Microparticle composition and methods for their preparing

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to a biologically active microparticles composition comprising microparticles that involve: (a) polymer taken among group consisting of poly-(α-hydroxyacid), polyhydroxybutyric acid, polycaprolactam, poly-ortho-ester, polyanhydride and polycyanoacrylate, and (b) the first part of detergent that is bound with polymer, and also complex adsorbed on microparticles complex that comprises: (a) biologically active macromolecule, and (b) the second part of detergent wherein the first part of detergent and the second part of detergent comprise the same detergent or different detergents and wherein biologically active macromolecule is taken above group consisting of polypeptide, polynucleotide, polynucleoside, antigen, pharmaceutical agent, hormone, enzyme, transcription or translation mediating agent, metabolite, an immunomodulating agent and adjuvant. Also, invention relates to methods for preparing the composition and its applying. Invention provides improvement of adsorption of biologically active agents on microparticles surface in delivery systems, especially, for medicinal agents that are characterized with high sensitivity and complexity in their preparing based on the composition proposed.

EFFECT: improved method preparing, improved and enhanced properties of composition.

44 cl, 1 tbl, 1 dwg, 7 ex

 

The status of related applications

The present invention relates to patent application serial No. 60/236077, filed September 28, 2000, the Specified application is fully incorporated into the present description by reference.

The technical field to which the invention relates.

The present invention relates to pharmaceutical compositions. In particular, the invention relates to microparticles with absorbent surfaces, methods of making such particles and to their use. In addition, the invention relates to compositions containing biodegradable microparticles, where on the surface of the microparticles adsorbed biologically active substances, such as therapeutically useful polynucleotide, polypeptides, antigens and adjuvants.

Background of invention

To achieve parenteral controlled delivery of therapeutic compounds used media in the form of particles. Such carriers develop to retain the active agent delivery system for a long period of time. Examples of carriers in the form of particles include media that are obtained from polymetylmetacrylate polymers, and also on the basis of microparticles derived from poly(lactides) (see, for example, U.S. patent No. 3773919), poly(latecollision), known as MRO (PLG) (see, for example, U.S. patent No. 4767628) is of polyethylene glycol, known as PEG (see, for example, U.S. patent No. 5648095). Polymetylmetacrylate polymers belong to degradiruem substances, whereas particles MRO decompose when non-directional non-enzymatic hydrolysis of ester groups on lactic and glycolic acid, which is separated by normal metabolic pathways.

For example, in U.S. patent No. 5648095 describes the use of microspheres with encapsulated in the composition of pharmaceutical agents as drug-delivery systems for nasal, oral, pulmonary and oral delivery. Were also described compositions with slow release, containing various polypeptide growth factors. See, for example, international publication number WO 94/12158, U.S. patent No. 5134122 and international publication number WO 96/37216.

Fattal et al., Journal of Controlled Release 53:137-143 (1998) describes the nanoparticles obtained from polyalkylacrylate (PAC), including adsorbed oligonucleotides.

Media in the form of particles with adsorbed or trapped in their composition antigens are also used in order to achieve an adequate immune responses. Such media presenterat immune system, multiple copies of the selected antigen and contribute to the capture and retention of antigens in the local lymph nodes. The particles may be subjected to phagocytosis with the no action macrophages, and they can increase prezentowanie antigen due to the release of the cytokine. For example, in concurrently pending application, joint ownership No. 09/015652, filed January 29, 1998, describes the use of microparticles with adsorbed antigen and encapsulated antigen to stimulate kletka-mediated immunological reactions, and methods of making such microparticles.

So, for example, simultaneously considering the serial application of joint ownership No. 09/015652, filed January 29, 1998, discloses a method of forming microparticles, which involves combining the polymer with an organic solvent, with subsequent addition of the emulsion stabilizer such as a surfactant polyvinyl alcohol (PVA), and evaporation of the organic solvent with the formation of the microparticles. The surface of the microparticles comprises a polymer and a stabilizer. On these surfaces can also be adsorbed macromolecules, such as DNA, polypeptides and antigens.

In U.S. patent No. 5814482 and 6015686 disclosed eukaryotic layered vector system initiation (ELVIS vectors), in particular those that are obtained and constructed on the basis of the genomes of alpha viruses (such as Sindbis virus), for use in stimulating the immune response to the antigen in the processes of inhibi the Finance pathogenic agents and the delivery of heterologous nucleotide sequences in eukaryotic cells and, among other cells in the animal.

In the international patent application of co-ownership PCT/US99/17308 and jointly consider the serial application for U.S. patent No. 09/715902 disclosed methods of making microparticles containing adsorbed macromolecule, such as a pharmaceutical agent, polynucleotide, polypeptide, protein, hormone, enzyme, the mediator of transcription or translation, intermediate metabolic pathway, an immunomodulator, an antigen, adjuvant, or combinations thereof, and other Microparticles include, for example, a polymer such as poly(alpha-hydroxycitrate) (e.g., MRO), polyhydroxyalkane acid, polycaprolactone, polychaetes, polyanhydride etc. you get with using, for example, cationic, anionic or nonionic detergents.

Although MRO microparticles with adsorbed antigen provides significant advantages in comparison with other more toxic systems, however, the adsorption of biologically active agents on the surface of the microparticles can be improved. For example, it is often difficult or impossible to carry out the adsorption of charged or volume of biologically active substances, such as polynucleotide, large polypeptides, etc. on the surface of microparticles. Thus, there is a continuing need for flexible delivery systems for such is exist and particularly for drugs which are characterized by high sensitivity and difficulties in the preparation on the basis of their composition.

A brief description of the invention

The authors of the present invention, it was found that the adsorption of macromolecules on the microparticles can be improved by ensuring the availability of detergent in the formation of complex molecules during the adsorption. This accessibility can be achieved, for example, by means of a separate add some detergent adsorption of macromolecules or by ensuring that the process of formation of microparticles will result in a product containing a significant amount of unbound detergent. This approach is contrary to the methods of the prior art, according to which the microparticles are thoroughly washed to remove residual quantities of detergent prior to the adsorption of macromolecules. Thus, in particular, in the above-cited document PCT/US99/17308 examples, according to which the microparticles are washed many times with water (i.e. they are washed with water four times, followed by centrifugation) before the introduction of the macromolecule of interest. Such stage of leaching removes essentially all unbound detergent, resulting in a finished product is more than 99% of the remaining detergent particles.

Thus, in accordance with the first aspect of the present invention features a composition of microparticles, which contains (1) microparticles that include a polymer and the first part of the detergent, which is associated with the polymer, and (2) a complex of biologically active macromolecules with the second part of the detergent, and the specified complex adsorbed on the surface of the microparticles. The first part of the detergent and the second part of the detergent may contain the same detergent or different detergents.

Preferred biologically active macromolecule selected from the group consisting of polypeptide, polynucleotide, polynucleotide, antigen, a pharmaceutical agent, a hormone, an enzyme, a mediator of transcription or translation, intermediate metabolic pathway, an immunomodulator and adjuvant.

Preferred polymers are poly(α-hydroxy acid), more preferably those selected from the group consisting of poly(L-lactide), poly(D,L-lactide) and poly(D,L-lacticacid). More preferred are poly(D,L-actigraphically) polymers. Preferred poly(D,L-actigraphically) polymers are those polymers which are characterized by a molar ratio of lactide/glycolide in the range from 30:70 to 70:30, more preferably from 40:60 to 60:40 and have molecular the th mass, ranging from 10000 to 100000 daltons, more preferably from 30,000 to 70,000 daltons daltons. A more preferred biologically active macromolecules include bacterial and viral antigens (e.g., HIV antigens such as gp120, gp140, p24gag and p55gag, meningitis antigens, antigens of Streptococcus In and antigens hemagglutinin of influenza a), and polynucleotide that encode antigens. The biologically active molecule can be, for example, in the form of plasmids, ELVIS vector or structure-based RNA vector. Particularly preferred biologically active macromolecule is pCMV-p55gag.

In some embodiments, the invention features a composition of microparticles with additional biologically active macromolecule, which may be in a bound or unbound form, and can even be captured by the polymer. So, for example, can be obtained composition of microparticles with an adjuvant, in particular, with Th1-stimulating adjuvant. Preferred adjuvants include CpG oligonucleotides, LTK63, LTR72, MPL and aluminum salts, including aluminum phosphate.

In some embodiments of the invention the first part of the detergent and the second part of the detergent containing the same detergent. Preferred for this purpose, the detergent is a cationic detergent, such as CTAB. In such Varian is Ah the invention, the first part of the detergent (which is associated with the polymer), preferably, is about 5-95% of the total detergent composition, more preferably about 10-90%, more preferably about 10-60% and, most preferably, about 25-40%.

In other embodiments of the invention the first part of the detergent and the second part of the detergent may contain a different detergents. For example, the first part of the detergent may include non-ionic detergent (e.g., PVA), and the second part of the detergent may include cationic detergent (e.g., CTAB).

In accordance with another aspect of the present invention to the above compositions of microparticles add pharmaceutically acceptable excipient.

Another aspect of the present invention relates to the delivery of macromolecules vertebral subject, which includes the introduction of the vertebral subject of the above compositions of microparticles.

In an additional aspect, the invention relates to a method manifestations of cellular and/or humoral immune response in the vertebrate subject, which includes the introduction of the vertebral subject a therapeutically effective amount of a composition of microparticles defined above.

Another aspect of the present invention relates to a method of immunization, which includes the introduction of the vertebral subject a therapeutically effective amount of the composition m is crocetin, defined above.

In other aspects of the present invention, the above composition of the microparticles used in the diagnosis of disease, in the treatment of diseases, in the composition of the vaccines and/or to enhance the immune system.

Still other aspects of the present invention relate to methods of producing compositions of microparticles. In General, these methods include: (a) obtaining emulsion comprising (i) a polymer selected from the group consisting of poly(α-hydroxyacids), polyhydroxyalkanoic acid, polycaprolactone, polyarteritis, polyanhydride and polycyanoacrylate, (ii) an organic solvent, (iii) a detergent and (iv) water; and then (b) removing the organic solvent. In the specified embodiment of the invention about 10-90% of the total detergent in the resulting composition, preferably, associated with microparticles, more preferably about 10-60% and, most preferably, about 25-40%. In General, the composition of the microparticles further incubated with a biologically active macromolecule, such as described above, with the formation of the biologically active composition.

Preferably, the emulsion is an emulsion of water-in-oil-in-water, which is formed by a process comprising: (a) emulsification of the organic phase containing the polymer and an organic solvent, with the first of the aqueous phase,

containing water, emulsion water-in-oil; and (b) emulsification of the second aqueous phase containing a cationic detergent and water emulsion formed in stage (a), to obtain the emulsion water - in - oil - in - water.

In some preferred versions of the invention, the detergent is a cationic detergent, which is provided in the form of an emulsion when the mass ratio of detergent to polymer of from approximately 0.05:1 to about 0.5:1. In these versions of the invention, the method preferably further includes filtering the particles in the cross-flow stage after removal of the solvent. In a specific embodiment of the invention the polymer is a poly(D,L-lacticacid), cationic detergent is a CTAB and cationic detergent is provided in the form of an emulsion when the mass ratio of detergent to polymer is from about 0.1:1 to about 0.5:1.

In other preferred embodiments of the invention, the detergent is a cationic detergent, which is provided in the form of an emulsion when the mass ratio of detergent to polymer, from about 0,001:1 to approximately 0.05:1. At these low levels there is usually no need to stage filtering or washing to remove excess detergent. In the specific embodiment of the invention the cationic detergent is a CTAB, the polymer is a poly(D,L-lacticacid), cationic detergent is provided in the form of an emulsion when the mass ratio of detergent to polymer of from about 0,002:1 to

around 0.04:1, and the particles are not subjected to the stage of removal of CTAB from the composition.

