DE69634993T2 - USE OF HYALURONAN IN GENE THERAPY - Google Patents

Description

BACKGROUND THE INVENTION

These Invention relates to the use of hyaluronic acid forms (hyaluronan forms) in gene therapy. This invention further relates to the use of hyaluronan as a targeting agent in gene therapy for the control or treatment of diseases and / or states to effect a purposeful control of active resources which minimize the function of genes that represent a target structure.

Hyaluronic acid poses a big, Complex oligosaccharide consisting of up to 50,000 pairs of the as Basic-building disaccharide glucuronic acid-β (1-3) -N-acetylglucosamine-β (1-4). In vivo, they are found to be the major constituent of the extracellular matrix. Your tertiary structure represents a random spiral with a Diameter of about 50 nm. Hyaluronic acid occurs in nature in the form their sodium salt. Hyaluronic acid and its pharmaceutical permissible or acceptable salts (such as sodium hyaluronate) are used herein referred to as HA.

HA has the ability size Binding amount of water, which makes it a viscous, in vivo, represents hydrated gel with viscoelastic properties. They are found in this form both in the vitreous and in the extracellular matrix of the mammalian eye.

HA was due to her ability to effect a targeted control of active agents at sites which the disease or condition is localized (International Patent Publications No. WO 91/04058 and No. WO 93/16733), both systemically and used topically for the treatment of diseases and conditions of the human body. For example, it has been shown that HA affects the injured carotid artery (in relation to to uninjured contralateral arteries), as well as developing ones colorectal tumors from experimental animals depots, and in the skin of humans and animals is stored. In all of these make Deposits make areas of high expression for HA receptors indicating that HA is specific to areas of For example, under-perfused and pathological tissue directed is, which express high levels of these receptors, and in particular aimed at tissue showing an unusual proliferation and migration completes, including Tissue indicative of injury, inflammation, development and tumorigenesis responds.

cell of the immune system, including Macrophages, neutrophils, T cells and natural killer cells play in the states described above a significant role. These cells rely on HA receptors, to develop into reactive cells that have the ability possess, growth factors and cytokines as well as oxidizing agents release, which subsequently initiate a repair process that results in tissue damage participates. additionally These cells rely on HA receptors to attach to cells such as for example, to bind those of the endothelium as well as to tumor cells, and about yourself move these receptors to the site of an injury (to migrate). Need tumor cells for migration, proliferation and metastasis also HA receptors. consequently Is it possible, by adding high concentrations of HA the infiltration of white blood cells, For example, in the injured rat carotid artery, to to inhibit a proliferation of the tumor cells and a movement of the to block smooth muscle. Reagents such as functionally blocking antibodies and peptides that mimic critical regions of HA receptors, are also able to exert this effect.

In summary show these results that HA is specific to certain receptors, which, for example, on the surface of a variety of damaged or carcinogenic cells, interacts.

The for HA specific cell surface receptors were identified, these being the histocompatibility antigen CD44 and the receptor for hyaluronan-mediated motility (RHAMM).

In all of the diseases and conditions described above, as well as others, researchers are now attempting to treat the diseases and conditions (for example restenosis) by means of gene therapy. The basic principle is to reduce a function of a specific gene, either by blocking the translation of messenger RNA into proteins by means of "antisense" oligonucleotides, or by gene expression by means of antisense cDNA. "To date, this has been largely accomplished by the use of these reagents by introduction in liposomes, as well as by direct application of antisense cDNA or antisense oligonucleotides to the site of the disease.Viruses (for example adenoviruses) have also been proposed However, it is difficult to effect targeted control of the viruses to specific disease sites, as evidenced by minimal uptake. For example, the process of using viruses attached to and taken up by cells to target DNA is inefficient, with the risk of using viruses being misguided and unacceptable in humans. If gene therapy involves the use of liposomes, it is difficult to target the liposomes efficiently to a target, and the uptake may be even lower than that of viruses.

A direct application of the oligonucleotides in the focus, such as for example, in a stenotic plaque, had a favorable effect in animals, being big Amounts of oligonucleotides needed and the method, for example, the balloon angioplasty operation extended, which increases the risk for increased the patient. Although, in theory, gene therapy is a viable approach to Treatment of diseases and conditions, these indicate Results indicate that the technical difficulties of a efficient focus on a target structure as well as an efficient one Recording must be improved.

In an article published on page 8 of the "The Toronto Star," a Toronto, Ontario, Canada newspaper, a newspaper article appeared on September 22, 1996 that addressed the use of gene therapy to treat cancer.
"The cancer gene therapy tries to address the problem of tumors, which arise from the fact that they contain faulty genes. There are a number of mutated genes associated with a variety of tumors, but one of the most important is a gene called p53.
In normal cells, p53 helps maintain the genome of the cell. If breaks in the DNA double helix occur, p53 switches on and initiates repair of the DNA. If things get out of hand and no repair is possible, p53 triggers the death of the cell. Failure to repair such genetic problems will result in abnormal cell growth - tumors.
Exactly the same happens when mutated or non-functional variants of p53 arise. Statistics prove this. At least half of all malignant tumors contain cells that have problems with their p53 genes. This includes 80 percent of colon cancer tumors, 50 percent of lung cancer tumors, and 40 percent of breast cancer tumors.
In a small-scale study reported in this month's issue of Nature Medicine, a team from the University of Texas treated nine lung cancer patients who failed all conventional treatments. All had multiple tumors, some of which had metastasized to other organs such as the brain. Gene therapy consisted of injecting intact copies of p53 into selected sites of the tumor.
The technology of introducing genes into tumors is amazing. The genes are placed inside inactivated viruses, and several tens of millions of viruses per milliliter are injected into the tumor, some of which enter the cancer cells and presumably integrate their gene bundles - including the intact p53 - into these cells.
As part of the study, two of the patients either failed to complete the study or died before it was completed. In fact, the gene therapy-treated tumor shrank in three of the remaining seven patients. In three other patients, although not shrinking, the tumor showed no signs of growth. In one patient, the tumor completely receded. Programmed cell death within the tumor, a process known to be initiated by active p53 genes, was observed in six of the patients.
It is recalled that these were patients who had no hope of using traditional cancer therapies. In fact, many of them died within months of the study due to the growth of other tumors that were not the target of gene therapy. "

The Suppression of the expression of genes encoding proteins, the one unwanted activity has been implicated in a variety of cases by introduction of "antisense" DN4 sequences in reaches the DNA of target cells. These antisense sequences are DNA sequences which lead to the formation of RNA after transcription, the Sequence to the protein coding sequence is antiparallel. such Antisense sequences have been tested in a number of viral diseases. Alternatively you can Antisense oligodeoxynucleotides are introduced into the target cells; such short sequences themselves are not transcribed, inhibited however, the transcription and / or the subsequent translation of the corresponding sense DNA sequence in the target cell.

