurate), Tween 40^®(Polyoxyethylene (20) sorbitan monopalmitate), Tween 80^®(Polyoxyethylene (20) sorbitan monooleate), Pluronic F68^®(polyoxyethylene polyoxypropylene block copolymer) and PEG (polyethylene glycol) may optionally be added to the formulation. These additives are not required to obtain the great advantages of the present invention, but may be useful if the formulation is in contact with plastic materials.

The invention also includes formulations of protamine salts of monomeric insulin analogs containing varying proportions of soluble ingredients. The non-specific conformation of the insulin molecule is required to stabilize the arginine-containing formulation, although excipients and preservatives like zinc (discussed above) normally added to insulin formulations may enhance stability with arginine. The concentration of arginine in the protamine-containing formulation is in the range of 1 to 100 mM. Most preferably the arginine concentration is in the range of 5 to 25 mM. Arginine can be added as a supplement to a solution or precipitated suspension already containing zinc ions and a phenolic preservative.

Administration may be by any route known to be effective by a physician of ordinary skill. Parenteral administration is preferred. Parenteral administration is generally understood to mean administration by the parenteral route. Preferred parenteral routes of administration for the formulations of the present invention include intravenous, intramuscular, subcutaneous, intraperitoneal, intraarterial, nasal, pulmonary and buccal routes. Intravenous, intraperitoneal, intramuscular, and subcutaneous routes of administration for the compounds of the invention are more preferred parenteral routes of administration. Intravenous, intraperitoneal and subcutaneous routes of administration of the formulations of the invention are more preferred routes.

Administration by a certain parenteral route may involve introducing the formulation of the invention into the patient via a sterile syringe or other mechanical device such as a needle or catheter propelled by a continuous infusion system. The formulations provided herein may be administered using a syringe, pump, or any other parenteral administration device known in the art. The formulations of the present invention may also be administered as an aerosol for absorption in the lung or nasal cavity. The formulation may also be administered by mucosal absorption, for example buccally.

The amount of the formulation of the invention administered to treat diabetes or hyperglycemia depends on a variety of factors including, but not limited to, the sex, weight and age of the patient, the underlying cause of the disorder or disease being treated, the route of administration and bioavailability, the persistence of in vivo administration of the monomeric insulin analog, the formulation and the potency of the monomeric insulin analog. When administered intermittently, the amount administered per time should also take into account the interval between the two doses and the bioavailability of the monomeric insulin analogue from the formulation. Administration of the formulation of the invention may be continuous. The titration amount and infusion rate or frequency of administration of the formulations of the present invention to achieve the desired clinical result are within the skill of the ordinary practitioner.

Monomeric insulin analogs for use in the present invention can be prepared by a variety of known peptide synthesis techniques including traditional solution methods, solid phase methods, semi-synthetic methods, and recombinant DNA methods. U.S. patent No. 5,514,646 issued to Chance et al, 5/7/1996, discloses the preparation of a variety of monomeric insulin analogs, which is sufficiently detailed that one skilled in the art can prepare any monomeric insulin analog useful in the present invention.

Zinc and phenol preservatives are essential to obtain a complex which is stable and which dissolves and takes effect rapidly. The hexamer complex consists of two zinc ions per one human insulin analog hexamer and at least three molecules of a phenolic preservative selected from chlorocresol, m-cresol, phenol, and mixtures thereof.

The soluble monomeric insulin analogue is converted to a hexameric complex by dissolving the monomeric insulin analogue in a diluent containing an appropriate amount of a phenolic preservative at a pH of about 7 to about 8, followed by the addition of zinc. Zinc such as (but not limited to) zinc acetate, zinc bromide, zinc chloride, zinc fluoride, zinc iodide and zinc sulfate is preferably added in the form of a zinc salt. The skilled person will recognise that there are many other zinc salts which may also be used to prepare monomeric insulin analogue complexes as part of the invention. Preferably, zinc acetate, zinc oxide or zinc chloride is used, as these compounds do not add new chemical ions for an industrially acceptable process.

Dissolving the monomeric insulin analog can be accomplished by a process commonly referred to as "acid dissolution". For acid dissolution, the pH of the water-soluble solvent is lowered to about 3.0 to 3.5 with a physiologically tolerated acid (preferably HCl) to aid in dissolution of the monomeric analog. Other physiologically tolerated acids include, without limitation, acetic acid, citric acid, and sulfuric acid. Phosphoric acid is preferably not used to adjust the pH in the preparation of the formulations of the present invention. The pH is then adjusted to about pH 7.3 to 7.5 with a physiologically tolerable base, preferably sodium hydroxide. Other physiologically tolerated bases include, without limitation, potassium hydroxide and ammonium hydroxide. Thereafter, a phenolic preservative and zinc were added.

