HUT76846A - Hyaluronic acid and derivatives for modulation of cellular activity - Google Patents

Description

FIELD OF THE INVENTION The present invention relates to modifications of cellular activity, in particular human cellular activity, using specific molecular amounts and fractions of hyaluronic acid forms, including, for example, sodium hyaluronate and various molecules that mimic the hyaluronic acid forms.

Cell adhesion molecules, originally thought to serve only to keep cells in tissues together, are now increasingly believed to play a more complex role in the functioning of the human body [see, e.g., Biotech

260 (1993)].

New therapeutic methods based on cell adhesion molecules have been developed. One class of endothelial adhesion molecules is ICAM-1 endothelial adhesion molecule. It has been found that this ICAM-1 molecule delivers human rhinoviruses, which cause about 5% of colds, to cells in the body. Rhinoviruses enter the body cells by attaching to the ICAM-1 adhesion molecule.

Previously, it has been proposed to block cell infection using soluble ICAM-1. The soluble ICAM, on the other hand, had poor adhesion, so this idea was not found for market use.

In one of the attempts to prevent common colds, genetic engineering techniques have been used to attach antibody fragments to the rhinovirus binding portions of ICAM-1.

Another adhesion molecule, called CD44, is normally found on lymphocytes. However, the CD44 adhesion molecule also occurs on pancreatic tumor cells that are metastatic. CD44 molecules can mistakenly mask tumor cells and thus allow the free circulation of tumor cells in the bloodstream.

In our studies, we have discovered that ICAM-1 acts as a cell surface receptor for hyaluronic acid (hyaluronan). ICAM-1 is expressed (produced and delivered to the cell surface) in liver endothelial cells and corneal epithelial cells. In inflammation, cancer, and infection, ICAM-1 is overexpressed.

Hyaluronan also functions as a cell surface receptor called HARLEC (Hyaluronic Acid (Hyaluronan) Receptors Liver Endothelial Cells; liver endothelial cell hyaluronic acid (hyaluronan) receptors], which is also expressed (produced and delivered to the cell surface) in liver endothelial cells and corneal epithelial cells. (In our opinion, the two are one and the same adhesion molecule, or at least so closely related to one another that the two are almost the same molecule.)

The CD44 and RHAMM (HA-Mediated Motility Receptor) molecules are also cell surface receptors for hyaluronic acid (hyaluronan). THE

RHAMM is a regulatory molecule. CD44, on the other hand, is an adhesion molecule.

The objects of the present invention can be summarized as follows: novel methods for treating diseases and conditions; new uses of the hyaluronate forms (hyaluronan, HA); new dosage amounts of pharmaceutical compositions; and novel pharmaceutical compositions suitable for use in said treatment methods and said uses of the HA forms.

In addition, the present invention also provides novel methods for treating diseases and conditions which employ molecules that mimic hyaluronic acid and its pharmaceutically acceptable salts (e.g., sodium hyaluronate) in the sense that they bind to the same receptors (e.g. bind to high affinity cell surface receptors for hyaluronic acid), to which the forms of hyaluronic acid bind; new uses of these hyaluronic acid mimetics; new dosage amounts of pharmaceutical compositions containing such molecules; and novel pharmaceutical compositions suitable for use in said treatment methods and said uses of mimicking molecules.

Based on the following detailed teaching and the abstract of the invention, one of ordinary skill in the art will appreciate the additional advantages of this invention.

For the modification of the cellular activity of tissues and cells expressing a high affinity cell surface receptor for hyaluronic acid, such as an adhesion molecule (such as ICAM-1, HARLEC and CD44) and a regulatory molecule (such as RHAMM). The method of the invention provides the human patient with a non-toxic, effective amount of a medicament tolerated by the human body. .

A form of hyaluronic acid, e.g., hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, e.g., less than 750,000 daltons, such as 50,000, is administered in an art vehicle or vehicle, such as sterile water. sodium hyaluronate having an average molecular weight of less than about 100,000 daltons and having an average molecular weight of about 150,000 to about 225,000 daltons from Hyal Pharmaceutical Corporation; sodium hyaluronates as well as various molecular weight fractions of a sodium hyaluronate form, such as those described in U.S. Patent No. 1,205,031. (Fidia), for example

Fractions of a molecular weight of from 000 to 100,000 Daltons, 250,000 to 350,000 Daltons, and 500,000 to 730,000 Daltons, or other molecular weight fractions, such as less than 50,000 Daltons or from 100,000 to 150,000 Daltons, or homologues, analogs, derivatives, complexes of hyaluronic acid, esters, fragments and / or subunits thereof and / or combinations thereof, preferably a hyaluronic acid having a molecular weight of less than 750,000 daltons, a pharmaceutically acceptable salt thereof, such as sodium hyaluronate and combinations thereof, and molecules which mimic the aforementioned forms of hyaluronic acid that they are capable of binding to the same receptors as the hyaluronic acid form to modify the cellular activity of tissues and / or cells that express a molecule that functions as a high affinity cell surface receptor for hyaluronic acid, · · • · · · · · · · · · · · * ·· • ··· "· · ·« »* ·····

β is, for example, an adhesion or regulatory molecule.

Where an inflammatory response may occur in an area of injury or injury, inflammatory cells (such as neutrophils, macrophages, or other white blood cells) may migrate to said area. Damage caused by an inflammatory response may be greater than actual injury or trauma injury. This is the case, for example, with fibrosis. Upon administration of the hyaluronic acid form, the hyaluronic acid form binds to the HA receptor (the high affinity cell surface receptor) of the molecule, for example, an 'adhesion molecule' or a regulator molecule, thereby modifying the body's response and subsequent damage. Similarly, when molecules which mimic the hyaluronic acid form are administered in the sense that they are capable of binding to the same receptors as the hyaluronic acid form binds are administered, the mimicking molecules bind to the HA receptor. Examples of molecules that mimic the hyaluronic acid form include, but are not limited to, antibodies that bind to the active site of the receptor, peptides that attach to the receptor site, and synthetic compounds that attach to the receptor site.

