KR20070017374A - A fast dissolving dried hyaluronic acid product - Google Patents

Abstract

Spray products containing high solubility in aqueous solvents and comprising hyaluronic acid or salts thereof, various compositions and articles comprising the products or compositions of the present invention, methods of making the products of the present invention, and use thereof.

Spray, hyaluronic acid, gram-positive bacterium, Bacillus, hyaluronate, lactose, hair loss, baldness, cancer, arthritis, borate esters.

Description

Rapidly Dissolving Dry Hyaluronic Acid Product {A FAST DISSOLVING DRIED HYALURONIC ACID PRODUCT}

The present invention relates to various compositions and articles comprising the products or compositions of the invention, as well as to rapidly dissolving dry products comprising hyaluronic acid or salts thereof, to methods of making the products of the invention, and to their use. will be.

The most abundant heteropolysaccharide in the body is glycosaminoglycans. Glycosaminoglycans are unbranched carbohydrate polymers composed of repeating disaccharide units (only keratan sulfate is branched in the key areas of carbohydrates). Disaccharide units are generally the first monosaccharide unit and comprise one of two modified sugars-N-acetylgalactosamine (GaINAc) or N-acetylglucosamine (GlcNAc). The second unit is usually uronic acid, such as glucuronic acid (GICUA) or iduronate.

Glycosaminoglycans are negatively charged molecules and have an extended conformation that confers high viscosity in solution. Glycosaminoglycans are located primarily on the cell surface or in the extracellular matrix. Glycosaminoglycans also have low compressibility in solution, and as a result are ideal as physiological lubricants, for example joints. The stiffness of glycosaminoglycans provides structural integrity to cells and provides pathways between cells, allowing cell migration. Glycosaminoglycans of highest physiological importance are hyaluronan, chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Most glycosaminoglycans bind proteoglycan core proteins covalently through specific oligosaccharide structures. Hyaluronan forms large aggregates with certain proteoglycans, with the exception of free carbohydrate chains forming non-covalent complexes with proteoglycans.

Numerous roles of hyaluronan have been identified in the body (see Laurent TC and Fraser JRE, 1992, FASEB J. 6: 2397-2404; and Toole BP, 1991, "Proteoglycans and hyaluronan in morphogeneis and differentiation." In: Cell Biology of the Extracellular Matrix, pp. 305-341, Hay ED, ed., Plenum, New York). Hyaluronan is present in both hyaluronic cartilage, synovial joint fluid, and skin tissue, dermis and epidermis. Hyaluronan is also believed to play a role in numerous physiological functions such as adhesion, development, cell motility, cancer, angiogenesis, and wound healing. Because of the unique physical and biological properties of hyaluronan, it has been adopted for eye and joint surgery and is being evaluated in other medical procedures.

The term "hyaluronan" or "hyaluronic acid" occurs naturally at the cell surface, in the basal extracellular substance of connective tissue of vertebrates, synovial fluid of joints, intraocular fluid of the eye, human umbilical cord tissue and cock's crests, D-glucuronic acid N-acetyl-D-glucosamine acid is used in the literature to mean acidic polysaccharides having different molecular weights composed of residues.

The term "hyaluronic acid" is used to mean a disintegrated fragment of the entire series of polysaccharides or even the same having alternating residues of D-glucuronic acid N-acetyl-D-glucosamine acid having substantially varying molecular weights in nature. It seems more accurate to use the plural term "Lonsan". However, the singular terms all of which are used identically herein; In addition, the abbreviation "HA" will often be used in place of this collective term.

HA plays an important role in biological organs as a mechanical support for cells of many tissues such as skin, tendons, muscles and cartilage, which is a major component of the matrix between cells. HA also serves as another important part of biological processes such as tissue hydration, and lubrication.

Although it is desirable to manufacture it today by microbiological methods that minimize the potential risk of transferring infectious disease pathogens and increase product uniformity, quality and availability, HA can be extracted from the natural tissues mentioned above.

HA and its various molecular size fragments and their salts, respectively, are particularly useful as agents in the treatment of arthrosis, especially as adjuvant and / or replacement for natural organs and tissues in ophthalmology and plastic surgery, and as pharmaceuticals in cosmetic manufacture Was used. Products of hyaluronan have also been developed for use in orthopedics, rheumatology, and dermatology.

HA can also be used as an additive for various polymer materials used in hygiene and surgical articles, such as polyurethanes, polyesters, etc., with the effect of making these materials biocompatible.

Water soluble gel forming materials comprising hyaluronic acid or salts thereof are well known and widely used in the pharmaceutical and cosmetic industries as well as in the health care sector, such as, for example, in ophthalmology, the treatment of osteoarthritis. These materials are sold, transported and stored in ideally dried form. However, dried products comprising hyaluronic acid or salts thereof are notorious for slowly dissolving.

The time it takes for the dried HA-product to dissolve completely is an important parameter, especially in the health care industry, and therefore it is of considerable interest to minimize the total dissolution time, ie the time taken to disperse and solubilize the dried HA product. The present invention is useful for preparing products of fast dissolving hyaluronic acid or salts thereof and similar water soluble gel forming materials.

Summary of the Invention

The problem to be solved by the present invention is a method for providing highly water-soluble dry HA products.

The present invention shows that the flocculation of the spray-dried HA product greatly improves the solubility of the product and thus achieves very fast dissolution in an aqueous solvent, preferably water or saline.

Thus, in a first aspect the present invention relates to a spray product comprising hyaluronic acid or a salt thereof, which product is preferably less than 60 minutes, preferably 45 in an aqueous solvent, as determined herein. It has a solubility of less than minutes, more preferably less than 40 minutes, even more preferably less than 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, less than 10 minutes, most preferably less than 5 minutes.

In a second aspect, the invention relates to a composition comprising a product and an active ingredient as defined in the first embodiment, preferably the active ingredient is pharmacologically active.

A third aspect of the invention relates to a pharmaceutical composition comprising an effective amount of a product as defined in the first aspect, together with a pharmaceutically acceptable carrier, excipient or diluent.

A fourth aspect relates to a pharmaceutical composition comprising an active amount of the product as defined in the first aspect, as a vehicle, with the pharmacologically active agent.

The fifth aspect relates to a cosmetic comprising a product as defined in the first aspect or an composition as defined in any of the second, third or fourth aspect, in an amount effective as an active ingredient.

In a sixth aspect the invention relates to a hygiene, medical or surgical article comprising a product as defined in the first aspect or a composition as defined in any of the second, third or fourth aspect. Preferably, the article is a portion included in a diaper, sanitary napkin, surgical sponge, wound healing sponge, or band-aid or other wound bandage material.

An important aspect relates to pharmaceutical capsules or microcapsules comprising a product as defined in the first aspect or a composition as defined in any of the second, third or fourth aspects.

Another important aspect of the invention relates to a process for the preparation of a spray product comprising hyaluronic acid or a salt thereof, which product is less than 60 minutes, preferably less than 45 minutes, more preferably less than 40 minutes in an aqueous solvent. And even more preferably solubility of less than 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, less than 10 minutes, most preferably less than 5 minutes, the method comprises the following steps:

a) spraying a product comprising hyaluronic acid or a salt thereof.

The final aspect of the present invention relates to a method of performing a procedure in the treatment of osteoarthritis or cancer, hair loss or baldness, a method of treating a wound, a skin or transdermal administration of an active agent, or a skin administration of a cosmetic, in ophthalmology. The improvement relates to the use of a product as defined in the first aspect or a composition as defined in any of the second, third or fourth aspects.

Numerous embodiments include osteoarthritis, the manufacture of a medicament for the treatment of cancer, the manufacture of a medicament for ophthalmological treatment, the manufacture of a medicament for wound healing, the manufacture of a medicament for angiogenesis, the preparation of a medicament for the treatment of hair loss or baldness Or to the use of a product as defined in the first aspect or a composition as defined in any of the aspects of the invention for the preparation of a humectant.

1: The skin penetration of four different average molecular weight fragments of radiolabeled hyaluronic acid was investigated as described in Example 7. FIG. The results are shown in Table 5 and shown in FIG. 1. They suggest that better penetration was apparent for lower molecular weight species, and there is an apparent dependency of transdermal transport (flow rate: Flux) on HA molecular weight. There was no significant difference between the apparent flow rates measured at 5 and 22 hours for any of the HA size fractions studied.

2: Skin uptake and distribution of two different average molecular weight fragments of fluorescently labeled hyaluronic acid were investigated in Example 8 using laser scanning confocal microscopy. The average molecular weight of the HA fractions was 300,000 and 50,000 Da. Fluorescently labeled HA of 50,000 Da (F-HA 0.05) was only sparse and apparently visible on the surface of the stratum corneum, but the labeled HA fragment was clearly visible around and / or within the hair follicles. 2, Panel A shows the skin surface (layered stratum corneum), hair follicles and hair shafts; Panel B clearly shows that green fluorescence from HA is mainly visible and / or around or around the vesicle.

