MXPA99011446A - Stable insulin formulations - Google Patents

Abstract

The present invention provides a monomeric insulin analog formulation stabilized against aggregation in which the buffering agent is either TRIS or arginine. The stable formulations of the present invention are useful for treating diabetes, and are particularly advantageous in treatment regimes requiring lengthy chemical and physical stability, such as, in continuous infusion systems.

Description

STABLE INSULIN FORMULATIONS Field of the Invention The present invention relates to the field of human medicine, particularly in the treatment of diabetes and hyperglycemia by the administration of monomeric insulin analogues. More specifically, the present invention relates to monomeric formulations of insulin analogs that have superior long-term physical stability when exposed to a large mechanical energy input and at an elevated temperature.

Background of the Invention Stable formulations of therapeutic agents are particularly required for use in delivery devices that expose these agents to elevated temperatures and / or mechanical stress. For example, stable insulin formulations are required for use in continuous infusion systems and feather delivery devices. Common formulations provide only limited stability in these types of delivery devices. Ref.032200 In continuous infusion systems, a fluid containing a therapeutic agent is pumped from a reservoir, usually to a subcutaneous, intravenous, or intraperitoneal reservoir. The reservoir, which must be filled periodically, is fixed to the patient's body, or is implanted in the patient's body. In any case, the patient's body heat and body movement plus turbulence in the tubing and pump impart a relatively high amount of thermomechanical energy to the formulation. In the interest of minimizing the frequency with which the deposit is filled, and of minimizing the size of the deposit, formulations having a relatively high concentration of the therapeutic agent are highly advantageous. Masse and Sheliga, U.S. Patent No. 4,839,341 (Eli Lilly and Company, 1989) describes the challenges involved in providing stable insulin formulations for continuous infusion, and presents a complete review of the technique since about 1984. The challenges are even greater in the present time because Insulin formulations that are stable for 1 to 3 months are now demanded. Injection pens have also been developed to help diabetics in the measurement and administration of an exact and controlled dose of insulin. In general, these pens are fixed or secured on a cartridge having a particular amount of liquid medication sealed therein. The cartridge includes a plunger and a mechanism for advancing the plunger in the cartridge such that the medicament is dispensed. Injector pens can be reusable or disposable. In reusable pens, a user can change an exhausted cartridge and readjust the boom advance screw back to its initial position. In a disposable pen, the cartridge is permanently captured in the pen which is discarded after the contents of the cartridge have been exhausted. The insulin formulations used in these pens are exposed to physical stress and usually limited stability is observed. With the introduction of new monomeric insulin analogs for the treatment of diabetes, there is a need to use these compounds in treatment regimens that can compromise the inherent stability of the formulations. Rapid acting insulins, known as monomeric insulin analogues, are well known in the art, and are described in Chance, et al., U.S. Pat. No. 5,514,646, issued May 7, 1996; Brems, et al., Protein Engineering, 6: 527-533 (1992); Brange, et al., EPO publication No. 214,826 (published March 18, 1987); and Brange, et al., Current Opinion in Structural Biology 1: 934-940 1991). Monomeric insulin analogs are absorbed much faster than insulin is absorbed, and are ideally suited for postprandial control of blood glucose levels in patients who have a need for it. They are also very suitable especially for administration by continuous infusion for both prandial and basal control of blood glucose levels because of their rapid absorption from the site of administration. Unfortunately, monomeric insulin analog formulations have a propensity to aggregate and become unstable when exposed to thermomechanical stress [Bakaysa, et al., Pat. U.S. No. 5,474,978, issued December 12, 1995]. Aggregation can still be manifested as the precipitation of higher order insulin species. In this way, aggregation can prevent the reproducible delivery of effective therapeutic doses of monomeric insulin analogues, and may also result in irritation at the site of administration or a more systematic unological response. Formulations of insulin analogs stabilized against aggregation are desirable. Formulations of monomeric insulin analogs for use in continuous infusion systems must remain soluble and substantially free of aggregation, even when subjected to heat and movement of the patient's body for periods ranging from a few days to several months. Instability is promoted by the higher protein concentrations that are desirable for continuous infusion systems and by the thermomechanical stress at which the formulations are exposed in continuous infusion systems. Therefore, the improvement in the physical and chemical stability of concentrated insulin analog formulations is urgently needed to enable them to be used successfully in continuous infusion systems. The improvement in the stability of the monomeric insulin formulations for different uses than in the continuous infusion is also beneficial. Stabilized formulations of insulin analogs that are fast acting are already known. Bakaysa, et al., In U.S. Pat. No. 5,474,978 describes and claims a complex of human insulin analogs comprising six molecules of a human insulin analogue (hexamer complex), two zinc atoms, and at least three molecules of a phenolic preservative, formulations comprising the complex of hexamer, and methods of treating diabetes mellitus by the administration of the formulation. Bakaysa, et al., Also claims formulations of the hexamer complex which further comprise an isotonicity agent and a physiologically tolerated buffer. The specification of U.S. Pat. Do not. ,474,978 discloses that zinc complexes of monomeric insulin analogs can be formulated in the presence of a "physiologically tolerated buffer". Among the buffers mentioned for use in the formulations are sodium phosphate, sodium acetate, sodium citrate, and TRIS. The examples in U.S. Pat. No. 5,474,978 only describes formulations wherein the buffer is sodium phosphate, and only the sodium phosphate buffer is required in a claim (Claim 5). None of the examples in U.S. Pat. No. 5,474,978 specifically describes the use of a Tris buffer in formulations of monomeric-zinc insulin analogues.