Until recently, there was widespread belief that the minimum length of sequence necessary to effect antisense inhibition of gene expression is 12 to 14 nucleotides (Wagner, 1994). However, it has now been shown that the specificity of binding to the target sequence by use modified oligonucleotides comprising C-5 propinopyrimidines and phosphorothioate internucleotide linkages can be sufficiently increased so that sequences which are only 7 or 8 nucleotides in length have a gene-selective, mismatch-sensitive, ribonuclease H-dependent inhibition flanking sequences of the target RNA are important for determining specificity (Wagner, et al, 1996).

In vivo is considered to counteract the expression of a gene successful use of antisense nucleotides, however Factors such as the need for cosuppression of expression mutant genes (Milan, 1993, McInnes and Bascom, 1992), or by the need for high concentrations of antisense nucleotides (Akhtar and Ivinson, 1993).

To Today, this form of therapy largely covers the use of antisense sequences packaged in liposomes, or a direct application of antisense cDNA or antisense oligonucleotides into the illness (Review 14.14a). Consequently, attempts were the inclusion of antisense sequences in the target cell by encapsulation to increase these sequences in liposomes, mostly unsuccessful. Furthermore It turns out to be difficult to liposomes in an efficient manner to focus on a target, with the recording even more less than viruses. Direct application of the oligonucleotides into a disease focus, such as stenotic plaque cheap in animals but with big ones Amounts of oligonucleotides needed and the procedure lengthens the balloon angioplasty procedure, thereby the danger for increased the patient.

The Targeted gene therapy was also used of viruses, such as an adenovirus, to give one to achieve virus-mediated DNA transfer.

adenoviruses were not dividing in themselves for the purpose of gene transfer Cells, as well as in proliferating cells on a large scale used. You can Absorb DNA up to 7.5 kb, providing efficient transfection cause and have a high viral titer. The essential Disadvantages of adenoviruses are their short-term gene expression as well as the risk of their use. This is a result of lacking Integration of the transgene into the cell genome. Consequently, this is Procedure inefficient, whereby the use of viruses may be an undesirable high risk for iatrogenic diseases, which is usually the case.

It Accordingly, it is an object of this invention to provide improved methods for one Targeted control of gene therapy in humans for treatment of diseases and conditions, the preparation of suitable compositions which are a hyaluronan form for the Use in such treatment include improved uses of the product / composition containing a hyaluronan form for include targeted control of gene therapy, and pharmaceutical Compositions comprising hyaluronan forms suitable for use in gene therapy are suitable to provide.

Further, and other objects of this invention will be appreciated by those skilled in the art following summary of the invention, as well as from the detailed Description of corresponding embodiments taken.

SUMMARY THE INVENTION

Whether gene therapy is used to treat a disease or condition involving the treatment of cells expressing receptors for hyaluronan, the gene therapy-mediated treatment improves by combining the therapy with an effective amount of one Hyaluronan form (hyaluronic acid form) such as sodium hyaluronate, which is preferably present in an amount of at least about 50 mg of the hyaluronan form per 70 kg of person, such as at least 500 mg of a hyaluronan form per 70 kg of subject (e.g. when gene therapy involves the treatment of cells expressing receptors for hyaluronan forms). The effective amount of gene therapeutic agent may now be an effective amount, due to the increased delivery of the hyaluronan form to the cell nuclei, below the level considered by those skilled in the art to be routine or generally accepted for treatment. The calculation of these amounts of hyaluronan form is based on the test results obtained here (within the scope of the tests, use of 100 μg / ml and smaller amounts, such as 10 μg / ml, of hyaluronan form - see page 22 of this document) and on The fact that an average person (70 kg person) has about 5 liters of blood. The total amount of water in an average weight person (70 kg) is approximately 40 liters (ie 0.55 L / kg), which is an average of 57% of his / her total body weight. About 25 liters of the 40 liters of body fluid are located within approximately 750000000000 cells of the body, and collectively called "intracellular fluid".

The extracellular liquid (the remaining liquid) can be in interstitial fluid, Plasma, cerebral / spinal fluid, liquid of the inner eye, and liquids of the gastrointestinal tract. The total amount liquid in the extracellular Compartment is an average of 15 L / 70 kg adult (i.e., 0.2 L / kg).

blood contains both extracellular liquid (the liquid of the plasma) as well as intracellular fluid (the fluid the red blood cells). The average blood volume of a normal adult is quite exactly 5 liters. On average, approximately 3 liters of this plasma, and the remaining 2 liters are red blood cells.

professionals will take that from changes in the weight of the patients the effective amounts of hyaluronic acid form, which in each treatment of taking a gene therapy Patients used change. It is assumed that the adjustment is proportional to the weight the person being treated. A person, for example 100 kg, preferably at least 71.4 mg, for example about 714 mg of hyaluronic acid form be administered. Such low levels of hyaluronic acid form per person can do something be reduced and continue to be effective (for example, at 49 or 48 mg / 70 kg person). The bigger the Reduction, the lower the effectiveness will be. However, in the frame the treatment of the patient a lot of, for example, 500 mg increased in hyaluronic acid form, so may such amount is 1000 mg or more. Since hyaluronic acid has no toxicity, the Hyaluronic acid forms without negative effects in larger quantities be used. Such amounts may be 1500 mg or more, such as about 3000 mg, exceed.