FIG. 3: A sparse fluorescence on the surface of the stratum corneum layer shown in FIG. 2 was observed in another skin sample, which was free of vesicles. 3, Panel A shows the skin surface; Figure 3 Panel B shows that only limited fluorescence from the labeled HA is visible at its surface.

Figure 4: A similar observation was made with fluorescence labeled HA of 300,000 Da (F-HA 0.30), which was also clearly visible around the hair follicles. 4, Panel A shows the skin surface (layered stratum corneum), hair follicles and hair shafts; Panel B shows green fluorescence from labeled HA around vesicles.

Figure 5: However, labeled HA fragments of 300,000 Da were also only sparse and seemingly visible at the skin surface, but it clearly showed differential accumulation between keratinocytes at the skin surface. 5, Panel A shows the skin surface; Panel B shows that the limited fluorescence from labeled HA is visible at its surface, but it accumulates between keratinocytes.

Justice

Nucleic Acid Constructs

A “nucleic acid construct” is defined herein as one of single- or double-stranded, separated from a naturally occurring gene as a nucleic acid molecule or modified to contain segments of nucleic acid that are combined and juxtaposed in a manner that would not otherwise exist in nature. . The term nucleic acid construct may be synonymous with the term expression cassette when the nucleic acid construct contains all control sequences necessary for the expression of the coding sequence. The term “coding sequence” is defined herein as a sequence that, when placed under the control of a control sequence mentioned below, is transcribed into mRNA and translated into the enzyme of interest. The boundaries of the coding sequence are generally determined by the ribosome binding site located immediately upstream of the open reading frame at the 5 'end of the mRNA and the transcription terminator sequence located immediately downstream of the open reading frame at the 3' end of the mRNA. Coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

Techniques used to isolate or clone nucleic acid sequences encoding polypeptides are well known in the art and include, for example, isolation from genomic DNA, preparation from cDNA, or combinations thereof. Cloning of nucleic acid sequences from such genomic DNA can be accomplished, for example, by using antibody screening of expression libraries to detect cloned DNA fragments of shared structural features or by well known polymerase chain reaction (PCR). See, eg, Innis et al., 1990, PCR Protocols: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification processes can be used, such as ligase chain reaction, ligated activation transcription, and nucleic acid sequence-based amplification. The cloning process may involve excision and isolation of the desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation into a Bacillus cell where a clone of the nucleic acid sequence of the recombinant vector will be replicated. The nucleic acid sequence can be genome, cDNA, RNA, semi-synthetic, synthetic origin, or any combination thereof.

Isolated nucleic acid sequences encoding enzymes can be engineered in a variety of ways to feed expression of the enzyme. Manipulation of the nucleic acid sequence prior to its insertion into a construct or vector may be desirable or necessary depending on the expression vector or Bacillus host cell. Techniques for modifying nucleic acid sequences using cloning methods are well known in the art. It will be appreciated that the nucleic acid sequences can also be engineered in host cells in vivo using methods well known in the art.

Numerous enzymes are involved in the biosynthesis of hyaluronic acid. These enzymes include hyaluronan synthase, UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase, hexokinase , Phosphoglucomutase, amidotransferase, mutase, and acetyl transferase. Hyaluronan synthase is a key enzyme in the production of hyaluronic acid.

"Hyaluronan synthase" is defined herein as a synthase that promotes elongation of the hyaluronan chain by the addition of GICUA and GIcNAc sugar precursors. The amino acid sequences of streptococcal hyaluronan synthase, vertebrate hyaluronan synthase, and viral hyaluronan synthase are distinguished from Pasteurella hyaluronan synthase and are classified as group I and group II hyaluronan synthase. Suggested group I hyaluronan synthase includes streptococcal hyaluronan synthase (DeAngelis, 1999). For the production of hyaluronan in Bacillus host cells, hyaluronan synthase of eukaryotic origin, such as mammalian hyaluronan synthase, is less preferred.

The hyaluronan synthase encoding the sequence can be any nucleic acid sequence that can be expressed in Bacillus host cells. The nucleic acid sequence can be of any origin. Preferred hyaluronan synthase genes are Streptococcus equisimilis , Streptococcus pyogenes, Streptococcus uberis , and Streptococcus equi subsp. I Group I hyaluronate synthase gene from zooepidemicus, or Pasturella group I or group II, such as the group II hyaluronan synthase gene of multocida .

The construct in which the precursor sugar of hyaluronan is thus supplied to the host cell is preferably a culture medium in the production of the HA of the invention, or by an endogenous gene in a Bacillus host cell or by a non-endogenous gene. Or by a combination of endogenous and non-endogenous genes. The precursor sugar may be D-glucuronic acid or N-acetyl-glucosamine.

In the method of the invention, the nucleic acid construct may further comprise one or more genes encoding enzymes in the biosynthesis of the precursor sugar of hyaluronan. Alternatively, the Bacillus host cell may further comprise one or more second nucleic acid constructs comprising one or more genes encoding enzymes in biosynthesis of the precursor sugar. Hyaluronan production is improved by the use of a construct having a nucleic acid sequence or sequences encoding a gene or genes that directs steps in the synthetic pathway of the precursor sugar of hyaluronan. "Indicating a step in the synthetic pathway of the precursor sugar of hyaluronan" means that the expressed protein of the gene is either N-acetyl-glucosamine or D-glucuronic acid, or N-acetyl-glucosamine and D-glucuronic acid. It is active in the formation of sugar, a precursor.

In a preferred method for supplying a precursor sugar, a host cell having a recombinant construct having a heterologous promoter region operably linked to a nucleic acid sequence encoding a gene indicative of a step in the synthetic pathway of a hyaluronan precursor sugar is provided. By culturing, constructs are provided to improve hyaluronan production in host cells with hyaluronan synthase. In a preferred method the host cell also comprises a recombinant construct having a promoter region operably linked to hyaluronan synthase, which is identical to or different from the nucleic acid sequence for the synthase involved in the biosynthesis of N-acetyl-glucosamine The promoter area can be used. In a preferred embodiment, the host cell may have a recombinant construct having a promoter region operably linked to another nucleic acid sequence encoding a second gene involved in the synthesis of the precursor sugar of hyaluronan.

Accordingly, the present invention also relates to constructs for improving hyaluronan production by the use of constructs having nucleic acid sequences encoding genes that direct steps in the synthetic pathway of the precursor sugar of hyaluronan. The nucleic acid sequence for the precursor sugar may be expressed from the same or different promoter as the nucleic acid sequence encoding hyaluronan synthase.

Genes involved in biosynthesis of precursor sugars for the production of hyaluronic acid include the UDP-glucose 6-dehydrogenase gene, the UDP-glucose pyrophosphorylase gene, the UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6 Phosphate isomerase gene, hexokinase gene, phosphoglucomutase gene, amidotransferase gene, mutase gene, and acetyl transferase gene.

In cells containing hyaluronan synthase, any one or a combination of two or more of hasB, hasC and hasD, or homology thereof, such as Bacillus subtilis tuaD, gtaB, and gcaD, respectively, as well as hasE, may be It can be expressed to increase the pool of precursor sugars available to the aze. The Bacillus subtilis genome is described in Kunst, et al., Nature 390,249-256, "Complete Genome Sequence of Gram-positive Bacterium Bacillus subtilis" (November 20, 1997). In some cases, such as when the host cell does not have native hyaluronan synthase activity, the construct may comprise the hasA gene.

Nucleic acid sequences encoding biosynthetic enzymes may be innate to the host cell, while in other cases heterologous sequences may be used. as hasA, hasB, hasC and genes of the HAS operon of S treptococcus equisimilis including the hasD, if two or more genes, they may be genes that are united with each other in a natural operon. In other cases, it may be desirable to use some combination of precursor gene sequences that does not include each element of the operon. The use of some genes innate in the host cell, and others that are exogenous, may also be desirable in other cases. The choices include the available pool of sugars in a given host cell, the ability of the cell to control overproduction without disrupting other functions of the host cell, and whether the cell regulates expression from its native genes other than exogenous genes. Will depend on.