Formulations of the monomeric insulin analog containing protamine have also been developed to give, during use, an intermediate duration of action. Protamine-monomeric insulin analog formulations are described in U.S. Pat. No. 5,461,031. Methods for crystallization of the monomeric insulin analogs with the protamine of the basic peptide to give a neutral protamine suspension are already known in the art. In addition, biphasic mixtures containing a solution of the monomeric insulin analogue and a protamine-monomeric insulin analogue suspension can be prepared. These mixtures have the optimal time-action properties of the analog in combination with the basal activity. Mixtures of the monomeric insulin analog are also described in U.S. Pat. No. 5,461,031. The formulations of the protamine-monomeric insulin analogue and biphasic mixtures are suitable for use in the presentation of the cartridge container. Still, because these devices require frequent manipulation of the patient, an increased tension for the preparation results. The formulations of the protamine salt in particular have limited stability when exposed to a thermomechanical stress. Accordingly, there is a need to develop stable, intermediate acting monomeric insulin protamine-analog formulations as well as formulations of the biphasic mixture. It has now been discovered that when certain physiologically tolerated buffers different from phosphate are employed in formulations of monomeric-zinc insulin analogue complexes, protamine salt formulations, or biphasic mixtures of the monomeric insulin analogue, the physical stability of the formulations is unexpected. and considerably larger than when the phosphate buffer is used. More advantageous is the discovery of the invention that, while the soluble formulations of the monomeric-zinc insulin analog complexes form a complex with a phosphate buffer, such as those specifically exemplified in U.S. Pat. No. 5,474,978, are not physically stable enough for long-term administration using continuous infusion pumping systems, the soluble formulations provided by the present invention are sufficiently stable to be used safely for prolonged periods of insulin infusion. It has also been found that the addition of arginine to the protamine salt formulations of the monomeric insulin analogues leads to dramatic improvements in the chemical and physical stability of the formulation. Accordingly, the present invention provides a solution formulation comprising a physiologically tolerated buffer selected from the group consisting of TRIS and arginine; a monomeric insulin analog, and a phenolic preservative. The invention also encompasses an insulin analog formulation comprising a monomeric insulin analogue; zinc; a phenolic preservative; protamine; and a buffer selected from the group consisting of TRIS and arginine. The invention further provides methods of using the insulin analog formulations to treat diabetes and hyperglycemia in a patient in need thereof., which comprises administering to the patient a stable formulation of the present invention. For the purposes of the present invention, as described and claimed herein, the following terms and abbreviations have the following meanings. The term "administer" means to introduce a formulation of the present invention into the body of a patient who has a need to treat a disease or condition. The various forms of the verb "add" refer to a process by which complexes or individual molecules associate to form aggregates. An aggregate is a polymeric assembly comprising molecules or complexes of the monomeric insulin analogue. For the purpose of the present invention, the hexamer of the monomeric insulin analog is not an aggregate, but a complex. The monomeric insulin analogs, and the hexamer complexes thereof, have a propensity to aggregate when exposed to thermomechanical stress. Aggregation can proceed to the extent that a visible precipitate forms. The term "arginine" refers to the amino acid and encompasses the D and L enantiomers as well as the mixtures thereof. The term also includes any pharmacologically acceptable salts thereof. Arginine is also known in the art as 1-amino-4-guanidinovaleric acid. Arginine easily forms salts, such as the hydrochloride salt. 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, among many other compounds. The monomeric insulin analog may be a divalent zinc ion ligand. The term "continuous infusion system" refers to a device for continuously delivering a fluid to a patient parenterally for an extended or extended period of time or for intermittently administering a fluid to a patient parenterally for an extended period of time without having to establish a new administration site each time the fluid is administered. The fluid contains a therapeutic agent or agents. The device comprises a reservoir for storing the fluid before it is infused, a pump, a catheter, or other tubing for connecting the reservoir to the administration site by means of the pump, and control elements for regulating the pump. The device can be constructed for implantation, in the usual manner subcutaneously. In such a case, the insulin reservoir will usually be adapted for percutaneous filling. Obviously, when the device is implanted, the content of the reservoir will be at body temperature, and subject to the movement of the patient's body. An "isotonicity agent" is a compound that is physiologically tolerated and imparts a suitable tonicity to a formulation to prevent the net flow of water through the membranes that are in contact with the formulation. Compounds, such as glycerin, are commonly used for such purposes at known concentrations. Other possible isotonicity agents include the salts, for example, sodium chloride, dextrose, and lactose. The terms "monomeric human insulin analog", "monomeric insulin analogue" and "human insulin analogue" are well known in the art, and generally refer to fast-acting analogues of human insulin, which include: insulin human, where Pro in position B28 is replaced with Asp, Lys, Leu, Val, or Ala, and where position B29 is Lys or is replaced with Pro; AlaB26-human insulin des (B28-B30) human insulin; and des (B27) human insulin. Such monomeric insulin analogs are described in Chance, et al., U.S. Pat. No. 5,514,646, issued May 7, 1996; 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 March 18, 1987); and Brange, et al., Current Opinion in Structural Biology 1: 934-940 (1991). The monomeric insulin analogues employed in the present formulations are appropriately crosslinked. An appropriately cross-linked insulin analog contains three disulfide bridges: one between position 7 of chain A and position 7 of chain B, one second between position 20 of chain A and position 19 of chain B, and a third between positions 6 and 11 of the chain A. The term "phenolic preservative" as used herein, refers to chlorocresol, m-cresol, phenol, or mixtures thereof. When used herein, the word "stability" refers to the physical stability of formulations of monomeric insulin analogues. The physical instability of a protein formulation can be caused by the aggregation of protein molecules to form higher order or even precipitated polymers. A "stable" formulation is one in which the degree of aggregation of the proteins therein is acceptably controlled, and does not increase unacceptably with the passage of time. Formulations of the monomeric insulin analogue have a propensity to aggregate when exposed to a thermomechanical stress. The physical stability can be evaluated by methods well known in the art, including the measurement of an apparent attenuation of the light of the sample (adsorbency, or optical density). Such a measurement of light attenuation refers to the turbidity of a formulation. Turbidity is produced by the aggregation or precipitation of the proteins or complexes in the formulation. Other methods for evaluating physical stability are well known in the art. The term "treatment" refers to the management and care of a patient having diabetes or hyperglycemia, or another condition for which the administration of insulin is indicated for the purpose of combating or alleviating the symptoms and complications of these conditions. The treatment includes administering a formulation of the present invention to prevent the onset of symptoms or complications, alleviating symptoms or complications, or eliminating the disease, condition, or disorder. The term "TRIS" refers to 2-amino-2-hydroxymethyl-1,3-propanediol, and to any pharmacologically acceptable salt thereof. The free base and the hydrochloride form are two common forms of TRIS. TRIS is also known in the art as trimethylol aminomethane, tromethamine, and tris (hydroxymethyl) -aminomethane.

