MXPA05009494A - Delivery system for drug and cell therapy - Google Patents

Abstract

A method of lowering blood glucose in a mammal includes administering orally or by injection or inhalation 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 or other therapeutic agents.

Description

ADMINISTRATION SYSTEM FOR MEDICATIONS AND CELL THERAPY FIELD OF THE INVENTION This application claims benefit of the following Provisional Applications from the US. 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 the development of biomaterials for the systemic and local administration of therapeutic agents directly within or over body tissues and for tissue engineering. In particular, the present invention relates to the manufacture of biocompatible implants containing cells, biologically active substances or combinations of the above and to microparticulate matrix materials for the administration of proteins, peptides and nucleic acids.

BACKGROUND OF THE INVENTION The term "biomaterial" has been used alternatively to describe materials derived from biological sources or to describe materials used for treatments in the human body. The present invention relates to the latter. The term "biocompatible" is used herein to indicate that the material itself or in combination with therapeutic agents, including living cells, does not react against foreign bodies or fibrosis. 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 administering orally or by injection or inhalation 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 or other therapeutic agents.

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 microparticles shown in Figure 1. Figure 2B is a photograph of a section of a microparticle 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 a subcutaneously injected implant consisting of crystallized dextran microparticles shown in Figure 3. Figure 5 is a photograph of an implant injected intramuscularly consisting of crystallized dextran microparticles. shown in Figure 3. Figures 6A, 6B and 6C are photographs of a mouse muscle cut with an injected implant consisting of crystallized dextran micro-particles (at 1st, 2nd and 28th days after injection, respectively) . Figures 7A and 7B are photographs of a muscle cut of mouse with injected implant consisting of crystallized dextran micro-particles (180 days after injection). Figures 8A, 8B, 8C, 8D, 8E, 8F and 8G are photographs of a mouse skin cut with an injected implant consisting of crystallized dextran microparticles (1st day, 4th day, 28th day, 180 days, 180 days, one year and one year after the injection, respectively). Figure 9 is a photograph of the slow release of the fluorescently labeled macro molecules of the implant, including micro-particles of crystallized dextran in mouse muscle tissue at day 14 after intramuscular injection.

Figure 10 is a photograph of a reporter gene expression in mouse muscle tissue after the release of plasmid DNA from the implant. Figure 11 is a photograph of an aqueous solution emulsion of PEG in aqueous dextran solution (Molecular Weight 500 kDa), containing the crystallized dextran microparticles shown in Figure 1. Figure 12 is a photograph of an emulsion of aqueous dextran solution (Molecular Weight 500 kDa), containing the crystallized dextran micro-particles shown in Figure 1, in aqueous PEG solution. Figure 13 is a photograph of an intramuscular injection of aqueous PEG aqueous solution emulsion in dextran aqueous solution (Molecular Weight 500 kDa), containing the crystallized dextran micro-particles shown in Figure 1. Figure 14 is a photograph of a subcutaneous injection of aqueous solution of PEG emulsion in aqueous dextran solution (Molecular Weight 500 kDa), containing the crystallized dextran micro-particles shown in Figure 1. Figures 15A and 15C, schematically illustrate the behavior of partition of different types of particles and phases in a two-phase aqueous system. Figure 15B is a photograph of a cut of an implant structure based on the two-phase system.

Figures 16A, 16B, 16C and 16D are photographs of HeLa cell partitioning and crystallized dextran microparticles in a two-phase system. Figures 17 and 18 schematically illustrate cell therapy methods in accordance with embodiments of the present invention. Figures 19, 20 and 21 schematically illustrate methods of administering therapeutic agents in accordance with embodiments of the present invention. Figures 22A and 22B are graphs of relatively normalized blood glucose concentrations for various compositions containing insulin vs. insulin. weather.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES A. Micro-particle Formation The present inventor has discovered, experimentally, that crystallized dextran micro-particles with an average diameter ranging from 0.5 to 3.5 microns, are formed spontaneously in concentrated aqueous solutions of dextrans (40-65% by 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 micro-particles (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 sterile distilled water, or by filtration of diluted suspension of micro-particles, (3 x 250 ml of 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.

B. Implants Based on Micro-particles of Crystallized Dextran. Concentrated suspensions of the crystallized dextran microparticles and aggregates thereof were tested as implants in experiments with mice to test their biocompatibility after injection into the body of the animal.

Figures 4 and 5 show the tissue implant after subcutaneous (Figure 4) and intramuscular injections (Figure 5) in experimental animals (mice). No inflammation reactions were detected in the animal's tissue for 180 days. In Figures 6A, 6B and 6C, the implant is shown in muscle tissue of a mouse 1, 4 and 28 days, respectively, after the injection. In Figures 7A and 7B, the implant is shown in muscle tissue of a mouse 180 days after injection. In Figures 8A, 8B and 8C, the implant is shown at the 4th, 11th and 28th day, respectively, after subcutaneous injection. In Figures 8D and 8E the implant is shown 180 days after the subcutaneous injection. In Figures 8F and 8G, the implant is shown one year after the subcutaneous injection. As shown in Figure 8G, normal tissue is formed rather than scar tissue at the site of the implant. 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. Figures 9 and 10 show the implant containing fluorescently labeled macro-molecules (FITC-dextran, Molecular Weight 500 kDa) and the slow release of the macro molecules from the implant to the muscle tissue of a mouse at day 14 after the intermuscular injection (Figure 9) and the expression of the reporter gene in the muscle tissue of a mouse after the release of the plasmid DNA from the implant (Figure 10). That is, the crystallized dextran microparticles can be used for the controlled time release of a label which can be a fluorescent label alone or in combination with a therapeutic agent.

C. Implants Based on 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, although 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% water and can have a buffer and become isotonic. If you add a suspension of cells or particles to a system like 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. Walter, 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 11 and 12. Figure 11 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 11, 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 12 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 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 dissipates in tissue fluids). As can be seen in Figure 12, the dextran phase of smaller volume (droplet) is formed as a large, spherical nucleus of dextran / dextran microparticles (lower right part of Figure 12) 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. The injection of two-phase aqueous systems containing micro-particles of crystallized dextran in tissues of experimental animals, revealed the formation of implants with the capsule structure shown in Figures 13 and 14. The volume of the dextran phase is greater than the volume of the PEG phase in the two-phase system. Both Figures 13 and 14 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 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 crystallized dextran micro-particles are added. 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. Moreover, 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 in that it allows a more uniform and prolonged release of the therapeutic agent from the core 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 15A, 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 15A. 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 15A, 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. Compared, 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 15A, then a capsule-like structure comprising discrete spheres of the second phase is formed. , 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 at the interface of phases 16 and 18 and act as a 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. It should be noted that the structure formed for the oral administration of two-phase systems can generally be described as a structured suspension containing a dispersed phase of PEG and a continuous phase of dextran. Moreover, the particles (ie molecular aggregates) 10, 12 and 14 can be replaced by a liquid material (eg oils) or selective-partition 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 15B illustrates an electron microscopy image of a cut of an implant structure based on the two-phase system illustrated schematically in Figure 15A. 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 15B 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 Fig. 15B is formed by self-assembly, as schematically shown in Fig. 15C. 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 to a material that restricts the free flow of phases 16 and 18, like the tissue of a mammal or a substrate material, such as a gel that simulates tissue, the composition 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 15C. 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 15C. 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.