As a result, is according to one Aspect of the invention a hyaluronan form (for example, hyaluronic acid and / or pharmaceutically acceptable salts thereof, such as sodium hyaluronate) bound to an agent (for example non-covalently bound) / associated, which is an antisense orientation to (against) a gene, a cDNA or RNA DNA oligonucleotide or an RNA-DNA oligonucleotide hybrid or a messenger RNA which communicates with a disease or condition is brought, the product to a patient in a suitable thinner and in any suitable administration form, such as intravenous Administration, injection, etc., administered in an effective amount is used to determine the function of the target gene (s) Genes in the control or treatment of the disease and / or of the condition, for example in the treatment of the cells, and the cell nuclei, whose genetic composition is modified will thus express HA receptors such as RHAMM or CD-44. Such treatment is given as long as for treatment the disease or condition is required. A suitable Amount of hyaluronan form for each single treatment includes more than about 50 mg / 70 kg of person, preferably about 500 mg / 70 kg person, and will be according to the weight of the Patient to whom the composition is administered or adjusted below. The appropriate amount of HA in gene therapy is dictated by the time it takes to get the disease or to treat the condition, such as through time, which is required to dissolve the disease or condition. The Amount of gene therapy agent may be greater than the amount in the absence a hyaluronan form in ordinary Gene therapy usually is used due to the mediated by the hyaluronan form increased Transports are reduced.

• Synthesis of Sulfonated Hyaluronan Derivatives containing Nucleic Acid Bases, Wada, T., Chirachanchai, S., Izawa, N., Takemoto, K., Chemistry Letters 1994 (11), 2027–2030 (1994), und
• Transport performance of nucleosides through nucleic acid base conjugated hyaluronan, Chirachanchai, S., Wada, T., Inaki, Y., Takemoto, K.: Chemistry Letters 1995 (2), 121–122 (1995, Artikel der Februarausgabe, Computerzusammenfassung).

There are many possible methods of attachment / attachment of hyaluronan to the antisense agent (DNA), which are exemplified in two articles discussed below:

• Synthesis of Sulfonated Hyaluronan Derivatives containing Nucleic Acid Bases, Wada, T., Chirachanchai, S., Izawa, N., Takemoto, K., Chemistry Letters 1994 (11), 2027-2030 (1994), and
• Hyaluronan, Chirachanchai, S., Wada, T., Inaki, Y., Takemoto, K .: Chemistry Letters 1995 (2), 121-122 (1995, February Article, Computer Abstract). ,

We have now shown in this application that the interaction of HA (labeled with a fluorochrome) with the cell receptors results in the uptake of HA into the cytoplasm as well as into the nucleus of the cells. This process is rapid, takes place within minutes, and culminates in the accumulation of receptor / hyaluronan complexes within the nucleus and around the cell nucleus where the genome is located. Thus, hyaluronan, which associates with the gene therapeutic agent (such as antisense DNA), interacts with cell receptors, resulting in the uptake of HA and the gene therapy agent into the cell and into the nucleus.

According to one Another aspect of the invention may be the cDNA or the oligonucleotide or oligonucleotide hybrid which is (s) to a gene that is in a People should be treated (neutralized), antisense orientation having, with the hyaluronic acid form (Hyaluronan form) combined (such as bound), and the combination then the patient intravenous be administered, which is different from those in the prior art submitted proposals represents the use of viruses, liposomes or a include direct application of oligonucleotides, all of them have a low uptake efficiency, not specific to a target structure are or not for recommended for use in humans (such as Viruses).

According to one Another aspect of the invention therefore relates to this invention the production of cDNA or oligonucleotides or oligonucleotide hybrids, which opposite the gene or messenger RNA that has a disease to treat is related to a condition to be treated (in connection brought), have an antisense orientation, wherein the Antisense cDNA or the antisense oligonucleotide or antisense oligonucleotide hybrid is associated / bound to a hyaluronan form (as described above), and administering the combination to the patient for neutralization of the gene becomes. Because hyaluronic acid is absorbed by receptors, which are in areas of the disease or the condition, such as injury or inflammation, in high levels are expressed, the antisense cDNA or the Antisense oligonucleotide or antisense oligonucleotide hybrid by receptor endocytosis of the antisense cDNA or the antisense oligonucleotide or antisense oligonucleotide hybrid in the nucleus or in the vicinity of the cell nucleus, which then allows the antisense agent to to neutralize the relevant gene, and thus, for example, the In any case, the production of the protein produced by this gene increases prevention.

According to one Another aspect of the invention thus becomes novel compositions provided the product of a hyaluronan form and a Antisense agents which are associated / bound to one another, with a suitable diluent (such as sterile water), the antisense agent across from a gene that is related to diseases or conditions of the human body has antisense orientation.

A Form of gene therapy is the use of RNA DNA oligonucleotides. In this therapy treatment, the correction of a mutant Form of an extrachromosomal gene (such as in sickle cell anemia) or of the plasmid by using a composite of DNA and RNA residues chimeric Achieved oligonucleotide. For example, a short, double-stranded vector becomes activated by incorporation of RNA residues for recombination and by capping both ends before exonucleolytic decomposition protected. The 2'-O-methyl-modified Ribose of RNA resulted to protect against cleavage due to ribonuclease H (RNase H) activity.

To effect a correction of the β ^s mutation (for example, for sickle cell anemia), the chimeric oligonucleotide (SC1) was designed as a single molecule (with two sequences that were reversed and complementary) that is capable of self-formation to form a duplex structure refold. The molecule was composed of DNA residues with two intervening blocks of 10 2'-O-methyl-RNA residues flanking a short range of five DNA residues. When the molecule was folded into the duplex conformation, the sequence of one strand encompassed all the DNA residues, while the other strand contained the RNA-DNA blocks. In this case, the internal sequence is complementary to the β ^s globin sequence over a range of 25 residues extending across the site of the β ^s mutation except for a single base (T). The five DNA residues flanked by RNA residues were aligned around the mutant T residue in the β ^s -coding sequence. A chimeric control oligonucleotide (SC2) was designed in the same way except for a base.