Other aspects of the present invention relate to methods of producing compositions of microparticles, these methods include: (1) preparation of microparticles in the process of emulsification, and these microparticles comprise a polymer and the first part of the detergent that is associated with the microparticles; and (2) the adsorption is carried complex biologically active macromolecule and the second part of the detergent on the surface of the microparticles. The first part of the detergent and the second part of the detergent may include the same detergent or different detergents. The polymer is preferably selected from the group consisting of poly(α-hydroxyacids), polyhydroxyalkanoic acid, polycaprolactone, polyarteritis, polyanhydride and polycyanoacrylate.

In some embodiments of the invention the first and second parts of the detergent include the same detergent. The specified detergent, preferably, is a cationic detergent, for example, CTAB. In such embodiments of the invention about 10-90%, more preferably about 10-60%, and most ol doctitle, 25-40% of the total detergent in the composition of the microparticles present in the form of the first part of the detergent, which is associated with microparticles. In a typical case, all the detergent is added during the process of emulsification.

In other embodiments of the invention the first part of the detergent includes a first detergent, and the second part of the detergent

includes a second detergent, different from the first detergent. In a typical case, the first detergent is added during the process of emulsification and the second detergent is added after emulsification, preferably, simultaneously with the addition of biologically active macromolecules. Preferably, the first part of the detergent includes a non-ionic detergent, such as PVA, and the second part of the cleaning agent includes a cationic detergent, such as CTAB.

These and other embodiments of the present invention will be clear to experts in this field from the following disclosure.

The drawing shows a schematic diagram of apparatus suitable for obtaining microparticles according to the present invention.

Detailed description of the invention

The practical implementation of the present invention includes, unless otherwise indicated, conventional methods of chemistry, polymer chemistry, biochemistry, molecular biology, immunology and pharmacology,all of which are available to professionals in this field. Such techniques are well presented in the literature. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Handbook of Surface and Colloidal Chemistry (Birdi, for K.S., ed. CRC Press, 1997) and Seymour/Carraher''s Polymer Chemistry (4th edition, Marcel Dekker Inc., 1996).

All publications, patents and patent applications cited in the present description previously or hereafter included in the present description by reference.

In the context of the present description and the proposed claims, the term "microparticle" (singular) refers to one or more microparticles, etc.

A. Definitions

In the course of describing the present invention will use the following terms should be understood as described below.

Unless specifically stated otherwise, all percentages and ratios are calculated based on the mass of the parts.

The term "microparticle" in the context of the present description refers to a particle diameter from about 10 nm to about 150 microns, more preferably with a diameter from about 200 nm to about 30 μm and, most preferably, a diameter of about 500 nm to about 10 μm. Preferably, the microparticles have a diameter that allows injecting or introducing chere is C mucosa without jamming needles and capillaries. The size of the microparticles can be easily identified known in the art methods such as photon-correlation spectroscopy, laser diffractometry and/or scanning electron microscopy. The term "particle" can also be used to refer to particles defined in the present description.

Polymeric microparticles for use according to the present description are derived from materials that can be sterilized, are non-toxic and biodegradable. Such materials include, but are not limited to, poly(α-gidrokshikislotu), polyhydroxyalkanoic acid, polycaprolactone, polychaetes, polyanhydride, PAC and polycyanoacrylate. Preferably, the microparticles used according to the present invention are microparticles of a polymer derived from poly(α-hydroxy acid), in particular, from a poly(lactide) ("PLA") or of a copolymer of D,L-lactide and glycolide or glycolic acid, such as poly(D,L-lacticacid) ("MRO" or "PLGA"), or a copolymer of D,L-lactide and caprolactone. The polymer microparticles may be obtained from any of a variety of source of polymeric materials characterized by different molecular weights, and in the case of copolymers, such as MRO, characterized by different ratios of lactide:glycolide, the choice is which determines, mainly, the specific situation, depending, inter alia, on the nature of jointly entered macromolecules. These options are discussed more fully below.

The term "detergent" in the context of the present description includes surfactants, dispersing agents, suspendresume agents and stabilizers of emulsions. Anionic detergents include, but are not limited to, LTOs (sodium dodecyl sulphate), LSN (sodium lauryl sulphate), BCA (desulfosarcina), sulfated fatty alcohols, and other Cationic detergents include, but are not limited to cetrimide (cetyltrimethylammonium bromide or CTAB"), benzalkonium chloride, DDA (bromide of dimethyldioctadecylammonium), DOTAP (dioleoyl-3-trimethylammonium) and other non-ionic detergents include, but are not limited to, nonionic surfactants such as PVA, povidone (also known as polyvinylpyrrolidone or PVP), esters sorbitan, Polysorbate, polyoxyethylene glycol monoether, polyoxyethylene ALKYLPHENOLS, poloxamer and other

After the formation of the particulate detergent can be associated or not associated with them. In the case of a tie, the detergent may be attached to the microparticles by any mechanism, including, but not limited to, ionic bond, hydrogen bond, covalent bond, physical Zech is at, bond van der Waals forces, as well as the communication type hydrophilic/hydrophobic interactions.

The term "macromolecule" in the context of the present description refers without limitation to a pharmaceutical agent, polynucleotide, polypeptide, hormone, enzyme, the mediator of transcription or translation, intermediate metabolic pathways, immunomodulator, an antigen, adjuvant or their combinations. Below in more detail the specific macromolecule used in accordance with the present invention. "Komplikovana" (in the form of a complex macromolecule is a macromolecule, which has formed an Association with detergent and which then became capable of adsorption on the particle.

The term "pharmaceutical agent" refers to biologically active compounds such as antibiotics, antiviral agents, growth factors, hormones, etc. that are discussed below in more detail.

The term "adjuvant" refers to any substance that promotes the activity of the pharmaceutical agent or modifies it, including, but not limited to, immunological adjuvants that enhance or diversify the immune response to the antigen.

"Polynucleotide" is a nucleic acid polymer, which typically encodes a biologically active (e.g., immunogenic or terapeuticas the th) protein or polypeptide. Depending on the nature of the polypeptide encoded by polynucleotides, polynucleotide may include only 10 nucleotides, for example, when polynucleotide encodes the antigen. Next, polynucleotide" can include double-stranded and single-stranded sequences and include, but not limited to cDNA on the basis of viral, prokaryotic or eukaryotic mRNA, genomic sequences of RNA and DNA from viral (e.g., RNA and DNA viruses and retroviruses) or prokaryotic DNA, and in particular, synthetic DNA sequences. The term also encompasses sequences that include any of the known analogues of the bases of DNA and RNA. The term also includes modifications, such as deletions, additions and substitutions (generally conservative in nature) on the basis of natural sequences are preferably such that when the nucleic acid molecule encodes a therapeutic or antigenic protein. These modifications can be implemented through site-directed mutagenesis, and the random events, such as the effects of the mutations host cells that produce antigens.

The terms "polypeptide" and "protein" refer to polymers of amino acid residues, and without limiting the minimum length of the product. Thus, peptides, oligopeptides, dimers, multimer and the m like included in this definition. This definition covers and proteins full length and fragments thereof. These terms also include modifications, such as deletions, additions and substitutions (generally conservative in nature) natural sequences are preferably such that when the protein retains the ability to cause an immunological reaction or to provide a therapeutic effect in the subject, which is injected protein.

The term "antigen" refers to a molecule that contains one or more epitopes capable of stimulating the immune system of the host cell by generating cell antigen specific immune response when the antigen is present in accordance with the present invention, or humoral response mediated by antibodies. The antigen may possess the ability to generate cellular or humoral response either by itself, or when it is present in combination with another molecule. OK epitope will include from about 3 to 15, mainly, about 5 to 15 amino acids. The epitopes of the corresponding protein can be identified using a variety of methods mapping of epitopes are well known in the art. See, for example, an epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. So, for example, linear epitopes can be determined, for example, through the om simultaneous synthesis of large numbers of peptides on solid substrates, the peptides correspond to the parts of the protein molecule, and the interaction of the peptides with antibodies, when the peptides are still attached to the substrate. Such techniques are known in the art and described in U.S. patent No. 4701871 and Geysen et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 3998-4002; Geysen et al. (1986) Molec. Immunol. 23, 709-715, which are fully included in the present description by reference. Similarly, can easily be identified conformational epitopes by identifying spatial patterns of amino acids, for example, by x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, an epitope Mapping Protocols, described above.

The term "antigen" in the context of the present description denotes both subunit antigens, i.e. antigens that have been purified and isolated from the whole organism with which the antigen is associated in nature, and killed, attenzione or inactivated bacteria, viruses, parasites or other microbes. Antibodies such as antiidiotypic antibodies, or fragments thereof, and synthetic peptide mimotope that can mimic an antigen or antigenic determinant, are also covered by the definition of antigen in the context of the present description. Similarly, in the determination of antigen in the context of the present description includes oligonucleotide or polynucleotide that expr serout therapeutic or immunogenic protein, or antigenic determinant in vivo, of those that find application in gene therapy and immunization nucleic acid.

In addition, for the purposes of the present invention, antigens can be derived from any representative of a number of known viruses, bacteria, parasites and fungi, as well as from any of a variety of tumor antigens. In addition, for the purposes of the present invention, the term "antigen" refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), natural sequences with the proviso that the protein retains the ability to induce an immunological response. These modifications can be carried out either by site-directed mutagenesis, or they may be the result of random events, such as, for example, which arise when mutations are master cells that produce antigens.

The term "immunological response" to an antigen or composition is the development in a subject of a humoral and/or cellular immune response to molecules present in the desired composition. In the framework of the present invention, the term "humoral immune response" refers to an immune response mediated by antibody molecules, while the concept of "cellular immune response" is immune to implement the function, mediated by T-lymphocytes and/or other leukocytes. One important aspect of cellular immunity involves antigen reaction cytolytic T cells (CFC). TCA have specificity for peptide antigens that are present in combination with the protein encoded by the main histochemistry complex (WGC [MHC]) and expressed on the cell surface. TCA contribute to the induction and increased intracellular degradation of intracellular microbes or lysis of cells infected with these germs. Another aspect of cellular immunity involves antigen specific response of T-helper cells. T-helpers operate in the direction of the stimulation function and activity concentrations of nonspecific effector cells against cells bearing on its surface a peptide antigens in combination with molecules of OGK. The term "cellular immune response" also refers to the formation of cytokines, chemokines and other molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T cells.

Composition, such as an immunogenic composition or a vaccine that induces a cellular immune response may serve to spinal sensitization of the subject by prezentowania antigen in combination with molecules of OGK on the cell surface. Opposed the bathroom across the cells of the immune reaction is directed at cells bearing on its surface an antigen, or to areas near them. In addition, antigen specific T lymphocytes can be generated to implement further protection of an immunized host.

The ability of a particular antigen or composition to stimulate mediated through cells of the immunological reaction can be detected using a variety of tests, such as tests lymphoproliferation (activated lymphocytes), tests for cytotoxicity of CTLs by examining the specificity of T-lymphocytes to the specified antigen in sensitized subject, or by measuring the formation of cytokines by T-cells in response to re-stimulation with antigen. Such tests are well known in the art. See, for example, Erickson et al., J. Immunol (1993) 151: 4189-4199; Doe et al., Eur. J. Immunol. (1994) 24, 2369-2376; and the following examples.