D. Cell Therapy Experiments performed with mammalian cells have demonstrated the possibilities of using the technique just described for administering to a body the cells adsorbed by the aggregates surface of crystallized dextran microparticles (see Figures 3 and 12). ) or absorbed by the same surface that is inside of macro-porous structures (see figure 11) or capsules (see figures 13 and 14). In general, a self-organizing material for treatments based on spontaneous organization in colloidal systems, may contain micro-particles of crystallized dextran or other particles such as PLA, PLGA, PMMA, alginate, cells, etc., for implants. For example, Figures 16A-16D illustrate a partition of HeLa cells and crystallized dextran microparticles in a dextran-PEG two-phase system. A two-phase system is illustrated in Figure 16A. The core comprising the PEG phase containing the cells is shown at the top of the photograph and the dextran / dextran micro-particle shell ("CDM") is shown at the bottom. The droplets of the PEG phase that segregate outside the shell and into the core are shown at the boundary of the core and shell regions. Figure 16D shows the partition of dextran particles in a PEG / dextran two-phase system. The PEG 16 phase is located above and the dextran 18 phase, which contains the dextran microparticles, is at the bottom. Figures 16C and 16D show HeLa cells in a dextran phase containing a surface of crystallized dextran microparticles. The implants can be "reservoir" type implants and can be used for treatment by cell replacement. "Deposit" type implants can improve pharmacokinetics (without "surge effects") and ensure a sustained release of biologically active substances. The "core" of the "reservoir" type implant can contain any type of active substances, in addition to the structure of the implant that is "loaded" with cells. The nucleus may contain peptides and therapeutic proteins, nucleic acids, vaccines, viruses and cells. Figure 17 schematically illustrates an example of treatment by cellular replacement based on self-organizing material for the beginning of therapy. For example, closed pore implants, based on crystallized dextran micro-particles, can be used for the treatment of type 1 diabetes, cancer treatment, treatment of Parkinson's disease and other suitable treatments, based on the use of proteins. and therapeutic peptides. In Figure 17, the implant 40 contains a microparticle of crystallized dextran, 12, 14, capsule or shell that encapsulates the transplanted cells 42. Any suitable cell can be used, such as insulin producing beta cells, K cells, cells pancreatic or stem cells. As shown in Figure 17, the capsule of crystallized dextran microparticles is porous to blood glucose 44 (as well as to oxygen, nutrients and other stimuli), however it is impermeable to immune system cells that are too large to enter the capsule and attack the insulin-producing cells. Blood glucose 44 is permeated through the crystallized dextran microparticle capsule and stimulates the production of insulin 46 from the insulin producing cells. The insulin is then disseminated through the capsule of crystallized dextran microparticles to the blood 48. The insulin producing cells encapsulated in crystallized dextran microparticles can be implanted in a mammal, like a human being, to treat the diabetes. The crystallized dextran microparticle capsule can be formed in vivo and does not need a surgical operation. This technique is known as "injectable tissue engineering". Even more, the crystallized dextran microparticle capsule does not require the use of toxic chemicals for crosslinking. The implants can preferably be used to provide a long-acting insulin preparation for suppressing hepatic glucose production and preserving an almost normal glycemia in the fasted state with an action profile over time lacking a peak. It should be noted that cell therapy can be used to treat other diseases. For example, dopamine-producing cells encapsulated in crystallized dextran microparticles can be used to treat Parkinson's disease, as schematically illustrated in Figure 18. Moreover, as shown in Figure 18, the structure of the capsule of crystallized dextran microparticles can be inverted in such a way that the cells 42 form the shell that encapsulates the microparticles 12, 14 in the tissue of a mammal 50. For example, one can locate the patient's own cells or stem cells out of the micro-particles. In comparison, cells from a source other than the patient, for example a donor, can be located inside the micro-particle housing as shown in Figure 17 to protect the cells from the patient's autoimmune reaction. This opens possibilities of tissue engineering in vivo for the treatment of conditions such as diabetes and others.

E. Bone Graft Substitutes Bone graft procedures involve the use of either autograft tissue, where the tissue is obtained from the patient, or allograft tissue, which is taken from donors or cadavers. The basic criterion that is used to judge a successful graft is the osteo-conduction and the osteo-induction. Harvesting tissue for autografts requires surgery at the donation site which can result in its own complications, such as inflammation, infection and chronic pain. The quantities of bone tissue that can be harvested are also limited, which also generates a supply problem. Allografts avoid some of the disadvantages of autografts by eliminating morbidity at the donor site and limited supply issues. However, allografts also present risks. Although there are many methods that can reduce the risk of disease transmission, the treatments used to stabilize the tissue eliminate proteins and factors, reducing or eliminating the bone induction of the tissue. Moreover, several alternatives to autografts and allografts have been developed. Many of these alternatives use various materials, including natural and synthetic polymers, ceramics and composites. Other alternative methods incorporate strategies based on factors or cells that are used either alone or in combination with other materials. Many substitutes for vocal grafts are natural or synthetic and can be used on their own or in combination with recombinant growth factors such as transforming growth factor (TGF), platelet derived growth factor (PDGF), growth factor fibroblasts (FGF) and bone morphogenic protein (BMP). Cell-based bone graft substitutes use cells to generate new tissues on their own or are sown to a support matrix (eg, mesenchymal stem cells). With polymer-based bone graft substitutes, both degradable and non-degradable polymers are used, either alone or in combination with other materials such as Cortoss®, open-porosity polylactic acid (OPLA) polymer from Orthovita, Inc. Degradable synthetic polymers, like natural polymers, are re-absorbed by the body. The benefit of having the implant reabsorbed by the body is that the body can heal itself completely without foreign body debris. To this end, the companies have used degradable polymers such as polylactic acid and polylactic-co-glycolic acid (PLGA), as independent devices and as extenders of auto-grafts and allo-grafts. For example, Bone Tec, Inc., has developed a porous PLGA matrix using a particle leaching process to induce porosity. OsteoBiologics, Inc., has the Immix Extenders, a PLGA product in particles that is used as a graft extender. In a preferred aspect of this embodiment, the porous crystallized dextran micro-particles described in the above embodiment are used as bone graft substitutes, in combination with a therapeutic agent that provides bone formation at special sites of said body. Any suitable therapeutic agent for bone formation, such as a peptide, stem cell or proteins, can be used, including without limitation the aforementioned mesenchymal stem cells, TGF, PDGF, FGF and BMP. The therapeutic agent can be located in the pores of the porous microparticles. For example, the therapeutic agent can be provided in the pores of the crystallized dextran microparticles after crystallization. In another preferred aspect of this embodiment, a method for the formation of an in vivo implant includes the preparation of a microparticle suspension in a first liquid phase, preparing a microparticle suspension in a second liquid phase immiscible with the first liquid phase, preparing an emulsion wherein the first liquid phase is a continuous phase and the second liquid phase is a dispersed phase and injecting the emulsion into the body of a mammal. Preferably the emulsion is used as a bone graft substitute. Preferably, the microparticles of the first and second phases are different. For example, the microparticles of the first phase may be non-polymeric microparticles, such as porous ceramic microparticles, while the microparticles of the second phase may be microparticles of polymers such as microparticles. crystallized dextran particles mentioned above. If desired, the second liquid phase may contain the therapeutic agent that provides bone formation at special sites in the body. The therapeutic agent can be located in the pores of the porous microparticles.