The agents for treating the disease or condition may also be used in combination with the hyaluronic acid form and the molecules that mimic the hyaluronic acid form; in this case, the active ingredient enhances the modulation of the HA form and the molecule that mimics HA activity. In addition, the form HA directs the active ingredient to the adhesion molecule (e.g., ICAM-1, HARLEC, and CD44) or the lamina to the RHAMM regulator molecule.

Suitable agents include, but are not limited to, free radical scavengers (e.g., ascorbic acid (vitamin C)), anticancer agents, chemotherapeutic agents, antiviral agents such as nonoxynol in non-ionic surfactant such as Delien ™. -9 (nonylphenoxy-polyethoxyethanol), anionic surfactants such as cetylpyridinium chloride, cationic surfactants such as benzalkonium chloride, non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, naproxen, diclofenac and (+/-) - ketorolac tromethamine salt sold under the trademark Toadol ™, steroidal anti-inflammatory drugs, antifungal agents, detoxifying agents such as by enema, rectal administration, analgesics, bronchodilators, antibacterial agents, ischemia (e.g., dia betes and Berger's disease), monoclonal antibodies, topical minoxidil for hair growth, diuretics such as furosemide under the trademark Lasix ™, immunosuppressants such as cyclosporins, lymphokines such as interleukin-2, etc., alpha. - and interferon beta, etc.

Another object of the invention is a molecular fraction of a form of hyaluronic acid, such as hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, e.g., less than 750,000 daltons, such as sodium hyaluronate having an average molecular weight of 225,000 daltons, such as Hyal Pharmaceuti. ·· - *

· .. · ··: · ... ·.

An average molecular weight fraction of 150,000 to 225,000 Daltons from 8 Cal Corporation, as described in Canadian Patent No. 1,205,031, Fidia, e.g. 50,000 to 100,000 Daltons, 250,000 to 350,000 Daltons, and 500,000, Or other fractions or homologues, analogues, derivatives, complexes, esters, fragments and / or subunits of hyaluronic acid and / or combinations thereof, and molecules which mimic the aforesaid forms of hyaluronic acid in the sense that: they are capable of binding to the same receptors as the hyaluronic acid form to use to modify the cellular activity of tissues and / or cells expressing high affinity cell surface receptors in the human body. The hyaluronic acid form may be used in association with a pharmaceutically acceptable carrier or carrier (e.g., sterile water). Tissue or cellular modulation is increased in patients and / or patients. The HA and / or HA mimicking molecule binds to the HA receptors of the molecule, such as an adhesion or regulatory molecule, such as the high affinity cell surface receptor for the hyaluronic acid form. The HA form and the HA mimicking molecule may also be used with a suitable active ingredient (as described above). The HA form, the HA mimicking molecule, and the active ingredient are used in effective non-toxic amounts.

The HA form and the HA mimicking molecule can also be used to prevent a disease or condition (e.g., a cold) in which the HA form and / or the HA mimicking molecule is 9 bound to ICAM-1 adhesion molecules (not rhinovirus bound or linked to ICAM-1). -1 adhesion molecules), we prevent the entry of human rhinovirus into body cells.

Accordingly, the present invention provides a novel method of preventing a disease and / or condition in a human by modifying the cellular activity of a high affinity cell surface receptor for hyaluronic acid, such as by expressing an adherent or regulatory molecule and / or cellular activity in a human. a non-toxic effective amount of a hyaluronic acid form, e.g., hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, e.g., less than 750,000 daltons, such as less than 50,000 daltons, in a pharmaceutically acceptable carrier or vehicle such as sterile water. Sodium hyaluronate having an average molecular weight of 100,000 daltons and 150,000 or 225,000 daltons and an average molecular weight of 150,000-225,000 daltons from Hyal Pharmaceutical Corporation, and on 08/143983. Sodium hyaluronates and various molecular weight fractions of a sodium hyaluronate form, e.g.

Fractions described in Canadian Patent No. 205,031 (Fidia), for example between 50,000-100,000 daltons, 250,000-350,000 daltons, and 500,000-730,000 daltons or other fractions, or homologues, analogs, derivatives, complexes of hyaluronic acid, esters, fragments and / or subunits thereof and / or · · · ·

Combinations thereof, and molecules that mimic the aforementioned forms of hyaluronic acid in the sense that they are capable of binding to the same receptors as the form of hyaluronic acid. The HA form or the mimicking molecule is preferably administered in a pharmaceutically acceptable carrier (e.g., sterile water). In this way, for example, common colds can be prevented. The agents for the prevention of the disease and / or condition may also be administered with the hyaluronic acid form and / or the hyaluronic acid mimetic. For example, acetylsalicylic acid may be administered together with the hyaluronic acid form and / or the hyaluronic acid mimetic.

For example, administration of the hyaluronic acid form and the hyaluronic acid mimicking molecule in the human body inhibits cellular adhesion and / or modifies cellular activity. The HA and the mimicking molecule bind to the HA receptors at the high affinity cell surface receptor for hyaluronic acid, such as an adhesion molecule (such as ICAM-1, HARLEC and CD44) and / or a regulatory molecule (such as RHAMM). . Upon administration of the hyaluronic acid form and the mimetic molecule, the hyaluronic acid form and the mimetic molecule bind to the HA receptor of the molecule, thereby preventing the disease or condition (e.g., preventing the rhinovirus from binding to the ICAM-1 adhesion molecule and thus preventing the rhinovirus bound to ICAM-1, enter cells of the body).