Thus, the immunological response in the context of the present description can represent this reaction, which stimulates the production of CTLs and/or the products or the activation of T-helper cells. The desired antigen can induce an immune response mediated by antibodies. Hence, an immunological response may include one or more of the following effects: the production of antibodies by b-cells and/or the activation of suppressor T-cells and/or γδ-T cells, the special is automatic directed to an antigen or antigens, present in any composition or vaccine. These reactions may serve to neutralize the infectivity and/or to play the role of mediator in the system of the antibody - complement or antibody-dependent cellular cytotoxicity (OCC) to ensure the protection of an immunized host. Such reactions can be determined using standard immunological methods of analysis and methods of neutralization, known in this technical field.

The composition that contains the selected antigen is adsorbed to the microparticle, shows "enhanced immunogenicity", which is characterized by the fact that it has higher ability to induce an immune response compared to the immune response caused by the equivalent amount of antigen in the case of delivery without combining it with the microparticle. Thus, the composition may be "enhanced immunogenicity", because the antigen is characterized by a higher immunogenicity due to its adsorption on the particle, or as required a lower dose of antigen for the manifestation of the immune response in the subject, which introduces the song. Such enhanced immunogenicity can be determined by administration of a composition of the microparticle/antigen and antigen controls to animals and comparing antibody titer in both cases with ISOE what Itanium standard methods of analysis, such as radioimmunoassay and ELISA (IFTF), known in this technical field.

The terms "effective amount" or "pharmaceutically effective amount" of a composition containing microparticles with adsorbed macromolecules, in the context of the present description refers to a nontoxic but sufficient amount of the composition of the particle/macromolecule for the treatment or diagnosis of this condition. For example, the above expression may be sufficient to provide the desired reaction, such as an immunological reaction, and the corresponding therapeutic effect, or in case of delivery of a therapeutic protein to a quantity sufficient for effective treatment of the subject, as indicated below. As will also be described below, the exact required amount will vary from subject to subject, depending on the species, age and General health of the subject, the severity of the condition to be treated, the specific nature of interest of the macromolecule, the method of administration and other Appropriate "effective" amount in any individual case may be determined by the specialist in the art using routine experimentation.

The term "vertebrate subject" refers to any representative vertebrates, including, without limiting the Oia, mammals, such as cattle, sheep, pigs, goats, horses and people; domestic animals such as dogs and cats; birds, including domestic, wild and singing birds such as cocks and hens including chickens, turkeys, and other representatives of the group of chicken. The term does not indicate a specific age of the subject. Thus, both adult and newborn animals are covered by the term.

The term "pharmaceutically acceptable" or "pharmacologically acceptable" refers to a material that is not biologically or for any other reason, undesirable, that is, the material may be administered to an individual in combination with a composition of microparticles, without causing any undesirable biological effects at the individual or not engaging in harmful interactions with any of the components of the composition in which they are located.

The term "excipient" refers to substances that are usually part of a finished dosage forms and which include carriers, binders, disintegrant, fillers (diluents), lubricants, glidant (amps fluidity), additives for extrusion, dyes, sweeteners, preservatives, suspendida/dispersing tools, forming tools/coatings, flavorings and marking paint.

The term "physiological pH" or "p is in the physiological range" refers to the pH value in the range of about 7,2-8,0, inclusive, more typically in the range of about 7,2-7,6, inclusive.

In the context of the present description, the term "treatment" (including its variations, such as "cure" or "treat"refers to any of the following definitions: (i) prevention of infection or re-infection, as in the case of traditional vaccines; (ii) the reduction or elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen or the disease. The treatment may be carried out prophylactically (prior to infection) or therapeutically (during infection).

In the context of the present description, the expression "nucleic acid" refers to DNA, RNA or chimeric forms, formed on the basis of them.

In the context of the present description the terms "oligonucleotide comprising at least one CpG motif"refers to polynucleotide comprising at least one CpG dinucleotide. Oligonucleotides comprising at least one CpG motif may include multiple CpG motifs. These oligonucleotides are known in the art as "CpG oligonucleotides". In the context of the present description, the expression "CpG motif" refers to a dimer portion of the oligonucleotide that includes casinoby nucleotide, followed by guanosine nucleotide. Instead of cytosine can also be used 5-Metelitsa is.

In the context of the present description, the term "replicon vector alpha viral RNA", "design RNA vector" and "replicon" refers to a RNA molecule, which is able to exercise its own amplification or replication in vivo, within the target cells. The replicon vector alpha-viral origin RNA should contain the following sequence sequence of elements: the viral 5'sequences required in CIS-form for replication (also referred to as 5'-CSE), sequences and their expression encode biologically active non-structural proteins alpha viruses (e.g., nsP1, nsP2, nsP3, nsP4), viral 3'sequences required in CIS-form for replication (also referred to as 3'-CSE), and polyadenylates plot. The replicon vector alpha-viral origin RNA may also contain a viral promoter subgenomic "hub site", which represents the sequence of the genes encoding one or more structural proteins, or portions thereof, the molecule(s) alien nucleic acid(s) has(have) a size large enough to produce viable virus and heterologous(s) sequence(s) for subsequent expression.

In the context of the present description, the expression "eukaryotic layered vector system initiation", "ELVIS" or "ELVIS-the sector" refers to the construction, which is able to carry out the expression of interest(s) sequence(s) or interest(s) gene(s). Eukaryotic layered vector system of initiation must contain a 5'promoter which is capable of initiating in vivo (within a cell) synthesis of RNA from cDNA, and viral vector sequence, which is able to exercise its own replication in eukaryotic cells, and to Express the heterologous sequence. In preferred variants of the invention, the vector nucleic acid sequence is a sequence of alpha-virus and includes a 5'sequence capable of initiating transcription of alpha-viral RNA (also referred to as 5'-CSE), as well as sequences which, when their expression encode biologically active non-structural proteins alpha viruses (e.g., nsP1, nsP2, nsP3, nsP4), and a recognizable RNA polymerase, a sequence of alpha-virus (also referred to as 3'-CSE). In addition, the vector sequence may include viral promoter subgenomic "hub site", which represents the sequence of the genes encoding one or more structural proteins, or parts thereof, the molecule(s) alien nucleic acid(s) has(have) a size large enough to best-fit the second amplification, heterologous sequence for subsequent expression, one or more restriction sites for insertion of heterologous sequences, as well as polyadenylate sequence. Eukaryotic layered vector system initiation may also contain sequence cleavage recognition sequence of the catalytic processing by ribozymes, nuclear export signal, and the sequence termination of transcription.

The concept of "design vector alpha virus" refers to a structure, which is capable of expression of the desired sequence or gene. Such vector constructs comprise a 5'sequence which is capable of initiating transcription of alpha-viral RNA (also denoted as 5'-CSE), as well as sequences which, when their expression encode biologically active non-structural proteins alpha viruses (e.g., nsP1, nsP2, nsP3, nsP4), a recognizable RNA polymerase, a sequence of alpha-virus (also referred to as 3'-CSE) and polyadenylates plot. In addition, the vector construct may include a promoter viral subgenomic hub section, the sequence of the genes encoding one or more structural proteins, or portions thereof, the molecule(s) alien nucleic acid(s) has the(s) size, sufficient for the production of viable virus, the 5'promoter which is capable of initiating the synthesis of viral RNA from cDNA in vitro or in vivo heterologous sequence for subsequent expression and one or more restriction sites for insertion of heterologous sequences.

In the context of the present description, the expression "vector construct" refers mainly to ELVIS-vectors, which include the cDNA complement to the RNA vector constructs themselves RNA vector constructs, alpha virus vector design and the like.

In accordance with some of the options for implementation of the present invention provides compositions and methods that can be used for treatment, including prophylactic and/or therapeutic immunization of an animal host against viral, fungal, mycoplasmal, bacterial or protozoal infections, as well as against tumors. The methods according to the present invention are used for producing a prophylactic and/or therapeutic immunity in mammals, preferably in humans. The methods according to the present invention can also be applied to mammals other than humans, including biomedical research.

C. General methods

The authors of the present invention found that the adsorption Mac is molecul on the microparticles can be improved by creating conditions, when the detergent is able to form complex molecules during the adsorption. In addition, it may be adsorbed many different molecules, including charged and/or volume of the macromolecule. Thus, the composition of the particle/macromolecule according to the present invention can be used as a delivery system for delivery of biologically active ingredients for the treatment, prevention and/or diagnosis of a large number of diseases.

The present invention can be used to deliver a variety of macromolecules, including, but not limited to, pharmaceutical agents, such as antibiotics and antiviral agents, non-steroidal anti-inflammatory drugs, analgesics, vasodilators, cardiovascular drugs, psychotropic drugs, neuroleptics, antidepressants, treatment of Parkinson's disease, beta-blockers, calcium channel blockers, inhibitors of bradykinin, ache-inhibitors, vasodilators, inhibitors of prolactin, steroids, hormone antagonists, antihistamines, serotonin antagonists, heparin, chemotherapeutic drugs, antineoplastics funds and growth factors, including, but not limited to, TRF (growth factors derived from platelets), EGF (epidermal growth factor), EFE (growth factor, according to the scientists from erythroblasts), IGF-1 (insulin-like growth factor 1), IGF-2, FGF (fibroblast growth factor), polynucleotide that encode therapeutic or immunogenic proteins, immunogenic proteins and their epitopes used in vaccines, hormones, including peptide hormones, such as insulin, proinsulin, growth hormone, RING (hormone-releasing factor, growth hormone), HLG (releasing factor, luteinizing hormone), EGF (epidermal growth factor), somatostatin, SNX-111, BNP, insulinotropic And-norprogesterone, FSH (follicle stimulating hormone), LH (luteinizing hormone), Fund (factor stimulating persistent cells) and hCG (human chorionic gonadotropin), sex steroid hormones (androgens, estrogens and progesterone), thyroid-stimulating hormone, inhibin, cholecystokinin, ACTH (adrenocorticotropic hormone), was made the KRF (corticotropin-releasing factor), dynorphin, endorphins, endothelin, fragments of fibronectin, Galanin, gastrin, insulinotropic, glucagon, fragments of the GTP-binding protein, guanylin, leukocidin, magainin, mastoparan, dermaseptin, systemin, neuromedin, neurotensin, pancreastatin, pancreatic polypeptide, substance P, secretin, thymosin and the like, enzymes, mediators of transcription or translation, the intermediates of metabolic pathways, immune modulators, such as any of various cytokines, including interleukin-1, Intel icin-2, interleukin-3, interleukin-4 and gamma interferon, antigens and adjuvants.

In a preferred embodiment of the invention, the macromolecule is an antigen. A particular advantage of the present invention is the ability of microparticles with adsorbed antigen to generate cell-mediated immune response in the vertebrate subject. The ability of antigen/microparticles according to the present invention to induce cell-mediated immune response against a selected antigen provides a powerful tool for influencing the infection caused by a wide range of pathogens. Accordingly, the specified complex antigen/microparticles according to the present invention may be included in a vaccine composition.