F. Vehicle for Oral Insulin Administration In another preferred embodiment of the present invention, the present inventor discovered that a porous (i.e., micro-porous) microparticle composition can be used as a vehicle for the oral administration of proteins. , like insulin. The porous microparticles can be any porous microparticle that allows oral administration of insulin with a significant reduction in blood glucose, such as a 30% reduction in no more than 60 minutes of oral administration. Preferably, the microparticles are bio-adhesive particles, such as particles that adhere at least temporarily to the intestinal walls of mammals, to allow administration of insulin through the intestinal wall. More preferably, the porous microparticles comprise the crystallized dextran microparticles described above. In a preferred embodiment of the present invention shown in Figure 19, the present inventor has discovered that an aqueous suspension of crystallized dextran microparticles 12, 14 and insulin 46, administered orally to mammals 53, such as rabbits , was about as effective in reducing glucose levels as an intramuscular injection of insulin alone. Figure 19 schematically illustrates insulin 46 permeating through the intestinal walls 52 of a mammal 53, from the orally administered composition 54 comprising the microparticles. Since rabbits have a common model for humans in drug testing, the present inventor considers that a liquid or solid composition 54 comprising the microparticles of crystallized dextran and insulin, such as an aqueous suspension, a solution, a tablet or a capsule, would also be effective in reducing blood glucose levels in humans when administered orally. The following examples illustrate oral insulin administration using crystallized dextran microparticles. The study involved Chinchilla rabbits (2.3 + 0.2 kg) and observation of their responses to aqueous suspensions administered orally consisting of crystallized dextran microparticles prepared according to the method described herein and human recombinant insulin. 3.0 g of Dextran T20 (Pharmacia, Uppsala, Sweden) were dissolved in 2.0 g of water and placed in a box at a temperature of 60 ° C. Three hours later, crystallized dextran micro-particles were washed by centrifugation at 3,000 g with 2 × 5.0 ml of water. Then the crystallized dextran micro-particles were suspended in 2.0 ml of water and allowed to dry at room temperature. The resulting dry powder was used to prepare a suspension containing insulin for the oral insulin administration experiment. The suspensions with insulin content were prepared by mixing 250 mg of the micro-particles; 0.3 ml (12 IU) or 0.6 ml (24 IU) of insulin (NovoNordisk Actrapid HM Penfill, 40 IU / ml) and distilled water to reach a volume of 2.0 ml.

Samples of the suspension (2.0 ml) were introduced into the rabbits' throats by catheter, followed by the introduction of 10.0 ml of drinking water. The animals were not fed for 3 hours before the introduction of the suspension. Blood samples were obtained from the rabbit auditory vein and its glucose concentration was analyzed. Blood glucose was measured using the glucosoxidase method in a "One Touch System Glucose Analyzer" (Lifescan Johnson &Johnson, Milpitas, CA, USA) after adequate calibration. Examples 1 to 8 are comparative examples involving eight rabbits. Examples 9 to 14 are examples of compliance with the present invention involving five rabbits. In Comparative Examples 1 and 2 (control experiment # 1 summarized in Table I) an aqueous solution of human recombinant insulin was introduced intramuscularly into two rabbits at a dose of 12 IU per animal. In comparative examples 3 and 4 (control experiment # 2 summarized in Table II), the tips were kept intact (ie, no insulin or other injection was given to the two rabbits). In comparative examples 5 and 6 (control experiment # 3 summarized in Table III), a suspension of crystallized dextran microparticles without insulin was orally administered to two rabbits. In comparative examples 7 and 8 (control experiment # 4, summarized in Table IV), a suspension of commercially obtained microparticles Sephadex G-150 with insulin (24 IU) was orally administered to two rabbits. In accordance with the Amersham Biosciences website, the Sephadex® G-150 microparticles are spherical gel microparticles having a diameter of 20 to 150 microns, prepared by cross-linking dextran with epichlorohydrin. In Examples 9-14 according to a preferred embodiment of the present invention (summarized in Table V) a suspension of dextran microparticles crystallized with insulin (24 IU) was administered orally to five rabbits. The results are summarized in Tables l-V below.