Where therapeutic agents are used to prevent a disease and / or condition, the active compounds are in the form of hyaluronic acid.

- can be used with 11 to prevent a disease or condition. For example, people today take effective amounts of acetylsalicylic acid (aspirin) to prevent stroke. The active ingredient may be associated with an effective non-toxic amount of a suitable form of hyaluronic acid (e.g., a sodium hyaluronate having a molecular weight of less than 750,000 daltons) and / or an effective non-toxic amount of a mimicking molecule. In our opinion, the combination of the two active ingredients reduces the risk of stroke to a greater extent than the individual receiving aspirin alone. In addition, the hyaluronic acid form directs the active agent to areas requiring treatment because the hyaluronic acid form binds to a high affinity cell surface receptor for the hyaluronic acid form, such as an adhesion molecule (e.g., ICAM-1). , HARLEC and CD44) and a regulator molecule (e.g. RHAMM) and thus modifies their activity.

Administration can be, inter alia, intravenous, intraarterial, intraperitoneal, intrapleural and percutaneous. In addition, the active ingredient may be administered intradermally to the skin, for example, to target the epidermis, or may be applied topically to an effective amount, for example, to the mucous membrane, whereby the active ingredient is administered intranasally or rectally; in the latter case, for example, by enema or topical application in specific areas of the rectum. In addition, the active ingredient may be administered by direct injection.

- Also in 12 forms.

The form of hyaluronic acid to be administered may be administered in various effective doses, for example, in the range of from 10 mg to 1000 mg for a person weighing 70 kg. Optimal dosages for a person weighing 70 kg are between 50 mg and 500 mg. Because hyaluronic acid has no toxic effect in humans, large amounts of hyaluronic acid (e.g. up to 3,000 mg for a 70 kg person) can be used without adverse effects.

When applied topically, for example when applied to the skin or mucosa, the hyaluronic acid form should be administered in an amount of at least 5 mg per square centimeter of skin or tissue (including mucous membrane).

In the case of analgesia, the hyaluronic acid form should preferably be applied topically in an amount of at least 10 mg / cm.

The HA mimicking molecules may be administered in effective dosage amounts known to those skilled in the art.

For modes of administration other than topical administration, an effective dosage amount of the hyaluronic acid form in a subject weighing 70 kg may be at least about 10 mg and may be greater than 1000 mg. When a person weighing 70 kg is administered an NSAID, such as indomethacin (dissolved in N-methylglucamine), together with more than 200 mg of hyaluronic acid such as sodium hyaluronate, during which the NSAID (e.g., indomethacin dissolved in N-methylglucamine) amount of person 1 ·· · ···· ··

1-2 mg / kg body weight does not cause any major toxic side effects such as gastrointestinal disorder, neurological abnormality, depression, etc., even when indomethacin is used in larger amounts when needed. If the amount of hyaluronic acid is reduced below this value, the usual side effects of using NSIAD compounds may reappear. For topical application, the dosage amounts should contain at least about 5 'mg of hyaluronic acid form per 1 square centimeter of skin, mucosa, or tissue to be treated.

The active compounds used in combination with the hyaluronic acid form and / or the HA mimicking molecule are formulated into a formulation containing the active ingredient in an effective non-toxic amount for the particular treatment.

One form of hyaluronic acid and / or pharmaceutically acceptable salts thereof (e.g., sodium salt) and homologues, analogs, derivatives, complexes, esters, fragments and subunits thereof, preferably hyaluronic acid and salts thereof, for use in the present invention. , is a form sold by Hyal Pharmaceutical Corporation. This form contains sodium hyaluronate at a concentration of 20 mg / ml in a 15 ml vial (300 mg / vial - 2F3). The form corresponds to a 2% solution of sodium hyaluronate having a molecular weight of about 225,000 daltons. The vial contains three times the desired amount of distilled water for injection.

- Contains sterile water that complies with 14 U.S. Pharmacopoeia regulations. Vials for hyaluronic acid and / or salts thereof are vials made of borosilicate glass type I, closed with a butyl stopper which does not react with the contents of the vial.

The fraction / amount of hyaluronic acid and / or its salts (e.g., sodium salt) can be described by the following characteristics:

purified, substantially pyrogen-free, hyaluronic acid fraction obtained from natural sources and having at least one of the following characteristics:

(I) a molecular weight of between 150,000 and 225,000; (Ii) about 1.25% by weight of the total weight, less than sulfated mucopolysaccharides; (Iii) about 0.6% by weight of the total weight, less than protein; (Arc) at about 150 ppm based on total weight contains less iron; (V) at about 15 ppm based on total weight contains less lead; (Vi) Less than 0.0025% by weight of glucosamine; (Vii) Less than 0.025% glucuronic acid; (Viii) Less than 0.025% by weight of ΔΓ-acetylglucosamine; (Ix) Less than 0.0025% by weight of amino acids; (X) UV extinction less than about 0.275 at 257 nm has a coefficient;

(xi) has a UV extinction coefficient of less than about 0.25 at 280 nm; and (xii) a pH of between 7.3 and 7.9.

Preferably, the hyaluronic acid is mixed with water and the hyaluronic acid fraction has a molecular weight of between 150,000 and 225,000. More preferably, the hyaluronic acid fraction has at least one characteristic selected from the group consisting of:

(i) less than about 1% by weight of sulfated mucopolysaccharides based on total weight;

(ii) less than about 0.4% protein by weight;

(iii) less than about 100 ppm iron by weight;

(iv) less than about 10 ppm lead by weight;

(v) less than 0.00166% by weight of glucosamine;

(vi) less than 0.0166% by weight of glucuronic acid;

(vii) less than 0.0166% by weight of W-acetylglucosamine;

(viii) less than 0.00166% by weight of amino acids;

(ix) having a UV extinction coefficient of less than about 0.23 at 257 nm;

(x) has a UV extinction coefficient of less than about 0.19 at 280 nm; and (xi) a pH of between 7.5 and 7.7.