The effectiveness of various options for the use of plasmid vectors and ELVIS-vectors, described in the prior art, can be enhanced by adsorption of selected plasmids and ELVIS-vectors on the microparticles with absorbent surfaces, which facilitates the introduction of the vector and the heterologous nucleic acid sequences included in the vector in animal cells. Alternatively, a structure-based RNA vectors can be adsorbed on the polymer particles or emulsions of submicron size in accordance with the present invention with the purpose of effective the th delivery of heterologous sequences of nucleic acids in animal cells. Provisional application for U.S. patent joint ownership, filed September 28, 2000 (dossier of a patent attorney No. CHIR-0270; serial number 60/236105), discloses the use of such molecules adsorbed on some of the microparticles. Thus, in the preferred embodiment, the macromolecule is polynucleotide, such as a plasmid, ELVIS-vector or RNA vector construct. The particular advantage of the present invention is the ability of microparticles with adsorbed ELVIS-vector to generate cell-mediated immune response in the vertebrate subject. Patent application, serial number 60/236105, also discloses the adsorption of polypeptide antigens, including polypeptide antigens of HIV, on the microparticles. The ability of the antigen/microparticles according to the present invention to induce cell-mediated immune response against a selected antigen provides a powerful tool for combating infections caused by a vast array of pathogens. Accordingly, the complex antigen/microparticles according to the present invention may be included in a vaccine composition.

Thus, in addition to the traditional or antibody-based test reactions described in the present invention, the system may provide, for example, the Association expressed antigens with OGK-molecularmass I, so occur in vivo cellular immune response to the desired antigen can be enhanced, which stimulates the formation of TCA followed readable antigen. In addition, these methods can detect antigen specific response helper T cells. Accordingly, methods according to the present invention will be useful with any of the macromolecule, in respect of which it is desirable to generate cellular and/or humoral immune response, preferably against antigens derived from viral pathogens that can induce the formation of antibodies, epitopes, T-cell helper cells and cytotoxic epitopes for T-cells. Such antigens include, but are not limited to, those that encode viruses of humans and animals and can match any structural or non-structural proteins.

Microparticles according to the present invention is particularly useful for immunization against intracellular viruses that normally cause a weak immune response. For example, the present invention can be used to stimulate the immune system against a large number of proteins from the family of herpes viruses, including proteins derived from herpes simplex virus (HSV) type 1 and type 2, such as glycoproteins gB, gD and gH from HSV-1 and HSV-2; antigens derived from Varicella zoster (VZV), Epstein-B is the PPA (EBV) and cytomegalovirus (CMV), including CMV gB and gH; and antigens derived from other herpes viruses in humans, such as HHV6 and HHV7. (See, for example, Chee et al., Cytomegaloviruses (J.K. McDougall, ed., Springer-Verlag 1990), pp. 125-169, containing a review of the encoding proteins of cytomegalovirus; McGeoch et al., J. Gen. Virol. (1988) 69: 1531-1574, in this work we present a discussion of the different proteins encoded by HSV-1; U.S. patent No. 5171568, which are discussed gB and gD proteins HVS-1 and HVS-2 and encode their genes; Baer et al., Nature (1984) 310: 207-211, which contains an identification of protein coding sequences in the genome of EBV; and Davison and Scott, J. Gen. Virol. (1986) 67: 1759-1816, which provides an overview of VZV).

Antigens from the family of hepatitis viruses, including hepatitis a virus (HAV), hepatitis b virus (HBV), hepatitis C virus (HCV), hepatitis Delta (HDV), hepatitis E virus (HEV) and hepatitis G (HGV), can also easily be used as described in this application techniques. As an example, you can specify on what is known as the genome sequence of the HCV virus, and methods of obtaining the specified sequence. See, for example, international application number WO 89/04669; WO 90/11089 and WO 90/14436. The genome of HCV encodes several viral proteins, including E1 (also known as E) and E2 (also known as E2/NSI), and N-terminal nucleocapsid protein (called "core") (see Houghton et al., Hepatology (1991) 14: 381-388, which contains a discussion of the HCV proteins, including E1 and E2). Each of these proteins, and their antigenic fragments can be used in compositions and methods according to the present invention.

Similarly, there is also information about the sequence δ-antigen from HDV (see, for example, U.S. patent No. 5378814), and the antigen can also be successfully used in compositions and methods according to the present invention. Additionally, antigens derived from HBV, such as nuclear antigen, surface antigen, sAg, as well as "pripoverkhnostnye" sequence pre-S1 and pre-S2 (previously called pre-S), as well as their combinations, such as sAg/pre-S1, sAg/pre-S2, sAg/pre-S1/pre-S2 and pre-S1/pre-S2, can find application in the framework of the present invention. See, for example, "HBV Vaccines - from the laboratory to license: a case study" in Mackett, M. and Williamson, J.D., Human Vaccines and Vaccination, pp. 159-176, which contains a discussion of the patterns of HBV, as well as U.S. patent No. 4722840, 5098704, 5324513 included in this description fully as references; Beames, et al., J. Virol. (1995) 69: 6833-6838, Birnbaum et al., J. Virol. (1990) 64: 3319-3330 and Zhou et al., J. Virol. (1991) 65: 5457-5464.

In the claimed compositions and methods can also find the use of antigens derived from other viruses, such as without limitation, proteins representatives of the families Picornaviridae (e.g. polio viruses, and other), Caliciviridae, Togaviridae (e.g., virus, rubella virus, Dengue fever and others), Flaviviridae, Coronaviridae, Reoviridae, Birnaviridae, Rhabodoviridae (for example, virus basin the TBA and others), Filoviridae, Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, and others), Orthomyxoviridae (e.g., influenza virus types a, b and C and others), Bunyaviridae, Arenaviridae, Retroviridae (e.g., HTLV-I, HTLV-II, HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.)), including, but not limited to antigens from the isolates of HIVIIIb, HIVSF2, HIVLAV, HIVLAI, HIVMN, HIV-1CM235, HIV-1US4, HIV-2, human immunodeficiency virus monkeys (SIV). Additionally, antigens can be derived from human papilloma virus (HPV) and viruses of tick-borne encephalitis. See, for example, Virology, 3rd Edition (W.K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B.N. Fields and D.M. Knipe, eds. 1991), which contains a description of these and other viruses.

More specifically, membrane proteins gp120 or gp140 from any of the above HIV-isolates, including members of the various genetic subtypes of HIV, are known and described in the literature (see, for example, Myers et al., Los Alamos Database, Los Alamos National Laboratory, Los Alamos, New Mexico (1992); Myers et al., Human Retroviruses and Aids, 1990, Los Alamos, New Mexico: Los Alamos National Laboratory; and Modrow et al., J. Virol. (1987) 61: 570-578, which contains a comparison of the sequences of shells of different HIV-isolates), with antigens derived from any of these isolates can be used in the methods according to the present invention. In addition, the present invention is equally applicable to others who shM immunogenic proteins, obtained from any of the various HIV-isolates, including any of the various coat proteins, such as gp160 and gp41, gag antigens such as p24gag and p55gag, as well as proteins derived from the pol and tat sections.

The influenza virus is another example of the virus, which is particularly suitable for use within the present invention. Specifically, the envelope glycoproteins HA and NA of influenza a virus are of particular interest from the point of view of generating an immune response. Were identified various HA subtypes of influenza a virus (Kawaoka et al., Virology (1990) 179: 759-767; Webster et al., "Antigenic variation among type A influenza viruses", p. 127-168, In P. Palese and D.W. Kingsbury (ed.), Genetics of influenza viruses. Springer-Verlag, New York). Thus, proteins derived from any of these isolates may also find use in the compositions and methods described in the present description.

Disclosed in the present description the compositions and methods also can be used against many bacterial antigens, such as antigens derived from organisms that cause diphtheria, cholera, tuberculosis, tetanus, whooping cough, meningitis, and other pathogenic States, including, without limitation, Bordetella pertussis, Neisseria meningitidis (A, B, C, Y), Neisseria gonorrhoeae, Helicobacter pylori, Haemophilus influenzae type a, Haemophilus influenzae type b (HIB), and Helicobacter pylori and their combinations. Examples of antigens from Neisseria meningitidis B were opened the following bids for the n conjunction patent: PCT/US99/09346, PCT IB98/01665 and PCT IB99/00103. Examples of parasitic antigens include antigens that are derived from organisms that cause malaria and Lyme disease.

For more antigens suitable for use according to the present invention, some of which are also listed in this application include the following list (link below):

- protein antigen from N. meningitidis sero-groups, such as specified in references 1-7 below;

- preparation of outer membrane vesicles (OMV) from N. meningitidis sero-groups, such as specified in references 8, 9, 10, 11, etc. below;

- charigny antigen from N. meningitidis sero-groups A, C, W135 and/or Y, such as the oligosaccharide specified in the link 12 below, from serological groups (see also reference 13);

- charigny antigen from Streptococcus pneumoniae [e.g., references 14, 15, 16];

- an antigen from N. gonorrhoeae [e.g., references 1, 2, 3];

- an antigen from Chlamydia pneumoniae [e.g., links 17, 18, 19, 20, 21, 22, 23];

- an antigen from Chlamydia trachomatis [e.g. 24];

- an antigen from hepatitis a virus, such as inactivated virus [e.g., reference 25, 26];

- an antigen from hepatitis b virus, such as surface and/or nuclear antigens [e.g., references 26, 27];

- an antigen from hepatitis C virus [e.g., Ref 28];

- an antigen from Bordetella pertussis, such as holotoxin pertussis (PT)and filamentous hemagglutinin (FHA) from B. pertussis, neo is Astelin in combination with pertactin and/or agglutinogens 2 and 3 [for example, references 29 and 30];

- diphtheria antigen, such as diphtheria toxoid [e.g., Chapter 3 references 31], for example, the mutant CRM197[for example, the link 32];

- antigen tetanus, such as the tetanus toxoid [e.g., Chapter 4 references 31];

- protein antigen from Helicobacter pylori such as CagA [for example, the link 33], VacA [for example, the link 33], NAP [for example, the link 34], HopX [for example, the link 35], HopY [for example, the link 35] and/or urease;

- charigny antigen from Haemophilus influenzae B [e.g., reference 13];

- an antigen from Porphyramonas gingivalis [for example, the link 36];

antigen(s) polio [for example, the links 37, 38], such as IPV or OPV;

antigen(s) rabies [for example, the link 39], such as liofilizovannye inactivated virus [e.g., the link 40, Rabavert™];

- antigen measles, mumps and/or rubella [e.g., chapters 9, 10 and 11 references 31];

antigen(s) of influenza [e.g., Chapter 19 references 31], such as proteins hemagglutinin and/or surface neuraminidase;

- an antigen from Moraxella catarrhalis [for example, the link 41];

- an antigen from Streptococcus agalactiae (group b Streptococcus) [e.g., links 42, 43];

- an antigen from Streptococcus pyogenes (group a Streptococcus) [e.g., links 43, 44, 45];

- an antigen from Staphylococcus aureus [for example, the link 46];

compositions comprising one or more of these antigens.

In the case when used charigny or carbohydrate antigen, preference is sustained fashion to carry out the conjugation with protein carrier to increase immunogenicity [for example, links to 47, 56]. Preferred protein-carriers are bacterial toxins or toxoid, such as toxoid diphtheria or tetanus. Especially preferred diphtheria toxoid CRM197. Other acceptable carrier proteins include a protein of the outer membrane of N. meningitidis [for example, the link 57], synthetic peptides [e.g., links 58, 59], heat shock proteins [e.g., the link 60], the proteins of the virus whooping cough [for example, the links 61, 62], protein D from H. influenzae [for example, the link 63], toxin a or b from C. difficile [for example, the link 64] and others In the case when the mixture comprises capsular saccharides from both serological groups a and C, it is preferable that the mass ratio of saccharide MenA: MenC saccharide was greater than 1 (e.g., 2:1, 3:1, 4:1, 5:1, 10:1 or above). The saccharides of different serological groups of N. meningitidis can be conjugated with the same or with different protein-carriers.

Can be any acceptable reactions of conjugation in combination with any appropriate linkers, where necessary.

If necessary, the toxic protein antigens may be subjected to detoxification (e.g., detoxification of pertussis toxin by chemical and/or other means [e.g., reference 30]).