As an example, depending on the metabolic requirements and growth conditions of the cells, and available precursor sugar pools, UDP-N-acetylglucosamine pyrophosphorylase, such as the hasD gene, Bacillus gcaD gene, and homology thereof, may be used. By expression of the encoding nucleic acid sequence, it may be desirable to increase the production of N-acetyl-glucosamine. As an alternative, the precursor sugar may be D-glucuronic acid. In one such embodiment, the nucleic acid sequence encodes UDP-glucose 6-dehydrogenase. Such nucleic acid sequences include the Bacillus tuaD gene, the hasB gene of Streptococcus , and homology thereof. The nucleic acid sequence may also encode UDP-glucose pyrophosphorylase, as in the Bacillus gtaB gene, hasC of Streptococcus , and homology thereof. In the method of the present invention, the UDP-glucose 6-dehydrogenase gene may comprise a hasB gene or tuaD gene; Or its homologue.

In the present invention, it is contemplated that the hyaluronan synthase gene and one or more genes encoding the precursor sugar are under the control of the same promoter. As an alternative, one or more genes encoding precursor sugars are under the control of the same promoter while other promoters drive the hyaluronan synthase gene. A further alternative is that each of the hyaluronan synthase gene and the gene encoding the precursor sugar are under the control of a different promoter. In a preferred embodiment, the hyaluronan synthase gene and one or more genes encoding precursor sugars are under the control of the same promoter.

The invention also provides a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally a UDP-glucose pyrophosphorylase gene, a UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6 A nucleic acid construct comprising an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising at least one gene selected from the group consisting of phosphate isomerase genes.

In some cases, the host cell will have a recombinant construct with a heterologous promoter region operably linked to a nucleic acid sequence encoding a gene that directs a step in the synthetic pathway of the precursor sugar of hyaluronan, which is from the recombinant construct. Can cooperate with the expression of synthase. Hyaluronan synthase may be expressed from a promoter region that is the same as or different from the nucleic acid sequence encoding the enzyme involved in the biosynthesis of the precursor. In another preferred embodiment, the host cell may have a recombinant construct having a promoter region operably linked to another nucleic acid sequence encoding a second gene involved in the synthesis of the precursor sugar of hyaluronan.

The nucleic acid sequence encoding the enzyme involved in the biosynthesis of the precursor sugar (s) may be expressed from the same or different promoter as the nucleic acid sequence encoding the hyaluronan synthase. In the former sense, an “artificial operon” is constructed, which can mimic each of hasA, hasB, hasC and hasD, or the operon of Streptococcus equisimilis in that it has homology, or alternatively exist in the Streptococcus equisimilis operon Less than all complements may be used. Artificial operon ”also includes glucose-6-, as well as one or more genes selected from the group consisting of hexokinase gene, phosphoglucomutase gene, amidotransferase gene, mutase gene, and acetyl transferase gene. Phosphate isomerase gene (hasE) In an artificial operon, at least one of the elements is heterologous relative to each other, such that the promoter region is heterologous to the coding sequence.

In a preferred embodiment, the nucleic acid construct comprises hasA, tuaD, and gtaB. In another preferred embodiment, the nucleic acid construct comprises hasA, tuaD, gtaB, and gcaD. In another preferred embodiment, the nucleic acid construct comprises hasA and tuaD. In another preferred embodiment, the nucleic acid construct comprises hasA. In another preferred embodiment, the nucleic acid construct comprises hasA, tuaD, gtaB, gcaD, and hasE. In another preferred embodiment, the nucleic acid construct comprises hasA, hasB, hasC, and hasD. In another preferred embodiment, the nucleic acid construct comprises hasA, hasB, hasC, hasD, and hasE. Based on this preferred embodiment, the specified gene can be substituted with its homologue.

In the methods of the invention, the nucleic acid construct comprises a hyaluronan synthase coding sequence operably linked to a promoter sequence heterologous to the hyaluronan synthase coding sequence. The promoter sequence can be, for example, a single promoter or a single line promoter.

A "promoter" is defined herein as a nucleic acid sequence involved in the binding of an RNA polymerase to initiate transcription of a gene. A "line promoter" is defined herein as two or more promoter sequences, each of which is operably linked to a coding sequence and mediates the transcription of the coding sequence into mRNA. “Operably linked” is defined herein as a configuration in which a control sequence, eg, a promoter sequence, is appropriately positioned at a position relative to a coding sequence such that the control sequence directs the preparation of the polypeptide encoded by the coding sequence. . As indicated above, a "coding sequence" is as defined herein as a nucleic acid sequence that is transcribed into mRNA and translated into a polypeptide when placed under the control of an appropriate control sequence. The border of the coding sequence is generally determined by the ribosome binding site located immediately upstream of the open reading frame at the 5 'end of the mRNA and the transcription terminator sequence located immediately downstream of the open reading frame at the 3' end of the mRNA. Coding sequences may include, but are not limited to, genomic DNA, cDNA, semisynthetic, synthetic, and recombinant nucleic acid sequences.

In a preferred embodiment, the promoter sequence can be obtained from a bacterial source. In a more preferred embodiment, the promoter sequence is a Bacillus strain, e.g., Bacillus agaradherens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium From a gram positive bacterium such as Bacillus pumilus , Bacillus stearothermophilus , Bacillus subtilis , or Bacillus thuringiensis or Streptomyces strains, eg Streptomyces lividans or Streptomyces murinus ; Or gram negative bacterium, for example E. coli or Pseudomonas sp.

Examples of suitable promoters for directing transcription of nucleic acid sequences in the methods of the invention include E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus lentus or Bacillus clausii alkaline protease gene (aprH), Bacillus licheniformis alkaline protease gene (subtilisin). Carlsberg gene, Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis alpha-amylase gene (amyE), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis subsp. tenebrionis CryIIIA gene (cryIIIA) or part thereof, a promoter obtained from the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731). Other examples are spo1 bacterial phage promoters and tac promoters (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Further promoters include "Useful Proteins from Recombinant Bacteria" in Scientific American, 1980,242: 74-94; And Sambrook, Fritsch, and Maniatus, 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor, New York.

The promoter may also be a "consensus" promoter with sequence TTGACA for the "-35" region and TATAAT for the "-10" region. Consensus promoters can be obtained from any promoter capable of functioning in Bacillus host cells. The construction of the "consensus" promoter is directed to site-directed mutations that produce promoters that more accurately match the consensus sequences established for the "10" and "-35" regions of the proliferative "sigma A-type" promoter for Bacillus subtilis. (Voskuil et al., 1995, Molecular Microbiology 17: 271-279).

In a preferred embodiment, the "consensus" promoter is the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus clausii or Bacillus lentus alkaline protease gene (aprH), Bacillus licheniformis alkaline protease gene, Bacillus subtilis levansucra Gene (sacB), Bacillus subtilis alpha-amylase gene (amyE), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniaseis gene penP), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis subsp. a promoter obtained from the tenebrioniscryIIIA gene (cryIIIA) or a portion thereof, or from the prokaryotic beta-lactamase gene spo1 bacterial phage promoter. In a more preferred embodiment, the "consensus" promoter is obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ).

Widner, et al., US Patent Nos. 6,255,076 and 5,955,310 describe in-line promoters and constructs and methods for use in expression in Bacillus cells, including the short consensus amyQ promoter (also called scBAN). Also described herein are the use of cryIIIA stabilizer sequences, and constructs using the sequences for improved manufacture of Bacillus.

Each promoter sequence of the in-line promoter may be any nucleic acid sequence that exhibits transcriptional activity in Bacillus cells of choice, including mutations, truncated, and hybrid promoters, and may be extracellular or of either homologous or heterologous to Bacillus cells. It can be obtained from a gene encoding an intracellular polypeptide. Each promoter sequence can be native or foreign to the nucleic acid sequence encoding the polypeptide and native or foreign to Bacillus cells. The promoter sequence can be the same promoter sequence or another promoter sequence.

Two or more promoter sequences of the in-line promoter can simultaneously promote transcription of the nucleic acid sequence. Alternatively, one or more of the promoter sequences of the line promoter may promote transcription of the nucleic acid sequence at different stages of growth of Bacillus cells.

In a preferred embodiment, the in-line promoter contains at least amyQ promoter of the Bacillus amyloliquefaciens alpha-amylase gene. In another preferred embodiment, the in-line promoter contains at least a "consensus" promoter having the sequence TTGACA for the "-35" region and TATAAT for the "-10" region. In another preferred embodiment, the line promoter contains at least amyL promoter of the Bacillus licheniformis alpha-amylase gene. In another preferred embodiment, the in-line promoter contains at least a cryIIIA promoter or part thereof (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107).

In a more preferred embodiment, the in-line promoter contains at least an amyL promoter and a cryIIIA promoter. In another more preferred embodiment, the in-line promoter contains at least an amyQ promoter and a cryIIIA promoter. In another more preferred embodiment, the in-line promoter contains a cryIIIA promoter and a "consensus" promoter with sequence TTGACA for at least the "-35" region and TATAAT for the "-10" region. In another more preferred embodiment, the in-line promoter contains at least two copies of the amyL promoter. In another more preferred embodiment, the in-line promoter contains at least two copies of the amyQ promoter. In another more preferred embodiment, the in-line promoter contains at least two copies of the "consensus" promoter having the sequence TTGACA for the "-35" region and TATAAT for the "-10" region. In another more preferred embodiment, the in-line promoter contains at least two copies of the cryIIIA promoter.