Additionally, we may recommend sodium hyaluronate sodium, for example, manufactured and marketed by LifeCore ' ^IM Biomedical, Inc., which has the following characteristics,

Typical appearance odor viscosity average molecular weight

UV / Vis spectrum (190-820 nm) Optical Density (OD) (260 nm) Hyaluronidase Sensitivity

IR spectrum pH (10 mg / g solution) water content protein content (1 mg sodium hyaluronate) acetate content (1 mg has:

DETAILED DESCRIPTION White-cream granules no detectable odor <750,000 Dalton corresponding to known spectrum <0.25 OD unit positive response according to known spectrum

6.2-7.8 Up to 8% <0.3 gg <0.3 gg in sodium hyaluronate) ······· · ···

- 17 maximum heavy metal content (ppm)

As Cd Cr Co Cu Fe Pb Hg Ni

2.0 5.0 5.0 10, 0 10.0 25.0 10.0 10, 0 5.0

microbial load no observable amount endotoxin content (1 mg <0.07 EU Sodium hyaluronate)

biosecurity enough for the rabbit eye testing toxicity test Skymart Enterprises, Inc. One called Hyaluronan HA-M5070 another sodium hyaluronate - sells a format that follows - has initial features:

Specification test

Results

serial HG1004 pH 6.12 chondroitin sulphate is not detected protein 0.05% heavy metals up to 20 ppm arsenic up to 2 ppm drying loss 2.07% combustion residue 16, 69% '·

intrinsic viscosity

12.75 dl / s (XW: 679,000)

nitrogen analysis microbiological

E. coli mold and yeast

3.14%

104.1%

80 / g negative up to 50 / g

Other forms of hyaluronic acid and / or salts (e.g., sodium salt) and homologues, analogs, derivatives, complexes, esters, fragments and subunits of hyaluronic acid, preferably hyaluronic acid and salts thereof, available from other vendors, such as those known in the art, may also be selected. The following references refer to hyaluronic acid as well as sources, methods of production and recovery of hyaluronic acid.

U.S. Patent 4,141,973 discloses hyaluronic acid fractions (including the corresponding sodium salts) having the following characteristics:

(a) having an average molecular weight of greater than about 750,000, preferably greater than about 1,200,000, i.e., having an intrinsic viscosity of about. Greater than 1400 cm ³ / g, and preferably greater than about 2000 cm / g;

(b) having a protein content of less than 0.5% by weight;

(c) an ultraviolet absorbance of a 1% sodium hyaluronate solution of less than 3.0 at 3.07 and less than 2.0 at 257 nanometers;

- 19 (d) 1% physiological buffered sodium hyaluronate3 2

kinetic viscosity of the solution is greater than about 1 x 10 m / s2 (1000 centistokes), preferably greater than 1 x 10 m / s (10,000 centistokes);

(e) a molar optical rotation of a 0.1-0.2% physiological buffered sodium hyaluronate solution at less than 220 nanometers less than -11x10 degrees-cm / mol disaccharide;

(f) if approximately half of the vitreous monkey vitreous humor is replaced by ml of 1% physiological buffered saline, there is no significant cellular infiltration in the vitreous humor and anterior chamber, no opacification or opacification in the aqueous humor. light reflection, and no pathological changes in the cornea, lens, iris, retina, and choroid;

(g) sterile and pyrogen-free; and (h) is not antigenic.

Canadian Patent No. 1,205,031 (U.S. Patent No. 4,141,937, which discloses prior art) discloses 50,000 and

Describes hyaluronic acid fractions of average molecular weight of 100,000, 250,000 to 350,000, and 500,000 to 730,000, as well as methods for preparing the fractions.

If high molecular weight hyaluronic acid (or en ··· · ··

- 20 -, -, - or other forms of the same), dilution should be used to permit administration and dilution to avoid intramuscular coagulation. .

The present invention relates to a dosage amount of a pharmaceutical composition which expresses tissues expressing a high affinity cell surface receptor for hyaluronic acid, such as an adhesion molecule (such as ICAM-1, HARLEC and CD44) and a regulatory molecule (such as RHAMM). and to modify the cellular activity of cells in the human body, which is a non-toxic, effective amount of a hyaluronic acid form, e.g., hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, e.g., 750, in a human carrier tolerated pharmaceutical carrier, e.g. Sodium hyaluronate having an average molecular weight of less than 000 Daltons, such as an average molecular weight of 225,000 Daltons, a sodium hyaluronate having an average molecular weight of 150,000 to 225,000 Daltons from Hyal Pharmaceutical Corporation, and U.S. Pat. sodium hyaluronates, as well as various molecular weight fractions of a sodium hyaluronate form, such as those described in Canadian Patent No. 1,205,031 (Fidia), e.g.

Fractions of molecular weight between 250,000 and 350,000 daltons and between 500,000 and 730,000 daltons, or other molecular weights, e.g., less than 50,000 daltons or between 100,000 and 150,000 daltons, or or subunits and / or combinations thereof and containing HA mimicking molecules that mimic the aforementioned forms of hyaluronic acid in the sense that they are capable of binding to the same receptors as the form of hyaluronic acid binds, so that the dosage amount modify the cellular activity of tissues and / or cells that express a molecule that functions as a high affinity cell surface receptor for hyaluronic acid, such as an adhesion or regulatory molecule.