In that case, when the composition is included diphtheria antigen, it is preferable to include also the antigen tetanus and antigens to clusa. Similarly, in the case where the composition includes an antigen tetanus, preferably also include antigens diphtheria and pertussis. And similarly, when the composition includes the pertussis antigen, preferably also include antigens diphtheria and tetanus.

It is obvious that the object of the present invention can be used to deliver a huge variety of macromolecules and, therefore, for the treatment and/or diagnosis of a large number of diseases. In some embodiments of the invention the composition of the macromolecule/microparticle according to the present invention can be used for site-specific targeted delivery. So, for example, intravenous administration of compositions of the macromolecule/microparticle can be used to target delivery to the lung, liver, spleen, blood stream or bone marrow.

Adsorption of macromolecules on the surface of the adsorbent particles is carried out by means of any binding interaction, including, but not limited to, ionic bond, hydrogen bond, covalent bond, bond van der Waals and communication through hydrophilic/hydrophobic interactions. Specialist in the art can easily choose detergents that are acceptable for the particular type of adsorbed macromole the uly.

For example, microparticles formed in the presence of charged detergents such as anionic or cationic detergents may constitute microparticles with a surface having a negative or positive charge, which can adsorb a wide variety of molecules.

For example, microparticles obtained using anionic detergents such as sodium dodecyl sulphate (LTOs), for example, LTO-MRO microparticles adsorb positively charged antigens, such as proteins. Similarly, microparticles, obtained using a cationic detergent, such as CTAB, for example, MRO/CTAB microparticles adsorb negatively charged molecules such as DNA. In those cases, when adsorbed macromolecules contain areas with positive and negative charge, reasonable can be either cationic or anionic, or nonionic detergents.

Biodegradable polymers suitable for the production of microparticles with a view to their use in the framework of the present invention can easily be obtained from commercial sources, for example, from Boehringer (Boehringer Ingelheim, Germany and Birmingham Polymers, Inc., Birmingham, AL.). For example, the polymers suitable for the formation considered in this description of the microparticles include homopolymers, copolymers and polymer mixtures obtained from EBUSY components: polyhydroxyalkanoic acid (also known as polyoxometalate), polyhydroxyvalerate acid (also known as polyoxometalate), polyglycolic acid (PGA) (also known as polyglycolide), polylactic acid (PLA) (also known as polylactide), polydioxanone, polycaprolactone, polyarthra and polyanhydride. More preferred are poly(α-hydroxy acid, such as poly(L-lactide), poly(D,L-lactide) (both indicated in the present description as "PLA"), poly(hydroxybutyrate), copolymers of D,L-lactide and glycolide, such as poly(D,L-lacticacid) (referred to in this description as "MRO" or "PLGC"), or a copolymer of D,L-lactide and caprolactone. Especially preferred for use according to the present invention polymers are polymers PLA and MRO. These polymers are available in the form of polymers with different molecular weights, and the appropriate molecular weight for a particular use can be easily determined by the person skilled in the art. For example, for the SQP suitable molecular weight may be the weight of the order of from about 2000 to 5000. For MRO suitable molecular weight is mostly from about 10,000 to about 200,000, preferably from about 15,000 to about 150000.

If for the formation of microparticles using such a copolymer, as the MRO, you may be using what about the large number of different ratios of lactide:glycolide, the ratio is determined mainly by the choice associated partially with jointly entered by the macromolecule and with the desired degree of degradation. For example, the polymer MRO 50:50 containing 50% D,L-lactide and 50% of glycolide, will lead to the formation of the copolymer with the rapid resorption, whereas the polymer MRO 75:25 decomposes much slower, and the polymer with a ratio of 85:15 and 90:10 is even slower due to increased lactides component. It is seen that the ratio of lactide:glycolide can be easily determined by the person skilled in the art based on, for example, the nature of the antigen and the disease. In addition, mixtures of particles with different ratios of lactide:glycolide can find application in order to achieve the desired release kinetics for this type of macromolecule and for generating primary and secondary immune response. The rate of decomposition of microparticles according to the present invention may also be controlled by such factors as the molecular weight of the polymer and the crystallinity of the polymer. Copolymers MRO with different ratios of lactide:glycolide and with different molecular weights can be obtained from commercially available sources, including the company Boehringer (Boehringer Ingelheim, Germany and Birmingham Polymers, Inc., Birmingham, AL.). These polymers can also be with nasirovna through simple polycondensation component of lactic acid using well known in the art methods, such as the technique described by Tabata et al., J. Biomed. Mater. Res. (1998) 22: 837-858.

Microparticles obtained using any of several methods known in the art. For example, in some embodiments of the present invention to obtain microparticles can be used in methods of evaporation of the double emulsion/solvent as described in U.S. patent No. 3523907 and in the work of Ogawa et al., Chem. Pharm. Bull. (1988) 36: 1095-1103. These techniques include the formation of the primary emulsion consisting of droplets of polymer solution, which is then mixed with a continuous aqueous phase containing a stabilizer particles/surface-active substance.

In other embodiments the invention, the microparticles can also be formed using spray drying and koatservatsii, as described in the literature (for example, Thomasin et al., J. Controlled Release (1996) 41: 131; U.S. patent No. 2800457; Masters, K. (1976) Spray Drying 2nd Ed. Wiley, New York), technologies for applying a slurry coating air, such as coverage in the drum and Wurster coating, as described in the work of Hall et al., (1980), The "Wurster Process" in Controlled Release Technologies: Methods, Theory, and Applications (A.F. Kydonieus, ed.), Vol. 2, pp. 133-154 CRC Press, Boca Raton, Florida and Deasy, P.B., Crit. Rev. Ther. Drug Carrier Syst. (1988) S(2): 99-139, and ionic gelation, as described, for example, Lim et al., Science (1980) 210: 908-910.

In preferred embodiments of the invention d is I the formation of microparticles can be used in the system of evaporation of solvent from a mixture of water-in-oil-in-water (W/m/in) according to the process, described O Hagan et al., Vaccine (1993) 11: 965-969; PCT/US99/177308 (WO 00/06123) (O Hagan et al.) and Jeffery et al., Pharm. Res. (1993) 10:362.

In General, the specific polymer is dissolved in an organic solvent such as ethyl acetate, dimethylgold (also called methylene chloride and dichloromethane), acetonitrile, acetone, chloroform and the like. The specified polymer will be about 1-30%, preferably about 2-15%, more preferably about 3-10% and, most preferably, about 4-6% solution in an organic solvent. The resulting polymer solution is then combined with the aqueous solution and emuleret with the formation of emulsion m/C. the Aqueous solution may represent, for example, deionized water, normal saline or a buffered solution such as phosphate buffer solution (FBI), or buffer solution comprising sodium citrate/ethylenediaminetetraacetic acid (sodium citrate/EDTA). Preferably, the volume ratio of polymer solution and the aqueous solution ranges from about 5:1 to about 20:1 and, more preferably, is approximately 10:1. Emulsification is performed using any suitable for this task, equipment, and in a typical case, it is a device with a high degree of shear, such as, for example, a homogenizer.

A certain amount of m/V of the emulsion is then, preferably, the volume of dineout with a large volume of aqueous solution, which preferably contains a cationic, anionic or non-ionic detergent. The volumetric ratio of the aqueous solution and m/in the emulsion is in General from about 2:1 to 10:1 and, typically, is about 4:1. Examples of anionic, cationic and nonionic detergents suitable for use in the practice of implementation of the present invention listed above and include LTOs, CTAB and PVA, respectively. Some of the macromolecule can more easily adsorbed on microparticles, which includes a combination of detergents, for example a combination of PVA and DOTAP. In addition, in some cases it may be desirable to add detergent to the above organic solution. In the case of non-ionic detergent, such as PVA, it is usually applied in the form of approximately 2-15% solution, more typically about 4-10% solution. In the case of cationic or anionic detergent it is usually applied in the form of about 0.05 to 5% solution, more typically, about 0.25 to 1% solution. Mainly will be used to weight detergent to polymer mass in the range from approximately within 0.00001:1 to about 0.5:1, more preferably in the range from about to 0.0001:1 to about 0.5:1, more preferably from about 0,001:1 to about 0.5:1 and even more preferably approximately from 0.005:1 to about 0.5:1.

The mixture is homoge serout with stable double emulsion/m/C. After that, the organic solvent is evaporated. By manipulating the parameters of the prepared composition can be obtained the drug from small particulate size of about 0.05 microns (50 nm) to larger particles size of 50 μm or even more. See, for example, Jeffery et al., Pharm. Res. (1993) 10: 362-368; McGee et al., J. Microencap (1996). So, for example, reducing the intensity of mixing results in particles of larger size, as well as the increase in the volume of the internal phase. Fine particles are formed when small volumes of the aqueous phase with high concentrations of emulsion stabilizers.

The figure schematically shows a preferred apparatus for implementing the above stages. The drawing shows the design of the production tank, indicated generally by the number 102. The design of the tank 102 is designated as a "closed system"so that the processing of stored aseptic conditions. All parts and components of the equipment, preferably, are selected so that they can be cleaned by location and autoclaved. All filters 104a-d, preferably represent a fluoropolymer filters, such as fully fluoropolymer filters Super-Cheminert™ from Pall Corporation. First, an aqueous solution such as a buffer system, sodium citrate/EDTA (106), and the body of the ical solution of the polymer, such as MRO solution in methylene chloride (108), filtered and injected into the reservoir 110, where there is a continuous mixing using a mixer 112. The mixture is then passed through a flow homogenizer 114 (e.g., high speed, high shear, autoclavable flow homogenizer, such as Kinematica MT 5000) emulsion m/C. the Obtained emulsion after its exit from the flow homogenizer 114 is cooled, for example, using a water-cooled condenser 116, and then return it to the reservoir 110. After all content is brought to the desired degree of emulsification, the reservoir 110 add an aqueous solution of a detergent, such as CTAB solution in water 118, then get the emulsion type in the/m/in through re-transmission of the entire contents of the tank via a flow mixer 114. After reaching a sufficient degree of emulsification obtained through emulsion/m/with the help of the distributor 119 miss nitrogen to remove organic solvent. Solvent vapours with filtered nitrogen and cooled in the condenser 120, accumulating the solvent in the container 122.

In those embodiments that use a relatively high ratio of the mass of the detergent by weight of polymer (for example, the mass ratio of detergent and polymer ranges from approximately 0.05:1 to about 0.5:1, more FAV is preferably, from about 0.10 to:1 to about 0,50:1 and, most preferably, from approximately 0.2:1 to about 0.4:1), it is desirable to wash the particles to remove excess amounts of detergent. In a typical case, the specified stage of washing is carried out after removal of the finished emulsion of an organic solvent, for example, by evaporation of the solvent (as shown in the drawing), by means of solvent extraction or both of these ways.

In some embodiments the invention, the microparticles are washed with subsequent centrifugation. This method reduces the total amount of detergent and leads to a finished composition which contains a relatively small amount of unbound detergent compared with the associated detergent. For example, in the example below, 2 the implementation of the above mentioned stages of the washing cycle (i.e. four times washing with water followed by centrifugation) leads to the production of microparticles containing about 1 wt.% CTAB, more than 99% of which is associated with the microparticles, and less than 1% found in unbound form.