An “mRNA processing / stabilization sequence” refers to one or more promoter sequences downstream of one or more promoter sequences, such that all mRNA synthesized from each promoter sequence can be processed to produce mRNA transcription with a stabilizer sequence at the 5 ′ end of transcription. It is defined herein as a sequence located upstream of a coding sequence to which each is operably linked. The presence of such stabilizer sequences at the 5 ′ end of mRNA transcription increases their half-life (Agaisse and Lereclus, 1994, supra, Hue et al., 1995, Journal of Bacteriology 177: 3465-3471). The mRNA processing / stabilization sequence is complementary to the 3 'end of bacterial 16S ribosomal RNA. In a preferred embodiment, the mRNA processing / stabilization sequence produces essentially single-size transcription with a stabilizing sequence at the 5 'end of transcription. The mRNA processing / stabilization sequence is preferably one, which is complementary to the 3 'end of bacterial 16S ribosomal RNA. See, US Patent Nos. 6,255,076 and 5,955,310.

In a more preferred embodiment, the mRNA processing / stabilizing sequence is a Bacillus thuringiensiscryIIIA mRNA processing / stabilizing sequence disclosed in WO 94/25612 and Agaisse and Lereclus, 1994 or part thereof having mRNA processing / stabilizing function. In another more preferred embodiment, the mRNA processing / stabilizing sequence is Hue et al., 1995, Bacillus subtilis SP82 mRNA processing / stabilizing sequence disclosed above or part thereof having mRNA processing / stabilizing function.

When the cryIIIA promoter and its mRNA processing / stabilization sequence are employed in the method of the present invention, the sequence or promoters disclosed in WO 94/25612 and Agaisse and Lereclus, 1994 and parts thereof retaining mRNA processing / stabilizing action DNA fragments containing can be used. Furthermore, DNA fragments containing only cryIIIA promoter or only cryIIIA mRNA processing / stabilizing sequences can be prepared using methods well known in the art to construct various line promoters and mRNA processing / stabilizing sequence combinations. In this embodiment, the cryIIIA promoter and its mRNA processing / stabilization sequence are preferably located downstream of the other promoter sequence (s) constituting the line promoter and upstream of the coding sequence of the gene of interest.

Isolated nucleic acid sequences encoding the desired enzyme (s) involved in hyaluronic acid preparation can be further promoted to improve expression of the nucleic acid sequence. Expression is to be understood as including, but not limited to, any steps involved in the preparation of a polypeptide, including transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Techniques for modifying nucleic acid sequences using cloning methods are well known in the art.

Nucleic acid constructs comprising a nucleic acid sequence encoding an enzyme may be operably linked to one or more control sequences capable of directing the expression of the coding sequence in Bacillus cells under conditions compatible with the control sequence.

The term "control sequence" is defined herein to include all components necessary or beneficial for the expression of the coding sequence of the nucleic acid sequence. Each control sequence can be natural or foreign to the nucleic acid sequence encoding the enzyme. In addition to the promoter sequences detailed above, such control sequences include, but are not limited to, leader, signal sequence, and transcription termination sites. At least, the control sequence comprises a promoter and a transcriptional and translational stop signal. The control sequence may be provided with a linker for the purpose of introducing a specific restriction site that facilitates ligation of the control sequence with the coding region of the nucleic acid sequence encoding the enzyme.

Control sequences may also be appropriate transcription terminator sequences, sequences recognized by Bacillus cells to terminate transcription. The terminator sequence is operably linked to the 3 'end of the nucleic acid sequence encoding the enzyme or last enzyme of the operon. Any terminator that acts on the selected Bacillus cells can be used in the present invention.

Control sequences are also suitable leader sequences, untranslated regions of mRNA that are important for translation by cells. The leader sequence is operably linked to the 5 'end of the nucleic acid sequence encoding the enzyme. Any leader sequence that acts on the selected Bacillus cells can be used in the present invention.

The control sequence can also be a signal peptide coding region, which encodes for an amino acid sequence linked to an amino terminus of the polypeptide that can direct the expressed polypeptide to the cell's secretory pathway. The signal peptide coding region can be native to the polypeptide or can be obtained from a foreign source. The 5 'end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region inherently linked in a translation reading frame having a segment of the coding region encoding the secreted polypeptide. As an alternative, the 5 'end of the coding sequence may contain a signal peptide coding region that is heterologous to the portion of the coding sequence encoding the secreted polypeptide. A heterologous (foreign) signal peptide coding region may be necessary in which the coding sequence does not normally contain the signal peptide coding region. Alternatively, the heterologous signal peptide coding region may simply replace the native signal peptide coding region to obtain secretion of the enhanced polypeptide relative to the native signal peptide coding region normally associated with the coding sequence. Signal peptide coding regions can be obtained from amylase or protease genes from Bacillus species. However, any signal peptide coding region capable of directing the expressed polypeptide into the secretory pathway of the selected Bacillus cells can be used in the present invention.

Effective signal peptide coding regions for Bacillus cells include the maltogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subtilisin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral protease gene nprM), and a signal peptide coding region obtained from the Bacillus subtilis prsA gene. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

The control sequence may also be a propeptide coding region that encodes for an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is known as an enzyme precursor or propolypeptide (or in some cases an enzyme source). Propolypeptides can generally be converted from propolypeptide to inactive and mature active polypeptides by catalytic or autocatalytic cleavage of the propeptide. The propeptide coding region can be obtained from genes for Bacillus subtilis alkaline protease (aprE) and Bacillus subtilis neutral protease (nprT).

When both the signal peptide and the propeptide region are present at the amino terminus of a polypeptide, the propeptide region is located next to the amino terminus of the polypeptide and the signal peptide region is located next to the amino terminus of the propeptide region.

It may be desirable to add regulatory sequences that allow control of expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that start or stop the expression of genes in response to chemical or physical stimuli involving the presence of regulatory compounds. Regulatory systems in prokaryotic systems include lac, tac, and trp operating systems.

Produce

In the method of the invention, the host cell is cultured in a nutrient medium suitable for the production of hyaluronic acid using methods known in the art. For example, cells can be shake flask cultured, small or large-scale fermentation (continuous, batch, feed) in a laboratory or industrial incubator performed in a suitable medium and under conditions that express an enzyme involved in hyaluronic acid synthesis and isolate hyaluronic acid. -Batch, or solid state fermentation). Cultivation takes place in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using procedures known in the art. Appropriate media from commercial suppliers are available or can be prepared according to published compositions (eg in the catalog of the American Type Culture Collection). Secreted hyaluronic acid can be recovered directly from the medium.

The resulting hyaluronic acid can be separated by methods known in the art. For example, hyaluronic acid can be separated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The separated hyaluronic acid is then, but not limited to, chromatographic (eg, ion exchange, affinity, hydrophobicity, chromatofocusing, and size exclusion), electrophoretic processes (eg, preliminary equipotential focusing), differential solubility (Eg, ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). It can be further purified by the procedure.

details

Hyaluronic acid

A "hyaluronic acid" is an unsulfated glyco consisting of repeating disaccharide units of N-acetylglucosamine (GIcNAc) and glucuronic acid (GICUA) linked together by alternating beta-1,4 and beta-1,3 glycosidic bonds. Defined herein as samminoglycans. Hyaluronic acid is also known as hyaluronan, hyaluronate, or HA. The terms hyaluronan and hyaluronic acid are used interchangeably herein.

Rooster crests are an important commercial source for hyaluronan. Microorganisms are an alternative source. U.S. Patent No. 4,801,539 discloses a fermentation method for producing hyaluronic acid involving a strain of Streptococcus zooepidemicus with a reported yield of about 3.6 g of hyaluronic acid per liter. European Patent No. EP0694616 discloses a fermentation process using an improved strain of Streptococcus zooepidemicus with a reported yield of about 3.5 g of hyaluronic acid per liter. As disclosed in WO 03/054163 (Novozymes), which is incorporated herein in its entirety, hyaluronic acid or a salt thereof can be produced recombinantly, for example, in a Gram-positive Bacillus host.