As a result of treatment with the dosage amount, tissue and cellular modulation in the individual suffering from the disease or condition is enhanced. For example, modulation and / or regulation of cellular adhesion and / or cellular activity is inhibited as a result of administration of the hyaluronic acid form (HA) or the HA mimicking molecule. Both the hyaluronic acid and the HA mimicking molecule bind to the HA receptors of the molecule (e.g., adhesion molecule, regulator molecule, etc.). In this way, a disease or condition can be prevented. Unexpectedly, high affinity between HA and the HA mimicking molecule and the cell surface HA receptors allows modulation of the disease or condition, such as modulation of the inflammatory process, fibrosis, and oncogenic control (preventing cancer and metastasis).

The dosage amount of the pharmaceutical composition may also comprise, in an effective, non-toxic amount, a pharmaceutical agent or therapeutic agent containing a hyaluronic acid form or a hyaluronic acid form for the prevention of a disease or condition.

- 22 For treating a disease and / or condition associated with a mimicking molecule. The active ingredient can thus enhance the modulation of the activity of the HA form and the molecule mimicking the HA form. In addition, the HA form directs the drug to the cell surface receptor of the molecule, such as ICAM-1, HARLEC and CD44, and the RHAMM regulatory molecule.

According to the present invention, the dosage amount of a pharmaceutical composition may comprise the following components:

i) a non-toxic, therapeutically effective amount of a pharmaceutical and / or therapeutic agent for treating a disease or condition;

ii) a non-toxic effective amount of a hyaluronic acid form, such as hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, e.g., sodium hyaluronate having an average molecular weight of less than 750,000 daltons, such as 225,000 daltons, from Hyal Pharmaceutical Corporation; dalton sodium hyaluronate average molecular weight fraction; Sodium hyaluronates and various molecular weight fractions of a sodium hyaluronate form, e.g.

U.S. Patent No. 205,031 (Fidia), e.g., 50,000 to 100,000 Daltons,

250,000 - 350,000 Daltons and 500,000 - 730,000 Daltons or other, such as less than 50,000 Daltons

4444 · *

- 23 • ν · • 4 · ·

4 ··· »• 4 · · ·

4 «« ·

Fractions having a molecular weight of 4 or 100,000 to 150,000 Daltons, or homologues, analogs, derivatives, complexes, esters, fragments and / or subunits of hyaluronic acid and / or combinations thereof, and HA mimicking molecules as defined above The hyaluronic acid forms are mimicked in the sense that they bind to the same receptors as the hyaluronic acid form, so that the component modifies the cellular activity of tissues and / or cells in the human body that express a molecule. functions as a high affinity cell surface receptor for hyaluronic acid, such as an adhesion or regulatory molecule; and iii) a pharmaceutically acceptable carrier (e.g., sterile water);

wherein component (ii) is in such a form that when administered in a dosage amount of the pharmaceutical composition, component (ii) is capable of modifying the cellular activity of tissues and cells that, for example, have an adhesion molecule (e.g., ICAM-1, et CD44et) and a regulatory molecule (RHAMM), wherein the modification of cellular activity is based on the fact that component ii) binds to a receptor for the hyaluronic acid form and / or HA mimicking molecules, and component ii) it is capable of transporting component (i) to the body or skin immediately if:. component ii) is a hyaluronic acid for * ··· ··

- 24 ma.

Suitable agents include, but are not limited to, free radical scavengers (e.g., ascorbic acid (vitamin C)), anticancer agents, chemotherapeutic agents, antiviral agents, such as a nonionic surfactant such as nonoxyn in the Delien ™ contraceptive cream. 1-9 (nonylphenoxypolyethoxyethanol), anionic surfactants such as cetylpyridinium chloride, cationic surfactants such as benzalkonium chloride, non-steroidal anti-inflammatory drugs (NSAIDs), e.g. indomethacin, naproxen, diclofenac and (+/-) - ketorolac tromethamine salt sold under the trademark Toadol ™, steroidal anti-inflammatory drugs, antifungal agents, detoxifying agents such as enema, rectal administration, analgesics, bronchodilator , vascular ischemia (e.g., di abetes and Berger disease), anti-monoclonal agents, topical minoxidil for hair growth, diuretics such as furosemide under the trademark Lasix ™, immunosuppressants such as cyclosporins, lymphokines such as interleukin-2, etc., alpha and interferon beta, etc.

Dosage amounts of the pharmaceutical compositions may be stored in multi-dose containers, from which the individual dosage amount may be withdrawn.

The invention is illustrated by the accompanying drawings.

Figure 1 a) Immunohistochemical staining for CCI 218 tumor.

25 shows the use of polyclonal antibodies to the HARLEC HA receptor. Figure 1 (b) depicts a control obtained using preimmune antibodies. (See Materials and Methods for details).

Figure 2 shows the clearance of radioactivity from the blood following intravenous injection of 125 µg of I-T-HA into rats. Inner graph: Radioactivity in various organs of the rat 10 minutes after intravenous injection of 5 µg of I-T-HA. KC is a Kuppfer cell; PC is a parenchymal cell; and LEC is a liver endothelial cell.

Figure 3 shows a scintigraphic view of a nude rat 3 weeks after a CCL in one of the rats

218 human colon cancers were inoculated and 24 hours after injection of 1.5 mg HA containing 2.5 MBq of I-T-HA into 12 5 rats. In Figure 3 (b), the liver is covered with a lead shield. The arrow points to the tumor.

Figure 4 a) depicts immunohistochemical staining of a CCI 218 tumor after intravenous injection of I-T-HA using polyclonal antibodies to the HARLEC HA receptor.

Figure 4 (b) shows immunohistochemical staining at 4 hours after treatment with hyaluronidase.

Figure 4 (c) and (d) are the same as Figure (a) and (b), but preimmune (control) antibodies were used in these cases.

Figures 4 (e) and (f) are the tumors of Figures (a) to (d)

- 26 shows the staining of HA using biotinylated HA binding protein.

Figure 5 (a) shows a scintigraphic image of a naked rat carrying CCI 218-derived tumor that received 3.5 µg (0.75 MBq) of ¹²⁵ IT-HA intratumoral one day prior to capture.

