MXPA05009495A - Long acting injectable insulin composition and methods of making and using thereof - Google Patents

Abstract

A method of lowering blood glucose in a mammal includes injecting a therapeutically effective amount of crystallized dextran microparticles and insulin to the mammal to lower blood glucose of the mammal. The composition may be a one phase or a structured multi-phase composition for controlled release of insulin over an extended period of time.

Description

INJECTABLE INSULIN COMPOSITION OF PROLONGED ACTION AND METHODS FOR PRODUCING AND USING IT FIELD OF THE INVENTION This application claims the benefit of the following Provisional Applications from the U.S. Nos. Of Series 60/451, 245, filed on March 4, 2003; 60 / 467,601, filed May 5, 2003; 60 / 469,017, filed May 9, 2003 and 60 / 495,097, filed August 15, 2003, the descriptions of which are hereby incorporated by reference in their entirety. The present invention relates in general to insulin compositions and specifically to an injectable insulin composition containing insulin and crystallized dextran microparticles.

BACKGROUND OF THE INVENTION Dextrans are high molecular weight polysaccharides synthesized by some micro-organisms or by biochemical methods. Dextran with an average molecular weight of approximately 75 kDa has a colloidal osmotic pressure similar to that of blood plasma, therefore its aqueous solutions are used clinically as plasma expanders. Dextrans with crosslinking in the form of beads are the basis of "Sephadex" ® that is used in the GPC of proteins and of "Cytodex" ® developed by Pharmacia to meet the special requirements of a micro-vehicle cell culture. For example, in the US Patents. Nos. 6,395,302 and 6,303,148 (Hennink et al.) Describe the annexation of different biomaterials to dextran particles with crosslinking. However, dextran-based beads with crosslinking generally can not be used for the manufacture of implants due to their potential toxicity due to the application of crosslinking agents (Blain Jf, Maghi K., Pelletier S. and Sirois P. Inflamm. Res. 48 (1999): 386-392). In the US Patent. No. 4,713,249 (Schroder) describes a method for producing a deposit matrix for biologically active substances. According to this patent, the deposit matrix presumably consists of micro-particles of carbohydrates, stabilized by crystallization, which involves using non-covalent bonds. Schroder describes the following procedure to produce the presumed micro-particles of crystallized carbohydrates. A solution of polymeric carbohydrate and a biologically active substance is formed in one or more hydrophilic solvents. The mixture of the carbohydrate and the biologically active substance is then emulsified in a liquid hydrophobic medium to form spherical droplets. The emulsion is then introduced into a crystallization medium comprising acetone, ethanol or methanol to form spheres having a crystalline polymeric carbohydrate matrix with non-covalent crosslinking, said matrix incorporating 0.001-50% by weight of the biologically active substance. That is, the biologically active substance is supplied in the solution before the spheres crystallize. Schroder does not describe the micro structure of micro-particles made by this multi-step method. Schroder's multi-step method is complex and uses organic solvents that are potentially toxic to cells and need to be removed.

BRIEF DESCRIPTION OF THE INVENTION A method for reducing blood glucose in a mammal includes injecting a therapeutically effective amount of crystallized dextran microparticles and insulin to the mammal to reduce the blood glucose of the mammal. The composition may be a single-phase or multi-phase composition structured for the controlled release of insulin over a prolonged period of time.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a photograph of the crystallized dextran microparticles formed spontaneously in an aqueous solution at 55.0% by weight on dextran weight with Molecular Weight of 70.0 kDa. Figure 2A is a photograph of a section of the crystallized dextran micro-particles shown in Figure 1.

The Figure 2B is a photograph of a section of a micro-particle shown in Figure 2A. The micro-porous structure of the micro-particle can be observed. Figure 3 is a photograph of aggregates of the crystallized dextran microparticles. Figure 4 is a photograph of the slow release of fluorescence-labeled macromolecules from an implant including dextran microparticles in mouse muscle tissue at day 14 after intramuscular injection. Figure 5 is a photograph of an emulsion of aqueous PEG solution in aqueous dextran solution (Molecular Weight 500 kDa), which contains the crystallized dextran micro-particles shown in Figure 1. Figure 6 is a photograph of an emulsion of aqueous dextran solution (Molecular Weight 500 kDa), containing the crystallized dextran micro-particles shown in FIG. Figure 1, in aqueous solution of PEG. Figure 7 is a photograph of an intramuscular injection of PEG aqueous solution emulsion in aqueous dextran solution (Molecular Weight 500 kDa), containing the crystallized dextran micro-particles shown in Figure 1. Figure 8 is a photograph of a subcutaneous emulsion injection of aqueous PEG solution in aqueous dextran solution (Molecular Weight 500 kDa), containing the crystallized dextran microparticles shown in Figure 1. Figures 9A and 9C schematically illustrate the behavior of partition of different types of particles and phases in a two-phase aqueous system. Figure 9B is a photograph of a section of an implant structure based on the two-phase system. Figures 10 and 11 schematically illustrate methods of administering therapeutic agents in accordance with embodiments of the present invention. Figures 12A and 12B are graphs of relatively normalized blood glucose concentrations, for various compositions containing insulin vs. weather.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The present inventor has discovered that a crystallized dextran microparticle composition and insulin, injected into mammals, unexpectedly extended the duration of insulin efficacy as compared to injections of the same dose of the same insulin alone. The composition may be a single-phase composition or a multi-phase composition that forms a structured implant in a mammal.

The first section below describes the micro-particles of crystallized dextran, the second section describes the formation of the structured implant from a multi-phase composition and the following sections describe specific examples of the injection of the composition to mammals and methods for making the injectable composition.