This chimeric molecule was prepared in advance (see "Correction of the Mutation Responsible for Sickle Cell Anemia" by RNA-DNA Oligonucleotides, Cole-Strauss, Allyson et al., Science, Vol. 273, September 6, 1996, pp. 1386-8) of a commercial liposome formulation into lymphoblastoid cells (β-cells) which were homozygous for the β ⁵ allele.

The chimeric molecule can now be introduced by means of an effective amount of a hyaluronan form. Thus, according to another aspect of this invention, this chimeric molecule can now be introduced into the human being to be treated, the chimeric molecule being accompanied by a hyaluronic acid form which introduces the chimeric molecule into each cell to accumulate in the nucleus and around the nucleus where the genome is located, subsequently affecting the genetic material within the cell.

A effective amount of, for example, by intravenous administration in a suitable excipient (for example, sterile Water) hyaluronan form is greater than about 50 mg / 70 kg person, for example more than 500 mg / 70 kg person, where the amount is administered as long as necessary.

• a) eine Zusammensetzung, umfassend eine Hyaluronan-Form assoziiert mit/gebunden an Antisense-Nukleinsäure,
• b) ein Behandlungsverfahren, umfassend den Schritt der Verabreichens von Antisense-Nukleinsäure assoziiert mit/gebunden an eine Hyaluronsäure-Form, und
• c) die Verwendung einer Hyaluronan-Form (wie etwa Hyaluronsäure und pharmazeutisch annehmbare Salze hiervon {wie beispielsweise Natriumhyaluronat}) und Antisense-Nukleinsäure zur Herstellung einer pharmazeutischen Zusammensetzung, umfassend eine Kombination aus Hyaluronan-Form und Antisense-Nukleinsäure, wie etwa ein Antisense-Nukleinsäure-Hyaluronsäure-Komplex in Verbindung mit einem geeigneten Verdünnungsmittel, zur Behandlung eines Menschen, welcher an einer Erkrankung oder an einem Zustand leidet, die/der durch Anwendung der Gentherapie behandelbar ist.

Accordingly, in accordance with various aspects of the invention:

• a) a composition comprising a hyaluronan form associated with / linked to antisense nucleic acid,
• b) a method of treatment comprising the step of administering antisense nucleic acid associated with / attached to a hyaluronic acid form, and
• c) the use of a hyaluronan form (such as hyaluronic acid and pharmaceutically acceptable salts thereof (such as sodium hyaluronate)) and antisense nucleic acid for the preparation of a pharmaceutical composition comprising a combination of hyaluronan form and antisense nucleic acid, such as an antisense A nucleic acid-hyaluronic acid complex in association with a suitable diluent for the treatment of a human suffering from a disease or condition treatable by the use of gene therapy.

Lots Hyaluronan forms can for the although it may be preferred, although preferred are, which are described below. In particular are suitable Hyaluronan forms with a molecular weight between about 150000 Dalton and about 750000 daltons, prepared in sterile water with a viscosity for intravenous administration.

A special pharmaceutical grade form is a 1% sterile sodium hyaluronate solution (50 ml vial) provided by Hyal Pharmaceutical Corporation and having the following properties: Testing specifications 1. Description of the container 150 ml flint glass vial with red or gray rubber stopper and 20 mm aluminum seal 2. Description of the product clear, colorless, odorless, transparent, slightly viscous liquid 3. filling volume 50.0 to 52.0 ml 4. pH 5.0 to 7.0 at 25 ° C 5. Specific density 0.990 to 1.010 at 25 ° C 6. Intrinsic viscosity 4.5 to 11.0 dL / g 7. Molecular weight 178,000 to 562,000 daltons 8. Sodium hyaluronate assay and identification 9.0 to 11.0 mg / ml. positive 9. suspended matter no visible suspended matter 10. Sterility meets sterility requirements, USP 23 11. Bacterial endotoxins (LAL) complies with the requirements for bacterial endotoxins, USP 23

This pharmaceutical grade 1% sterile solution of hyaluronan can be prepared from granules / powder having the following properties: Testing specifications 1. Description white or off-white granules or powder, odorless 2. Identification (IR spectrum) must comply with the reference standard range 3. pH (1% solution) between 5.0 and 7.0 at 25 ° C 4. loss of drying NMT 10.0% at 102 ° C for 6 hours 5. Glue residue between 15.0 and 19.0% 6. Protein content NMT 0.10% 7. Heavy metals NMT 20 ppm (according to USP 23, p. 1727) 8. Arsenic NMT 2 ppm (according to USP 23, p. 1724) 9. Solvent residues a) acetone: NMT 0.1 b) ethanol: NMT 2.0% c) formaldehyde: NMT 100 ppm 10. Sodium hyaluronate assay 97.0 to 102.0% (dry basis) 11. Intrinsic viscosity between 10.0 to 14.5 deciliters per gram 12. Molecular weight (calculated using the Laurent formula) between 500,000 and 800,000 daltons (based on intrinsic viscosity) 13. Total number of aerobic germs NMT 50 microorganisms / gram (according to USP 23, p. 1684) 14. Test for Escherichia coli Escherichia coli not present (according to USP 23, p. 1685) 15. Yeasts & molds NMT 50 microorganisms / gram (according to USP 23, p. 1686) 16. Endotoxins (LAL) NMT 0.07 EU / mg (according to USP 23, p. 1696)