Hyaluronan synthase is described from vertebrates, bacterial pathogens, and avian viruses (DeAngelis, PL, 1999, Cell. Mol. Life Sci. 56: 670-682). WO 99/23227 describes group I hyaluronate synthase from Streptococcus equisimilis . WO 99/51265 and WO 00/27437 describe Pasturella. Group II hyaluronate synthase is described from multocida . Ferretti et al. Are hyaluronan synthas of Streptococcus pyogene , consisting of three genes, hasA, hasB, and hasC, encoding hyaluronate synthase, UDP glucose dehydrogenase, and UDP-glucose pyrophosphorylase, respectively. Aze operons are disclosed (Proc. Natl. Acad. Sci. USA. 98,4658-4663, 2001). WO 99/51265 describes nucleic acid segments having coding regions for Streptococcus equisimilis hyaluronan synthase.

Since hyaluronan of recombinant Bacillus cells is expressed directly in the culture medium, a simple process can be used to separate hyaluronan from the culture medium. First, Bacillus cells and cell debris are physically removed from the culture medium. The culture medium is first diluted, if desired, to reduce the viscosity of the medium. Many methods for removing cells from the culture medium, such as centrifugation or microfiltration, are known to those skilled in the art. If desired, the remaining supernatant can then be filtered by ultrafiltration or the like to concentrate and remove small molecular contaminants from the hyaluronan. After removal of cells and cell debris, simple precipitation of hyaluronan from the medium is carried out by known mechanisms. Salts, alcohols, or a combination of salts and alcohols may be used to precipitate hyaluronan from the filtrate. Once reduced to a precipitate, hyaluronan can be easily separated from the solution by physical means. Hyaluronan can be dried or concentrated from the filtrate solution by using evaporation techniques known in the art such as spray drying.

A first aspect of the invention relates to a spray product comprising hyaluronic acid or a salt thereof, the product having a solubility of less than 60 minutes in an aqueous solvent.

Host cell

Preferred embodiments relate to the product of the first aspect, wherein the hyaluronic acid or salt thereof is preferably by gram-positive bacterium or host cell, more preferably by bacterium of genus Bacillus, It is made recombinantly.

The host cell can be any Bacillus cell suitable for recombinant production of hyaluronic acid. Bacillus host cells can be wild type Bacillus cells or mutations thereof. Useful Bacillus cell in the practice of this invention include, but are not limited to, Bacillus agaraderhens, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium , Bacillus pumilus , Bacillus stearothermophilus , Bacillus subtilis, and Bacillus thuringiensis cells. In particular mutant Bacillus subtilis cells adapted to recombinant expression are described in WO 98/22598. Non-encapsulated Bacillus cells are particularly useful in the present invention.

In a preferred embodiment, the Bacillus host cells are Bacillus amyloliquefaciens , Bacillus clausii , Bacillus lentus , Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cells. In a more preferred embodiment, the Bacillus cells are Bacillus amyloliquefaciens cells. In another more preferred embodiment, the Bacillus cells are Bacillus clausii cells. In another more preferred embodiment, the Bacillus cells are Bacillus lentus cells. In another more preferred embodiment, the Bacillus cells are Bacillus licheniformis cells. In another more preferred embodiment, the Bacillus cells are Bacillus subtilis cells. In the most preferred embodiment, the Bacillus host cell is Bacillus subtilis A164Δ5 (see US Pat. No. 5,891,701) or Bacillus subtilis168Δ4.

Transformation of Bacillus host cells with the nucleic acid constructs of the invention is accomplished by, for example, protoplast transformation (see, eg, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115) using competent cells. (See, eg, Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, eg For example, Shigekawa and Dower, 1988, Biotechnology 6: 742-751), or by conjugation (see, eg, Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278).

Molecular Weight

The level of hyaluronic acid can be determined according to the modified carbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334). Furthermore, Ueno et al., 1988, Chem. Pharm. Bull. 36, 4971-4975; Wyatt, 1993, Anal. Chim. Acta 272: 1-40; And average molecular weights of hyaluronic acid using standard methods in the art, such as those described by Wyatt Technologies, 1999, "Light Scattering University DAWN Course Manual" and "DAWN EOS Manual" Wyatt Technology Corporation, Santa Barbara, California. Can be determined.

In a preferred embodiment, the hyaluronic acid obtained by the method of the present invention has a molecular weight of about 10,000 to about 10,000,000 Da. In a more preferred embodiment, the hyaluronic acid obtained by the method of the present invention has a molecular weight of about 25,000 to about 5,000,000 Da. In the most preferred embodiment, the hyaluronic acid obtained by the method of the present invention has a molecular weight of about 50,000 to about 3,000,000 Da.

Preferred embodiments relate to the product of the first aspect, wherein hyaluronic acid or a salt thereof is in the range of 300,000 to 3,000,000; Preferably in the range of 400,000 to 2,500,000; More preferably in the range of 500,000 to 2,000,000; And most preferably have a molecular weight ranging from 600,000 to 1,800,000 Da.

When recombinantly prepared hyaluronic acid or a salt thereof is used in the method of the present invention to prepare a product or composition of the present invention, it may be beneficial for certain uses to first reduce the average molecular weight of the hyaluronic acid or salt thereof. . For example, by some manufacturers of so-called low-molecular weight fragments of hyaluronic acid, it can penetrate the skin barrier to restore the natural content of hyaluronic acid in the skin, thus such fragments can be anti-skin-aging and anti It is mentioned that it is particularly suitable for cosmetic compositions sold as wrinkles. For food use, low MW hyaluronic acid has been shown to penetrate the gastrointestinal tract barrier, thereby increasing its bioavailability. Finally, low MW hyaluronic acid exhibits an anti-inflammatory effect and has potential applications in the treatment of inflammatory diseases. Reduction of the average molecular weight of hyaluronic acid or its salts can be achieved by standard methods in the art, such as simple heat treatment, enzymatic digestion, sonication, or acid hydrolysis. For example, US Pat. No. 6,020,484, and T. Miyazaki et al., Which describe ultrasonication techniques of HA comprising NaOCI as additive. (2001) Polymer Degradation and Stability, 74: 77-85.

Thus, a preferred embodiment relates to the product of the first aspect, wherein hyaluronic acid or a salt thereof is in the range of 10,000 to 800,000 Da; Preferably in the range of 20,000 to 600,000 Da; More preferably in the range of 30,000 to 500,000 Da; Even more preferably in the range of 40,000 to 400,000 Da; Most preferably it has a low average molecular weight in the range of 50,000 to 300,000 Da.

Salt and Crosslinking  HA

Preferred embodiments include inorganic salts of hyaluronic acid, preferably sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, or cobalt hyaluronate It relates to the product of the first aspect.

The preparation of crosslinked HA or salts thereof, prepared by crosslinking HA with a multifunctional epoxy compound, is disclosed in EP 0 161 887 B1. Total or partially crosslinked esters of HA with fatty alcohols, and salts of inorganic or organic bases of such partial esters are disclosed in US Pat. No. 4,957,744. Another method of cross-linked HA is described in US Pat. 5,616,568,5,652,347, and 5,874,417. Crosslinked HA can also be prepared by treating HA with boric acid, as follows:

Dried sodium hyaluronate (Na-HA, 203 mg), prepared recombinantly from Bacillus subtilis (WO 03/054163; Novozymes) by fermentation, is dissolved in 0.2 M NaOH to give a 4% solution. Boric acid (35 mg (approximately 1 equivalent of HA disaccharide) was added and the sample was stirred for 1.5 hours at room temperature and then stored for about 2.5 days at 5 ° C. Exactly as described above, but without boric acid, the control sample was It prepared in parallel.

The viscosity of the resulting HA-borate hydrogel was measured using a Carrimed CSL controlled stress rheometer (conical appearance: 6 cm, 2 °) at 25 ° C. Viscosity was dependent on shear rate and increased by at least 4.2- to 8.4-fold in HA-borate hydrogel compared to control samples, indicating the formation of crosslinked networks.

Compared to the standard spectrum of Na-HA, in the FT-IR spectrum of HA-borate hydrogels, new peaks were observed at 1200 and 945 cm −1, indicating that of the newly formed borate esters in the crosslinked HA-borate hydrogels. Corresponds to existence.

Thus, a preferred embodiment relates to the product of the first aspect comprising crosslinked hyaluronic acid or salts thereof, preferably hyaluronic acid is crosslinked with boric acid, more preferably crosslinked hyaluronic acid is borate Esters.

Determination of moisture

The moisture content of the dried product powder according to the invention is the loss of weight after drying the powder to constant weight at 102 ° C. ± 2 ° C., expressed as a percentage. Empty glass metering dishes with ground lids were dried in an oven, then cooled and weighed in an assay balance with a sensitivity of at least 0.1 mg. Approximately 3 g dried product powder is placed in a dish and weighed. Put the dish containing the powder into the oven without a lid and dry for 2 hours at a temperature of 102 ℃ ± 2 ℃; It is then placed in a dryer and cooled to room temperature before weighing it again. The dish with powder is placed in an oven without a lid and dried for at least 1 hour, then cooled and weighed as previously described; This is repeated until the weight remains constant, ie, two consecutive weighings are not different by more than 0.5 mg.