A. Crystallized Dextran Microparticles The present inventor has discovered, experimentally, that the crystallized dextran microparticles with an average diameter ranging from 0.5 to 3.5 microns, are formed spontaneously in concentrated aqueous solutions of dextrans (40-65%) in weight on weight), with molecular weights ranging from 1.0 to 200.0 kDa, at temperatures ranging between 20 and 90 ° C. If it is desired to form the microparticles at room temperature, dextran solutions of 2 to 18 kDa can be used. Of course, microparticles can also be formed with solutions of 2 to 18 kDa at temperatures above room temperature, if desired. The microparticles can be formed spontaneously from dextran solutions of higher molecular weight, such as solutions of 20 to 75 kDa, at temperatures higher than room temperature, for example about 40 ° C to about 70 ° C. The microparticles may have any suitable shape, which may be regular or irregular shape, but preferably are spherical in shape and preferably with a diameter of 10 microns or less, such as 0.5 to 5 microns.

Transmission electron microscopy revealed the micro-porous structure of the crystallized dextran microparticles (see Figures 2A, 2B). Preferably, the porosity of the microparticles is at least 10% by volume, such as from about 10% to about 50%, most preferably about 20% to about 40%. That is, the structure comprises micro-porous micro-particles with areas of macro-porosity located between the particles. Aerosol drying of the aqueous suspensions of the crystallized dextran microparticles has demonstrated the possibility of producing substantially spherical aggregates of crystallized dextran microparticles, with a diameter ranging from 10.0 to 150.0 microns (see Figure 3). A non-limiting example of a method for forming the dextran microparticles is as follows. 50.0 grams of T40 dextran (40 kDa molecular weight) from Amersham Biosciences is added to 50.0 g of sterile distilled water in a 500 ml laboratory flask to obtain a 50 wt% solution on weight under laminar flow. The mixture is stirred at 60 ° C (water bath) on a magnetic stirrer at 50 rpm until the dextran is completely dissolved and a clear solution is obtained. The solution can be subjected to vacuum to eliminate air inclusions. The clear solution is placed in a laboratory oven at 60 ° C, under a Tyvek® lid. 3.5 hours later, a viscous, cloudy suspension develops as a result of the formation of crystallized dextran microparticles.

To remove the non-crystallized dextran, the microparticles are washed by centrifugation, for example 3000 g, 30 min., With 3 x 250 ml of sterile distilled water, or by filtration of diluted suspension of micro-particles, (3 x 250 my sterile distilled water through sterilization filter). The centrifugation / washing is done under laminar flow. The micro-particles are placed in a 500 ml laboratory flask under a Tyvek® cap and dried at 60 ° C in a laboratory oven for eight hours to reach a moisture level of approximately 5%. The resulting dry powder consists of particles with an average diameter of about two microns. The crystallized microparticles, preferably comprise dextran molecules (ie, polymer molecules), which are held together by various hydrogen bonds, Van Der Waals forces and / or ionic bonds and which have, substantially, an absence of non-covalent bonds between the dextran molecules. That is, preferably, the molecules of the microparticles do not have intentional crosslinking (ie the crosslinking step is not performed) and the microparticles do not contain bonds, covalent between the molecules or have less than 10% bonds, covalent between molecules. The slow release of macro-molecules from the implants has been demonstrated in experiments in which macro molecules were dissolved in aqueous suspensions of crystallized dextran particles or their aggregates before injections. Figure 4 shows an implant containing fluorescence-labeled macro-molecules (FITC-dextran, 500 kDa Molecular Weight) and the slow release of the macro-molecules from the implant to the mouse muscle tissue on day 14 after intermuscular injection .

B. Two Phase Systems. Self-assembled implant structures based on crystallized dextran micro-particles and their aggregates can be formed based on two-phase systems. Colloidal systems, such as oil droplets, liposomes, micro- and nanoparticles can be dispersed in a suspension of crystallized dextran microparticles and injected to form an implant that releases therapeutic agents after administration to the body of a mammal. For example, in the case of oil, a special type implant structure can be formed wherein the oil core is surrounded by a shell composed of crystallized dextran micro-particles, or aggregates thereof, dispersed in water or solutions aqueous polymers such as polysaccharides (e.g., dextrans). The structure described can be called a capsule. It should be noted that the shell may comprise a substantially spherical shell which is the result when the capsule is surrounded by tissue. However, when the capsule is located near a barrier, such as a substrate, bone or intestinal wall, the capsule may comprise a core located between one or more walls of micro-particles on one side and the barrier on the other side. Moreover, ough oil is used as an illustrative example, the core may comprise other materials such as other polymers, cells, and the like. Two-phase aqueous systems are applied to form the structure of the capsule. When aqueous solutions of different polymers are mixed above certain concentrations, they often form two-phase immiscible liquid solutions. Each of the phases usually consists of more than 90% or water and can have a buffer and become isotonic. If a suspension of cells or particles is added to a system such as this, it is often found that the cells or particles have been unevenly partitioned between the phases. This preferential partition behavior can be used as a basis for separation procedures for the purpose of differentiating populations of cells or particles since the partitioning in these systems is directly determined by the surface properties of the cell or particle. Cells or particles that do not have identical surface properties exhibit a sufficiently different partition behavior. The competitive adsorption of the two phases of the polymer depends on the chemical nature of the polymers. A two-phase polymer method has been applied to separate or partition cells, proteins, nucleic acids and minerals ("Partitioning in Aqueuous Two Phase Systems", 1985, eds., H. Wr, D. Brooks and D. Fisher, pubis Academic Press).