Hyaluronan of topical quality may be suitable in certain circumstances, which may be prepared from the following granules / powder having the following properties: Testing specifications 1. Description white or off-white granules or powder, odorless 2. Identification (IR spectrum) must comply with the reference standard range 3. pH (1% solution) between 6.0 and 8.0 at 25 ° C 4. loss of drying NMT 10.0% at 102 ° C for 6 hours 5. Glue residue between 15.0 and 19.0% 6. Protein content NMT 0.40% 7. Heavy metals NMT 20 ppm (according to USP 23, p. 1727) 8. Arsenic NMT 2 ppm (according to USP 23, p. 1724) 9. Solvent residues a) acetone: NMT 0.1 b) ethanol: NMT 2.0% c) formaldehyde: NMT 100 ppm 10. Sodium hyaluronate assay 97.0 to 102.0% (dry basis) 11. Intrinsic viscosity between 11.5 to 14.5 deciliters per gram 12. Molecular weight (calculated using the Laurent formula) between 600,000 to 800,000 daltons (based on intrinsic viscosity) 13. Total number of aerobic germs NMT 100 microorganisms / gram (according to USP 23, p. 1684) 14. Test for Staphylococcus aureus Staphylococcus aureus not present (according to USP 23, p. 1684) 15. Test for Pseudomonas aeruginosa Pseudomonas aeruginosa not present (according to USP 23, p. 1684) 16. Yeasts & molds NMT 200 CFU / gram (according to USP 23, p. 1686)

This Material in topical quality can subsequently be sterilized.

Other Forms, such as a hyaluronic acid form and / or salts thereof (for example, sodium salt), which also in an amount offered by Hyal Pharmaceutical Corporation can, can also be suitable. Such a quantity constitutes a 15 ml vial with sodium hyaluronate 20 mg / ml (300 mg / vial - lot 2F3). The sodium hyaluronate fraction is a 2% solution with a mean average molecular weight of about 225000. The amount contains about that In addition, water q.s., which in accordance with U.S. P. for Formulations for injection is triple-distilled and sterile. The vials with hyaluronic acid and / or salts thereof in a type 1 borosilicate glass vial be included, which is closed with a butyl stopper, not with the contents of the vial responding.

• i) einem Molekulargewicht im Bereich von 150000–225000,
• ii) weniger als etwa 1.25% an sulfatierten Mucopolysacchariden, bezogen auf das Gesamtgewicht,
• iii) weniger als etwa 0.6% an Protein, bezogen auf das Gesamtgewicht,
• iv) weniger als etwa 150 ppm an Eisen, bezogen auf das Gesamtgewicht,
• v) weniger als etwa 15 ppm an Blei, bezogen auf das Gesamtgewicht,
• vi) weniger als 0.0025% an Glucosamin,
• vii) weniger als 0.025% an Glucuronsäure,
• viii) weniger als 0.025% an N-Acetylglucosamin,
• ix) weniger als 0.0025% an Aminosäuren,
• x) einem UV-Extinktionskoeffizienten von weniger als etwa 0.275 bei 257 nm,
• xi) einem UV-Extinktionskoeffizienten von weniger als etwa 0.25 bei 280 nm, und
• xii) einem pH im Bereich von 7.3–7.9. Bevorzugt wird die Hyaluronsäure mit Wasser vermischt, wobei die Fraktion der Hyaluronsäure-Fraktion ein mittleres durchschnittliches Molekulargewicht im Bereich von 150000–225000 aufweist.

The amount of hyaluronic acid and / or salts thereof (for example, sodium salt) may include hyaluronic acid and / or salts thereof, which have the following properties:
a purified, essentially pyrogen-free, natural-source hyaluronic acid fraction having at least one property selected from the group consisting of the following properties:

• i) a molecular weight in the range of 150000-225000,
• ii) less than about 1.25% of sulfated mucopolysaccharides by total weight,
• iii) less than about 0.6% of protein by total weight,
• iv) less than about 150 ppm of iron by total weight,
• v) less than about 15 ppm of lead, based on the total weight,
• vi) less than 0.0025% of glucosamine,
• vii) less than 0.025% of glucuronic acid,
• viii) less than 0.025% of N-acetylglucosamine,
• ix) less than 0.0025% of amino acids,
• x) a UV extinction coefficient of less than about 0.275 at 257 nm,
• xi) has a UV extinction coefficient of less than about 0.25 at 280 nm, and
• xii) a pH in the range of 7.3-7.9. Preferably, the hyaluronic acid is mixed with water, the fraction of the hyaluronic acid fraction having a weight average molecular weight in the range of 150000-225000.

• i) weniger als etwa 1% an sulfatierten Mucopolysacchariden, bezogen auf das Gesamtgewicht,
• ii) weniger als etwa 0.4% an Protein, bezogen auf das Gesamtgewicht,
• iii) weniger als etwa 100 ppm an Eisen, bezogen auf das Gesamtgewicht,
• iv) weniger als etwa 10 ppm an Blei, bezogen auf das Gesamtgewicht,
• v) weniger als 0.00166% an Glucosamin,
• vi) weniger als 0.0166% an Glucuronsäure,
• vii) weniger als 0.016% an N-Acetylglucosamin,
• viii) weniger als 0.00166% an Aminosäuren,
• ix) einem UV-Extinktionskoeffizienten von weniger als etwa 0.23 bei 257 nm,
• x) einem UV-Extinktionskoeffizienten von weniger als etwa 0.19 bei 280 nm, und
• xi) einem pH im Bereich von 7.5–7.7.

Preferably, this amount of hyaluronic acid comprises at least one property selected from the group consisting of the following properties:

• i) less than about 1% of sulfated mucopolysaccharides by total weight,
• ii) less than about 0.4% of protein by total weight,
• iii) less than about 100 ppm of iron by total weight,
• iv) less than about 10 ppm of lead, by total weight,
• v) less than 0.00166% of glucosamine,
• vi) less than 0.0166% of glucuronic acid,
• vii) less than 0.016% of N-acetylglucosamine,
• viii) less than 0.00166% of amino acids,
• ix) a UV extinction coefficient of less than about 0.23 at 257 nm,
• x) a UV extinction coefficient of less than about 0.19 at 280 nm, and
• xi) has a pH in the range of 7.5-7.7.