TABLE I TABLE II TABLE III TABLE IV TABLE V The data in Tables IV show that the average reduction in sugar (ie glucose) in the blood of animals is comparable when 12 IU insulin is administered by intramuscular injection (examples 1-2) and 24 IU of insulin are administered per week. os (ie orally), with crystallized dextran micro-particles (examples 9-14). The maximum glucose reduction was from about 35% to about 60% at 60 minutes after oral administration. The glucose concentration profile is practically the same in the modes of oral administration and injection. It should be noted that other amounts of insulin can be administered. For example, 30 IU of insulin can be administered. In general, oral administration of two or three times the insulin compared to the amount of insulin injected produces a similar reduction in blood sugar for up to three hours. It is a well-known fact that insulin itself is degraded by intestinal enzymes and is not absorbed intact through the gastrointestinal mucosa (Amidon GL, Lee HJ, Absorption of peptide and peptidomimetic drugs, Ann Rev. Pharmacol Toxicol, 1994; 34: 321-41). However, examples 9-14 show that crystallized dextran microparticles can be used as a vehicle for oral administration of proteins, such as insulin, because the hypoglycemia effect obtained was significant. If wishing to be limited by a particular theory or mode of action, the present inventor considers that the use of porous, crystallized dextran micro-particles as a matrix for the administration of insulin in aqueous suspension protected the insulin from significant degradation. by intestinal enzymes and allowed insulin to be absorbed intact through the gastrointestinal mucosa. The insulin can be located in micro pores in the micro-porous micro-particles and / or in macro pores between the micro-particles. In comparison, the use of dextran microparticles with Sephadex G-150 crosslinking with insulin (Table IV, examples 7-8), did not produce an appreciable reduction in blood glucose concentration. As provided in Examples 9-14, the concentration of blood glucose in the mammal is reduced by at least 5 percent, preferably at least 30%, 60 minutes after administering the composition containing the microbes. crystallized dextran particles and insulin to the mammal (i.e., the blood glucose value in the mammal at 60 minutes after administration of the suspension is at least 5%, preferably 30% less than that measured just before of the administration of the suspension). Preferably, the concentration of blood glucose in the mammal is reduced by at least 5%, such as at least 30%, preferably by about 35% to about 40%, 30 minutes after administering the composition to the mammal . Preferably, the concentration of blood glucose in the mammal is reduced by about 35% to about 60%, for example 35% to 45%, 60 minutes after the suspension is administered to the mammal. More preferably, the concentration of blood glucose in the mammal is reduced throughout the period ranging from 30 to 240 minutes such as 30 to 120 minutes, after administering the composition to the mammal compared to the blood glucose concentration just before of the administration. For example, the concentration of blood glucose in the mammal is preferably reduced by at least 10%, preferably at least 30% and most preferably by at least 35%, such as 35% to 45% during a period ranging from 30 to 240 minutes, preferably 30 to 120 minutes after administering the composition to the mammal. That is, a preferred embodiment of the present invention provides a method for reducing blood glucose in a mammal by orally administering a therapeutically effective amount of a composition comprised of crystallized dextran microparticles and insulin. A "therapeutically effective" amount of the compositions can be determined by preventing or diminishing adverse conditions or symptoms of diseases, injuries or disorders that are treated. Preferably, the composition comprises an aqueous suspension of crystallized dextran microparticles having an average diameter of about 0.5 to about 5 microns and insulin. Moreover, the microparticles are preferably porous microparticles that are crystallized before adding the insulin to the suspension, such that the insulin is placed in contact with a surface of the microparticles and / or in pores of the micro-particles. 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 no, 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. Although a one-phase composition, 54, comprising insulin and micro-particles in Figure 19 is illustrated, a two-phase composition, described above, which is illustrated in Figures 13, 14, 15A, 15B, can also be used. 15C and 16B. For example, a two phase composition comprising a dextran phase, a PEG phase, crystallized dextran microparticles and insulin can be used. In vivo, the composition has a self-assembled capsule structure comprising a micro-particle of crystallized dextran containing wall or shell and a core containing PEG and insulin. Preferably, the mammal receiving the oral administration of insulin comprises a human being who needs to reduce blood glucose, such as a human being suffering from diabetes. That is, the preferred embodiment of the present invention provides a method for treating diabetes in a human being in need of treatment by oral administration of the insulin suspension and crystallized dextran micro-particles described above. Any therapeutically effective amount of insulin can be administered to the mammal. The amount of insulin may vary depending on the type of mammal (i.e., human or rabbit), the weight of the mammal, the composition of the suspension, the amount of desired reduction in blood glucose and other factors. A non-limiting example of insulin content in the suspension is from about 10 international units to about 2500 international units of recombinant human insulin per gram of suspension, such as about 12 international units to about 30 international units, such as 24 international units of insulin recombinant human. However, this amount may vary as desired. The present invention should not be considered limited to the preferred embodiments described above. Other matrix material can be used for the administration of oral insulin, such as micro-porous, organic or inorganic particles. Preferably, the particles are microparticles that improve the penetration of insulin through the gastrointestinal mucosa and / or stabilize the composition. Moreover, although the suspension preferably contains only one water solvent, one matrix and one insulin solution or suspension, the delivery system may also contain additional materials. For example, the composition may contain a second phase such as a PEG phase of a two-phase system. That is, another preferred aspect of the present invention includes a method for reducing blood glucose in a mammal, comprising orally administering a composition comprising a therapeutically effective amount of insulin and a matrix material to the mammal to reduce glucose in blood to reduce the blood glucose of the mammal by at least 30%60 min. after administering the suspension to the mammal. In another preferred aspect of the present invention, a method for administering a suspension to a mammal comprises orally administering an aqueous suspension of crystallized dextran microparticles and a therapeutically effective amount of insulin to the mammal.

As indicated above, the crystallized dextran microparticles which are used as a matrix material for the oral administration of insulin or other protein-based drugs can be made by any suitable method (see Figure 1, for example). Preferably, the microparticles are made by the process of any of the preferred embodiments described herein. Preferably, but not necessarily, the microparticles are formed in an aqueous solution without using an organic solvent. That is, in a preferred aspect of the present invention, a therapeutically effective amount of insulin and crystallized dextran particles are combined in water, after the microparticles have been crystallized to form an aqueous suspension of insulin and micro - crystallized dextran particles. The micro-particles can be added to the water before, at the same time and / or after adding the insulin to the water. Moreover, the microparticles can be administered orally to a mammal in the solvent in which they were formed. Alternatively, they can be removed from the solvent in which they were formed and placed in water or other aqueous solutions for oral administration, or dried and provided in solid form as in a tablet or capsule, for oral administration. The aqueous suspension of crystallized dextran microparticles and a therapeutically effective amount of insulin (or other suitable suspensions of insulin and a matrix material, such as an insulin suspension and micro-porous microparticles) are preferably provided. as a dosage pharmaceutical composition, which is dosed for oral administration to a human being. In a preferred aspect of the present invention, the composition is located in a container in a dosed amount for a single oral administration to a human being. The container may comprise any container that can retain a suspension, such as a plastic or glass bottle, a tube, a dropper, a spray nozzle, a bag and / or other suitable containers. This container contains a sufficient amount of suspension for a single oral administration of the composition. In another preferred aspect of the present invention, the composition is located in any suitable container, in an amount suitable for various doses of oral administration. The container contains an instruction for oral administration to a human being. The instruction may be printed on the container, for example by printing it directly on the container or on a label attached to the container, or included together with the container, printing it on a sheet of paper that is included with the container in a cardboard box or in a pharmacy envelope The instructions can describe the amount of the composition to be taken with each shot, the frequency with which the dose should be taken, how to measure the dose of the composition for oral administration and / or any other instruction appropriate to the medication, for a health care practitioner and / or a patient who needs the medication. Alternatively, the instructions may include the addresses for electronic or auditory access to the dosing and administration instructions, which may be a link to a website containing the instructions, a telephone number or a recording where the instructions will be from auditory way. In another preferred aspect of the present invention, the aqueous suspension of crystallized dextran particles and a therapeutically effective amount of insulin, located in a container, are provided in a kit of pharmaceutical composition with instructions. The kit may comprise printed instructions on the container or on a label affixed to the container or sheet of paper included with the container, such as a cardboard box or pharmacy envelope that includes a bottle (ie container) and instruction sheet. It should be noted that the composition for oral administration may be in the form of an aqueous suspensionhowever, other forms of administration can be used to reduce blood glucose in a mammal. For example, porous crystallized dextran micro-particles and insulin can be administered orally in the form of a tablet or capsule. To orally administer the composition in solid form to a mammal, such as a human, the solution of crystallized dextran microparticles and insulin are first dried, for example by freezing, to form a powder. The powder can be compressed into a tablet, together with optional pharmaceutically acceptable excipients or placed in a pharmaceutically acceptable capsule.