Experiments with the distribution of crystallized dextran microparticles in phase systems derived from, for example, mixtures of dextran / polyethylene glycol (PEG), revealed that dextran microparticles prefer to be in the dextran phase, while that other PEG phases can be dispersed in this dextran phase to form a weight-on-weight emulation and vice versa in the case where the volume of the PEG phase is greater than the volume of the dextran phase, as shown in Figures 5 and 6. Figure 5 is a photograph of an aqueous solution emulsion of PEG in dextran aqueous solution containing micro-particles of crystallized dextran. In the structure of Figure 5, the volume of the PEG phase is less than the volume of the dextran phase. The dextran phase contains the dextran and the crystallized dextran micro-particles. That is, the PEG phase is formed as one or more spherical nuclei, surrounded by a closed pore structure). Figure 6 is a photograph of an emulsion of aqueous dextran solution containing crystallized dextran microparticles in aqueous PEG solution, wherein the volume of the PEG phase is greater than the volume of the dextran phase. In this case, the dextran phase is formed as one or more spherical-shaped nuclei containing the dextran micro-particles surrounded by a PEG phase (i.e., an open pore structure that is formed in vivo while the PEG is dissipated in liquids). of tissues). As can be seen in Figure 6, the dextran phase of smaller volume (droplet) is formed as a large, spherical nucleus of dextran / dextran micro-particles (lower right part of Figure 6) to which they are attached and smaller spheres containing dextran / dextran micro-particles merge with it. That is, when the ratio of the volume of the first phase (such as the PEG phase and its inclusions, such as the therapeutic agent) to the volume of the second phase (such as the dextran phase and its inclusions, such as the micro - dextran particles) is less than one, then the capsule is formed by assembling with a first phase core surrounded by a second phase shell. If the composition contains a therapeutic agent, such as insulin, which prefers to partition in the PEG phase, and the dextran microparticles that prefer to partition in the dextran phase, then the therapeutic agent is selectively partitioned into the PEG core while the micro-particles are selectively partitioned into, and form the shell surrounding the PEG core by self-assembly. The emulsion can be prepared by mixing phases prepared separately from dextran and PEG and both can be suspensions of different types of particles that prefer to be in the PEG phase or in the dextran phase, respectively. The principle is that the partition of molecules (such as macro-molecules, DNA, plasmids, etc.) or molecular aggregates (such as micro-particles, cells, liposomes, proteins, etc.) into different polymer phases depends on their surface structure and the interfacial energy of the particles in the polymer solutions. Injection of two-phase aqueous systems containing crystallized dextran microparticles in tissues from experimental animals revealed the formation of implants with the capsule structure shown in Figures 7 and 8. The volume of the dextran phase is greater than the volume of the PEG phase in the two-phase system. Both Figures 7 and 8 show that a capsule with a PEG core and a dextran / dextran microparticle shell is formed by intimate self-assembly (i.e., after injection into the tissue of a mammal). The shell comprises macro-porous regions between adjacent micro-particles as well as micro-porous regions in the micro-particles themselves. A non-limiting example of a method for forming a capsule structure from a two-phase system is as follows. 10 g of dextran T40 (Molecular Weight of 40 kDa) and 2 g of PEG are dissolved in 88 ml of (Actrapid®), insulin solution containing 1, 000 IU to which 25 g of dextran micro-particles are added. crystallized These steps are performed under laminar flow conditions. The mixture is stirred on a magnetic stirrer at 100 rpm at room temperature for 30 minutes to form a homogeneous mixture (i.e., a suspension). 1.0 g of the suspension contains 8 IU of insulin. It should be noted that the dextran microparticles can be prepared from a dextran solution of different molecular weight to the dextran solution that is provided in the two-phase system. That is, the crystallized dextran microparticles can be formed in a dextran solution of lower molecular weight, such as a 2 to 20 kDa solution, than the dextran solution that is provided in the two-phase system, which can be a dextran solution of 40 to 500 kDa, such as a 40 to 75 kDa solution. This is advantageous since dextran solutions of higher molecular weight, such as solutions of 40 to 70 kDa, have received wider approval at the regulatory level and can be used to form a shell or capsule at lower concentrations. Lower molecular weight solutions can be used to reduce the crystallization time without the lower molecular weight dextran solution actually being provided in vivo. Even more, the micro-particles of lower molecular weight can be dissolved more easily in vivo. The capsule structure formed from the two-phase system is advantageous since it allows a more uniform and prolonged release of the therapeutic core agent than from a composition comprising a single phase containing the micro-particles. Moreover, it is considered that by using the capsule structure, fewer micro-particles may be needed to achieve the same release or a lower release, with controlled time, of a therapeutic agent than if a single-phase system is used. Moreover, by controlling the amount of micro-particles in the two-phase system, it is believed that the thickness of the micro-particle shell can be controlled. A thicker shell is the result of a larger amount of micro-particles in the two-phase system. That is, the amount, and / or time of the release of the therapeutic agent from the core of the capsule can be controlled by controlling the thickness of the shell. Therefore, the release profile of the therapeutic agent can be adapted for each patient or group of patients. It should be noted that, although PEG and dextran are used as examples of the materials of the two phases, any other suitable material showing the following partition compartment can be used. In Figure 9A, the partition behavior of different types of particles in a two-phase aqueous system is shown schematically. For example, three types of molecules or molecular aggregates are shown, which are preferably particles 10, 12 and 14 and the two phases 16 and 18 are shown in Figure 9A. However, there may be two or more than three types of particles. The particles can be micro-particles such as microspheres or nano-spheres, prepared from organic and / or inorganic materials, liposomes, living cells, viruses and macro-molecules. The particles of the first type 10, preferably segregate in the first phase 16. The particles of the second type 12, preferably segregate to the limits of the first phase 16 and of the second phase 18. The third type of particles 14, preferably they segregate into the second phase 18. That is, by analogy with the previous, non-limiting example, the first particles 10 may comprise a therapeutic agent, the second 12 and / or the third particles 14 may comprise micro-particles of crystallized dextran, the first phase 16 may comprise a PEG phase and the second phase 18 may comprise a dextran phase. If a smaller amount of the first phase 16 is provided to a larger amount of the second phase 18, as shown in area 20 of Figure 9A, then a capsule-like structure is formed comprising discrete spheres of the first phase 16 which it contains a concentration of the particles of the first type 10, located in the second phase 18. The particles of the second type 12 can be located at the interface of the phases 16 and 18 and act as a shell of the capsule. The particles 14 are dispersed in the second phase 18 and / or form a shell of the capsule. In comparison, if a smaller amount of the second phase 18 is provided in a larger amount of the first phase 16, as shown in area 22 of Figure 9A, then a capsule-like structure comprising discrete spheres of the second is formed. phase 18, which contain a concentration of the particles of the third type 14, located in a first phase 16. The particles of the second type 12 can be located in the phase phase of phases 16 and 18 and act as shell of the capsule. The particles 10 are dispersed in the first phase 16 and / or form a shell of the capsule. The two-phase systems 10 and 22 can be used as an implant, for example when being injected, surgically implanted or administered orally to a mammal that can be an animal or a human being, that is, the capsule forms a structured implant, three-dimensional, with the nucleus acting as a reservoir for the controlled release of the therapeutic agent through the shell. In comparison, an implant with a uniform distribution of micro-particles is an unstructured implant. Moreover, the particles 10, 12 and 14 can be replaced by a liquid material (for example oils) or selective separation macro-molecules in one of the phases. For example, a therapeutic agent, such as insulin, can be partitioned into the PEG phase of the PEG / dextran two-phase system. As insulin selectively partitiones in the PEG phase, the PEG phase forms a nucleus of the capsule structure containing insulin. It should be noted that although certain particles and therapeutic agents selectively partition, the term "selectively partitioned" does not necessarily mean that 100% of the particles or therapeutic agent will partition in one of the phases. However, most of the species with selective partitioning, preferably 80% of the partitioned species, are partitioned into one of the phases. For example, although most insulin partitions in the PEG phase, a portion of the insulin may remain in the dextran phase. Figure 9B illustrates an electron microscopy image of a cut of an implant structure based on the two-phase system illustrated schematically in Figure 9A. A two-phase aqueous composition, comprising a first dextran phase, a second PEG phase and crystallized dextran microparticles, was injected into sepharose gel. This gel composition simulates the tissue of a mammal by stopping the diffusion of crystallized dextran microparticles from the injection site. The image of Figure 9B illustrates the formation of a core-shell implant structure. The core comprises regions 30 and 32, surrounded by a shell 34. Region 30 is a vacuum that is filled with a PEG phase before cutting the gel for the electron microscopy image of the cut. The region of the PEG phase leaves the gel when the gel is cut during cutting. Region 32 is an outer portion of the core comprising droplets of PEG located in the crystallized dextran microparticles. Region 34 is the shell comprising the crystallized dextran microparticles which surrounds and holds the core containing PEG in place. Without intending to be limited to a particular theory, the present inventor considers that the core-shell structure shown in Figure 9B is formed by self-assembly, as schematically shown in Figure 9C. While the first phase 16 and the second phase 18, such as different, incompatible aqueous solutions of polymers, are found in the appropriate storage container 19, which may be a flask or vial, a phase 16, rises above the other phase 18. When the two-phase composition is injected into a material that restricts the free flow of phases 16 and 18, such as the tissue of a mammal or a substrate material, such as a gel that simulates the tissue, the composition It is self-assembled in the core-shell structure. First, the phase that is present in the smaller volume is formed approximately spherically, as shown in the central part of Figure 9C. Then the spherical shapes come together to form approximately one-phase spherical nuclei, surrounded by the shells of the other phase, as shown in the lower part of Figure 9C. Although an example has been illustrated in a two-phase system, of a multi-phase system, the multiple-phase system can have more than two phases, if desired.