Schwermetalle, maximale Menge in ppm Gesamtzahl mikrobieller Keime wird nicht beobachtet Endotoxin < 0.07 EU/mg NaHy Biologischer Sicherheitstest besteht den Kaninchenaugen-Toxizitätstest Other hyaluronic acid forms and / or their salts offered by other suppliers may also be selected, such as those described in prior art documents, which include hyaluronic acid forms having lower molecular weights between about 150000 daltons and 750,000 daltons such as 1-2% solutions in sterile water for intravenous administration. In addition, the sodium hyaluronate manufactured and offered by LifeCore ^™ Biomedical, Inc. may also be suitable, having the following properties (if sterile): properties specification appearance white to off-white particles odor no noticeable smell Viscosity average molecular weight <750000 daltons UV / Vis scan, 190-820 nm corresponds to the reference scan OD, 260 nm <0.25 OD units Hyaluronidase Sensitivity positive answer IR scan corresponds to the reference pH, 10 mg / g solution 6.2-7.8 water maximum 8% protein <0.3 mcg / mg NaHy acetate <10.0 mcg / mg NaHy

Heavy metals, maximum amount in ppm Total number of microbial germs is not observed endotoxin <0.07 EU / mg NaHy Biological safety test passes the rabbit eye toxicity test

The The following references report hyaluronic acid, sources thereof, and processes for their production and recovery.

The Canadian Patent 1,205,031 (which is assigned to U.S. Patent 4,141,973 referred to as state of the art) refers to hyaluronic acid fractions with an average molecular weight of 50,000 to 100,000, 250,000 to 350,000, and 500,000 to 730000, and explains procedures for their production.

When high molecular weight hyaluronic acid (or salts or other forms thereof) is used, it must be diluted before use, for example in conjunction with the antisense agent, to allow for administration and to ensure prevention of intramuscular coagulation. It has recently been discovered that high molecular weight hyaluronic acid, which has a molecular weight in excess of 1,000,000 daltons, forms its own aggregates and thus does not interact well with HA receptors. Consequently, higher molecular weight hyaluronic acid (such as Healon ^™ ) should be avoided.

• (a) einem durchschnittlichen Molekulargewicht, welches größer als etwa 750000, bevorzugt größer als etwa 1200000, ist – d.h. eine begrenzende Viskositätszahl besitzt, welche größer als etwa 1400 cm³/g, und bevorzugt größer als etwa 2000 cm³/g, ist,
• (b) einem Proteingehalt von weniger als 0.5 Gew.-%,
• (c) einer Absorption von ultraviolettem Licht von weniger als 3.0 bei einer Wellenlänge von 257 Nanometern und weniger als 2.0 bei einer Wellenlänge von 280 Nanometern für eine 1%ige Natriumhyaluronat-Lösung
• (d) einer kinematischen Viskosität von mehr als etwa 1000 Centistokes, bevorzugt mehr als etwa 10000 Centistokes, für eine 1%ige Natriumhyaluronat-Lösung in physiologischem Puffer,
• (e) einer molaren optischen Rotation von weniger als –11 × 10³ Gradcm²/mol (an Disaccharid), gemessen bei 220 Nanometer, für eine 0.1–0.2%ige Natriumhyaluronat-Lösung in physiologischem Puffer,
• (f) keine signifikante zelluläre Infiltration des Glaskörpers und der vorderen Augenkammer, kein Lichtreflex im Kammerwasser, keine Trübung oder kein Lichtreflex im Glaskörper, und keine pathologischen Veränderungen in der Hornhaut, Linse, Iris, Retina und Aderhaut des Eulenaffenauges, wenn ein Milliliter einer 1%igen Natriumhyaluronat-Lösung, welche in physiologischem Puffer gelöst ist, in den Glaskörper eingebracht wird, wobei annähernd die Hälfte des vorliegenden flüssigen Glaskörpers ausgetauscht wird, wobei die HUA
• (g) steril und pyrogenfrei, und
• (h) nicht-antigen ist.

U.S. Patent 4,141,973 describes hyaluronic acid fractions (including sodium salts) with:

• (a) an average molecular weight which is greater than about 750,000, preferably than about 1,200,000, is larger - that is, a limiting viscosity number has which is greater cm ³ / g than about 1400, and preferably greater than about 2000 cm ³ / g, is,
• (b) a protein content of less than 0.5% by weight,
• (c) an absorbance of ultraviolet light of less than 3.0 at a wavelength of 257 nanometers and less than 2.0 at a wavelength of 280 nanometers for a 1% sodium hyaluronate solution
• (d) a kinematic viscosity of greater than about 1000 centistokes, preferably greater than about 10,000 centistokes, for a 1% sodium hyaluronate solution in physiological buffer,
• (e) a molar optical rotation of less than -11 x 10 ³ degrees cm ² / mol (on disaccharide), measured at 220 nanometers, for a 0.1-0.2% sodium hyaluronate solution in physiological buffer,
• (f) no significant cellular infiltration of the vitreous body and anterior chamber, no light reflex in the aqueous humor, no haze or light reflex in the vitreous, and no pathological changes in the cornea, lens, iris, retina and choroid of the Owl's eye when one milliliter of a 1 % sodium hyaluronate solution dissolved in physiological buffer is introduced into the vitreous body, with approximately half of the present liquid vitreous being exchanged, the HUA
• (g) sterile and pyrogen free, and
• (h) is non-antigenic.

According to one Aspect of the invention thus provides the invention a composition ready to use a gene therapy agent containing hyaluronic acid or a derivative thereof is bound / associated in association with a derivative thereof pharmaceutically acceptable diluent includes.