The parenteral formulations of the present invention can be prepared using conventional dissolution and mixing methods. To prepare a suitable formulation, for example, the amount of monomeric insulin analogue measured in water is mixed with the required preservatives, zinc compound and buffer in sufficient water to prepare the hexameric complex. The formulations are typically filter sterilized prior to administration. Variations of this process will be recognized by those of ordinary skill in the art. For example, the order of addition of the ingredients, the order of adjustment of the pH, if any, and the temperature and ionic strength at the time of preparation of the formulation may be optimized for the concentration and method of administration used.

The following examples and preparations are provided to further illustrate the preparation of the formulations of the present invention. The scope of the present invention is not limited to the following examples.

Example 1

Containing Lys^B28Pro^B29Preparation of U100 soluble formulations of human insulin analogues and TRIS

Lys in an amount calculated to give 100 units of insulin activity per ml in the final formulation^B28Pro^B29-human insulin analogue-zinc crystals were suspended in an aqueous solution containing 0.715mg/mL phenol, 1.76mg/mL m-cresol, 16mg/mL glycerol and zinc oxide. The insulin analogue-zinc crystals contain about 0.36% by weight of zinc. The concentration of zinc oxide in the aqueous diluent solution is replenished so that the final zinc ion concentration of the formulation is up to about 0.025mg per 100 units of insulin activity. The pH was adjusted to 2.8 to 3.0 by the addition of 10% hydrochloric acid. After dissolving the crystals with stirring, 10% sodium hydroxide solution was carefully added to adjust the pH to 7.4 to 7.7. The calculated TRIS stock solution (50mg/mL, pH 7.4, measured at room temperature, i.e., about 22 ℃) giving a TRIS concentration of 2mg/mL in the final formulation was added to the insulin analog solution. The formulation was diluted to final volume with water. The preparation was filter sterilized using a 0.2 micron filter.

Example 2

Containing Lys^B28Pro^B29Preparation of U100 soluble formulations of human insulin analogues and L-arginine

The procedure described in example 1 was followed until the addition of buffer. The calculated L-arginine stock (200mM, pH 7.4) was then added to the insulin analogue solution in place of the amount of TRIS stock solution, the concentration of L-arginine in the final formulation being 20 mM. The formulation was diluted to final volume with water. The preparation was filter sterilized using a 0.2 micron filter.

Example 3

Containing Lys^B28Pro^B29Preparation of U400 soluble formulations of human insulin analogues and TRIS

Converting the calculated Lys^B28Pro^B29-human insulin analogue-zinc crystals suspended in a suspension containing 0.715mg/mL phenol, 1.76mg/mL m-cresol, 16mg/mL glycerol and zinc oxide in water to give 400 units per mL of insulin activity in the final formulation. The insulin analogue-zinc crystals contain about 0.36% by weight of zinc. The concentration of zinc oxide in the aqueous diluent solution is replenished such that the final zinc ion concentration of the formulation is about 0.025mg per 100 units of insulin activity. The pH was adjusted to 2.8 to 3.0 by the addition of 10% hydrochloric acid. After dissolving the crystals with stirring, 10% sodium hydroxide solution was carefully added to adjust the pH to 7.4 to 7.7. The calculated TRIS stock solution (50mg/mL, pH 7.4, measured at room temperature, i.e., at about 22 ℃) was added to the insulin analog solution to give a concentration of TRIS of 2mg/mL in the final formulation. The formulation was diluted to final volume with water. The preparation was sterile filtered using a 0.2 micron filter.

Example 4

Containing Lys^B28Pro^B29Preparation of U400 soluble formulations of human insulin analogues and phosphate

Converting the calculated Lys^B28Pro^B29-human insulin analogue-zinc crystals were suspended in an aqueous solution containing 0.715mg/mL phenol, 1.76mg/mL m-cresol, 16mg/mL glycerol and zinc oxide such that 400 units of insulin activity per mL in the final formulation. The insulin analogue-zinc crystals contain about 0.36% by weight of zinc. The concentration of zinc oxide in the aqueous diluent solution is replenished so that the final zinc ion concentration of the formulation is about 0.025mg per 100 units of insulin activity. 10% hydrochloric acid was added to adjust the pH to 2.8 to 3.0. After dissolving the crystals with stirring, the pH was adjusted to 7.4 to 7.7 by careful addition of 10% sodium hydroxide solution. The calculated disodium hydrogen phosphate stock solution was added to the insulin analogue solution such that the concentration of disodium hydrogen phosphate in the final formulation was 3.78mg/mL (pH 7.4 ± 0.1). The formulation was diluted to final volume with water. The preparation was filter sterilized using a 0.2 micron filter.