C. Vehicle for Injectable Insulin Administration The present inventor has discovered that a composition of crystallized dextran microparticles and insulin, injected into mammals, such as rats and rabbits, unexpectedly extended the duration of insulin efficacy in comparison with injections of the same dose of the same insulin alone. Figure 10 illustrates schematically the formation of an implant 40 in a mammal 53, by injection of a single-phase composition comprising the microparticles 12, 14 and insulin 46, using a syringe 56. Figure 11 illustrates schematically the formation of a structured implant 40 in a mammal 53, by injection of a two-phase composition comprising a dextran phase 18, containing crystallized dextran microparticles, selectively partitioned, 12, 14 and a PEG 16 phase, which contains the selectively partitioned therapeutic agent 10, which comprises insulin. The dextran phase 18 forms a shell around the PEG phase core 16. As rats and rabbits are a common model for humans in drug testing, the present inventor considers that the composition comprising micro-particles of crystallized dextran and insulin would also be effective in extending the duration of insulin efficacy when injected to adult humans and children. Examples 1-8 illustrate the advantage of using crystallized dextran microparticles as a vehicle for the administration of injectable insulin, compared to the injection of insulin alone. The experiment involved mice and their response was observed to an aqueous suspension injected subcutaneously consisting of crystallized dextran micro-particles and human recombinant insulin (NovoNordisk Actrapid HM Penfill®, 40 IU / ml). The suspension was prepared as follows. 5.0 g. of Dextran T10 (Pharmacia, Uppsala, Sweden) were dissolved in 20.0 g of water. The solution was filtered through a 0.22 μm filter (Millipore, Bedford, MA) and freeze dried. 3.0 g of the resulting powder were dissolved in 3.0 g of sterile water and placed in a box at a temperature of 60 ° C. 6 hours later, the crystallized dextran micro-particles were washed by centrifugation at 3,000 g with 3 x 5.0 ml of sterile water. Finally, the suspension of crystallized dextran microparticles was mixed with an aqueous solution of insulin and used in the experiment with mice. Samples of the suspension were introduced into the legs of the mice and samples of the animal's blood were taken from the tail of each animal and the glucose concentration was analyzed. Blood glucose was measured using the glucosoxidase method in a One-touch glucose analyzer system (Lifescan, Johnson &Johnson, Milpitas, CA, USA) after proper calibration. In comparative example 1, the mouse was not injected with insulin. In comparative examples 2, 3 and 7, insulin alone (0.5 IU) was injected into the three mice. In Examples 4-6 and 8, insulin (0.5 IU) and a crystallized dextran micro-particle implant were injected into the four mice. The results are summarized in Table I.