In another preferred aspect of the present invention, the composition comprises crystallized dextran microparticles and a therapeutically effective amount of insulin that can be administered to a mammal, such as a human, by inhalation. In this case, the composition is placed in a container adapted for the administration of a pharmaceutical composition to a mammal by inhalation, which may be an inhaler that provides a metered dose of a composition when tightened or pressed. Preferably, the composition is provided to a mammal through the mouth by spraying the composition in solution or suspension from the inhaler. Preferably, the composition is administered to the lungs of the mammal (i.e., pulmonary administration).

G. Vehicle for Injectable Insulin Administration The present inventor has discovered that a composition of crystallized dextran micro-particles 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 20 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 21 schematically illustrates 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 it 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 the efficacy of insulin when injected to adult humans and children. Examples 15-22 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 15, the mouse was not injected with insulin. In comparative examples 16, 17 and 21, insulin alone (0.5 IU) was injected into the three mice. In Examples 18-20 and 22, insulin (0.5 IU) and a crystallized dextran micro-particle implant were injected into the four mice. The results are summarized in Table VI.

TABLE VI 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 VI, the glucose level in the mice of comparative examples 16, 17 and 21 is approximately equal to or less than the glucose level in mice of Examples 18-20 and 22 during the first 45 minutes after of the injection. The glucose level is approximately the same in mice of 5 the comparative examples 16, 17 and 21 and the examples 18-20 and 22, 120 minutes after the injection. However, the glucose level in the mice of Comparative Examples 16, 17 and 21 is approximately three times higher than the glucose level in Examples 18-20 and 22 from 210 to 390 minutes after injection. In fact, the blood glucose level in mice in Examples 18-20 and 22 did not increase substantially (ie did not increase by more than 10%, remained the same or decreased) from 120 to 390 minutes after the injection . In comparison, the blood glucose level in the mice of comparative examples 16, 17 and 21, 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 22A and 22B, the composition comprising the short-acting insulin Actrapid HM® and the crystallized dextran micro-particles prolonged the absorption of insulin and extended the hypoglycemic effect (i.e. the duration of 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 22B) and approximately 17 to approximately 24 hours for the insulin "Monotard HM® "alone (Figure 22A). 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 22A and 22B, the upper line illustrates the control line for intact rabbits to which insulin was not administered. The y-axis of Figures 22A and 22B is a relative scale, normalized blood glucose concentration for the same dose of 8 IU of insulin. 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 22A and 22B 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 23 and 24, two intact rabbits did not receive insulin. In comparative examples 25 and 26, an aqueous solution of long-acting insulin was introduced subcutaneously into two rabbits in a dose of 8 IU. In examples 27-29 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 VII.

TABLE VII The above examples 27-29 illustrate that the Actrapid HM® short-acting 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 the 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 23 to 29, 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% 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 insulin in blood and blood glucose concentration at least 30% longer, such as 100% to 400% longer, than the same dose of ia. 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. Even more, the composition comprises a phase one, which can flow, or a colloidal system of several phases (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 30, 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 HIV.

TABLE VIII As can be seen in Table VIII, the duration of the effectiveness 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 effectiveness 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). As mice and rabbits are a common model for humans in drug tests, the data shown in the above tables VI to VIII suggest that the use of implants based on crystallized dextran micro-particles makes it possible to develop delivery administration systems controlled with better pharmacokinetic and dynamic characteristics and that better satisfy the initial insulin needs in patients such as humans.