That the present invention provides formulations of monomeric insulin analogs that have an increased physical stability widely relative to those known in the art will be readily appreciated from the following data. Formulations comprising a monomeric insulin analogue, the analogue of LysB28ProB29-Human Insulin, and TRIS, prepared essentially as described in Example 3 here, were subjected to an accelerated stability test as described below. Samples of the prepared formulations were placed in pre-prepared, 2 ml capacity, glass self-sampling vials for HPLC. Each vial contained three Teflon® balls of approximately 0.47 cm (3/16 inches) in diameter. The air was completely displaced from the vial by the sample of the formulation. After sealing, the ampoules were agitated continuously at 40 Hz (20 xg, average linear acceleration) at a peak-to-peak amplitude of 12 mm, and at 37 ° C to provide a relatively high level of mechanical energy input to the formulations at a temperature that favors aggregation and physical instability. The ampules were placed on the agitator in such a way that their long dimension (ie from top to bottom) was oriented parallel to the direction of linear acceleration - ie, they remain on their sides on the surface of the agitator. It has been shown for insulin formulations that the increased stability under the accelerated conditions described above correlates with the widely increased stability in use. The optical density at 450 nm of the sample formulations and control formulations was periodically measured using a Shimadzu 1201 spectrophotometer. The control formulations were prepared in the same manner as the sample formulations, but were stored at 4 ° C. without agitation. The net optical density was calculated for a sample by subtracting the optical density of the control density from the optical density of the sample. The values in Table 1 are the average net optical density and the standard deviation for the given number of samples (n). The sampling and control formulations containing the phosphate as the buffer (pH 7.4 + 0.1) were prepared essentially as described in Example Table 1. Effects of the Shock Absorber and the Exposure Time at a High Mechanical Energy Input at 37 ° C on Turbidity (Optical Density at 450 nm) of the Formulations of the Human Insulin Analog-LysB28ProB29 16 hours 70 hours 100 hours 500 hours Example 3 0. 02 + 0 03 + 0 01 + 0 04 + (TRIS) 0. 01 0. 02 0. 01 0. 01 n = 5 n = 5 n = 5 n = 4 Example 4 0.81 + N.D. N.D, N.D. (Phosphate) 0.71 n = 5 N.D. = not determined Under the conditions described above, turbidity in formulations having a phosphate buffer reached very high, unacceptable levels for 16 hours (Table 1, Example 4) compared to control formulations containing phosphate which were stored at 4 ° C. C without agitation. On the other hand, the optical density of the formulations having TRIS as the buffer remained virtually identical to the optical density in the control for 500 hours for the formulations containing TRIS (Example 3). The data in Table 1 clearly demonstrate that the replacement of the phosphate buffer with the TRIS buffer in the formulations of Analog of Human Insulin-LysB28ProB29 drastically increases the stability of the formulations. Based on observations with other insulin formulations, it is believed that the surprising and significant stability of the monomeric insulin analog formulations in the TRIS buffer in the accelerated test will result in an "in use" stability well in excess of 500 hours because the energy input is greater in the accelerated test than during the expected uses. Formulations comprising a monomeric insulin analog, the Insulin Analog Hu ana-Lys B28 Pro B29, and either TRIS, phosphate, or L-arginine as buffers, were prepared essentially as described in Examples 3, 4, and 5, respectively. Three batches of the Human Insulin Analog-LysB28ProB29 were used to prepare the formulations. For each combination of the batch of analogues and the buffer, six samples were subjected to the stability test as described above. Four different agitators were used to impart mechanical energy to the ampoules. Each agitator had at least one sample of each batch and buffer combination. The stability of the formulations was evaluated periodically by measuring the optical density of the samples and controls as described above. The results are in Table 2. The values in Table 2 are the average net optical density and the standard deviation of six samples for each batch and buffer.