TABLE I The average reduction of blood sugar (ie blood glucose) of the animals is very different when 0.5 Ul i.m. with and without crystallized dextran micro-particles. As shown in Table I, the glucose level in the mice of Comparative Examples 2, 3 and 7 is approximately equal to or less than the glucose level in mice of Examples 4-6 and 8 during the first 45 minutes after of the injection. The glucose level is approximately the same in the mice of Comparative Examples 2, 3 and 7 and Examples 4-6 and 8, 120 minutes after the injection. However, the glucose level in the mice of Comparative Examples 2, 3 and 7 is approximately three times higher than the glucose level in Examples 4-6 and 8 from 210 to 390 minutes after injection. In fact, the blood glucose level in mice in Examples 4-6 and 8 did not increase substantially (ie did not increase by more than 10% or, remained the same or decreased) from 120 to 390 minutes after injection. In comparison, the blood glucose level in the mice of Comparative Examples 2, 3 and 7, injected with the same amount of insulin if substantially increased from 120 to 390 minutes after injection. Injection with crystallized dextran micro-particles / insulin reduces blood glucose for a longer time than an injection of insulin of the same dose by itself. That is, the composition containing micro-particles of crystallized dextran and insulin can be dosed for injection. The following experiments in rabbits also demonstrate how the injection with crystallized dextran micro-particles / insulin lowers blood glucose and maintains an initial level of insulin in the blood for a longer time than an injection of the same insulin into the blood. same dose by itself. A subcutaneously injected composition comprising Actrapid HM®, short-acting insulin and crystallized dextran micro-particles was unexpectedly discovered that extended the duration of efficacy of this short-acting insulin and exceeded that of the long-acting insulin Monotartd HM ® injected subcutaneously on its own. The term duration of efficacy means reducing the blood glucose concentration and / or maintaining an initial level of blood insulin concentration at desired levels regardless of external events that cause spikes in blood glucose, such as eating. Thus, the term duration of efficacy is a relative term that compares the efficacy of insulin and the composition of microparticles with the same dose of the same insulin alone. In other words, the duration of efficacy is a duration of action or a duration of the pharmacological effect, which can be measured in a fasted patient, to compare the efficacy of insulin and the composition of microparticles with the same dose of the same insulin alone. As shown in Figures 12A and 12B, the composition comprising the short-acting insulin Actrapid HM® and the crystallized dextran micro-particles prolonged the absorption of the insulin and extended the hypoglycemic effect (ie the duration of the efficacy of insulin) for at least 24 hours, such as approximately 28 to 31 hours, compared with approximately two to approximately 8 hours for the Actrapid HM® insulin alone (Figure 12B) and approximately 17 to approximately 24 hours for the insulin "Monotard HM® "alone (Figure 12A). Both Actrapid HM® and Monotard HM® are insulins produced by Novo Nordisk and the duration of the advertised efficacy of these human insulin compositions obtained from the company's information is 8 and 24 hours, respectively. In Figures 12A and 12B, the upper line illustrates the control line for intact rabbits to which insulin was not administered. The y-axis of Figures 12A and 12B is a relative, normalized scale of the blood glucose concentration for the same 8 IU insulin dose. The data in the Figures were adjusted to show on a plot for each figure and show the blood glucose levels in the blood of the animals after the insulin injections. The data shown in Figures 12A and 12B were obtained as follows. Chinchilla rabbits (2.3 + 0.3 kg) were monitored for their responses to injections of a crystallized dextran micro-particle formulation and Actrapid HM® short acting insulin. Samples of the formulation were injected subcutaneously into the rabbits. Monotard HM® (40 IU / ml) and short-acting insulin Actrapid HM® were injected subcutaneously into separate rabbits without the micro-particles and used as controls. Blood samples were taken from the animal in the vein of the rabbits ear and the glucose concentration was analyzed. The blood glucose concentration was measured using a glucose analyzer (One Touch® Lifescan, Johnson &; Johnson, Milpitas, CA, USA) after proper calibration. In comparative examples 9 and 10, two intact rabbits did not receive insulin. In comparative examples 11 and 12, an aqueous solution of long-acting insulin was introduced subcutaneously into two rabbits in a dose of 8 IU. In Examples 13-15, a suspension of crystallized dextran microparticles was introduced subcutaneously. with Actrapid HM® short-acting insulin to three rabbits in a dose of 8 IU. The results of the experiments are shown in Table II.

TABLE II The above examples 13-15 illustrate that the Actrapid HM® short-acting insulin-crystallized dextran microparticle composition provides a prolonged effect that exceeds the effect of Monotard HM® long-acting insulin and is considered to be comparable to the effect of long-acting insulin (one shot per day) glargine Lantus® from Aventis (see www.aventis-us.com/Pls/lantus_TXT.html). In addition, Lantus® insulin should not be diluted or mixed with another insulin or solution. If Lantus® insulin is diluted or mixed, the pharmacodynamic profile (eg, onset of action, time to peak effect) of Lantus® and / or mixed insulin can be altered in an unpredictable manner. In comparison, the microparticulate composition of dextran crystallized with insulin does not have this limitation since any suitable insulin, such as human insulin, can be used. In the composition of the crystallized dextran microparticles and insulin, the ratio of insulin and microparticles can be varied as desired. Moreover, any suitable insulin can be used to adapt the insulin treatment to a patient individually. That is, Actrapid HM® was used in the composition as an illustrative example of a typical insulin and the composition is not limited to this brand of insulin. As shown in Examples 9 to 15, the composition containing the micro-particles of crystallized dextran and insulin is effective to maintain the duration of the efficacy of the insulin at least 30% longer, such as at least 100% more time, preferably 100% to 400% or more time than the same dose of the same insulin without the micro-particles. The microparticulate and insulin composition is effective to maintain an initial desired level of blood insulin and blood glucose concentration of at least 30% > more time, such as 100% to 400% longer, than the same dose of the same insulin without the micro-particles. That is, the duration of the effectiveness of the composition containing micro-particles is at least 24 hours, which allows its injection once a day to the mammal, such as a human being, who needs it. The composition of long-acting insulin and crystallized dextran micro-particles is safer than the long-acting insulin compositions of the prior art because it can achieve long-lasting efficacy without using a higher dose of insulin as the compositions of the art. previous. For example, if it has been determined that a dose of 8 IU of short-acting insulin is medically safe for a patient without significant risk of overdose, then the composition comprising the same short-acting insulin and the crystallized dextran micro-particles may provide an efficacy of prolonged duration at the same 8 IU of short-acting insulin dose without a significant risk of overdose, even if all the insulin is released to the patient at the same time. Moreover, this composition provides cost savings compared to the prior art compositions since it extends the efficacy without increasing the amount of insulin. The current long-acting diabetes treatments of the prior art are made with insulin analogs such as Lantus® insulin from Aventis. In comparison, the composition containing crystallized dextran microparticles preferably contains human recombinant insulin, whose safety profile is established. That is, this composition reduces the risk of adverse reactions and the number of injections to diabetics, thus improving the quality of life of diabetics. The injectable composition may comprise a single-phase system, comprising insulin and micro-particles or a two-phase system, which forms a core of PEG and insulin and a dextran and a dextran micro-particle shell for a duration of even greater efficiency. Moreover, the composition comprises a phase one, which can flow, or a multi-phase colloidal system (i.e., a suspension or an emulsion) which is relatively easy to inject into a mammal.