The percentage of moisture is then calculated as follows: (W2-W3) / (W2-W1) x100; W1 is the weight of the empty dish, W2 is the weight of the dish with powder, and W3 is the weight of the dish with dry powder. The result is calculated to 2 decimal places, and the reproducibility of this method is about ± 0.1%.

Preferably, when the spray product of the first aspect is determined herein, it comprises less than 5% moisture, preferably less than 2%, most preferably less than 1% moisture.

Particle size

The preferred product of the first embodiment has a particle size of 50% thereof, D ₅₀ , which is 10 to 1,000 microns, preferably 100, as determined by laser diffraction measurement of particles suspended in isopropanol, as shown in the examples below. To 1,000 microns, more preferably 150 to 900 microns, even more preferably 200 to 800 microns.

In a preferred embodiment, the polydispersity of the product of the first aspect is measured as the SPAN value, calculated according to the formula: SPAN = (D ₉₀ -D ₁₀ ) / D ₅₀ , wherein the SPAN value is from 1.0 to 2.5; Preferably the SPAN value is 1.2 to 2.2; More preferably the SPAN value is 1.5 to 1.9; Most preferably the SPAN value is 1.6 to 1.8.

Dispersibility

The dispersibility of the product of the invention is determined as follows: 0.1 g of spray product is added to a 100 ml glass bottle. When 50 ml of 0.9% (w / v) sodium chloride solution is added and the bottle is sealed and visually determined, 180 until all the added particles are dispersed as a single particle, ie no lumps remain. Also repeat the call by hand and record the time taken to perform the reversal in the data sheet. The time so recorded is the dispersibility of the product.

Preferred products of the first aspect, as determined herein, are less than 30 minutes, preferably less than 20 minutes, more preferably less than 15 minutes, more preferably less than 10 minutes, more preferably less than 5 minutes, in an aqueous solvent, More preferably less than 3 minutes, more preferably less than 2 minutes, most preferably less than 1 minute.

Solubility

The solubility in various solvents of a dry or spray product or composition comprising hyaluronic acid according to the invention can be determined as follows:

0.1 g of hyaluronan powder to be tested is added to a 100 ml sealable glass flask. 50 ml of 0.9% (w / v) sodium chloride is added to the flask containing the HA sample to be tested and grade water, anhydrous ethanol, acetone or ether is injected. When the flask is sealed and visually determined by inspection, a 180 degree inversion is performed until HA enters the solution, and the time taken to perform the inversion is recorded in the data sheet. The time recorded is the solubility of the sample.

The product according to the first aspect, as determined herein, is less than 60 minutes, preferably less than 45 minutes, more preferably less than 40 minutes, even more preferably 35 minutes, 30 minutes, 25 minutes, 20 minutes in an aqueous solvent. , Solubility of less than 15 minutes, less than 10 minutes, most preferably less than 5 minutes.

Other ingredients

In a preferred embodiment, the nebulized product of the invention may also comprise other components, preferably one or more active ingredients, preferably one or more pharmacologically active substances, and also preferably water-soluble excipients such as lactose. .

Non-limiting examples of active ingredients or pharmacologically active substances that can be used in the present invention include human growth hormone, bovine growth hormone, pig growth hormone, growth hormone releasing hormones / peptides, granulocyte-colony stimulating factor, granulocyte leukocytes Colony stimulating factor, macrophage-colony stimulating factor, erythropoietin, bone morphogenic protein, interferon or derivatives thereof, insulin or derivatives thereof, atriopeptin-III, monoclonal antibody, tumor necrosis factor, macrophage activation Protein and / or peptide drugs such as factors, interleukins, tumor altering factors, insulin-like growth factors, epidermal growth factors, tissue plasminogen activators, factor IIV, factor IIIV, and urokinase.

Water soluble excipients may be included for the purpose of stabilizing active ingredient (s), and such excipients may include proteins such as albumin or gelatin; Amino acids such as glycine, alanine, glutamic acid, arginine, lysine, and salts thereof; Carbohydrates such as glucose, lactose, xylose, galactose, fructose, maltose, saccharose, dextran, mannitol, sorbitol, trehalose and chondroitin sulfate; Inorganic salts such as phosphate; Surfactants such as TWEEN® (ICI), polyethylene glycol, and mixtures thereof. Excipients or stabilizers can be used in amounts ranging from 0.001 to 99% by weight of the product.

Some embodiments of the present invention relate to various compositions and medicaments comprising, among other ingredients, an effective amount of a product as defined in the first embodiment, and an active ingredient, preferably the active ingredient is pharmacologically active; Pharmaceutically acceptable carriers, excipients or diluents, preferably water-soluble excipients, and most preferably lactose.

Preferred embodiments of the invention included in the bubble tablets may be formulated differently as described in the art for the products or compositions of the invention. For example, bubble tablets may include citric acid, sodium bicarbonate, and oligosaccharides or other sugars. Foam tablets are products of the invention that are easy to store and that dissolve quickly, and they dissolve rapidly and thus provide an ideal means of oral administration.

Moreover, aspects of the present invention relate to articles, such as cosmetics, hygiene articles, medical or surgical articles, comprising a product as defined in the first aspect or a composition as defined in the above aspects and embodiments. . In a final aspect the invention relates to pharmaceutical capsules or microcapsules comprising a product as defined in the first aspect or a composition as defined in other aspects and embodiments of the invention.

Manufacturing method

In another embodiment the invention comprises hyaluronic acid or a salt thereof, and in an aqueous solvent less than 60 minutes, preferably less than 45 minutes, more preferably less than 40 minutes, even more preferably 35 minutes, 30 minutes, Provided are methods for preparing spray products having a solubility of less than 25 minutes, 20 minutes, 15 minutes, less than 10 minutes, most preferably less than 5 minutes.

a) spraying a product comprising hyaluronic acid or a salt thereof.

Spray

In a preferred embodiment of the method, the spraying step is carried out using a spray-dryer, preferably a Two-Fluid-Nozzle (TFN) or a rotary sprayer, preferably spray-drying using TFN and the following range of conditions Was:

Inlet temperature: 100-200 ℃

Outlet temperature: 40-90 ℃

Nozzle Spray Pressure: 2-10 bar

Nozzle Air Temperature: 40-100 ℃

Feed temperature: 40-100 ℃

How to Use a Product or Composition

Various aspects of the present invention relate to a method of carrying out a treatment procedure, for example in the medical field, using the product of the first aspect, or using the composition of the present invention.

One embodiment relates to a method of carrying out a process in ophthalmology, which comprises the use of a product or a composition of the invention as defined in the first embodiment.

Another aspect relates to a method of performing a procedure in the treatment of osteoarthritis, which comprises the use of a product or a composition of the invention as defined in the first aspect.

Yet another aspect relates to a method of performing a procedure in the treatment of cancer, which comprises the use of a product or a composition of the invention as defined in the first aspect.

Yet another aspect relates to a method of performing a procedure in the treatment of hair loss or baldness, and improvements include the use of a product or a composition of the invention as defined in the first aspect.

One aspect relates to a method of pharmacologically performing transdermal or skin administration of an active agent, which comprises the use of a product or a composition of the invention as defined in the first embodiment.

Another aspect relates to a method of performing skin administration of a cosmetic product, which comprises the use of a product or a composition of the invention as defined in the first aspect.

One aspect relates to an ophthalmological method using the product or composition of the invention as defined in the first aspect.

Another aspect relates to a method of treating osteoarthritis comprising administering to a mammal an effective amount of a product or a composition of the invention as defined in the first aspect, preferably the administration is to skin, transdermal, oral, or injection It is.

One aspect relates to a method of treating a wound, comprising administering to a mammal an effective amount of a product or a composition of the invention as defined in the first aspect.

Yet another aspect relates to a method of treating hair loss or baldness comprising administering to a mammal an effective amount of a product or a composition of the invention as defined in the first aspect, preferably the administration is skin, transdermal, oral, Or by injection.

Another aspect is the manufacture of a medicament for the treatment of osteoarthritis, a medicament for ophthalmological treatment, a medicament for the treatment of cancer, a medicament for the treatment of wounds, a medicament for angiogenesis, or a medicament for the treatment of hair loss or baldness , To the use of the product or composition of the invention as defined in the first aspect.

The last aspect relates to the use of the product or composition of the invention as defined in the first aspect for the preparation of a humectant.

Example   One

The liquid feed consisting of sodium hyaluronate solution containing about 8 g / liter was spray dried using two different types of spraying techniques: A) Two-Fluid-Nozzle (TFN) and B) rotary atomizer. Commercially available types of spray-drying techniques and equipment are also contemplated as being suitable for the purposes of the present invention. (See, eg Niro Sprayers Inc).