Example 5

Containing Lys^B28Pro^B29Preparation of U400 soluble formulations of human insulin analogues and L-arginine

The procedure described in example 3 was followed until the addition of buffer. The calculated L-arginine stock (200mM, pH 7.4) was then added to the insulin analogue solution in place of the TRIS stock solution to give a concentration of 20mM L-arginine in the final formulation. The formulation was diluted to final volume with water. The preparation was filter sterilized using a 0.2 micron filter.

Example 6

U100Lys containing L-arginine^B28Pro^B29Human insulin analogue high cocktail formulation (75% v/v soluble, 25% v/v neutral protamine Lys^B28Pro^B29) Preparation of

A. Neutral protamine Lys^B28Pro^B29Preparation of

The calculated amount of Lys contained 200U/mL^B28Pro^B29Zinc insulin crystals were suspended in an aqueous solution containing 0.715mg/mL phenol, 1.76mg/mL m-cresol, 16mg/mL glycerol and zinc oxide acidified with hydrochloric acid to replenish the final zinc ion concentration to 0.025 mg/100U. The pH of the solution was adjusted to 2.8 to 3.0 by the addition of 10% hydrochloric acid. After dissolution with stirring, the solution pH was adjusted to 7.4 to 7.7 by the addition of 10% sodium hydroxide solution. A75.6 mg/mL solution of disodium hydrogen phosphate at pH 7.2 was added, which corresponded to a final formulation concentration of 3.78 mg/mL. The precipitated solid was dissolved and the pH was maintained at 7.4 by titration, followed by dilution of the formulation to final volume with water and filtration of the solution.

The solid protamine sulfate calculated to contain 0.6mg/100U of protamine base was dissolved in an aqueous solution containing 0.715mg/mL phenol, 1.76mg/mL m-cresol and 16mg/mL glycerol. Solid disodium hydrogen phosphate salt was added to give a concentration of 3.78mg/mL of the formulation. The solution was adjusted to pH 7.4 with 10% hydrochloric acid, diluted to final volume with water and filtered.

200 units of Lys at 15 ℃^B28Pro^B29Both the solution and the protamine solution reach equilibrium. Adding protamine solution to Lys^B28Pro^B29In solution, and the resulting suspension was incubated at 15 ℃ for 24 hours without disruption.

B.Lys^B28Pro^B29Preparation of the high mixture

Lys containing L-arginine corresponding to 75% of the final volume prepared in example 2^B28Pro^B29100 units of solution was added to a calculated amount of 100U/mL neutral protamine Lys^B28Pro^B29In (1). The suspension was stirred at room temperature.

Example 7

U100Lys containing L-arginine^B28Pro^B29Human insulin analogue Low cocktail formulation (25% v/v soluble, 75% v/v neutral protamine Lys^B28Pro^B29) Preparation of

A. Neutral protamine Lys^B28Pro^B29Preparation of

The calculated amount of Lys contained 200U/mL^B28Pro^B29Zinc insulin crystals were suspended in an aqueous solution containing 0.715mg/mL phenol, 1.76mg/mL m-cresol, 16mg/mL glycerol and zinc oxide acidified with hydrochloric acid to replenish the final zinc ion concentration to 0.025 mg/100U. The pH of the solution was adjusted to 2.8 to 3.0 by the addition of 10% hydrochloric acid. After dissolution with stirring, the solution pH was adjusted to 7.4 to 7.7 by the addition of 10% sodium hydroxide solution. A solution of 75.6mg/mL of the sodium dihydrogen phosphate salt at pH 7.2 was added, which corresponds to a final formulation concentration of 3.78 mg/mL. The precipitated solid was dissolved and the pH was maintained at 7.4 by titration, followed by dilution of the formulation to final volume with water and filtration of the solution.

The solid protamine sulfate calculated to contain 0.6mg/100U of protamine base was dissolved in an aqueous solution containing 0.715mg/mL phenol, 1.76mg/mL m-cresol and 16mg/mL glycerol. Solid sodium dihydrogen phosphate salt was added to give a concentration of 3.78mg/mL of the formulation. The solution was adjusted to pH 7.4 with 10% hydrochloric acid, diluted to final volume with water and filtered.