H. Materials The term "insulin" should be interpreted to include insulin analogues, naturally-extracted human insulin, recombinantly produced human insulin, insulin extracted from bovines and / or pigs, porcine and bovine insulin produced recombinantly and mixtures of any 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. Moreover, the therapeutic agents described herein are not limited to insulin. Any other suitable therapeutic agent can be used in conjunction with the microparticles for oral administration, administration by inhalation, administration by injection and administration by surgical implant. For example, a suitable therapeutic agent may comprise a peptide, polypeptide or protein ranging from 0.5 K Daltons to 150 K Daltons of molecular size. In particular, peptide, polypeptide or protein therapeutics may include aids for diabetics such as those mentioned above, insulin and insulin analogues, amylin, glucagon, surfactants, peptides and immunomodulatory proteins such as cytokines, chemokines, lymphokines, taxol, Interleukins such as interleukin-1, interleukin-2 and interferons, erythropoietins, thrombolytics and heparins, anti-proteases, entitrypsins and amiloride, rhDNase, antibiotics and other anti-infectives, hormones and growth factors such as parathyroid hormones, LH-RH and analogues of GnRH, nucleic acids, DDAVP, calcitonins, cyclosporins, ribavirin, enzymes, heparins, hematopoietic factors, cyclosporins, vaccines, immunoglobulins, vasoactive peptides, antisense agents, oligonucleotides and nucleotide analogues. Other suitable therapeutic agents include viruses, cells, genes and other agents that have therapeutic properties such as other suitable vaccines and molecular therapeutic agents. The term "amylin" includes human native amylin, bovine amylin, porcine, rat, rabbit as well as synthetic, semi-synthetic or recombinant amylin or amylin analogs including pramlintide and other amylin agonists. The term "immunomodulatory proteins" includes cytokines, chemokines, lymphokines, complement components, growth hormones, accessory and adhesion molecules of the immune system and their receptors of human or non-human animal specificity. Useful examples include GM-CSF, G-CSF, IL-2, IL-12, OX40, OX40L (gp34), lymphotactin, CD40, CD40L. Useful examples include interleukins, for example interleukins 1 to 15, interferon alpha, beta or gamma, tumor necrosis factor, granulocyte-macrophage colony stimulation factor (GM-CSF) macrophage colony stimulation factor ( M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP) macrophage chemo-attractor and activation factor (MCAF), RANTES, macrophage inflammatory peptides MIP- 1a and MIP-1b, complement components and their receptors or an accessory molecule such as B7.1, B7.2, ICAM-1, 2 or 3 and cytokine receptors. OX40 and OX40 ligand (gp34) are other useful examples of immunomodulatory proteins. The immunomodulatory proteins can, for various purposes, be of human or non-human animal specificity and can be represented, for the present purposes, as the case may be and as appropriate, by extracellular domains and other fragments with the binding activity of the proteins. proteins and their muteins and their fusion proteins with other polypeptide sequences, i.e. with the constant domains of heavy chain immunoglobulin. When nucleotide sequences that encode more than one protein are inserted, they can, for example, comprising more than one cytokine or a combination of cytokines and accessory / adhesion molecules. The term "interferon" or "IFN", as used herein, means the family of highly homologous, species-specific proteins that inhibit viral replication and cell proliferation and modulate the immune response. Interferons are grouped into three classes based on their cellular origin and antigenicity, ie alpha-interferon (leukocytes), beta-interferon (fibroblasts) and gamma-interferon (immunocompetent cells). The recombinant and analogous forms of each group have been developed and are commercially available. The sub-types of each group are based on antigenic / structural characteristics. At least 24 alpha interferons (grouped into subtypes A through H) that have distinct amino acid sequences have been identified by isolating and sequencing DNA encoding these peptides. The terms "alpha-interferon", "alpha interferon", "interferon alpha", "human leukocyte interferon" and "IFN" are used interchangeably herein to describe the members of this group. The human leukocyte interferon prepared in this manner contains a mixture of human leukocyte interferons having different amino acid sequences. The term "erythropoietin" is applied to products of synthetic, semi-synthetic, recombinant, natural, human, monkey or other animal or microbiologically isolated polypeptides having a part of the primary structural conformation (ie, continuous sequence of amino acid residues). ) and one or more of the biological properties (ie, immunological properties and biological activity in vivo and in vitro) of natural erythropoietin, including its allelic variants. These polypeptides are also uniquely characterized as the prokaryotic or eukaryotic host expression product (ie, by bacterial, yeast and mammalian cells in culture) of exogenous DNA sequences obtained by genomic cloning or cDNA or by gene synthesis. Products of microbial expression in vertebrate cells (ie, mammals and birds) can also be characterized as being free from association with human proteins or other contaminants that can be associated with erythropoietin in their natural environment in mammalian cells or in extracellular fluids like plasma or urine. The products of the host cells typical of yeast (ie Saccaromyces cerevisiae) or prokaryotes (ie E. coll), are free of association with mammalian proteins. Depending on the host employed, the polypeptides of the invention may be glycosylated with mammalian carbohydrates or with other eukaryotic carbohydrates or may not be glycosylated. The polypeptides may also include an initial amino acid residue methionine (in the -1 position). The novel glycoprotein products of the invention include those that have a primary structural conformation sufficiently copied from natural (ie, human) erythropoietin to allow for the possession of one or more of the biological properties thereof and which has an average carbohydrate composition which differs from natural (ie human) erythropoietin. The terms "heparins" and "thrombolytics" include anticoagulant factors such as heparin, low molecular weight heparin, tissue plasminogen activator (TPA), urokinase (Abbokinase) and other factors used to control clots. The terms "anti-proteases" and "protease inhibitors" are used interchangeably and apply to synthetic, semi-synthetic, recombinant, natural or non-natural, soluble or immobilized agents, reactive with receptors or acting as antibodies, enzymes or acids nucleic These include receptors that modulate an immune humoral response, receptors that modulate a cellular immune response (eg, T lymphocyte receptors), and receptors that modulate a neurological response (eg, glutamate receptor)., glycine receptor, gamma-amino butyric acid receptor (GAMA)). These include cytokine receptors (involved in arthritis, septic shock, rejection of transplants, autoimmune disease and inflammatory diseases), the main histocompatibility receptors (MHC) Class I and II associated with the presentation of antigen to lymphocyte receptors. Cytotoxic T and / or T helper receptors (implicated in autoimmune diseases) and the thrombin receptor (involved in coagulation, cardiovascular diseases). Antibodies that recognize autoantigens such as antibodies involved in autoimmune disorders and antibodies that recognize viral antigens (e.g., HIV, herpes simplex virus) and / or microbial antigens are also included. The terms "hormones" and "growth factors" include hormone-releasing hormones such as growth hormone, thyroid hormone, thyroid-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), leuteinizing hormone, hormone-releasing hormone leutenizing (LHRH, including superagonists and antagonists, such as leuprolide, deltirelix, gosorellna, nafarelin, danazol, etc.) obtained from natural, human, porcine, bovine, ovine, synthetic, semi-synthetic or recombinant sources. These also include somatostatin analogues such as octreotide (Sandostatin). Other agents in this category of biotherapeutics include medications for uterine contraction (eg, oxytocin), diuresis (eg, vasopressin), neutropenia (eg, GCSF), medications for respiratory disorders (eg, superoxide dismutase). ), RDS (eg, surfactants that optionally include apoproterins) and the like. The term "enzymes" includes recombinant deoxyribonuclease as proteases DNase (Genentech) (eg serine proteases such as trypsin and thrombin), polymerases (eg, RNA polymerases, DNA polymerases), reverse transcriptases and cinases, enzymes involved in arthritis, osteoporosis, inflammatory diseases, diabetes, allergies, rejection of organ transplantation, activation of oncogenes (eg dihydrofolate reductase), signal transduction, regulation of auto-cycles, transcription, replication and DNA repair. The term "nucleic acids" includes any segment of the DNA or RNA containing natural or non-natural nucleosides or other proteinoid agents capable of specifically binding to other nucleic acids or oligonucleotides by complementary hydrogen bonds and also capable of binding ligands. which are not nucleic acids. The term "vaccines" refers to therapeutic compositions for stimulating humoral and cellular responses of the immune type, either in isolation or through an antigen-presenting cell, such as an activated dendritic cell, which can activate T-lymphocytes to produce a multivalent immune cellular response against a selected antigen.