Figure 5 (b) shows a scintigraphic image of a naked rat carrying CCI 218-derived tumor that was 3.5 gg (0.75 g) intraperitoneally 15 days prior to capture.

MBq) received ¹²⁵ IT-HA.

125

Figure 6 shows the inhibition of I-T-HA and LÉC cell association in culture and at 37 ° C. Results are mean ± standard deviation values; n = 3 (for control medium) and n = 6 (for 1A29 medium). (See Materials and Methods for details).

Figure 7 shows the clearance of radioactivity in tumor tissue and control muscle tissue 18-20 hours after 2 mg of I-T-HA by intravenous injection. Results are mean ± standard deviation values; n = 3. (See Materials and Methods for details).

Figure 8 shows frozen sections of tumor tissue stained for HA and HARLEC / ICAM-1, 18-20 hours after I-T-HA by intravenous injection. (See Materials and Methods for details).

Figure 8 (a): staining for HA;

Figure 8 (b): staining for HARLEC / ICAM-1;

• · • · · · · · · · · · · · · · · · · · · · · · · · · ·

Figure 8 c) Staining to HARLEC / ICAM-1 after treatment with hyaluronidase.

Further details of the invention are the following examples ^- is shown.

EXAMPLE 1

Hyaluronan (hyaluronic acid; HA or HYA) polysaccharide is rapidly cleared from the circulation, primarily by endothelial cells of the liver, via receptor-mediated endocytosis. The HA receptor on these cells has already been characterized; the receptor was purified from rat liver endothelial cells (LECs) and raised polyclonal antibodies. Other cell surface receptors for HA have also been described previously, such as the lymphocyte-guiding CD44 receptor and the HA-mediated receptor for fibroblasts (RHAMM). Many tumors have been reported to enrich HA and have also described HA binding sites on cells derived from such tumors. Previous observations have shown that transformed tissues such as murine mastocytomas and human breast carcinomas give positive staining for the 'HA receptors originally found on liver endothelial cells.

The present work was begun to determine whether HA binding sites are available in vivo in tumor tissue and to determine the relationship between these potential sites and the HA binding proteins described above.

- 28 MATERIALS AND METHODS

polysaccharides

HA used for labeling, uptake and cycle assays was extracted from avian tissue (Hyal Pharmaceutical Corporation, Toronto, Ontario, Canada). The molecular weight distribution of HA was determined by chromatography on a calibrated Sephacryl HR column of porosity 400, 1000 and 2000 (Pharmacia, Uppsala, Sweden), 0.25 M sodium chloride, 0.05% chlorobutanol.

The HA content of each fraction was monitored by measuring the absorbance at 214 nanometers. Radioactivity was measured by gamma counting on a Packard automotive gamma counter.

(The average molecular weight is about 450,000 daltons (derived from 500,000 to 800,000 medium molecular weight powders)).

Indication of HA

Following the activation of the polysaccharide in bromocyanide, HA labeling is described by Glabe et al., Preparation and Properties of Fluorescent Polysaccharides, Anal. Biochem. 130: 287-294 (1983)] with DL-tyrosine (Sigma Chemical

Company, St. Louis, Missouri, USA). At this time, 15 mg of HA was activated at pH 11 with 8 mg of bromocyanine per minute. The activated polysaccharide was equilibrated with Sephadex G25 (PD 10, 0.2 M borate buffer, pH 8.0).

Pharmacia, Uppsala, Sweden) was isolated from the reaction mixture by a short column. Activated HA was incubated overnight with 1 mg tyrosine (T). Tyrosine was bound to HA (T-HA) and then T-HA was separated from unbound tyrosine;

• · ··································································· · · ·

- 29 this separation was carried out on a PD10 column previously prepared with 8 g / l sodium chloride, 0.2 g / l potassium chloride, 0.2 g / l potassium dihydrogen phosphate and 1.15 g / l disodium equilibrated with phosphate buffered saline (pH 7.5) containing phosphate buffered saline (PBS) containing hydrogen phosphate.

The T-HA. part of it was iodinated with I. 100 µg T-HA and 0.5 mCi I were placed in a small test tube and 10 µg 1,3,4,6-tetrachloro-3a, 6a-diphenylglycouryl (Sigma Chemical Company, St. Louis, Missouri, USA). Unfiltered ¹²⁵ I was removed on a PD10 column equilibrated with PBS. The iodinated THA ( ¹²⁵ IT-HA) was stored at 5 ° C. The specific radioactivity is usually 1500-5000 dpm / ng.

125

I-T-HA retained its higher molecular weight profile during gel-permeation chromatography. We found that the 125

I-T-HA was cleared from circulation by the same kinetics and organ distribution as described previously for high molecular weight HA biosynthetically labeled. Isolated rat liver endothelial cells, 125 in vivo and in vitro, uptake the -I-labeled polysaccharide, indicating that binding did not affect binding to specific cell surface receptors on these cells (1,2).

immune Painting

All staining was performed using the ABC-elite method (Vectastain® Elite ABC). Frozen sections of 6 gm were prepared and the sections were placed on glass slides and filled with gelatine • · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- coated with 30-chromalun mixture (the gelatin-chromalun mixture was prepared by mixing 0.5 g of chromalun [KCr (SO ₄ ) ₂ ^X 12 H ₂ O) with 750 ml of distilled water, 5 g of gelatin and 250 ml of distilled water. water was heated to 56 ° C each and the two solutions were then mixed together. To remove non-specific binding to serum proteins, antiserum and preimmune serum were adsorbed on rat serum protein-coupled (6 mg protein / ml gel) Sepharose® 4B gel (Pharmacia LKB Technology, Uppsala, Sweden). The coupling was performed according to the manufacturer's specifications (Affinity Chromatography; principles &methods; Pharmacia LKB Technology, Uppsala, Sweden). Equal volumes of serum and gel were mixed and the resulting mixture was incubated for 6 hours at 4 ° C. Frozen sections were fixed in cold methanol for 10 minutes, dried for 10 minutes and washed with phosphate-buffered saline (PBS). The peroxidase was blocked with 0.3% hydrogen peroxide in methanol and the sections were washed again in PBS. An avidin / biotin blocking kit (Vector laboratories) was used to block endogenous biotin and biotin binding activity. The sections (S) were then in 4% goat serum in PBS (Vectastain