In other preferred versions of the invention, the microparticles are subjected to processing, aimed at reducing the level of detergent, which nevertheless leaves a significant number of the of emergent in the unconjugated form. So, for example, can be carried out stage filtration cross-flow to keep a significant amount of unbound detergent. In a typical case, the phase filter of the specified type leads to the formation of microparticles containing about 0.2 to 5 wt.% the detergent as a whole, of which from about 10 to 60% associated with microparticles and approximately 40-90% found in unbound form. More preferably, from about 25 to 40% of the detergent is associated with the microparticles, and approximately 60-75% is unbound form. For example, when implementing the procedure described in the example below, 5, are formed microparticles, comprising, in General, about 1 wt.% CTAB, of which approximately 30% are associated with microparticles and approximately 70% is unbound form.

In those embodiments of the invention, which uses a fairly low ratio of detergent and polymer (for example, the ratio of detergent to polymer is about from 0.001:1 to 0.05:1, more preferably, from about a 0.002:1 to around 0.04:1 and, more preferably, from about 0,006:1 to approximately 0.02:1), there is no need to wash the microparticles to remove excessive amounts of detergent. In a typical process of this type leads to the formation of microparticles containing approximately 0.2 is about 5 wt.% detergent, of which from about 10 to 60% associated with microparticles and approximately 40-90% is unbound form. More preferably, from about 25 to 40% of the detergent is associated with microparticles and approximately 60-75% is unbound form. For example, when implementing the procedure described in the example below 6, are formed microparticles containing approximately 1 wt.% CTAB, of which approximately 30% are associated with microparticles and approximately 70% is unbound form.

The particle size can be determined, for example, using laser light scattering using, for example, a spectrometer with built helium-neon laser. In General, the particle size is determined at room temperature, and this definition includes multiple analyses of a sample (e.g., 5-10), which gives the average value of the particle diameter. The particle size can be determined by scanning electron microscopy (SEM). After receiving drugs microparticles can be stored in the same form or lyophilizer for later use. In order to adsorbed macromolecules on the microparticles, the drug particles can be simply mixed with the interests of the macromolecules, and the resulting composition may be re-dried of tomoment use. However, as mentioned above, the authors of the present invention found that the adsorption of macromolecules on the polymeric microparticles can be improved if you create the conditions in which a significant amount of detergent adsorption of macromolecules is in the unconjugated form. In the case when the resulting composition of microparticles in the unconjugated form is a very small amount of detergent (for example, approximately 5% or less), it is preferable to carry out the incubation of these particles together with the macromolecule and the additional amount of detergent. Preferably, use a ratio of weight of the detergent to the mass of the macromolecule about from 0.002:1 to 0.05:1, more preferably about 0.005:1 to 0.02:1.

On the other hand, if in the resulting composition of microparticles of significant quantities of detergent in the unconjugated form (for example, approximately 50-90% of the unbound detergent, more preferably, approximately 60-75%), good results can be obtained by simple incubation of the particles with the target macromolecule, and the use of additional detergent becomes optional.

Not limiting myself to any theory, it is believed, in both the above cases that the unbound detergent available for the formation of a complex with interesting makr the molecule, making the macromolecule more capable of adsorption on the particle.

Basically, the macromolecule is added to the microparticles to obtain microparticles with adsorbed on them, macromolecules, characterized by the ratio of the mass of macromolecules to the mass of microparticles from some of 0.0001:1 to 0.25:1, preferably from 0.001:1 to 0.1:1, more preferably from 0.01:1 to 0.05:1. The content of macromolecules in the microparticles can be determined using standard techniques.

Microparticles according to the present invention may contain macromolecules, captured or encapsulated inside these particles, and may also contain macromolecules adsorbed on them. For example, the person skilled in the art can obtain on the basis of the present invention microparticles containing encapsulated adjuvants with adsorbed proteins on them, or microparticles containing encapsulated proteins adsorbed on them adjuvants.

The person skilled in the art can similarly be obtained on the basis of the present invention microparticles containing encapsulated adjuvants with complex adsorbed on them ELVIS-vectors, or microparticles containing encapsulated antigen adsorbed thereon nucleic acid plasmids. The present invention encompasses many acetaminophena macromolecules, adsorbed on microparticles or enclosed inside the particles, along with other macromolecules, such as antigenic molecules.

Immediately after receipt of microparticles with adsorbed macromolecules they can be included in the pharmaceutical compositions, including vaccines for the treatment and/or diagnosis of a large number of diseases mentioned above. Such compositions comprise one or more pharmaceutically acceptable excipients. For example, use media such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, and other such media may also be present other excipients, such as wetting or emulsifying agents, biological buffering agents and the like. Biological buffer can represent virtually any solution, which is pharmacologically acceptable and which allows to obtain a composition with the desired pH value, i.e. with a value of pH in the physiological range. Examples of buffer solutions include saline, phosphate buffer solution, buffered by Tricom saline, saline buffer Hanks and others In the finished dosage form may also include other known in the art, excepi the options including binders, disintegrant, fillers (diluents), lubricants, glidant (amps fluidity), additives for extrusion, dyes, sweeteners, preservatives, suspendida/dispersing tools, forming tools/coatings, flavorings and marking paint.

Adjuvants can be used to improve the efficiency of pharmaceutical compositions. Adjuvants may be introduced simultaneously with the microparticles according to the present invention, i.e. in the same composition or in separate compositions. Alternatively, the adjuvant may be administered before or after the compositions of microparticles according to the present invention. In another embodiment, the implementation of adjuvant, such as an immunological adjuvant, can be encapsulated in microparticles. Adjuvants as well as any macromolecule, can be encapsulated into microparticles using any of a number known in the art techniques. See, for example, U.S. patent No. 3523907; Ogawa et al., Chem. Pharm. Bull. (1988) 36: 1095-1103; O Hagan et al., Vaccine (1993) 11: 965-969; Jeffrey et al., Pharm. Res. (1993) 10: 362. Alternatively, adjuvants can be adsorbed on microparticles, as described above with respect to any macromolecule.

Immunological adjuvants include, but are not limited to: (1) compounds of aluminum (alum), such as the hydroxide and uminia, the aluminum phosphate, aluminum sulfate and other; (2) other emulsion compositions according to the type oil - in - water (with or without other specific immunostimulating means, such as muramylpeptide (see below) or components of the cell wall of bacteria), such as for example (a) MF59 (international publication number WO90/14837; Chapter 10 in Vaccine design: the subunit an adjuvant approach, eds. Powell &Newman, Plenum Press 1995), containing 5% squalene, 5% tween-80 and 0.5% span-85 (optionally containing various amounts ITF-PE (see below), although not required), which entered into submicron particles using microsatellites, such as Model 110Y microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% squalene, 0.4% tween-80, 5% pluronic-block-co-polymer L121, and thr-MDP (see below), which is subjected to spray mist with the formation of a submicron emulsion or processing in a vortex mixer with the formation of the emulsion particles of a larger size, and (C) adjuvant system Ribi™ (RAS), (Ribi Immunochem, Hamilton, MT)containing 2% squalene, 0.2% tween-80, and one or more components of the cell wall of bacteria from the group consisting of monophosphorylated And (IFF), the trehalose dimycolate (TDM) and cell wall skeleton (SCS), preferably, IFF+SCS (Detox™) (for more information on acceptable submicron emulsions of the type oil - in - water for use within the present invented what I can get in the patent application of co-ownership No. 09/015736, filed January 29, 1998); (3) saponine adjuvants, such as Quil A or QS21 (see Stimulon™ (Cambridge Bioscience, Worcester, MA), may be used or particles derived from them, such as Desired (immunostimulating complexes)with the Search may not require the introduction of additional detergent, see, for example, WO 00/07621; (4) complete adjuvant's adjuvant (CFA) and incomplete adjuvant's adjuvant (IFA); (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO 99/44636), etc.), interferons (e.g. gamma interferon), colony-stimulating factor macrophage (M-CSF), tumor necrosis factor cells (TNF), etc; (6) monophosphorylated And (IFF) or 3-O-describeany IFF (dmpl), see, for example, GB-2220221, EP-A-0689454, not necessarily in the absence of essentially, compounds of aluminum when used with pneumococcal saccharides, such as, for example, in WO 00/56358; (7) combination dmpl, for example, QS21 and/or emulsions of the type oil - in - water, see, for example, EP-A-0835318, EP-A-0735898, EP-A-0761231; (8) oligonucleotides comprising CpG motifs (Roman et al., Nat. Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al., J. Immunol. 1988, 160, 870-876; Chu et al., J. Exp. Med., 1997, 186, 1623-1631; Lipford et al., Eur. J. Immunol., 1997, 27, 2340-2344; Moldoveanu et al., Vaccine, 1988, 16, 1216-1224; Krieg et al., Nature, 1995, 374, 546-549; Klinman et la., PNAS USA, 1996, 93, 2879-2883; Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery et al., J. Immunol., 1996, 156, 4570-4575; Halpern et al., Cell Immunol., 1996, 167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al., J. Immunol., 1996, 157, 2116-122; Messina et al., J. Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996, 157, 4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al., J. Immunol., 1998, 160, 4755-4761; and Yi et al., J. Immunol., 1998, 160, 5898-5906; international patent application WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495, WO 98/37919 and WO 98/52581), i.e. containing at least one CG dinucleotide, thus instead of cytosine may not necessarily be used 5-methylcytosine; (9) a simple or complex polyoxyethylene ether, see, for example, WO 99/52549; (10) surface-active substance based on a complex ester of polyoxyethylenesorbitan in combination with octoxynol (WO 01/21207) or surfactant based on simple or complex polyoxyethyleneglycol ester in combination with at least one additional non-ionic surface-active agent, such as an octoxynol (WO 01/21152); (11) a saponin and an immunostimulating oligonucleotide (e.g. a CpG oligonucleotide) (WO 00/62800); (12) an immunostimulant and a particle of metal salt, see, for example, WO 00/23105; (13) a saponin and an emulsion of the type oil - in - water, see, for example, WO 99/11241; (14) a saponin (e.g., QS21) +tmpl+IL-12 (optionally +a Sterol)see, for example, WO 98/57659; (15) detoxificaton mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT) or thermo-labile toxin of E. coli (LT), particularly LT-K63 (where lysine replaces the amino acid in the wild-type molecule in polozhenie), LT-R72 include (in which arginine replaces the amino acid in the molecule wild type at position 72), CT-S109 (where serine replaces the amino acid in the molecule wild type at position 109), and PT-K9/G129 (where the molecule in the wild-type lysine replaces the amino acid at position 9 and glycine replaces the amino acid at position 129) (see, for example, international publication number WO 93/13202 and WO 92/19265); and (16) other substances that act as Immunostimulants to increase the efficiency of the composition. Aluminum compounds (particularly phosphate and/or aluminum hydroxide) and MF59 are preferred.

Muramylpeptide include, but are not limited to, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-soglo(drug of choice) (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanyl-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)ethylamine (MTP-PE), etc.

Information about other examples of adjuvants, see Vaccine Design, The Subunit and Adjuvant Approach, Powell, M.F. and Newman, M.J. eds., Plenium Press, 1995.

The composition will contain a "therapeutically effective amount" of interest to the macromolecule. I.e. the number of complex macromolecule/microparticle, which should be included in the composition and which forces the subject to produce a sufficient response for the purposes of prevention, reduction, elimination or diagnostic symptoms. Need the exact number is VA is to iravati depending for example, from the subject to be treated; the age and the ground state of the subject to be treated; the severity of this condition; in the case of immunological response from the immune system of a subject to synthesize antibodies; the desired degree of protection and the selected specific antigen, and the method of administration, among other factors. An appropriate effective amount can be readily determined by the person skilled in the art. Thus, a "therapeutically effective amount" in the typical case covers a relatively broad range that can be determined by conventional tests. For example, for the purposes of the present invention in the case when the macromolecule is polynucleotides, an effective dose will typically range from about 1 ng to about 1 mg, more preferably from about 10 ng to about 1 μg, and most preferably, from about 50 ng to about 500 ng of the macromolecule to be delivered in the form of dose; in that case, when the macromolecule is an antigen, an effective dose will typically range from about 1 μg to about 100 mg, more preferably, from about 10 μg to about 1 mg and most preferably, from about 50 μg to about 500 μg of the macromolecule to be delivered in the form of a dose.