The cell presenting the potent antigen is stimulated by exposing the cell in vitro to a polypeptide complex. The polypeptide complex may comprise a protein that binds to a dendritic cell and a polypeptide antigen, but preferably, the polypeptide antigen is either a tissue-specific tumor antigen or an oncogene gene product. However, it is appreciated that other antigens, such as viral antigens, can be used in said combination to produce immunostimulation responses. In another preferred embodiment, the dendritic cell binding protein that is part of the immunostimulatory polypeptide complex is GM-CSF. In a further preferred embodiment, the polypeptide antigen that is part of the complex is a tumor-specific antigen prostatic acid phosphatase. In other preferred embodiments, the polypeptide antigen can be any of the peptide antigens produced by oncogene. The polypeptide complex may also contain, between the dendritic cell binding protein and the polypeptide antigen, a linker peptide. The polypeptide complex can comprise a dendritic cell binding protein covalently linked to a polypeptide antigen, said polypeptide complex preferably being formed from a dendritic cell binding protein, preferably GM-CSF and an antigen of polypeptide. The polypeptide antigen is preferably a tissue-specific tumor antigen such as prostatic acid phosphatase (PAP) or an oncogene product, such as Her2, p21 RAS and p53, however, other modalities, such as viral antigens , they are also within the scope of the invention. The term "immunoglobulins" includes oligonucleotides of polypeptides involved in host defense mechanisms, such as encoding and encoding by one or more gene vectors, conjugating different nucleic acid binding portions in host defense cells or coupling vectors expressed to aid in the treatment of the human or animal subject. The drugs included in this class of polypeptides include IgG, EgE, EgM, IgD, either individually or in combination with one another. Other suitable therapeutic agents include adrenocorticotropic hormone, epidermis growth factor, platelet-derived growth factor (PDGF), prolactin, luliberin, luteinizing hormone-releasing hormone (LHRH) agonists, LHRH antagonists, tastrin, tetragastrin, pentagastrin, orogastrone. , secretin, enkephalins, endorphins, angiotensins, tumor necrosis factor (TNF), nerve growth factor (NGF), heparinase, bone morphogenic protein (BMP), hANP, glucagon-like peptide (GLP-1), interleukin-11 ( IL-11), VEG-F, recombinant hepatitis B surface antigen (rHBsAg), renin, bradykin, bacitracins, polymyxins, colistins, torocidin, gramicidin and synthetic analogs, modifications and pharmacologically active fragments thereof, enzymes, cytokines, antibodies and vaccines.

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 Like other polymer micro-particles, including polysaccharides, PLA, PLGA, PMMA, polyesters, 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 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.

I. 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 live 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 orally or 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 (62)

NOVELTY OF THE INVENTION CLAIMS

1. - A flowable pharmaceutical composition, comprising biodegradable and biocompatible components to be provided within or on the body of a mammal and adapted to form a biodegradable, biocompatible, three-dimensional, structured object, the composition comprising a multi-phase, flowable and colloidal system therapeutic agents to provide local or systemic therapeutic effects in or on said body.

2. A pharmaceutical composition to be provided within or on the body of a mammal, comprising a first phase, a second phase, first molecules or molecular aggregates that are adapted to preferentially partition in the second phase, wherein the Second molecules or molecular aggregates self-assemble a wall adjacent to the first molecules or molecular aggregates when the composition is provided to said body of a mammal.

3. The composition according to claim 1, further characterized in that the colloidal system is a liquid suspension or emulsion containing micro-particles.

4. The composition according to claim 2, further characterized in that the first molecules or molecular aggregates comprise a therapeutic agent, the second molecules or molecular aggregates comprise microparticles, the wall comprises a microparticle shell around a core which contains the therapeutic agent.

5. The composition according to claim 3 or 4, further characterized in that the microparticles are porous microparticles and wherein the composition is administered by injection into the body of the mammal.

6. The composition according to claim 5, further characterized in that the porous microparticles are polymer microparticles.

7. The composition according to claim 6, further characterized in that the porous microparticles are crystallized dextran microparticles having an average diameter ranging between 0.5 and 5.0 microns.

8. The composition according to any of claims 1 to 7, further characterized in that the composition is a two-phase liquid system containing a mixture of micro-particles.

9. The composition according to claim 8, further characterized in that the two-phase system forms a capsule structure when administered in vivo, the capsule structure comprises a first-phase core and a second-phase shell surrounding the capsule. core.

10. The composition according to claim 9, further characterized in that the therapeutic agent selectively partifies in the liquid phase of the core and the microparticles selectively partition in the liquid phase of the shell.

11. The composition according to any of claims 8 to 10, further characterized in that the therapeutic agent is selected from the group consisting of a peptide, a protein, a nucleic acid, a virus, a cell and a combination of the same.

12. The composition according to any of claims 9 to 11, further characterized in that the core comprises an aqueous phase of selective PEG and insulin and the shell comprises an aqueous phase of dextran and dextran microparticles crystallized with partition selective

13. The composition according to claim 2, further characterized in that the composition comprises a flowable composition or a dry composition that can be made to flow by hydration, the first molecules or molecular aggregates are selected from a group formed by micro-particles , macro-molecules, cells, liposomes, DNA, plasmids and protein, and the second molecules or molecular aggregates are selected from a group consisting of micro-particles, macro-molecules, cells, liposomes, DNA, plasmids and proteins.

14.- The use of a first phase, a second phase, first molecules or molecular aggregates that are adapted to preferentially partition in the first phase and second molecules or molecular aggregates that are adapted to selectively partition in the second phase, to prepare a pharmaceutical composition for providing said pharmaceutical composition within of or on the body of a mammal, wherein the second molecules or molecular aggregates self-assemble a wall adjacent to the first molecules or molecular aggregates when the composition is provided within or on said body of a mammal.

15. The use claimed in claim 14, wherein the pharmaceutical composition is a liquid suspension or emulsion containing microparticles when it is outside the mammalian hide.

16. The use claimed in claim 14, wherein the first molecules or molecular aggregates comprise a therapeutic agent, the second molecules or molecular aggregates comprise micro-particles, the wall comprises a micro-particle shell around a core which contains therapeutic agent.

17. The use claimed in claim 15 or 16, wherein the microparticles are polymer microparticles and the body of the mammal comprises an animal or human body.

18. The use claimed in claim 17, wherein the polymer microparticles are porous microparticles.

19. The use claimed in claim 18, wherein the porous microparticles are crystallized dextran microparticles with an average diameter ranging from 0.5 to 5.0 microns.

20. - The use claimed in any of claims 14 to 19, wherein the composition is a two-phase system containing micro-particles.

21, - The use claimed in claim 20, wherein the two-phase system forms a capsule structure when provided in vivo, the capsule structure comprising a first phase core and a second phase shell surrounding the capsule. core.

22. The use claimed in claim 21, wherein the therapeutic agent selectively partitioned into the core and the microparticles selectively partition in the shell.

23. The use claimed in claim 22, wherein the therapeutic agent is selected from a group consisting of a peptide, a protein, a nucleic acid, a virus and a cell.

24. The use claimed in any of claims 21 to 23, wherein the core comprises an aqueous phase of PEG and selective partition insulin and the shell comprises an aqueous phase of dextran and dextran microparticles crystallized with partition selective

25. The use claimed in claim 14, wherein the composition comprises a flowable composition or a dry composition that can be made to flow by hydration, before being provided to the mammalian body, the first molecules or molecular aggregates are selected to starting from a group formed by micro-particles, macro-molecules, cells, liposomes, DNA, plasmids and proteins, and the second molecules or molecular aggregates are selected from a group formed by micro-particles, macro-molecules, cells, liposomes, DNA, plasmids and proteins.