Table 2. Effects of the Shock Absorber, the Analog Batch, and the Exposure Time to a High Mechanical Energy Input at 37 ° C on the Turbidity (Optical Density at 450 nm) of the Insulin Analog Formulations Hurnana-LysB28ProB29 Optical Density at 450 nm Analogue Shock Absorber 23 hours 47 hours 87 hours 139 hours Lot 1 0.02 + 0.06 + 0.05 + 0.00 + 0.02 0.02 0.02 0.02 TRIS Lot 2 0.00 + 0.04 + 0.03 + 0.00 + 0.01 0.02 0.01 0.02 Lot 3 0.02 + 0.05 + 0.04 + 0.01 + 0.02 0.03 0.03 0.02 Lot 1 0.01 + 0.04 + 0.04 + 2.12 + 0.02 0.02 0.02 1.03 Arginine Lot 2 0.01 + 0.04 + 0.06 + 1.80 + 0.02 0.02 0.08 0.60 Lot 3 0.00 + 0.03 + 1.84 + N.D. 0.02 0.02 0.66 Phosphate Lot 1 0.13 + 2.68 + 2.61 + N.D. 0.06 0.17 0.11 Table 2 (Cont, Phosphate Lot 2 0.21 + 2.14 + 2.75 + N.D. 0.24 0.75 0.14 Lot 3 0.29 + 2.75 + 2.79 + N.D. 0.23 0.14 0.11 Under the conditions described above, the turbidity in the formulations having a phosphate buffer reached very high and unacceptable levels for 23 hours, regardless of the batch of the insulin analog used (Table 2). In contrast, turbidity in formulations that have TRIS as the buffer remained essentially unchanged for 139 hours, regardless of the batch of insulin used. The formulations containing the L-arginine buffer showed better physical stability compared to the phosphate-containing formulations, and the duration of its stability depends somewhat on the lot of the insulin analog used. The data in Table 2 clearly demonstrate that formulations of the Human Insulin Analog-LysB28ProB29 comprising the TRIS buffer or the L-arginine buffer at pH 7.4 remain stable against aggregation for markedly longer periods of time than the formulations comprising a phosphate buffer Again, it is believed that the surprising and significant stability of the monomeric insulin analogue formulations in the TRIS and L-arginine buffer will result in a much greater "in use" stability than that observed in the accelerated test because of that the energy input is greater in the accelerated test than during the expected uses. Susceptibility to changes in morphology and appearance for suspension formulations of LysB28ProB29 were evaluated by the Physical Stability Stress Test (PSST). In this thermomechanical method, the preparations were sealed without top space in a fixed volume container with glass beads. The containers were placed in a chamber at an elevated temperature (approximately 37 degrees centigrade), rotated or centrifuged at a fixed speed (approximately 30 rpm) for a defined time (approximately 4 hours) and then kept quiet for the remainder of a period of time. 24 hours. The containers were evaluated to verify the changes and were removed from the test when it was determined that the aggregation (formation of lumps) has occurred. The longest periods during the test without a failure, as well as larger numbers of the containers that remain in the test, were matched with increased physical stability. Two different mixtures containing LysB28ProB29 and protamine-LysB28ProB29 crystals were tested. The ratio of LysB28ProB29 with respect to protamine-LysB28ProB29 for the lower mixture was 25:75 and for the high mixture was 75:25. The mixtures were prepared as described in Examples 6 and 7. When the low mixture was tested using the PSST method, only the containers that had arginine containing the formulations remained for 18 days. Two of the test samples had containers that remained for 44 days. The PSST on the high mixtures showed similar results with the formulations containing arginine that have approximately 50% of the containers remaining after 36 days of testing while the control formulations containing the phosphate buffer had 0 to 5% of the containers that remain after 36 days. The preferred monomeric insulin analogs for use in the formulations of the present invention are human insulin-LysB28ProB29, human insulin-AspB28, and human insulin-AlaB26. The concentration of the monomeric insulin analogue in the present formulations ranges from 1.2 mg / ml to 50 mg / ml. A preferred range of analog concentration is from about 3.0 mg / ml to about 35 mg / ml. The most preferred concentrations are about 3.5 mg / ml, about 7 mg / ml, about 14 mg / ml, about 17.5 mg / ml, and about 35 mg / ml thereof. correspond roughly to formulations having approximately 100 units, approximately 200 units, approximately 400 units, approximately 500 units, and approximately 1000 units of insulin activity per ml, respectively. The concentration of zinc in the formulations ranges from about 4.5 mg / ml to about 370 mg / ml, and must be such that at least two zinc atoms are available to complex with the six insulin molecules in each hexamer. The ratio of total zinc (zinc that has formed a complex plus zinc that did not form a complex) to the hexamer of the insulin analog must be between 2 and 4. A ratio of about 3 to about 4 total zinc atoms per complex of the insulin analogue hexamer is preferred. A minimum concentration of the phenolic preservative is required to form the hexamer of the monomeric insulin analogue in the present formulations. For some purposes, such as satisfying the requirements of summarized preservative effectiveness for multiple-use formulations, the concentration of the phenolic preservative in the present formulations may be increased above that required to form hexamers up to an amount necessary to maintain the preservative effectiveness. The concentration of condoms necessary for effective preservation depends on the condom used, the pH of the formulation, and also whether the substances that bind or sequester the condom are present. In general, the amount needed can be found, for example, in WALLHAUSER, K.DH., DEVELOP. BIOL. STANDARD. 24, pp. 9-28 (Basel, S. Krager, 1974). When formulated, the insulin analogue hexamer complex used in the present formulation binds to as many as seven phenolic substances, although generally, only six phenolic substances are attached to the hexamer. A minima of approximately three phenolic substances is required for the formation of the hexamer. When a condom is required for antimicrobial effectiveness, the preferred phenolic concentration is about 23 mM to 35 mM. M-cresol and phenol, either separately or in mixtures, are the preferred condoms. The formulations may optionally contain an isotonicity agent. The formulations preferably contain an isotonicity agent, and glycerin is the most preferred isotonicity agent. The concentration of glycerin, when used, is in the range known in the art for insulin formulations, preferably approximately 16 mg / ml. The pH of the formulations is controlled by a buffer, such as TRIS or L-arginine. The concentration of the buffers is not thought to play a critical role in obtaining the subject of the invention, and should be such as to provide adequate buffering of the pH during storage to maintain the pH at a target pH or target of + 0.1. pH units. The preferred pH is between about 