As indicated, the gene therapy agent may be a nucleotide sequence which has an antisense orientation to a target sequence. The target sequence is a nucleic acid sequence which coincides with the Cause or aggravation of a pathological condition in combination is brought. This target nucleic acid sequence can be a gene, a genomic DNA, a cDNA, a messenger RNA, or an RNA-DNA oligonucleotide or RNA-DNA oligonucleotide hybrid. Is the target nucleic acid sequence a genomic DNA, it can be present in a coding region prefers to store RNA residues by capping both ends Protect exonucleolytic decomposition, or in a regulatory Region.

alternative For example, the gene therapy agent may comprise a nucleic acid sequence which to be transferred to a target cell. The target nucleic acid sequence may be a genomic DNA, a cDNA, a messenger RNA, or an RNA-DNA oligonucleotide or RNA-DNA oligonucleotide hybrid. In this case, the nucleic acid can either a sense sequence, which are introduced into a target cell should, to perform a function, or may be an antisense sequence which are introduced in order to inhibit the function of a nucleic acid present in the target cell.

The invention further provides a method for treating a pathological condition in a A subject in need of such treatment, comprising the step of administering to the subject an effective dose of a composition of the invention over a period of time which may be as long as necessary. In some cases, one dose may be effective, while in others, treatment may take months.

It It is clear that the dose and mode of administration of the depend on the condition to be treated, whereby the attending physician or veterinarian will easily be able to determine the appropriate doses and administration forms. It will considering pulled that the compositions of the invention by intravenous Administration, injection, topical administration, for example on gels or sponges absorbed, or by direct administration into the treatment Tissue, for example by intraocular or intratumoral injection, can be administered.

The The subject to be treated can be a human or an animal.

The The invention further provides a process for the preparation of a composition according to the invention comprising the step of combining as described above, for example, binding of a gene therapy agent with hyaluronan and / or a pharmaceutically acceptable salt thereof, isolating of the associate formed in this way, combining the associate with a diluent (such as sterile water), and formulating it appropriately pharmaceutically acceptable Form (which of course is not toxic).

SHORT DESCRIPTION THE FIGURES

The 1 - 4 (Figures labeled photographs A, B, C and D) illustrate the in vitro uptake of hyaluronan (HA) at different times in cells and nucleus, where hyaluronan is labeled with CY-3 fluorochrome covalently linked to hyaluronan and cell lines (Fibroblasts) was added.

Consequently 1 (denoted as Figure A) is a photograph of hyaluronan labeled with CY-3 fluorochrome and added to live cells at time t = 0, the photograph showing intracellular uptake of HA.

2 (labeled as Figure B) represents a photograph of the combination at time t = 5 minutes after addition.

3 (labeled as Figure C) represents a photograph of the combination at time t = 10 minutes after addition.

4 (labeled as Figure D) represents a photograph of the combination at time t = 20 minutes after addition.

5 Figure 4 is a graph illustrating the binding of hyaluronan to DNA.

6 is a graph entitled "Proliferation of Rat Hepatoma Cells".

From the review of photographs of 1 to 4 (Photographs A, B, C, and D), it becomes clear that, as in Photograph C ( 3 ), which has penetrated CY-3 fluorochrome-labeled hyaluronan into the cytoplasm or into the cell nucleus and surrounds the cell nucleus (the dark circle lying in the center of FIG. C is now light-colored 10 minutes after introduction of the fluorochrome-labeled hyaluronan , surrounded with fluorochrome-labeled hyaluronan).

To Hyaluronan has disappeared for 20 minutes, possibly by turning it off brought out of the cell or was decomposed. The cells were cultured using a confocal Microscope photographed using laser, which the cell layer cut so that the HA (hyaluronan) of the cytoplasm is recognizable.

Referring to 5 For example, hyaluronan-Sepharose, sepharose alone, and the transcription factor jun-sepharose (a positive control) were incubated with genomic DNA. Bound DNA was amplified by polymerase chain reaction (PCR) and the DNA run on an agarose gel. The gel was photographed and the optical density determined. From the result shown in the graph It is clear that HA binds to the DNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The Invention will now be with reference to the following non-limiting Examples are described.

The following tests were performed. The goal was to provide targeted control of antisense RNA / DNA on tumor cells as well as increase the uptake of these oligonucleotides, thereby reducing the concentration required for biological efficacy. In order to increase the uptake of nucleotides into the cell efficiently, as well as to effect a targeted control of the nucleotides on the nucleus and other intracellular compartments, the method uses for this the hyaluronan polysaccharide (see 1 - 4 , Photographs A, B, C and D).

In mixed form, hyaluronan binds to RNA / DNA, and shows high affinity for interaction with the HA receptors RHAMM and CD44 ^1,2 . The interaction of HA with these receptors causes rapid uptake of HA and its targeting agent into the nucleus and other parts of the cell (see 3 - Photograph "C.") This rapid uptake increases the availability of antisense oligonucleotides for inhibiting RNA translation or DNA transcription.

Results

Out the cell lines containing different amounts of RHAMM and CD44 became a rat-derived hepatoma cell line 1548 selected (see Table A, B). In relation to to primary Hepatocytes and other hepatocyte cell lines expressed the rat cell line 1548 the highest Amounts of RHAMM and CD44.

As in 6 As shown, myc antisense oligonucleotides inhibit the proliferation of cell line 1548 at 5000 nmol to myc antisense oligonucleotide by 17%. By adding 100 μg / ml of HA, the required concentration of oligonucleotide decreased to 500 nmol, at which concentration inhibition of proliferation increased further to 53%. (In other tests, lower levels, such as 10 μg / ml, were successfully used on HA). When the concentration of oligonucleotide was reduced even further to 200 nmol in the presence of 100 μg / ml of HA, the inhibition was 32%. For cell lines that did not express high levels of RHAMM or CD44, the results were far less drastic.

Around determine if the effects of HA on an interaction specific for the myc antisense oligonucleotide are due an antisense oligonucleotide was designed which interferes with the RHAMM synthesis causes. This HA receptor regulates the MAP kinase cascade, which is the proliferation and progression of the G1 phase of the cell cycle controlled. Was this antisense oligonucleotide used, such was the sole use of 1000 nmol of the RHAMM antisense oligonucleotide a 42% inhibition of proliferation achieved while in the presence of HA with 50-100 nmol of antisense oligonucleotide a 37% inhibition was obtained.