U200Lys at 15 ℃^B28Pro^B29Both the solution and the protamine solution reach equilibrium. Adding protamine solution to Lys^B28Pro^B29In solution, and the resulting suspension was incubated at 15 ℃ for 24 hours without disruption.

B.Lys^B28Pro^B29Preparation of the Low mixture

Lys containing L-arginine corresponding to 25% of the final volume prepared in example 2^B28Pro^B29The U100 solution was added to a calculated amount of 100U/mL neutral protamine Lys^B28Pro^B29In (1). The suspension was stirred at room temperature.

Claims (21)

1. A monomeric insulin analog solution formulation for parenteral administration comprising a physiologically tolerable buffer selected from TRIS and L-arginine, a monomeric insulin analog, zinc and a phenolic preservative, wherein the monomeric insulin analog is selected from Lys^B28Pro^B29-human insulin analogue and Asp^B28-a human insulin analogue, wherein the pH of the solution is between 7 and 8 when measured at about 22 ℃ and the concentration of the monomeric insulin analogue in the formulation is between 1.2mg/mL and 50 mg/mL.

2. The formulation of claim 1, wherein the monomeric insulin analog is Lys^B28Pro^B29-human insulin analogue, the buffer being TRIS.

3. The formulation of claim 1, wherein the monomeric insulin analog is Asp^B28-human insulin analogue, the buffer being TRIS.

4. The formulation of claim 2, further comprising an isotonic agent.

5. The formulation of claim 4, wherein said Lys^B28Pro^B29The concentration of human insulin analogue is between 1.2mg/mL and 50 mg/mL.

6. The formulation of claim 5, wherein said Lys^B28Pro^B29The concentration of human insulin analogue is between 3.0mg/mL and 35 mg/mL.

7. The formulation of claim 3, further comprising an isotonic agent.

8. The formulation of claim 7, wherein Asp^B28The concentration of human insulin analogue is between 1.2mg/mL and 50 mg/mL.

9. The formulation of claim 8, wherein Asp^B28The concentration of human insulin analogue is between 3.0mg/mL and 35 mg/mL.

10. The formulation of claim 6, wherein TRIS is present at a concentration of about 2 mg/mL; glycerol is an isotonic agent and is present at a concentration of about 16 mg/mL; and m-cresol is present at a concentration of about 1.76mg/mL and phenol is present at a concentration of about 0.715 mg/mL.

11. The formulation of claim 9, wherein TRIS is present at a concentration of about 2 mg/mL; glycerol is an isotonic agent and is present at a concentration of about 16 mg/mL; and m-cresol is present at a concentration of about 1.76mg/mL and phenol is present at a concentration of about 0.715 mg/mL.

12. Stable solution formulation of monomeric insulin analogue for continuous infusion systems, mainly comprising an isotonicity agent, a buffer selected from TRIS and L-arginine, monomeric insulin analogue, zinc and a phenolic preservative, wherein said monomeric insulin analogue is selected from Lys^B28Pro^B29-human insulin analogue and Asp^B28-a human insulin analogue, wherein the pH of the solution is between 7 and 8 when measured at about 22 ℃ and the concentration of the monomeric insulin analogue in the formulation is between 1.2mg/mL and 50 mg/mL.

13. The formulation of claim 12, wherein the insulin analog is Lys^B28Pro^B29Human insulin analogues.

14. The formulation of claim 12, wherein the insulin analog is Asp^B28Human insulin analogues.

15. The formulation of any one of claims 12 to 14, wherein the buffer is L-arginine.

16. The formulation of any one of claims 1 to 11, wherein the formulation is for a continuous infusion system.

17. Use of a monomeric insulin analogue solution formulation as described in any one of claims 1 to 15 for the preparation of a medicament for the treatment of diabetes.

18. Use of a monomeric insulin analogue solution formulation as described in any one of claims 1 to 15 for the preparation of a medicament for the treatment of hyperglycemia.

19. A method of preparing a monomeric insulin analog solution formulation of claim 1 comprising the step of admixing a physiologically tolerable buffer selected from TRIS and arginine with a monomeric insulin analog, zinc and a phenolic preservative, wherein the monomeric insulin analog is selected from Lys^B28Pro^B29-human insulin analogue and Asp^B28Human insulin analogues.

20. The method of claim 19, wherein the monomeric insulin analog is Lys^B28Pro^B29-human insulin analogue, the buffer being TRIS.

21. The method of claim 19, wherein the monomeric insulin analog is Asp^B28-human insulin analogue, the buffer being TRIS.