7 and about 8, when measured at a temperature of about 22 ° C. Other additives, such as pharmaceutically acceptable solubilizers similar to Tween 20® (polyethylene (20) sorbitan monolaurate), Tween 40® (polyethylene (20) sorbitan monopalmitate), Tween 80® (polyoxyethylene (20) sorbitan monooleate), Pluronic F68® (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) can optionally be added to the formulation. These additives are not required to achieve the great advantage of the present invention, but may be useful if the formulations will make contact with the plastic materials. The present invention also encompasses protamine salt preparations with varying proportions of soluble fractions of monomeric insulin analogues. No specific conformational requirement of the insulin molecule is required to stabilize the formulation with arginine, although zinc-like excipients and condoms normally added to insulin formulations (described above) can work in unison with arginine to improve stabilization . Arginine concentrations vary from 1 to 100 mM in formulations containing protamine. A range of arginine concentration of 5 to 25 mM is more preferred. Arginine can be added as a supplement to precipitated solutions or suspensions that already contain zinc ions and phenolic preservatives. Administration can be by any route known to the physician with ordinary skill in the art, which is effective. Parenteral administration is preferred. Parenteral administration is commonly understood as administration by a route other than the gastrointestinal route. Preferred parenteral routes for administration of the formulations of the present invention include intravenous, intramuscular, subcutaneous, intraperitoneal, intraarterial, nasal, pulmonary, and buccal routes. The intravenous, intraperitoneal, intramuscular, and subcutaneous routes of administration of the compounds used in the present invention are the most preferred parenteral routes of administration. The routes of intravenous, intraperitoneal, and subcutaneous administration of the formulations of the present invention are still more highly preferred. Administration by certain parenteral routes may involve introducing the formulations of the present invention into the body of a patient through a needle or catheter, driven by a sterile syringe or some other mechanical device such as a continuous infusion system. A formulation provided by the present invention can be administered using a syringe, injector, pump, or any other device recognized in the art for parenteral administration. A formulation of the present invention can also be administered as an aerosol for absorption in the lungs or a nasal cavity. The formulations may also be administered for absorption through mucous membranes, such as in buccal administration. The amount of a formulation of the present invention that is administered to treat diabetes or hyperglycemia depends on several factors, including, without limitation, the sex, weight and age of the patient, the underlying causes of the condition or disease to be treated, the route of administration and bioavailability, the persistence of the monomeric insulin analogue in the body, the formulation, and the potency of the monomeric insulin analogue. Where administration is intermittent, the amount per administration must also be taken into account in the interval between doses, and the bioavailability of the monomeric insulin analog of the formulation. The administration of the formulation of the present invention must be continuous. It is within the experience of the ordinary physician to graduate or titrate the dose and the rate of infusion or the frequency of administration of the formulation of the present invention to achieve the desired clinical result. The monomeric insulin analogs used in the present invention can be prepared by any of a variety of recognized peptide synthesis techniques including classical solution methods, solid phase methods, semi-synthetic methods, and recombinant methods. Chance, et al., U.S. Patent No. 5,514,646, issued May 7, 1996, describes the preparation of various monomeric insulin analogs in sufficient detail to enable one skilled in the art to prepare any of the monomeric insulin analogs used in the present invention. Both zinc and a phenolic preservative are essential to achieve a complex that is stable and capable of dissociation and rapid onset of action. The hexamer complex consists of two zinc ions per hexamer of the human insulin analog, and at least three molecules of a phenolic preservative selected from the group consisting of chlorocresol, m-cresol, phenol, and mixtures thereof. The monomeric insulin analog is converted to the hexamer complex by dissolving the monomeric insulin analog in a diluent containing the phenolic preservative in suitable amounts at a pH of about 7 to about 8 and then adding zinc. The zinc is preferably added as a zinc salt, such as, without limitation, zinc acetate, zinc bromide, zinc chloride, zinc fluoride, zinc iodide, and zinc sulfate. The skilled artisan will recognize that there are many other zinc salts which could also be used to make the monomeric insulin analog complexes that are part of the present invention. Preferably, zinc acetate, zinc oxide, or zinc chloride are used because these compounds do not add new chemical ions to t the commercially accepted processes. The dissolution of the monomeric insulin analogue can be aided by what is commonly known as an "acid solution". For the acid solution, the pH of the aqueous solvent is reduced to about 3.0 to 3.5 with a physiologically tolerated acid, preferably HCl, to aid in the dissolution of the monomeric analogue. Other physiologically tolerated acids include, without limitation, acetic acid, citric acid, and sulfuric acid. The 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 with a physiologically tolerated base, preferably sodium hydroxide, up to about pH 7.3 to 7.5. Other physiologically tolerated bases include, without limitation, potassium hydroxide and ammonium hydroxide. After this, the phenolic preservative and zinc are added. The parental formulations of the present invention can be prepared using conventional solutions and mixing procedures. To prepare a suitable formulation, for example, a measured amount of the monomeric insulin analogue in water is combined with the desired preservative, a zinc compound, and the buffering agent, in water in amounts sufficient to prepare the hexamer complex. The formulation is filtered in a generally sterile manner prior to administration. Variations of this process could be recognized by a person with ordinary skill in the art. For example, the order in which the components are added, the order in which the pH is adjusted, if any, the temperature and strength or ionic concentration at which the formulation is prepared, can be optimized for concentration and the means of administration used.