The following example illustrates the use of an injectable, two-phase composition comprising a dextran phase, a PEG phase, insulin and crystallized dextran microparticles. It is considered that when a mammal is injected, this composition forms a structured reservoir-type implant having a three-dimensional capsule structure. In the capsule structure, the microparticles selectively partition in the dextran phase and the insulin selectively partitiones in the PEG phase. The dextran phase containing the microparticles forms a shell around a core comprising the PEG phase containing the insulin. This structured implant allows controlled release from the core through the housing. In comparative example 16, 0.5 Ul of Actrapid HM® insulin (100 IU / ml) is injected subcutaneously into a mouse. In Example 31, 0.4 g of crystallized dextran microparticles are dispersed in 0.6 ml of a 20% by weight aqueous solution of dextran by weight, having a molecular weight of 70 kDa (Pharmacia, Sweden) to form a suspension . 10 mg of PEG having a molecular weight of 6 kDa (Fluka) are dissolved in 0.1 ml of Actrapid HM® insulin (100 IU / ml) to form a solution. 0.05 ml of the PEG solution and insulin are mixed with 0.15 ml of the micro-particle suspension and dextran to form a two-phase composition or mixture. 0.02 ml of the two-phase mixture containing 0.5 IU of insulin is injected subcutaneously into the mouse. The results are shown in Table III.

TABLE III As can be seen in Table III, the effectiveness duration of the two-phase composition was longer than that of insulin alone. Furthermore, the two-phase composition decreased blood glucose more gradually than insulin alone. Without wishing to be bound by a particular theory, these effects are considered to be due to the controlled release of insulin from the core of the capsule structure. Moreover, the composition containing micro-particles can be adapted to each patient individually by adjusting the amount of insulin and / or micro-particles to allow the patient to inject the composition at the same time each day (ie once a day). day every 24 hours, once every 48 hours, etc.). That is, the duration of the efficacy of the composition is adjustable for each patient. For a two-phase system, the insulin release profile from the core of the capsule can be adjusted by controlling the amount of micro-particles to control the thickness of the capsule shell. Although the inventor does not wish to be bound by any particular theory, it is considered that the long-lasting effect of the same insulin dose in mice and rabbits with crystallized dextran micro-particles can be explained by the diffusion of insulin molecules from the implant based on crystallized dextran micro-particles (ie a self-controlled insulin release). Since mice and rabbits are a common model for humans in drug tests, the data shown in the preceding tables I to III suggest that the use of implants based on crystallized dextran microparticles makes it possible to develop delivery delivery systems controlled with better pharmacokinetic and dynamic characteristics and that better satisfy the initial insulin needs in patients such as humans.

D. Materials In the preferred embodiments of the present invention, the therapeutic agent comprises insulin. In other words, the therapeutic agent may consist essentially of insulin alone or comprise insulin in combination with another agent. The term "insulin" should be interpreted as including insulin analogues, naturally-occurring human insulin, recombinantly produced human insulin, insulin extracted from bovines and / or pigs, porcine and bovine insulin produced recombinantly, and mixtures of either of these insulin products. The term is intended to include the polypeptide normally used in the treatment of diabetes in a substantially purified form but which includes the use of the term in its commercially available pharmaceutical form., which includes additional excipients. The insulin is preferably of recombinant production and may be dehydrated (dried completely) or in solution. The terms "insulin analogue", "monomeric insulin" and the like are used interchangeably herein and attempt to include any form of "insulin" as defined above, wherein one or more of the amino acids within the peptide chain has been replaced by an alternate amino acid and / or where one or more of the amino acids has been removed or where one or more additional amino acids has been added to the polypeptide chain or amino acid sequence, which acts as insulin in the reduction of glucose levels in blood. In general, the term "insulin analogues" of the embodiments of the present invention includes "insulin lispro analogues," as described in U.S. Patent No. 5,547,929, which is incorporated herein by reference in its entirety, analogs of insulin including LysPro insulin and humalog insulin and other "super insulin analogues", where the ability of the insulin analog to affect serum glucose levels is substantially improved compared to conventional insulin, as well as hepatoselective insulin analogs which are more active in the liver than in adipose tissue. Preferred analogs are monomeric insulin analogs that with insulin-like compounds used for the same general purpose as insulin, such as insulin lispro, ie compounds that are administered to reduce blood glucose levels.