The resulting dried powders were significantly different. The powder produced by TFN had a significantly smaller bulk density. The reason for this difference is revealed in further analysis shown below.

sprayer (Batch No.) Particle density (g / ml) Solubility time (minute) Particle size ( D ₅₀ ) A) TFN (04PBAC0016) 0.210 15 95 B) rotary atomizer (MAG30012) 0.430 31 45

Compared with rotary nebulizers, the TFN nebulization principle was used to show that the solubility time was significantly reduced. Despite the fact that the particle size is much larger, this will usually increase the solubility time. However, increased solubility times were found to be due to the air contained in the TFN spray dried particles. This can be seen from the particle density of the TFN product, which was less than half the particle density of the dry product produced by the rotary atomizer.

Example  2

A liquid feed formulation consisting of 75,000 g sodium hyaluronate solution, with about 8 g / liter sodium hyaluronate and 2400 g sodium chloride, was added to the spray dryer at a constant rate. Hyaluronate had a molecular weight of about 690.000 Da. The spray dryer had a diameter of about 1.8 m and a height of about 5 m. Two-Fluid-Nozzle (TFN) was selected for spraying the feed. Drying conditions were adjusted to provide a dry powder batch (batch nr. 03PBAC0035-1) with the desired particle size:

Inlet temperature: 160 ℃

Outlet temperature: 75 ℃

Nozzle Spray Pressure: 4 bar

Nozzle air temperature: 70 ℃

Feed temperature: 60 ℃

The resulting particle size distribution was determined using laser diffraction on a suspension of the powder in isopropanol:

D ₁₀ : 12 micron

D ₅₀ : 39 micron

D ₉₀ : 84 micron

Molecular Weight: 631.000 Da

SPAN: 1.77

The measure of polydispersity is the 'SPAN' value, which can be calculated according to the formula:

SPAN = (D ₉₀ -D ₁₀ ) / D ₅₀

The molecular weight hardly changed during the drying run, thus demonstrating that the spray drying process was very smooth, although high temperatures were applied. If redissolution of the product was attempted, it formed a wet dust, which was found to dissolve only slowly. This unwanted low solubility behavior is due to the too low dispersibility of the dried particles.

Example  3. Molecular weight and skin penetration

Side-by-side diffuse cells with a diameter of 2 cm were used to examine the skin penetration of four different average molecular weight fragments of radiolabeled hyaluronic acid (effective skin area of 3.14 cm ² ). The volume on the receptor was 8-8.5 ml and the donor compartment volume was 1-1.5 ml.

Skin segment skin (750 micrometers) from pig ears was thawed and placed on one side of the diffuse cells. All visible hair was carefully cut with scissors. The other side of the diffuse cells was closed with a silicon disk. The receptor was filled with phosphate-buffered saline (PBS) at pH 7.4. A magnetic stir bar was introduced.

Three 10 microliter samples (5.64 mg / ml cold to 22.56 micrograms / ml hot cold / hot ratio provided on the donor) were taken to determine specific activity. The donor phase was then filled with the solution provided: for 5 ml (volume received), only 3 or 4 cells could be provided.

The cells were placed in a stirrer plate and the experiment was performed at ambient temperature. After 5 hours, the entire receptor phase was recovered and radioactivity was determined there by liquid scintillation counting. The receptor chamber was then refilled with PBS and diffusion continued overnight.

After 22 hours (ie 5 hours + 17 hours), the receiver solution was recovered again and analyzed for radioactivity. Cells were degraded and the skin was incubated with 5 ml of SOLUENE® (PerkinElmer) for 48 hours or until completely dissolved before adding scintillation fluid and counting for radioactivity. The experimental results are summarized for four different molecular weight HA fragments in Table 2.

Typically, 3 or 4 copies (Table 5: R1, R2, R3, R4) were obtained. However, sometimes an experimental problem was evident (eg, apparent skin perforation, or leakage from diffuse cells), in which case only two copies were possible.

The overall recovery of radioactivity ('mass balance') was less than 100%, mainly because the viscosity on the donor prevented their complete removal from the diffuse cell compartment. The problem increased with increasing HA molecular weight.

These results are shown in FIG. 1, and they suggest that there is a clear dependence on the HA molecular weight of transdermal transport, with clear better penetration for lower molecular weight species. There was no significant difference between the apparent flow rates measured at 5 and 22 hours for any of the HA size subsections studied.

Overall, HA penetration therefore depends on HA molecular weight. The lower the molecular weight, the better the skin penetration.

However, the amount of penetration into the skin corresponds to a small fraction of the applied radiation.

Example  4. Local distribution of different molecular weight fragments of HA

Skin uptake and distribution of two different average molecular weight subregions of fluorescence labeled hyaluronic acid were investigated using laser scanning confocal microscopy and the average molecular weights of the HA fragments were 300,000 and 50,000 Da.

Fluorescent HA samples were aqueous solutions at concentrations of 1 mg / mL; Diluted 10-fold with non-labeled HA of the same molecular weight in distilled water before applying the fluorescent HA solution.

The skin used for HA application was obtained from pig ears. Each HA fragment was applied and tested on at least three separate samples of skin from different animals. Skin samples were taken as fresh and stored frozen for up to two weeks before use.

Prior to the experiment, thawed skin samples (approximately 0.8 cm ² ) were sandwiched between donor and receptor phases of simple free diffuse cells and maintained at 32 ° C. The donor phase was 0.1 ml of labeled HA solution and the receptor phase was pH 7.4 buffer.

The fluorescent HA solution was maintained in contact with the skin sample for 3 hours, after which excess donor solution was removed, the skin was washed with 3 hours pH 7.4 buffer and gently dried with tissue.

The skin was immediately immobilized on glass slides, layered stratum corneum side up and examined microscopically without further processing using the LSM 410 Invert Laser Scan Microscope (Carl Zeiss, Germany). The system was equipped with an argon-krypton laser with excitation wavelengths of 568 and 488 nm, which was used to examine skin and labeled HA separately.

Using Plan-neofluar 10x / 0.3 and 40x / 0.6 objectives, images were obtained and then color-coded digitally so that the fluorescence from the skin was red, and the fluorescence from labeled HA was green. Images were color-coded and overlaid (or stacked) using Zeiss LSM confocal software.

In each experiment, skin samples were examined in the absence of fluorescent probes. Autofluorescence from pig skin allowed structural features to be identified. To visualize the distribution of fluorescent HA, confocal images were obtained at the xy-plane (ie, parallel to the skin surface). The skin surface (z = 0 μm) was defined as the brightest fluorescent imaging face with the morphological properties of the layered stratum corneum. Using at least three separate pieces of skin, replicate images were obtained for each treatment.

 Fluorescently labeled HA of 50,000 Da (F-HA 0.05) was only sparse and apparently visible on the surface of the stratum corneum, but labeled HA fragments were clearly visible around and / or in the hair follicles. 2, Panel A shows the skin surface (layered stratum corneum), hair follicles and hair shafts; Panel B clearly shows that green fluorescence from HA is mainly visible around the vesicles and / or in the vesicles.

A sparse fluorescence on the surface of the stratum corneum was observed in another skin sample, which did not have vesicles. 3, Panel A shows the skin surface; Panel B shows that only limited fluorescence from the labeled HA is visible at its surface.

 Similar observations were made with fluorescence labeled HA of 300,000 Da (F-HA 0.30), which was also clearly visible around the hair follicles. 4, Panel A shows the skin surface (layered stratum corneum), hair follicles and hair shafts; Panel B shows green fluorescence from labeled HA around the vesicles.

However, the labeled HA fragment of 300,000 Da was also sparse and apparently visible at the skin surface, but it clearly showed differential accumulation between keratinocytes at the skin surface. 5, Panel A shows the skin surface; Panel B shows that the limited fluorescence from labeled HA is visible at its surface, but it accumulates between keratinocytes.

The conclusion is that low molecular weight HA shows some differential accumulations in vesicle tissue and in the skin. This could be one possible penetration route for the low MW HA product of the compositions of the invention, which would be of particular interest for the treatment of hair loss or baldness.

After removal of HA, only a few fluorescent species were found on the surface layer in sparse appearance, so there was no evidence for transport through the intact layer skin stratum corneum of low MW HA of 0.05 MDa. Finally, HA of 0.30 MDa shows differential accumulation between keratinocytes at the skin surface. This can contribute to improved skin hydration because the HA barrier could limit water loss through the passive epidermis from the skin.