Elite ABC) were incubated for 30 minutes. HARLEC antiserum was diluted 1: 300 in PBS containing 4% goat serum and incubated for one hour. The sections were washed with PBS and then diluted with PBS in a 1: 200 dilution with a second goat anti-rabbit antibody (Vectastain Elité ABC) for 30 min. · · · · · · · · · · · · · · · · · · · · · · · ·· · · · ·

- 31 incubated. After incubation with the second antibody, sections were washed and incubated with ABC-Elite Complex (Vectastain Elite ABC). For color development, a solution of 10 mg of 3-amino-9-ethylcarbazole in 6 ml of dimethylsulfoxide (DMSO) was mixed with 45 ml of 15 mM acetate buffer (pH 5) and 4 μΐ of Perhydroll (Merck) and then stirred for 7.5 minutes. incubations were performed. After washing the sections

Mayer was stained with hematoxylin. The glass slides were placed in Kelser glycerol gelatin (Merck).

Treatment of sections with hyaluronidase

After methanol fixation and washing in PBS, sections were incubated at 37 ° C for 2 hours with 5 U / ml Streptomyces hyaluronidase (Amano Pharmaceutical Co., Ltd, Japan), 1 mg / ml pepstatin (Sigma Chemical). Company, St. Louis, Missouri, United States), 0.1 M W ethyl ethyl maleimide, 0.1 M EDTA (Merck) and 5 KIE / ml

Trasylol (Bayer). Subsequently, we followed the usual staining procedures.

Examination of in vivo recording

14 to 30 days prior to the experiments, under ether anesthesia, four female naked (Nu / Nu) rats weighing 200 to 250 g were exposed to the CCL 218 human cell line (American β) in one hind paw.

Approximately 3x10 cells of Tissue Type Collection) were inoculated subcutaneously (in 0.5 mL RPMI medium). The animals were anesthetized with pentobarbital (45 mg / kg body weight) and injected with 3.4 to 1500 pg (5-15 to ^{10 6} cpm) of ¹²⁵ IT-HA into the tail vein 2 or directly into a tumor of approximately 1-2 cm. The specific • · ·

- Unlabeled HA was also occasionally administered to reduce 32 activity and to increase the chemical content of HA [0.05-1.0 ml of 0.15 M sodium chloride and 10 mM sodium dihydrogen phosphate (pH 7.4).

After the test, the animals were sacrificed and the tumor and organs were analyzed for radioactivity. Data were processed on a Macintosh SE / 30 or Macintosh Ilsi computer (Apple Computer Inc., Cupertion, California, USA) and charted using Cricket Graph (version 1.3, Cricket Software, Malvern, Pennsylvania, USA) and was created using Canvas (version 3.0.2, Deneba Systems Inc., Miami, Florida, USA).

Scintigraphic examinations

The rats were anesthetized and injected as described above. In dynamic assays, rats were injected on a standard medium resolution collimator of the gamma camera. Images were captured using a Gamma 11 system (Philips) with a 34 keV and 80% window setting, in word mode, in a 64x64 pixel (pixel) matrix. The dynamic sequence was preset at 1 fpm. Fifteen minutes later, still images were taken at various times after injection. The images were then transferred to the Hermes system (Nuclear Diagnostics, Hagersten, Sweden) and the regions of interest (ROI: 6) were drawn. In some cases, the area may be larger than the tumor or liver due to scattering

- 33 blanket ROIs had to be drawn.

RESULTS

Immunohistochemical studies have shown that polyclonal antibodies to HARLEC recognize structures in the tumors of the CCL 218 human colon cancer cell line in nude rats. Staining was mainly localized in vascular endothelium and tumor cells, where it was enhanced towards the edge of the tumor [1. Preimmune antibodies showed virtually no staining [Fig. 1 a]. Figure b), which indicated that HARLEC staining was specific to this system.

125

When tracer levels were injected intravenously with I-T-HA, the radioactivity was rapidly transferred from the blood to the liver and, after a few minutes, only a small amount of radioactivity remained in the blood (Figure 2).

Because of the high binding capacity of the liver, few tracers were left in the blood after passing through the liver and only very small amounts could reach other tissues. As a result, the animals were injected with amounts in excess of the hepatic binding capacity. At a dose of 1.5 mg HA, the substance was still present in the general circulation after 24 hours and, according to a scintigraphic examination, radioactivity accumulated in the tumors (Figure 3). Most of the radioactivity was detected above the liver, but significant background activity was also observed above the tumor [3. Figure a)]. The activity above the tumor was many times higher than would have been expected by the tumor size alone. Covering the liver with a lead radiation shield, we obtained scintigraphic images that showed 50% of the liver today. · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··

34 x levels were well matched to tumor size [Fig. 3 Figure b).

Immunohistochemical analysis of treated animals showed that treatment of sections with hyaluronidase caused a specific increase in HARLEC immunoreactivity [4. Figure a) and b) without increasing the preimmune response [Figure 4]. Figures c) and d)]. Extensive receptor staining coincides with staining for HA [4. (e) and (f)], HA staining was most pronounced in the marginal zone and in and around the vessels, although other receptor stained structures were found to be positive for HA.