The term "analogue" refers to a molecule that shares a common functional activity with the molecule to which it is considered comparable and typically also shares common structural characteristics. The term "recombinant" refers to any cloned therapeutic agent expressed in prokaryotic cells or a genetically engineered molecule or a library of combination molecules that can also be processed in another state to form a second combination library, especially molecules that contain protective groups that improve the physicochemical, pharmacological and clinical safety properties of the therapeutic agent. The term dextran microparticles includes unsubstituted dextran microparticles and substituted dextran microparticles. For example, substituted dextran microparticles include dextran substituted with a suitable group, such as a methyl group, to an extent that does not affect the crystallization of the dextran microparticles, such as up to 3.5 or less percent branching . The average microparticle diameter is preferably about 0.5 to about 5 microns, more preferably about 1 to about 2 microns. Furthermore, although dextran microparticles without porous crosslinking, such as crystallized microparticles, are preferably used with the therapeutic agent, other suitable microparticles, organic or inorganic, can be used in place of these, such as other polymer microparticles, including polysaccharides, PLA, PLGA, PMMA, polyimides, polyesters, acrylates, acrylamides, vinyl acetate or other polymeric materials, particles of biomaterials such as alginate and inorganic cells or particles, such as silica, glass or calcium phosphates. Preferably the microparticles are biodegradable. Preferably, porous microparticles are used. More preferably, the microparticles have sufficient porosity to contain the therapeutic agent within the pores and to provide a time release of the therapeutic agent from the pores. In other words, the therapeutic agent is released over time from the pores, such as in more than 5 minutes, preferably in more than 30 minutes, more preferably in more than one hour, such as in several hours to several days, instead of everything at once. Then, the material of the particles, the pore size and the pore volume can be selected based on the type of therapeutic agent used, the volume of therapeutic agent that is needed for its administration, the duration of the administration of the therapeutic agent. , the environment where the therapeutic agent will be administered and other factors. That is, in a preferred aspect of the present invention, the therapeutic agent is located, at least partially, in the pores of the porous microparticles. Preferably, the therapeutic agent is not encapsulated in the microparticle (ie, the microparticle does not act as a shell with a core of therapeutic agent inside the shell) and is not attached to the surface of the microparticle. However, if desired, a portion of the therapeutic agent can also be encapsulated in a microparticle shell and / or attached to the surface of the microparticle in addition to being located in the pores of the microparticle. The location of the therapeutic agent in the pores provides an optimal release of the therapeutic agent over time. In comparison, the therapeutic agent attached to the surface of the microparticle is often released too rapidly while the therapeutic agent encapsulated in the microparticle is often not released early enough and then all is released to the disintegrating Micro-particle case. In a two-phase system, at least 80% of the therapeutic agent is preferably located in a core surrounded by a wall or shell comprising the micro-particles.

E. Manufacturing Methods Microparticles can be formed by any suitable method. Preferably, the microparticles are combined with the therapeutic agent after forming the microparticles. That is, the microparticles such as the crystallized dextran microparticles are formed by any suitable method and then the therapeutic agent and the microparticles are combined with any suitable method. In comparison, in some methods of the prior art, the therapeutic agent is encapsulated in a microparticle shell providing the precursor material of particles and the therapeutic agent in a solution and then crystallize or cross-link the precursor material, such as a monomer or oligomer, for encapsulating a core of therapeutic agent in a microparticle shell. Preferably, the therapeutic agent is provided in the pores of the porous microparticles after forming the microparticles. That is, the porous microparticles are formed and then the therapeutic agent is provided in a solution containing the microparticles to allow the therapeutic agent to permeate into the pores of the microparticles. Of course, part of the therapeutic agent can also be attached to the surface of the microparticle in this procedure. That is, a method for the manufacture of porous crystallized dextran microparticles without crosslinking includes the preparation of a dextran solution, such as an aqueous dextran solution, conducting a crystallization process to form dextran microparticles. crystallized porous and, if desired, isolate crystallized porous dextran micro-particles from the solution. Then a therapeutic agent is allowed in the pores of the microparticles by providing the therapeutic agent in the crystallization solution containing the microparticles or by providing the isolated microparticles and the therapeutic agent in a second solution, such as a second solution watery For example, the crystallized dextran microparticles can be formed into a first aqueous low molecular weight dextran solution, such as a dextran solution of 2 to 20 kDa. The microparticles are then removed from the first solution and then placed in a second aqueous dextran solution having a higher molecular weight dextran, such as a 40 to 500 kDa solution, for example a 40 to 75 kDa solution . The second solution may comprise a first phase or a two-phase system, which is then combined with a second phase, such as a PEG phase containing a therapeutic agent. A similar method can be used with other porous microparticles, where a therapeutic agent is then permeated into the pores of the microparticles after the porous microparticles are formed by any suitable method of microparticle formation, including without limitation, crystallization. The components of the composition, such as insulin, micro-particles and one or more aqueous phases, can be combined in any suitable order, sequentially or simultaneously. Preferably, the microparticles are formed by self-assembly from a solution that does not contain organic solvents and organic reaction promoters that leave an organic residue in the micro-particles. That is, for example, the dextran microparticles are preferably formed by self-assembly from an aqueous dextran solution. However, if desired, organic solvents and / or organic promoters of the reaction can also be used. In this case, the micro-particles can be purified before the subsequent use to remove the harmful organic residue. As described above, the capsule structure having a first phase core and a second phase shell or shell, can be formed in vivo or in vitro from a two phase composition. The composition can be a dry powder, freeze-dried and stored as a porous powder or tablet. When the composition is ready to be administered to a mammal, it is hydrated and administered to a mammal by injection. Preferably, the composition that includes the microparticles and the therapeutic agent is a colloidal system that flows when the composition is dosed for injection. Examples of flowing colloidal systems include emulsions and suspensions that can be injected into a mammal using a common measuring syringe or needle without undue difficulty. In comparison, some prior art compositions include a therapeutic agent in a dextran hydrogel or in a crosslink dextran matrix. A dextran hydrogel and a dextran matrix with crosslinking are not compositions that flow if they are not specifically prepared. In another preferred aspect of the present invention, the microparticles comprise microparticles that are adhesive to the mucosa of the mammal. Preferably, the adhesive microparticles are the porous microparticles described above. This further improves the effective administration of the therapeutic agent. In another preferred aspect of the present invention, the microparticles comprise microparticles whose surface has been specially modified to improve the adhesion of the therapeutic agent to the surface of microparticles and optimize the administration of the therapeutic agent. The surface of the microparticles may contain any suitable modification that would increase adhesion of the therapeutic agent. The foregoing description of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form described and that modifications and variations are possible in the light of the above teachings or can be acquired from the practice of the invention. The drawings and description were selected in order to explain the principles of the invention and its practical application. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents. All publications and patent applications and patents cited in this description are hereby incorporated by reference in their entirety.