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

Example 1 Preparation of a U100 Soluble Formulation Containing the Human Insulin Analog-LysB28ProB29 and TRIS An amount of the Human Insulin Analog-LysB28ProB29-Zinc Crystals calculated to give 100 units of insulin activity per milliliter in the final formulation were suspended in an aqueous solution containing 0.715 mg / m? of phenol, 1.76 mg / ml of m-cresol, 16 mg / ml of glycerin, and zinc oxide. Zinc-analogue insulin crystals contained approximately 0.36% zinc on a weight basis. The concentration of the zinc oxide in the aqueous diluent was such that it supplements the concentration of the final zinc ion of the formulation to approximately 0.025 mg per 100 units of insulin activity. A volume of 10% hydrochloric acid was added to adjust the pH to 2.8 to 3.0. After stirring to dissolve the crystals, aliquots of a 10% sodium hydroxide solution were carefully added to adjust the pH from 7.4 to 7.7. One volume of a TRIS storage solution (50 mg / ml, pH 7.4, measured at room temperature, i.e., about 22 ° C) calculated to give a TRIS concentration of 2 mg / ml in the final formulation, was added to the insulin analog solution. Water was added to dilute the formulation to the final volume. The formulation was filtered sterile using a 0.2 micron filter.

Example 2 Preparation of a Soluble Formulation U100 Containing the Human Insulin Analog-Lys B28Ptro "B29 and L-Arginine The process described in Example 1 was followed until the addition of the buffer. Then, instead of adding a volume of a storage solution of TRIS, a volume of a storage solution of L-arginine (200 mM, pH 7.4), calculated to give a concentration of L-arginine of 20 mM in the formulation Finally, it was added to the insulin analog solution. Water is added to dilute the formulation to the final volume. The formulation was filtered under sterile conditions using a 0.2 micron filter.

Example 3 Preparation of a Soluble Formulation U400 Containing the Human Insulin Analog-LysB28ProB29 and TRIS An amount of the Humán Insulin AnalogLysB8ProB29-Zinc Crystals calculated to give 400 Units of insulin activity per milliliter in the final formulation were suspended in an aqueous solution containing 0.715 mg / ml phenol, 1.76 mg / ml m-cresol, 16 mg / ml glycerin, and zinc oxide. Zinc-analogue insulin crystals contained approximately 0.36% zinc based on weight. The concentration of the zinc oxide in the aqueous diluent was such that it supplements the concentration of the final zinc ion of the formulation at approximately 0.025 mg per 100 units of insulin activity. A volume of 10% hydrochloric acid was added to. adjust the pH to 2.8 to 3.0. After stirring to dissolve the crystals, aliquots of a 10% sodium hydroxide solution were carefully added to adjust the pH from 7.4 to 7.7. One volume of the TRIS storage solution (50 mg / ml, pH 7.4, measured at room temperature, i.e., about 22 ° C) calculated to give a TRIS concentration of 2 mg / ml in the final formulation was added to the insulin analog solution. Water was added to dilute the formulation to the final volume. The formulation was filtered sterile using a 0.2 micron filter.