26.- A flowable composition comprising a colloidal suspension or emulsion of biocompatible and biodegradable microparticles and a label in a fluid.

27. The composition according to claim 26, further characterized in that the micro-particles comprise micro-particles of crystallized dextran, the label comprises a fluorescent macromolecule that is released in a controlled manner from the composition when it is located in the body of a mammal.

28. The composition according to claim 26, further characterized in that the colloidal suspension comprises a capsule having a first phase shell comprising a selective partition label and a second phase core containing selective partition microparticles. .

29.- The use of cells embedded in or in contact with an outer surface of a capsule of crystallized dextran microparticles, to prepare a medicament for cells in a mammal.

30. The use claimed in claim 29, wherein the cells are embedded in the capsule such that the cells comprise a core of the capsule and the micro-particles comprise a shell that includes the core, and the shell It is porous to oxygen and nutrients but impermeable to the cells of the immune system.

31. The use claimed in claim 30, wherein the cells comprise insulin producing cells that produce and release insulin through the capsule into the blood of a mammal to reduce blood glucose in response to the permeation of blood glucose to the capsule.

32. The use claimed in claim 30, wherein the cells comprise cells that are not of the mammal to which the composition is administered.

33. The use claimed in claim 29, wherein the cells come into contact with the outer surface of the capsule and the cells comprise the cells of the mammal to which the composition is administered.

34.- A bone graft substitute comprising porous crystallized dextran microparticles.

35. The bone graft substitute according to claim 34, further characterized in that it additionally comprises a therapeutic agent located in the pores of the microparticles that provides bone formation in special areas of a body.

36.- The use of porous crystallized dextran microparticles to prepare a bone graft substitute to provide said bone graft substitute at a bone graft site in the body of a mammal.

37. - The use claimed in 36, further characterized in that it additionally comprises preparing a suspension of porous crystallized dextran microparticles in a first liquid phase, preparing a suspension of second microparticles in a second liquid phase immiscible with the first liquid phase; and preparing an emulsion wherein the second liquid phase is a continuous phase and the first liquid phase is a dispersed phase.

38.- The use claimed in claim 37, wherein the second micro-particles comprise ceramic micro-particles and a therapeutic agent that provides bone formation at special body sites is located in the pores of the micro-particles of dextran crystallized porous.

39.- A method comprising providing crystallized dextran microparticles and combining a therapeutically effective amount of insulin and the crystallized dextran microparticles in a solution after the microparticles have been crystallized to form an insulin suspension and micro-particles of crystallized dextran.

40. The method according to claim 39, further characterized in that the crystallized dextran micro-particles comprise micro-particles having an average diameter of 0.5 to 5.0 microns, the step of providing the micro-particles comprises forming the micro-particles. particles in a non-organic solvent, the solution comprises an aqueous solution and the mammal comprises a human.

41. The method according to claim 39, further characterized in that it further comprises drying the suspension to provide a composition comprising the micro-particles of crystallized dextran and insulin.

42.- A method for the manufacture of porous, non-crosslinked crystallized dextran microparticles, comprising (a) preparing an aqueous dextran solution lacking an organic solvent, (b) conducting a crystallization process to form the dextran micro-particles at a temperature above room temperature; and (c) isolating porous crystallized dextran microparticles, without crosslinking from the solution.

43. The method according to claim 42, further characterized in that the dextran solution comprises dextran having a molecular weight of 2 to 200 kDa and the crystallization process is carried out at a temperature of 40 ° C to 99 ° C.

44. The method according to claim 43, further characterized in that the microparticles are formed spontaneously from the solution, the dextran solution comprises dextran having a molecular weight of 20 to 75 kDa, and the crystallization process It is carried out at a temperature of 40 ° C to 70 ° C.

45. The method according to claim 42, further characterized in that it comprises combining the micro-particles with insulin.

46. - A . method for making porous crystallized dextran microparticles containing a therapeutic agent in the pores of the microparticles, which comprises providing a suspension comprising porous crystallized dextran microparticles and providing the therapeutic agent in the suspension in such a way that the therapeutic agent permeates the pores of the porous microparticles.

47. The method according to claim 46, further characterized in that the step of providing a suspension comprises preparing an aqueous solution of dextran, performing a crystallization process to form porous crystallized dextran microparticles, isolating the microparticles from the solution and provide the micro-particles in a colloidal system comprising the therapeutic agent.

48. The method according to claim 47, further characterized in that the therapeutic agent comprises insulin and the colloidal system comprises a suspension comprising insulin and the micro-particles.

49.- A composition comprising insulin and micro-particles of crystallized dextran, porous, wherein the micro-particles are formed before the combination of the insulin and the micro-particles in the composition.

50.- The composition according to claim 49, further characterized in that the composition comprises a flowable colloidal composition and the micro-particles comprise micro-particles having an average size of 0.5 to 5 microns.

51. The composition according to claim 49, further characterized in that the insulin is located in pores of the crystallized dextran micro-particles but is not embedded within each micro-particle.

52. The composition according to claim 49, further characterized in that the insulin selectively partitioned into a first polymer phase and the microparticles selectively partition in a second polymer phase such that the composition forms a structured implant to the introduction to the body of a mammal.

53.- A composition comprising insulin, a first polymer, a second polymer that is not compatible with the first polymer and micro-particles.

54. The composition according to claim 53, further characterized in that the composition comprises a flowable colloidal composition and the microparticles comprise crystallized dextran microparticles having an average size of 0.5 to 5 microns.

55.- The composition according to claim 54, further characterized in that the first polymer comprises a dextran and the second polymer comprises PEG.

56.- The composition according to claim 55, further characterized in that the insulin selectively partitiones into a PEG phase and the microparticles selectively partition into a dextran phase, such that the composition forms a structured implant comprising a PEG phase core and a dextran phase shell when introduced to the body of a mammal.

57.- The use of crystallized dextran micro-particles and insulin to prepare a 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.

58. The use claimed in claim 57, wherein the composition comprises a flowable colloidal composition and the microparticles comprise crystallized dextran microparticles having an average size of 0.5 to 5 microns.

59. The use claimed in claim 58, wherein the composition comprises a composition of two phases 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 introduction into the body of a mammal.

60. The use claimed in claim 59, further comprising controlling a thickness of the shell based on the body of the animal receiving the composition to control the release of insulin from the implant.

61. - The use claimed in claim 57, wherein the composition is for a human being suffering from diabetes to reduce the concentration of blood glucose in the human.

62. An inhaler comprising a container adapted to deliver a dose of a pharmaceutical composition to a mammal by inhalation and a pharmaceutical composition comprising crystallized dextran microparticles and a therapeutically effective amount of insulin located in the container. 63.- The use of crystallized dextran micro-particles and insulin, to prepare a composition for reducing blood glucose in a mammal, wherein the composition is administrable by inhalation.