Claims (46)

Hyaluronic acid or a salt thereof, and less than 60 minutes, preferably less than 45 minutes, more preferably less than 40 minutes, even more preferably 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 in an aqueous solvent. Spray product having a solubility of less than 10 minutes, most preferably less than 5 minutes. The product according to claim 1, wherein the hyaluronic acid or a salt thereof is produced recombinantly, preferably by gram-positive bacterium, more preferably by bacterium of the genus Bacillus. The method of claim 1 or 2, wherein the hyaluronic acid or a salt thereof is in the range of 300,000 to 3,000,000; Preferably in the range of 400,000 to 2,500,000; More preferably in the range of 500,000 to 2,000,000; And most preferably have a molecular weight in the range of 600,000 to 1,800,000. The method of claim 1 or 2, wherein the hyaluronic acid or a salt thereof is in the range of 10,000 to 800,000 Da; Preferably in the range of 20,000 to 600,000 Da; More preferably in the range of 30,000 to 500,000 Da; Even more preferably 40,000 to 400,000 Da; Most preferably a low average molecular weight in the range of 50,000 to 300,000 Da. The inorganic salt of hyaluronic acid, preferably sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate according to any of claims 1-4. Ronate, or a product comprising cobalt hyaluronate. The method according to any one of claims 1 to 5, comprising a crosslinked hyaluronic acid or a salt thereof, preferably the hyaluronic acid is crosslinked with boric acid, more preferably the crosslinked hyaluronic acid is borate. A product comprising an ester. 7. The product according to claim 1, comprising less than 5% moisture, preferably less than 2%, and most preferably less than 1% moisture, as determined herein. 8. 8. The method according to claim 1, wherein its 50 percentile, D ₅₀ , is determined by laser diffraction measurement of particles suspended in isopropanol, preferably from 100 to 1,000 microns. , More preferably from 150 to 900 microns, even more preferably from 200 to 800 microns. The method according to any one of claims 1 to 8, as determined herein, in an aqueous solvent of less than 30 minutes, preferably less than 20 minutes, more preferably less than 15 minutes, more preferably less than 10 minutes, more Preferably, the product has a dispersibility of less than 5 minutes, more preferably less than 3 minutes, more preferably less than 2 minutes, and most preferably less than 1 minute. The method of claim 1, wherein the spray-drying or drying is carried out in a rotary atomizer; Preferably the product is characterized in that the product is spray-dried. The product according to claim 1, further comprising an active ingredient. 12. The product according to claim 11, wherein the active ingredient is a pharmacologically active substance. The product according to claim 1, further comprising a water soluble excipient, preferably lactose. 14. A composition comprising a product as defined in any of claims 1 to 13 and an active ingredient, preferably the active ingredient is pharmacologically active. The composition according to claim 14, further comprising a water soluble excipient, preferably lactose. A pharmaceutical composition comprising an effective amount of a product as defined in any of claims 1 to 13 together with a pharmaceutically acceptable carrier, excipient or diluent. A pharmaceutical composition comprising a pharmaceutically acceptable agent in an effective amount of a product as defined in any one of claims 1 to 13 as a vehicle. A cosmetic comprising a product as defined in any of claims 1 to 13 or a composition as defined in any of claims 14 to 17 as an active ingredient in an effective amount. A hygiene, medical or surgical article comprising a product as defined in any one of claims 1 to 13 or a composition as defined in any one of claims 14 to 17, preferably The article of claim 1, wherein the article is a portion included in a diaper, sanitary napkin, surgical sponge, wound healing sponge, or band-aid or other wound bandage material. A pharmaceutical capsule or microcapsule comprising a product as defined in any of claims 1 to 13 or a composition as defined in any of claims 14 to 17. Hyaluronic acid or a salt thereof, and less than 60 minutes, preferably less than 45 minutes, more preferably less than 40 minutes, even more preferably 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 in an aqueous solvent. A process for preparing a spray product having a solubility of less than 10 minutes, most preferably less than 5 minutes, a) spraying a product comprising hyaluronic acid or a salt thereof. The method of claim 21, wherein the hyaluronic acid or a salt thereof is recombinantly produced preferably by gram-positive bacterium, more preferably by bacterium of the genus Bacillus. 23. The method of claim 21 or 22 wherein the hyaluronic acid or salt thereof is in the range of 300,000 to 3,000,000; Preferably in the range of 400,000 to 2,500,000; More preferably in the range of 500,000 to 2,000,000; Most preferably having a molecular weight in the range of 600,000 to 1,800,000. 23. The method of claim 21 or 22 wherein the hyaluronic acid or salt thereof is in the range of 10,000 to 800,000 Da; Preferably in the range of 20,000 to 600,000 Da; More preferably in the range of 30,000 to 500,000 Da; Even more preferably in the range of 40,000 to 400,000 Da; Most preferably having a low average molecular weight in the range of 50,000 to 300,000 Da. The process according to claim 21, wherein the product is an inorganic salt of hyaluronic acid, preferably sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, Zinc hyaluronate, or cobalt hyaluronate. 26. The hyaluronic acid according to any one of claims 21 to 25, wherein the product comprises crosslinked hyaluronic acid or a salt thereof, preferably hyaluronic acid is crosslinked with boric acid, more preferably crosslinked hyaluronic acid A method comprising this borate ester. 27. The process according to any of claims 21 to 26, wherein the spraying step is carried out using a spray-dryer or a rotary sprayer, preferably a Two-Fluid-Nozzle (TFN) spray-dryer. The method of claim 27, wherein the spraying step is performed using a TFN spray-dryer and the following range of conditions. Inlet temperature: 100-200 ℃ Outlet temperature: 40-90 ℃ Nozzle Spray Pressure: 2-10 bar Nozzle Air Temperature: 40-100 ℃ Feed temperature: 40-100 ℃ In a method of performing a procedure in ophthalmology, an improvement is the use of a product as defined in any of claims 1 to 13 or a composition as defined in any of claims 14 to 17. Method comprising a. In the method of carrying out the procedure in the treatment of osteoarthritis, the improvement is a product as defined in any of claims 1 to 13, or a composition as defined in any of claims 14 to 17. Characterized in that it comprises the use of a. In a method of carrying out a procedure in the treatment of cancer, an improvement is a product as defined in any of claims 1 to 13 or a composition as defined in any of claims 14 to 17. Method comprising the use of. In a method of pharmacologically performing transdermal administration of an active agent, an improvement is a product as defined in any of claims 1 to 13, or as defined in any of claims 14 to 17. A method comprising the use of the same composition. In a method of pharmacologically performing skin administration of an active agent, an improvement is a product as defined in any of claims 1 to 13, or as defined in any of claims 14 to 17. A method comprising the use of the same composition. In a method of carrying out skin administration of a cosmetic, an improvement is in the product as defined in any of claims 1 to 13, or in a composition as defined in any of claims 14 to 17. A method comprising use. In the method of carrying out the procedure in the treatment of hair loss or baldness, the improvement is a product as defined in any of claims 1 to 13, or as defined in any of claims 14 to 17. A method comprising the use of the same composition. An ophthalmological method using a product as defined in any of claims 1 to 13 or a composition as defined in any of claims 14 to 17. 18. Treatment of osteoarthritis comprising administering to a mammal an effective amount of a product as defined in any one of claims 1 to 13 or a composition as defined in any one of claims 14 to 17. Method, characterized in that the administration is preferably by skin, transdermal, oral, or injection. A method of treating a wound comprising administering to a mammal an effective amount of a product as defined in any one of claims 1 to 13 or a composition as defined in any one of claims 14 to 17. . Hair loss or baldness comprising administering to a mammal an effective amount of a product as defined in any one of claims 1 to 13 or a composition as defined in any one of claims 14 to 17. The method of treatment, wherein the administration is preferably by skin, transdermal, oral, or injection. Use of a product as defined in any one of claims 1 to 13 or a composition as defined in any one of claims 14 to 17 for the manufacture of a medicament for the treatment of osteoarthritis. Use of a product as defined in any one of claims 1 to 13 or a composition as defined in any one of claims 14 to 17 for the manufacture of a medicament for ophthalmic treatment. Use of a product as defined in any one of claims 1 to 13 or a composition as defined in any one of claims 14 to 17 for the manufacture of a medicament for the treatment of cancer. Use of a product as defined in any one of claims 1 to 13 or a composition as defined in any one of claims 14 to 17 for the manufacture of a medicament for the treatment of a wound. Use of a product as defined in any of claims 1 to 13 or a composition as defined in any of claims 14 to 17 for the manufacture of a medicament for angiogenesis. Use of a product as defined in any one of claims 1 to 13 or a composition as defined in any one of claims 14 to 17 for the preparation of a humectant. Use of a product as defined in any one of claims 1 to 13 or a composition as defined in any one of claims 14 to 17 for the manufacture of a medicament for the treatment of hair loss or baldness.

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