Example 4 Preparation of a Soluble Formulation U400 Containing the Human Insulin Analog-LysB28ProB29 and Phosphate An amount of the Human Insulin Analog-LysB28ProB 9-Zinc Crystals calculated to give 400 Insulin activity units per milliliter in the final formulation were suspended in an aqueous solution containing 0.715 mg / ml phenol, 1.76 mg / ml of m-cresol, 16 mg / ml of glycerin, and zinc oxide. Zinc-analogue insulin crystals contained approximately 0.36% zinc on a weight basis. The concentration of the zinc oxide in the aqueous diluent was such as to supplement the concentration of the final zinc ion of the formulation to approximately 0.025 mg per 100 units of insulin activity. A volume of 10% hydrochloric acid was added to adjust the pH from 2.8 to 3.0. After stirring to dissolve the crystals, aliquots of 10% sodium hydroxide solution were carefully added to adjust the pH from 7.4 to 7.7. a volume of a dibasic sodium phosphate storage solution calculated to give a dibasic sodium phosphate concentration of 3.78 mg / ml, pH 7.4 + 0.1 in the final formulation was added to the insulin analogue solution. Water was added to dilute the formulation to the final volume. The formulation was filtered under sterile conditions using a 0.2 micron filter.

Example 5 Preparation of a Soluble Formulation U400 Containing the Human Insulin Analog-LysB28ProB29 and L-Arginine The process described in Example 3 was followed until the addition of the buffer. Then, instead of adding a volume of a storage solution of TRIS, a volume of a storage solution of L-arginine (200 mM, pH 7.4) calculated to give a concentration of L-arginine of 20 mM in the final formulation, to the insulin analog solution.

Water is added to dilute the formulation to the final volume. The formulation was filtered sterile using a 0.2 micron filter.

Example 6 Preparation of a Mixing Formulation with a Level Elevated Insulin Analog Human-Lys BB2 ¿8aPr, BB2¿9a (75% v / v soluble, 25% v / v neutral protamine LysB28ProB29) Containing L-arginine A. Preparation of Neutral Protamine Lys B28Ptro. B29 An amount calculated to contain 200 U / ml of Zinc Insulin Crystals LysB28ProB29 was suspended in an aqueous solution containing 0.715 mg / ml of phenol, 1. 76 mg / ml of m-cresol, 16 mg / ml of glycerin, and zinc oxide acidified with hydrochloric acid to supplement the concentration of the final zinc ion to 0.025 mg / 100 U. A volume of 10% hydrochloric acid was added to adjust the solution to a pH of 2.8 to 3.0. After stirring to dissolve it, the 10% sodium hydroxide solution was added to adjust the solution to pH of 7. 4 to 7.7. A volume equivalent to a final formulation concentration of 3.78 mg / ml of a 75.6 mg / ml dibasic sodium phosphate solution at pH 7.2 was added. Following the dissolution of the precipitated solids and titration or concentration to maintain the pH at 7.4, water was added to dilute the formulation to the final volume, after which the solution was filtered. Solid protamine sulfate, calculated to contain 0.6 mg / 100 U of the protamine base, was dissolved in an aqueous solution containing 0.715 mg / ml phenol, 1.76 mg / ml m-cresol and 16 mg / ml glycerin . The solid dibasic sodium phosphate was added so that the concentration of the formulation was 3.78 mg / ml. The solution was adjusted to pH 7.4 with 10% hydrochloric acid, diluted to its final volume with water, and filtered. Both the 200-unit LysB28ProB29 solution and the protamine solution were equilibrated at 15 ° C. The protamine solution was added to the solution of LysB28ProB29 and the resulting suspension allowed to incubate without alteration at 15 ° C for 24 hours.

B. Preparation of a Mixture with a High Content of LysB28ProB29 An amount of the 100 unit Lys Pro solution containing L-argmin prepared in Example 2 corresponding to 75% of the final volume was added to a calculated volume of 100 U / ml neutral protamine LysB28ProB29. The suspension was stirred at room temperature. Example 7 Preparation of a Mixing Formulation with a Low Level of Human Insulin Analog-LysB28ProB29 (25% v / v soluble, 75% v / v of neutral protamine Lys Pro) Containing L-arginine A. Preparation of Neutral Protamine Lys B28Ppr_oB29 An amount calculated to contain 200 U / ml of Zinc Insulin Crystals LysB28ProB29 was suspended in an aqueous solution containing 0.715 mg / ml of phenol, 1. 76 mg / ml of m-cresol, 16 mg / ml of glycerin, and zinc oxide acidified with hydrochloric acid to supplement the concentration of the final zinc ion to 0.025 mg / 100 U. A volume of 10% hydrochloric acid was added to adjust the solution to pH 2.8 to 3.0. After stirring to dissolve it, the 10% sodium hydroxide solution was added to adjust the solution to pH of 7. 4 to 7.7. A volume equivalent to a final formulation concentration of 3.78 mg / ml of a 75.6 mg / ml dibasic sodium phosphate solution at pH 7.2 was added. Following the dissolution of the precipitated solids and titration or concentration to maintain the pH at 7.4, water was added to dilute the formulation to the final volume, after which the solution was filtered. Solid protamine sulfate, calculated to contain 0.6 mg / 100 U of protamine base, was dissolved in an aqueous solution containing 0.715 mg / ml of phenol, 1.76 mg / ml of m-cresol and 16 mg / ml of glycerin. The solid dibasic sodium phosphate was added so that the concentration of the formulation was 3.78 mg / ml. The solution was adjusted to pH 7.4 with 10% hydrochloric acid, diluted to its final volume with water, and filtered. Both the U200 LysB28ProB29 solution and the protamine solution were equilibrated at 15 ° C. The protamine solution was added to the solution of LysB28ProB29 and the resulting suspension allowed to incubate without alteration at 15 ° C for 24 hours.