Made of the composition is according to the present invention may be introduced parenterally, for example, by injection. The composition can be administered subcutaneously, intraperitoneally, intravenously or intramuscularly. Other routes of administration include nasal, mucosal, rectal, vaginal, oral and pulmonary routes of administration, the introduction of using suppositories and using percutaneous or crosslisted method.

The dosage for treatment may represent the introduction of single or multiple doses. The mode of administration of multiple doses is a mode in which a primary course of introduction may include 1-10 separate doses, after which you can enter other doses at certain intervals of time, chosen in such a way as to maintain and/or enhance therapeutic response, such as 1-4 months for a second dose, if required, in a few months for the introduction of follow-up(their) dose (dose). The dosage may also at least partially determined by the needs of the subject and the choice of the treating physician.

In addition, if desired prevention of disease, macromolecule in vaccines administered mainly to the manifestations of primary infection is considered a pathogen. If it is desirable to carry out treatment, i.e. to attenuate the symptoms of the disease or prevent its recurrence, macromolecules injected, mainly after developing ervices infection.

Experimental part

The following are specific embodiments of the present invention. The examples are given only for illustrative purposes and should not be construed as limiting the scope of the present invention.

Should take all measures to promote compliance with the accuracy of the experiments from the point of view used quantitative characteristics (e.g., amounts, temperatures, and other), although, of course, some experimental errors and deviations in data values may be admitted.

Example 1

The reference version of the microparticles using PVA

Control microparticles (i.e. without adsorbed or trapped macromolecules) is obtained using polyvinyl alcohol (PVA) as follows. Use solutions:

(1) 6% RG 504 MRO (Boehringer Ingelheim) in dichloromethane.

(2) 10% polyvinyl alcohol (PVA) (ICN) in the water.

In particular, the microparticles were obtained by combining 10 ml of polymer solution with 1.0 ml of distilled water followed by mixing for 3 minutes in a homogenizer Omni benchtop c 10 mm probe at 10 rpm with emulsion water/oil (W/m). Emulsion/m is added to 40 ml of 10% PVA solution and homogenized for 3 minutes with the formation of the emulsion water/oil/water (W/m/in). The above emulsion water/oil/water left overnight for the apareunia solvent, which leads to the formation of microparticles. The obtained microparticles are washed with water, centrifuger 4 times, and lyophilizers. Then determine the size of the microparticles on the meter Malvern Master for future use.

Example 2

The reference version of the microparticles using CTAB

Control microparticles obtained using CTAB as follows. Use solutions:

(1) 6% RG 504 MRO (Boehringer Ingelheim) in dimethylamide.

(2) a 0.5% CTAB (Sigma Chemical Co., St. Louis, MO) in water.

In particular, the microparticles were obtained by combining 12.5 ml of polymer solution with 1.25 ml of distilled water followed by mixing for 3 minutes in a homogenizer Omni benchtop c 10 mm probe at 10 rpm with emulsion water/oil (W/m). Emulsion/m is added to 50 ml of 0.5% solution of CTAB and homogenized for 3 minutes with the formation of the emulsion water/oil/water (W/m/in). The above emulsion water/oil/water left overnight to evaporate the solvent, which leads to the formation of microparticles. Next, the resulting microparticles are filtered through the sieve of 38 μm, washed with water, centrifuger 4 times, and lyophilizers. Determine the size of the microparticles on the meter Malvern Master for future use.

Example 3

The reference version of the microparticles using LTOs

Control microparticles which are square-using-ordinator as follows. Use solutions:

(1) 6% RG 504 MRO (Boehringer Ingelheim) in dimethylamide.

(2) 1% LTOs (Sigma Chemical Co., St. Louis, MO) in water.

In particular, the microparticles were obtained by combining 12.5 ml of polymer solution with 50 ml LTOs followed by homogenization for 3 minutes in a homogenizer Omni benchtop c 10 mm probe at 10 K/min Emulsion left overnight to evaporate the solvent. The obtained microparticles are filtered through the sieve of 38 μm, washed with water, centrifuger 4 times, and lyophilizers. Determine the size of the microparticles on the meter Malvern Master for future use.

Example 4

Microparticles with adsorbed complex DNA/CTAB

Pre-gain control microparticles MRO/PVS using the standard method of solvent evaporation. To get the party size 1 g 2 ml of deionized water (DI) homogenized with 16 ml of 6% (weight/volume) solution of RG 504 (polymer MRO) in dichloromethane (DHM). The above emulsion is homogenized for 2 minutes and to this mixture is added 60 ml of 10% (weight/volume) solution of polyvinyl alcohol (PVA). Multiple emulsion is then homogenized for 3 minutes and then put on a magnetic stirrer for evaporation of the solvent during the night. The obtained microparticles are washed twice with deionized water and dried by freezing. Determine the size of microcast is, equal to about 1 micron.

To obtain compositions with DNA 100 mg MRO/PVA microparticles incubated with 1 mg of CTAB and 1 mg DNA (pCMV-p55gag) in 5 ml of TE buffer. The suspension is gently stirred overnight at 4° to achieve full absorption. Next microparticles centrifuged once at 5000 rpm and the precipitate is washed once with 50 ml of TE buffer. The precipitate is suspended in 3 ml of DI and microparticles are dried by freezing.

The actual number of adsorbed DNA is determined according to the method of determining the decreasing number (measurement of supernatant) and in conducting basic hydrolysis of microparticles. It was found that the inclusion of DNA reaches of 0.91 wt.%, and effectiveness of the inclusion is 91%.

10 mg of the composition demonstrates not release any free DNA in vitro in 1 ml of TE buffer at day 1.

Example 5

The reference version of the microparticles using CTAB

Methodology crossflow filtration

Control microparticles obtained using CTAB as follows. Use solutions:

(1) 6% RG 504 MRO (Boehringer Ingelheim) in methylene chloride.

(2) a 0.5% CTAB (Sigma Chemical Co., St. Louis, MO) in water.

(3) sodium Citrate/EDTA in water.

Microparticles get in apparatus similar to that shown in the drawing when combining 80 ml of polymer solution with 10 ml of citrate/EDTA in the tank when the permanent stirring. The mixture is then homogenized in a flow homogenizer to obtain an emulsion m/in the presence of the dispersed phase (water phase)having an average particle size of 1-2 microns. At a specified point in a vessel with constant stirring add 310 ml CTAB. The mixture is homogenized in a flow homogenizer before the formation of a stable emulsion/m/in the presence of the dispersed phase (m/phase)having an average particle size of 1 micron. Through obtained as/m/in the emulsion flow of nitrogen to remove the organic solvent, and the thus obtained microparticles are filtered using a 0.1 μm crossflow filter cartridge, Millipore using the 4.0 liter with deionized water to remove excess CTAB. After the final crossflow filtration collect the suspension, which contains approximately 1% CTAB, of which 30% is present in bound form, and 70% is present in the unbound form.

Example 6

The reference version of the microparticles using CTAB

The technique without washing

Control microparticles obtained using CTAB as follows. Use solutions:

(1) 6% RG 504 MRO (Boehringer Ingelheim) in methylene chloride.

(2) 0,01825% CTAB (Sigma Chemical Co., St. Louis, MO) in water.

(3) sodium Citrate/EDTA in water.

Microparticles get in the apparatus, such that the image is n in the drawing, when combined with 300 ml of polymer solution with 60 ml of citrate/EDTA in the vessel with constant stirring. The mixture is then homogenized in a flow homogenizer to obtain an emulsion m/in the presence of the dispersed phase (water phase)having an average particle size of 1 micron. At a specified point in a vessel with constant stirring, 1.8 liters of a solution of CTAB. The mixture is homogenized in a flow homogenizer before the formation of a stable emulsion/m/in the presence of the dispersed phase (m/phase)having an average particle size of 1 micron. Through obtained as/m/in the emulsion flow of nitrogen to remove the organic solvent. Determine the size of the suspension of microparticles on the meter Malvern Master for future use. These particles contain about 1% CTAB, of which 30% is present in bound form, and 70% is present in the unbound form.

Example 7

Immunogenicity of microparticles with adsorbed P55 DNA

Composition is prepared DNA by incubating 100 mg of a suspension of microparticles (10 ml)obtained in example 6, 1.0 mg DNA (plasmid pCMVgag encoding a protein of the HIV p55 gag, under the control of the early promoter of cytomegalovirus) in the amount of 0.5 ll of Tris-EDTA buffer. The suspension is incubated at 4°C for 12 hours. After incubation, centrifuged DNA-containing microparticles are washed their Tris-EDTA buffer, suspended in deionized water and dried by freezing (lyophilizer). The DNA content in obrazovan the x microparticles is about 1 wt.%.

Next, the DNA-containing microparticles is injected intramuscularly to mice in two doses on the content of DNA. One DNA also injected in the same quantities as control. Each composition is injected ten mice. Mice again subjected to immunization after 28 days. Two weeks after the second immunization collect serum and determine the geometric mean of the titers (CPEs) for each of the investigated sera together with the value of standard error (SB). The results are shown in the table below:

TrackThe GTSCO
DNA P55 MRO-CTAB (content of 1 wt.%), 1 mcg15,5654,764
DNA P55 MRO-CTAB (content of 1 wt.%), 10 mg27,2775,693
1 μg DNA P553671,572
10 µg DNA P552,1851,652

Although in the present description in detail preferred embodiments of the present invention, it should be understood that it can be made obvious variations without departure from the principles and scope of the invention defined by the attached claims.

References:

Link 1 - the international patent application WO 99/24578

Link 2 - the international patent application WO 99/36544.

Link 3 - the international patent application WO 99/57280.

Link 4 - the international patent application WO 00/22430.

Link 5 - Tettelin et al. (2000) Science 287:1809-1815.

Link 6 - international patent application WO 96/29412.

Link 7 - Pizza et al. (2000) Science 287:1816-1820.

Link 8 - international patent application PCT/IB01/00166.

Link 9 - Bjune et al. (1991) Lancet 338(8775):1093-1096.

Link 10 - Fukasawa et al. (1990) Vaccine 17:2951-2958.

Link 11 - Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333.

Link 12 - Costantino et al. (1992) Vaccine 10:691-698.

Reference 13 - Costantino et al. (1999) Vaccine 17:1251-1263.

Reference 14 - Watson (2000) Padiatr Infect Dis J 19:331-332.

Link 15 - Rubin (2000) Pediatr Clin North Am 47:269-285, v.

Link 16 - Jedrzejas (2001) Environ Mol Biol Rev 65:187-207.

The link 17 is an international patent application filed July 3, 2001, with priority from GB-0016363.4].

Reference 18 - Kalman et al. (1999) Nature Genetics 21:385-389.

The link 19 is Read et al. (2000) Nucleic Acids Res 28:1397-406.

Link 20 - Shirai et al. (2000) J. Infect. Dis. 181(Suppl 3):S524-S527.

The link 21 is the international patent application WO 99/27105.

The link 22 is the international patent application WO 00/27994.

The link 23 is the international patent application WO 00/37494.

The link 24 is the international patent application WO 99/28475.

Reference 25 - Bell (2000) Pediatr Infect Dis J 19:1187-1188.