These results indicate that HA reduces the required concentration (and hence the amount) of antisense oligonucleotides, for example, in cells expressing HA receptors by 20-25 fold (100 μg / ml of HA). This effect was observed for both RHAMM and myc antisense oligonucleotides, suggesting that this is a general effect. We conclude that HA represents an effective targeting agent with respect to cells (both cytoplasmic and nucleus) expressing HA receptors. ^(1,2) It has been shown that HA receptors undergo upregulation during tumorigenesis, embryogenesis, and inflammatory processes. ¹

References:

• 1. Sherman, L., Sleeman, J., Herrlich, P., and Ponta, H. 1994 Curr. Opin. Cell Biol. 6, 726-733.
• 2. Entwistle, J., Hall, C.L. and Turley, E.A. 1996 J. Cell. Biochem. 61, 569-577.
• 3. Hall, C.L., Yang, B., Yang, X., Zhang, S., Turley, M., Samuel, S., Lange, L.A., Wang, C., Curpen, G.D., Savani, R.C., Greenberg, A.H. and Turley, E.A., 1995 Cell 82, 19-28.

Table A: RHAMM (V4)

Table B: CD44 (SD)

The use according to the invention from HA convicts that Gene therapy agents to cells expressing HA receptors in the cell, and thus into the cytoplasm and into the nucleus. This is on it attributed to that effective amounts of HA forms simultaneously on cell membranes and other molecules bind, penetrating the nucleus and the effective amount introduce gene therapy agents into the cells. Be sufficient Levels of HA are administered to a person, which is effective Quantities of gene therapy for effective treatment of all cells gets to be effective (being the amount less than due to the effectiveness of the HA forms the usual used and administered by a professional), so the treatment can be effective. However, adequate Quantities are administered to each agent. The ability of the HA molecule, simultaneously for example, the RNA-DNA oligonucleotide and the cell membrane to bind or transfer the RNA-DNA oligonucleotide increases the contact time of the gene therapy agent in the vicinity of the cell and the nucleus.

Claims (27)

Use of a hyaluronan form selected from Hyaluronan, pharmaceutically acceptable salts thereof and combinations thereof, in association with a gene therapy agent selected from a nucleic acid molecule having a Having sense or antisense orientation to a molecule or a sense or antisense molecule which is selected from a gene, a nucleic acid, a cDNA or RNA DNA oligonucleotide or RNA-DNA oligonucleotide hybrids or messenger RNA, for production a pharmaceutical composition for control or treatment a subject with a pathological condition, in which a gene, a cDNA or RNA DNA oligonucleotide or RNA-DNA oligonucleotide hybrids or messenger RNA involved, with the gene therapy agent transported to the nucleus and the pathological condition includes cells which Express hyaluronan receptors, and wherein the cells are not retinal cells are. Use according to claim 1, wherein the hyaluronan has a molecular weight of less than 1,000,000 daltons. Use according to claim 1, wherein the hyaluronan has a molecular weight of less than 750,000 daltons. Use according to any one of claims 1 to 3, wherein the amount the hyaluronan form exceeds about 50 mg. Use according to any one of claims 1 to 4, wherein the composition in a suitable dosage form is administered to a patient and the amount of hyaluronan form exceeds about 500 mg. Use according to one of the preceding claims, wherein the gene therapy agent from an antisense molecule to an RNA DNA oligonucleotide or an antisense molecule to RNA-DNA oligonucleotide hybrids selected which are associated / bound to the hyaluronan form. Use according to one of the preceding claims, wherein the excipient Water comprises, and the gene therapy agent from an antisense molecule to a RNA DNA oligonucleotide or an antisense molecule to RNA-DNA oligonucleotide hybrids selected becomes. Use according to one of the preceding claims, wherein the gene therapy agent is a nucleic acid molecule which is a sequence comprising, in an antisense orientation to a target sequence is. Use according to one of the preceding claims, wherein the target nucleic acid sequence a genomic DNA, a cDNA, a messenger RNA or an oligonucleotide or oligonucleotide hybrid. Use according to any one of claims 1 to 7, wherein the gene therapy agent comprises a nucleic acid molecule, which is to be transferred to the nucleus of a target cell. Use according to claim 10, wherein the to be transferred Nucleic acid molecule genomic DNA, a cDNA, a messenger RNA or an oligonucleotide or DNA oligonucleotide which is protected at both ends by RNA. Use according to claim 11, wherein the nucleic acid molecule comprises a sense sequence, which introduced into a target cell should be to perform a function. Use according to claim 12, wherein the nucleic acid molecule is an antisense sequence which are introduced into the nucleus of a target cell is intended to inhibit the function of a nucleic acid present in the nucleus of the target cell. Use according to any one of claims 1 to 13, wherein the composition for one systemic administration is formulated by injection. Use according to any one of claims 1 to 13, wherein the composition for one topical administration is formulated. Use according to any one of claims 1 to 13, wherein the composition for one direct administration into the tissue to be treated is formulated. Use according to any one of claims 1 to 14, wherein the composition for one intravenous administration is formulated. Use according to any one of claims 1 to 14, wherein the composition for one systemic administration is formulated. Use according to any one of claims 1 to 13, which further association or binding of the gene therapy agent to the hyaluronan form under formation of a complex, and further the isolation of the complex formed in this way and the combination of the complex with a pharmaceutical excipient. Use according to one of the preceding claims, wherein the hyaluronan form has a molecular weight of less than about 750,000 Dalton, but has more than about 150,000 daltons. Use according to one of the preceding claims, wherein the hyaluronan form is sodium hyaluronate. Use according to one of the preceding claims, wherein the pharmaceutical composition is sterile. Use according to claim 22, wherein the pharmaceutical Composition intravenously should be administered. Use according to any one of claims 1 to 23, wherein the pathological Condition is a tumorigenesis. Use according to any one of claims 1 to 23, wherein the pathological Condition an inflammatory process is. Use according to one of the preceding claims, wherein the subject is a human. Use according to any one of claims 1 to 24 or 26, wherein the cell is a tumor cell.