B. Preparation of a Mix with a Low Content of LysB8ProB29 An amount of the U100 solution of LysB28ProB29 containing L-arginine prepared in Example 2 corresponding to 25% of the final volume was added to a calculated volume of 100 U / ml of neutral protamine LysB28ProB29. The suspension was stirred at room temperature.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (33)

1. A solution formulation, characterized in that it comprises: a physiologically tolerated buffer selected from the group consisting of TRIS and arginine; a monomeric insulin analogue; zinc; and a phenolic preservative.

2. The formulation according to claim 1, characterized in that the monomeric insulin analogue is human insulin-LysB28ProB29 and the buffer is TRIS.

3. The formulation according to claim 1, characterized in that the monomeric insulin analogue is human insulin-AspB28 and the buffer is TRIS.

4. The formulation according to claim 2, characterized in that it also comprises an isotonicity agent and wherein the pH of the formulation is between pH 7.0 and pH 8.0 when measured at a temperature of 22 ° C.

5. The formulation according to claim 4, characterized in that the concentration of human insulin-LysB28ProB29 is between about 1.2 mg / ml and about 50 mg / ml.

6. The formulation according to claim 5, characterized in that the concentration of hu? T? Ana-LysB28ProB29 insulin is between about 3.0 mg / ml and about 35 mg / ml.

7. The formulation according to claim 3, characterized in that it also comprises an isotonicity agent and wherein the pH of the formulation is between pH 7.0 and pH 8.0 when measured at a temperature of 22 ° C.

8. The formulation according to claim 7, characterized in that the concentration of human insulin-AspB28 is between about 1.2 mg / ml and about 50 mg / ml.

9. The formulation according to claim 8, characterized in that the concentration of human insulin-AspB28 is between about 3.0 mg / ml and about 35 mg / ml.

10. The formulation according to claim 6, characterized in that the TRIS is present at a concentration of about 2 mg / ml; glycerol is the isotonicity agent and is present at a concentration of approximately 16 mg / ml; and m-cresol is present at a concentration of about 1.76 mg / ml and the phenol is present at a concentration of about 0.715 mg / ml.

11. The formulation according to claim 9, characterized in that the TRIS is present at a concentration of about 2 mg / ml; glycerol is the isotonicity agent and is present at a concentration of approximately 16 mg / ml; and -cresol is present at a concentration of approximately 1.76 mg / ml and the phenol is present at a concentration of approximately 0.715 mg / ml.

12. A soluble, stable formulation of a monomeric insulin analogue for use in a continuous infusion system, characterized in that it consists essentially of: an isotonicity agent; a buffer selected from the group consisting of TRIS and arginine; a monomeric insulin analogue; zinc; and a phenolic preservative.

13. The formulation according to claim 1, characterized in that it also comprises protamine.

14. The formulation according to claim 13, characterized in that the insulin analogue is LysB28ProB29.

15. The formulation according to claim 13, characterized in that the insulin analogue is Asp B28

16. The formulation according to any of claims 13 to 15, characterized in that the buffer is arginine.

17. The formulation according to any of claims 1 to 12, for use in a continuous infusion system.

18. A method for the treatment of diabetes, characterized in that it comprises administering an effective dose of the formulation of any of claims 1 to 16 to a patient in need thereof.

19. A method for the treatment of diabetes, characterized in that it comprises administering an effective dose of the formulation of any of claims 1 to 12, wherein the formulation is administered using a continuous infusion system.

20. A method for the treatment of hyperglycemia, characterized in that it comprises administering an effective dose of the formulation of any of claims 1 to 16 to a patient in need thereof.

21. A method for the treatment of hyperglycemia, characterized in that it comprises administering an effective dose of the formulation of any of claims 1 to 12, wherein the formulation is administered using a continuous infusion system.

22. The formulation of the monomeric insulin analogue according to any of claims 1 to 16, for use as a medicament for the treatment of diabetes.

23. The formulation of the monomeric insulin analogue according to any of claims 1 to 16, for use as a medicament for the treatment of hyperglycemia.

24. A monomeric insulin analog formulation, characterized in that it is as described hereinbefore with reference to any of the examples.

25. A process for preparing the monomeric insulin analog formulation according to claim 1, characterized in that it comprises the steps of mixing a physiologically tolerated buffer selected from the group consisting of TRIS and arginine with a monomeric insulin analogue; zinc; and a phenolic preservative.

26. The process according to claim 25, characterized in that the monomeric insulin analogue is human insulin-LysB28ProB29 and the buffer is TRIS.

27. The process according to claim 25, characterized in that the monomeric insulin analogue is human insulin-AspB28 and the buffer is TRIS.

28. A process for preparing a monomeric insulin analogue formulation, characterized in that it comprises the steps of mixing a buffer selected from the group consisting of TRIS and arginine with a monomeric insulin analogue; zinc; protamine; and a phenolic preservative.

29. The process according to claim 28, characterized in that the monomeric insulin analog is LysB28ProB29.

30. The process according to claim 28, characterized in that the monomeric insulin analog is Asp B28.

31. The process according to any of claims 28 to 30, characterized in that the buffer is arginine.

32. The formulation according to any of claims 1 to 12, characterized in that it is made by the process of claim 25.

33. The formulation according to any of claims 13 to 16, characterized in that it is made by the process of claim 28.