The link 26 is Iwarson (1995) APMIS 103:321-326.

The link 27 is Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.

Reference 28 - Hsu et al. (1999) Clin Liver Dis 3:901-915.

Reference 29 - Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355.

The link 31 - Vaccines (1988) eds. Plotkin &Mortimer. ISBN 0-7216-1946-0.

Reference 32 - Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70.

The link 33 is the international patent application WO 93/18150.

The link 34 international patent application WO 99/53310.

The link 35 international patent application WO 98/04702.

The link 36 - Ross et al. (2001) Vaccine 19:4135-4142.

Reference 37 - Sutter et al. (2000) Pediatr Clin North Am 47:287-308.

Reference 38 - Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126.

The link 39 is Dreesen (1997) Vaccine 15 Suppl:S2-6.

Reference 40 - MMWR Morb Mortal Wkly Rep 1998 Jan 16;47(1):12, 19.

The link 41 - McMichael (2000) Vaccine 19 Suppl 1:S101-107.

The link 42 is Schuchat (1999) Lancet 353(9146):51-6.

Reference 43 - Patent application GB 0026333.5, 0028727.6 & 0105640.7.

Reference 44 - Dale (1999) Infect Dis Clin North Am 13:227-43, viii.

Reference 45 - Ferretti et al. (2001) PNAS USA 98:4658-4663.

Reference 46 - Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also page 1218-1219.

The link 47 - Ramsay et al. (2001) Lancet 357(9251):195-196.

The link 48 - Lindberg (1999) Vaccine 17 Suppl 2:S28-36.

The link 49 is Buttery & Moxon (2000) J R Coll Physicians London 34:163-168.

Link 50 - Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii.

Link 51 - Goldblatt (1998) J. Med. Environ. 47:563-567.

The link 52 of the European patent 0477508.

Link 53 - U.S. Patent No. 5306492.

The link 54 - international patent application WO 98/42721.

The link 55 is Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326, especially vol.10:48-114.

The link 56 is Hermanson (1996) Bioconjugate Techniques ISBN: 0123423368 & 012342335X.

The link 57 European patent application 0372501.

Reference 58 - European is th patent application 0378881.

The link 59 European patent application 0427347.

The link 60 to the international patent application WO 93/17712.

The link 61 is the international patent application WO 98/58668.

The link 62 European patent application 0471177.

The link 63 international patent application WO 00/56360.

The link 64 is the international patent application WO 00/61761.

1. The biologically active composition of microparticles containing mikrochastitsy, which include (a) a polymer selected from the group consisting of poly(α-hydroxyacids), polyhydroxyalkanoic acid, polycaprolactone, polyarteritis, polyanhydride and polycyanoacrylate, and (b) the first part of the detergent, which is associated with the polymer; and adsorbed on the surface of the microparticles, the complex, and this complex includes (a) a biologically active macromolecule and (b) the second part of the detergent, where the first part of the detergent and the second part of the detergent include the same detergent or different detergents and where biologically active macromolecule selected from the group consisting of polypeptide, polynucleotide, polynucleotide, antigen, a pharmaceutical, a hormone, an enzyme, a mediator of transcription or translation, intermediate metabolic pathway, an immunomodulator and adjuvant.

2. The composition of the microparticles according to claim 1, in which the polymer includes poly(α-gidrokshikislotu)selected from the group, ostoja of poly(L-lactide), poly(D,L-lactide) and poly(D,L-lacticacid).

3. The composition of the microparticles according to claim 2, in which the polymer comprises poly(D,L-lacticacid)with (a) the molar ratio of lactide/glycolide in the range from 30:70 to 70:30 and a molecular weight of from 10000 to 100000 daltons, or (b) the molar ratio of lactide/glycolide in the range from 40:60 to 60/40 and the molecular weight of 30,000 to 70,000 daltons.

4. The composition of the microparticles according to any one of the preceding paragraphs, in which the first and second parts of the detergent include the same detergent.

5. The composition of the microparticles according to claim 4, in which the first and second parts of detergents include cationic detergents.

6. The composition of the microparticles according to any one of claims 1 to 3, in which the first and second parts of the detergent include different detergents.

7. The composition of the microparticles according to claim 6, in which the first part of the detergent includes a non-ionic detergent, and the second part of the detergent comprises a cationic detergent.

8. The composition of the microparticles according to claim 7, in which the first part of the detergent includes PVS and the second part of the detergent includes CTAB.

9. The composition of the microparticles according to any one of the preceding paragraphs, in which the biologically active macromolecule is polynucleotide that encodes an antigen selected from the group consisting of HIV antigens such as gp120, gp140, p24gag, p55gag, antigen meningitis, streptococcal antigen group and antig is on hemagglutinin of influenza A.

10. The composition of the microparticles according to any one of the preceding paragraphs, in which the biologically active macromolecule is a member of the group consisting of plasmids, ELVIS vector and designs RNA vector.

11. The composition of the microparticles of claim 10, in which the biologically active macromolecule is a pCMV-p55gag.

12. The composition of the microparticles according to any one of claims 1 to 8, in which the biologically active macromolecule is an antigen selected from HIV antigens, antigens meningitis, streptococcal antigens of group b and antigens hemagglutinin of influenza A.

13. The composition of the microparticles indicated in paragraph 12, in which the HIV antigen is selected from the group consisting of gp120, gp140, p24gag, p55gag.

14. The composition of the microparticles according to any one of the preceding paragraphs, further comprising a second biologically active macromolecule selected from the group consisting of a polypeptide, polynucleotide, polynucleotide, antigen, a pharmaceutical, a hormone, an enzyme, a mediator of transcription or translation, intermediate metabolic pathway, an immunomodulator and adjuvant.

15. The composition of the microparticles through 14, in which the second biologically active macromolecule is an adjuvant selected from the group consisting of CpG oligonucleotides, LTK63, LTR72, MPL and aluminum salts.

16. The composition of microparticles of clause 15, in which the aluminium salt is predstavljaet a phosphate of aluminum.

17. The composition of the microparticles according to any one of claims 4 to 16, in which the first part of the detergent, which is associated with the polymer comprises about 10-90% of the total detergent composition.

18. The composition of the microparticles according to 17, in which the first part of the detergent, which is associated with the polymer, approximately 10-60% of the total detergent composition.

19. The composition of the microparticles according to any one of pp.5-18, in which the cationic detergent is a CTAB.

20. The composition of the microparticles according to any one of the preceding paragraphs, optionally containing pharmaceutically acceptable excipient.

21. The composition of the microparticles according to claim 20, for use in the method for the diagnosis of disease or as a drug for the treatment of disease.

22. The composition of the microparticles according to claim 20, for use as a vaccine and/or to enhance the immune response.

23. The use of a composition of microparticles according to claims 1 to 22 as the active agent for the manufacture of a medicine for vertebrates.

24. A method of obtaining a composition of microparticles, including

(a) obtaining emulsion comprising (i) a polymer selected from the group consisting of poly(α-hydroxyacids), polyhydroxyalkanoic acid, polycaprolactone, polyarteritis, polyanhydride and polycyanoacrylate, (ii) an organic solvent, (iii) a detergent and (iv) water;

(b) in Alenia organic solvent from the emulsion to form microparticles;

where about 10-90% of the total detergent in the composition of the microparticles is associated with the microparticles and the remaining part is not connected and where these particles are not subjected to the stage of washing.

25. The method according to paragraph 24, in which the emulsion is an emulsion of water-in-oil-in-water, which is obtained by the method, including

(a) emulsification of the organic phase containing the polymer and an organic solvent with a first aqueous phase containing water, emulsion water-in-oil; and

(b) emulsification of the second aqueous phase containing a cationic detergent and water emulsion formed in stage (a), to obtain the emulsion water-in-oil-in-water.

26. The method according to paragraph 24 or 25, in which, after removal of the organic solvent conduct filtering in cross flow.

27. The method according to any of p-26, in which the detergent is a cationic detergent that is introduced into the emulsion at a mass ratio of detergent and polymer in the range of from about 0,001:1 to approximately 0.05:1, or (b) from approximately 0.05:1 to about 0.5:1.

28. The method according to item 27, in which the cationic detergent stage b) is injected into the emulsion at a mass ratio of detergent and polymer in the range from about 0.1:1 to about 0.5:1, where the polymer is a poly(D,L-lacticacid) and cationic detergent is a CTAB.

30. The method of obtaining biologically active composition of microparticles, including

(a) formation of a composition of microparticles method according to any one of PP-29

(b) incubating the composition of microparticles with a biologically active macromolecule.

31. The method according to item 30, in which the biologically active macromolecule is polynucleotide, pharmaceutical agent, a polypeptide, a hormone, an enzyme, a mediator of transcription or translation, intermediate metabolic pathway, an immunomodulator, an antigen, adjuvant, or combinations thereof.

32. A method of obtaining a composition of microparticles, including

obtaining microparticles process of emulsification, and these microparticles include (a) a polymer selected from the group consisting of poly(α-hydroxyacids), polyhydroxyalkanoic acid, polycaprolactone, polyarteritis, polyanhydride and polycyanoacrylate, and (b) the first part of the detergent, which is associated with the polymer; and the adsorption is carried complex biologically the active macromolecule and the second part of the detergent on the surface of the microparticles, where the first part of the detergent and the second part of the detergent include the same detergent or different detergents and biologically active macromolecule selected from the group consisting of polypeptide, polynucleotide, polynucleotide, antigen, a pharmaceutical, a hormone, an enzyme, a mediator of transcription or translation, intermediate metabolic pathway, an immunomodulator and adjuvant.

33. The method according to p, in which the first and second parts of the detergent include the same cationic detergent and polymer includes poly(α-gidrokshikislotu)selected from the group consisting of poly(L-lactide), poly(D,L-lactide) and poly(D,L-lacticacid), and biologically active macromolecule is polynucleotide.

34. The method according to p, in which the first part of the detergent, which is associated with the polymer comprises about 10-90% of the total detergent in the composition and the detergent corresponding to the first and second parts of detergent, add during the process of emulsification.

35. The method according to clause 34, in which the first part of the detergent, which is associated with the polymer, approximately 10-60% of the total detergent composition.

36. The method according to clause 34 or p, in which the process of emulsification includes

(a) emulsification of the organic phase containing the polymer and an organic solvent with a first aqueous phase containing in the u, with the formation of emulsions of the water-in-oil and

(b) the second emulsification of the aqueous phase containing the detergent and water with the emulsion obtained in stage (a), with the formation of emulsions of the water-in-oil-in-water.

37. The method according to any of PP-36, in which the cationic detergent is a CTAB.

38. The method according to p, in which the first part of the detergent includes a first detergent and the second part of the detergent comprises a second detergent, different from the first detergent.

39. The method according to § 38, in which the detergent is added in the process of emulsification and the second detergent is added after emulsification.

40. The method according to § 39, in which the second detergent is added simultaneously with the biologically active macromolecule.

41. The method according to any of PP-40, in which the polymer includes poly(α-gidrokshikislotu)selected from the group consisting of poly(L-lactide), poly(D,L-lactide) and poly(D,L-lacticacid), and the first detergent includes a non-ionic detergent and a second detergent include cationic detergent, and biologically active macromolecule is polynucleotide.

42. The method according to paragraph 41, in which the detergent is a PVA and a second detergent is a CTAB.

43. The method according to any of PP-42, in which the polymer is a poly(D,L-lacticacid), having a molar ratio of lactide/licopid in the range from 40:60 to 60:40 and a molecular weight of 30,000 to 70,000 daltons.

44. The composition of the microparticles obtained by the method according to any of PP-43.



 

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