Claims (21)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of crystallized dextran micro-particles and insulin in an injectable pharmaceutical composition for reducing blood glucose in a mammal, wherein the micro-particles are formed before the combination of insulin and micro-particles in the composition .

2. The use claimed in claim 1, wherein the composition comprises a colloidal composition that flows and the microparticles comprise crystallized dextran microparticles having an average size of 0.5 to 5 microns.

3. The use claimed in claim 2, wherein the composition comprises a composition of two phases comprising a dextran phase and a PEG phase, the insulin particulates selectively in the PEG phase and the microparticles partition selectively in the dextran phase, and the composition forms a structured implant comprising a PEG phase core and a dextran phase shell after being injected into the body of a mammal.

4. The use claimed in claim 3, wherein the thickness of the shell has the ability to be controlled based on the body of the animal receiving the composition to control the release of insulin from the implant.

5. - The use claimed in claim 1, wherein the composition is administrable to a human being suffering from diabetes to reduce the concentration of blood glucose in the human.

6. A dosed pharmaceutical composition comprising crystallized dextran microparticles and a therapeutically effective amount of insulin wherein the composition is dosed for injection to a human being and the microparticles are formed prior to the combination of insulin and insulin. micro-particles in the composition.

7. The composition according to claim 6, further characterized in that the composition comprises a colloidal composition that flows and the microparticles comprise crystallized dextran microparticles having an average diameter of 0.5 to 5 microns.

8. The composition according to claim 7, further characterized in that the composition comprises a two-phase composition comprising a dextran phase and a PEG phase, the insulin particulates selectively in the PEG phase and the microparticles partition selectively in the dextran phase, and the composition forms a structured implant comprising a PEG phase core and a dextran phase shell when injected into the human body.

9. A dosed pharmaceutical composition comprising crystallized dextran microparticles and a therapeutically effective amount of first insulin wherein the composition is dosed for injection into a mammal and the duration of efficacy of the composition when injected into a mammal is at least 30% longer than the duration of efficacy in the mammal of the same dose of the same first insulin without the micro-particles.

10. The composition according to claim 9, further characterized in that the composition comprises a colloidal composition that flows, the micro-particles comprise micro-particles of crystallized dextran having an average size of 0.5 to 5 microns and the micro-particles they are formed before the combination of the first insulin and the micro-particles in the composition.

11. The composition according to claim 10, further characterized in that the composition comprises a two-phase composition comprising a dextran phase and a PEG phase, the first insulin selectively partitioned into the PEG phase and the micro-particles selectively partition in the dextran phase and the composition forms an implant comprising a PEG phase core and a dextran phase shell when injected into the body of a mammal.

12. The composition according to claim 9, further characterized in that the duration of the efficacy of the composition when injected into the mammal is at least 24 hours and the duration of the efficacy of the composition when injected into a mammal. it is at least 100% or more prolonged than a duration of efficacy in a mammal of the same dose of the same first insulin without the micro-particles.

13. - The use of crystallized dextran micro-particles and first insulin to prepare an injectable pharmaceutical composition for reducing blood glucose in a mammal, wherein a duration of compositional efficacy in the mammal is at least 30% longer that a duration of efficacy in a mammal of the same dose of the same first insulin without the micro-particles.

14. The use claimed in claim 13, wherein the composition comprises a colloidal composition that flows, the micro-particles comprise micro-particles of crystallized dextran having an average diameter of 0.5 to 5 microns and the micro-particles they are formed before the combination of the first insulin and the micro-particles in the composition.

15. The use claimed in claim 14, wherein the composition comprises a two-phase composition comprising a dextran phase and a PEG phase, insulin selectively partitioned into the PEG phase and the microparticles partition. selectively in the dextran phase and the composition forms an implant comprising a PEG phase core and a dextran phase shell after being injected into the body of a mammal.

16. The use claimed in claim 13, wherein the duration of the efficacy of the composition when injected into the mammal is at least 24 hours and the duration of the efficacy of the composition in the mammal is at least less 100% longer than a duration of efficacy in a mammal of the same dose of the same first insulin without the micro-particles.

17. The use claimed in claim 16, wherein the duration of the efficacy of the composition in the mammal is 100% to 400% longer than a duration of efficacy in a mammal of the same dose of the same first insulin without the micro-particles.

18. A method for producing a dosed pharmaceutical composition comprising providing crystallized dextran microparticles, combining a therapeutically effective amount of insulin and the crystallized dextran particles in a solution after the microparticles have been crystallized to form a Insulin composition and crystallized dextran microparticles and dose the composition for injection to a mammal.

19. The method according to claim 18, further characterized in that the composition comprises a colloidal composition that flows and the microparticles comprise crystallized dextran microparticles having an average diameter of 0.5 to 5 microns.

20. The method according to claim 19, further characterized in that the composition comprises a two-phase composition comprising a dextran phase and a PEG phase, insulin selectively partitioned into the PEG phase and the microparticles partition selectively in the dextran phase and the composition forms a structured implant comprising a PEG phase core and a dextran phase shell after being injected into the body of a mammal.

21. The method according to claim 18, further characterized in that the duration of the effectiveness of the composition when injected into a mammal is at least 24 hours and the duration of the efficacy of the composition in the mammal is by at least 100% longer than a duration of efficacy in a mammal of the same dose of the same insulin without the micro-particles.