In order to reduce hepatic uptake and to attempt a therapeutic response, two animals received a single injection of mtratumoral I-T-HA. One rat received 3.4 pg of high specific activity (0.25 MBq / gg) labeled ΗΆ, more than 90% of which remained in the tumor. Small amounts reached the general circulation, which was then taken up by the liver [5. Figure a)]. Fifteen days after injection, the ratio of tumor to liver radioactivity was similar to that observed immediately after injection [5. Figure b). The animal's response was to reduce the size of the tumor from about 2 cm to about 1 cm within 7 days. The tumor gradually diminished and softened until day 14, when the tumor was no longer palpable. On day 15, the animal was sacrificed and subsequent histochemical analysis showed that the tumor was approximately

He was always present at a size of 0.5 cm. The other rat, although 250 µg, had only 0.025 MBq of HA

- 35 received intratumoral, no response, and approximately 5 cm tumor was found in the animal's body at the time of sacrifice (two rats killed at the same time).

DISCUSSION

The fact that HAR'LEC immunohistochemical staining was clear in tumors untreated with hyaluronidase (Figure 1) indicated that the binding sites were to some extent free, that is, that there was potential for uptake of circulating HA. This observation is consistent with that observed for liver receptors.

EXAMPLE 2

HA (hyaluronic acid and its pharmaceutically acceptable salts) is eliminated from the bloodstream by receptor endocytosis mediated primarily by hepatic endothelial cells (LECs). The HA receptor on the LEC (HAR) was characterized by HA affinity chromatography of surface-labeled rat LEC and purified from LEC membranes to homogeneity. This receptor (HARLEC for short) is in the range of 85 kD to 100 kD with an average molecular weight of about 90 kD and about

It has a pl value of 6.7. A monospecific polyclonal antibody (in short, anti-HARLEC) has been developed against the receptor which is capable of inhibiting the binding of HA to LEC and LEC membranes. Using this antibody, 85-90 kD protein specimens were also detected with the antibody in the kidney, spleen, thymus and lymph nodes. The same tissues - with the liver - are anti-· · ·

- 36 -HARLECs give positive staining, immunohistochemical staining is mainly restricted to vascular regions such as sinusoids in the liver, spleen and lymph nodes, as well as in small intestine capillaries, although it appears in certain specific structures such as thymicus reticular cells . CEC staining is inhibited if the corneas are treated with HA prior to staining, while staining intensity can be restored by treatment with hyaluronidase.

HARLEC was purified from rat rat liver by a series of affinity chromatography steps. In the final step, virtually all HA bound to HA-Sepharose, and specific elution using HA oligosaccharides was performed. Trypsin digestion of purified HARLEC resulted in several peptides which were separated by reverse-phase chromatography. For four peptides, the sequence was determined and found to be identical

Intracellular adhesion molecule-1 (ICAM-1). ICAM-1 is a glycosylated, single-chain 80-114 kD protein having a 55 kD polypeptide nucleus. It is normally expressed only in small amounts, but ICAM-1 has already been detected on the endothelium of the normal liver at the sinusoids and on the endothelium of certain lymph nodes, spleen and kidney, and on the corneal endothelial cells. The immunohistochemical localization of ICAM-1 is in good agreement with known results previously obtained with HARLEC staining. ICAM-1 is also localized in tissues in which the former • · · • · · · ····

- 37 bans showed HA binding and HA uptake.

HARLEC / ICAM-1 is expressed in tumor endothelium in mouse mastocytomas and is capable of binding to intravenously radiolabelled HA. Significant increases (approximately 5 times the control) in tumor tissue were observed.

During immunohistochemical examination, we found that HA is localized in areas that also give positive staining for ICAM-1, i.e., mainly in blood vessels. After treatment of the sections with hyaluronidase, ICAM-1 immunoreactivity increased dramatically.

Further evidence of the HA receptor function of HARLEC / ICAM-1 is that HA also targets human tumors in nude rats and that control is mainly via binding to HARLEC / ICAM-1 on the tumor endothelium.

MATERIALS AND METHODS

polysaccharides

HA used for labeling, uptake and cycle assays was extracted from avian tissue (Hyal Pharmaceutical Corporation, Toronto, Ontario, Canada). The average molecular weight was approximately 450,000 daltons.

Indication of HA

HA was labeled with DL-tyrosine (Sigma Chemical Company, St. Louis, Missouri, USA).

• ·

- 38 Monoclonal antibodies

The rat monoclonal antibody to ICAM-1 (clone 1A29) was obtained as a gift from Professor M. Miyasaka (Osaka University, Japan).

Cultivation of endithelial cells

Rat liver endothelial cells (LEC) were isolated after perfusion of rat liver collagenase and cultured in RPMI medium without serum.

Antibody inhibition experiments

100,000 to 200,000 liver endothelial cells cultured for 4-5 hours were incubated with 0.5 μg / ml IT-HA, whereas in competing experiments, they were performed with unlabeled HA or 1: 2 diluted anti-ICAM-1 supernatant medium. incubation. Since at anti-ICAM-1 contained 115 ng / ml HA, a control medium containing 115 ng / ml HA was also prepared and used as a control at 1: 2 dilution to inhibit HA-related binding activity in the antibody supernatant. . Competitors were added prior to the addition of radiolabeled HA. Total incubation time was 35-40 minutes. After incubation was completed, the media was removed, the cells were washed, and the radioactivity was analyzed as described previously (10).

immune Painting

All staining was performed using the ABC elite method (Vectastain Elite ABC). Frozen sections of 6 μιη were prepared, and the sections were placed on glass slides and coated with a gelatin-chromalun mixture. Serum Protein Link • «· · · ·

- HARLEC antiserum was adsorbed on Sepharose 4B gel (Pharmacia LKB Technology, Uppsala, Sweden) coupled with rat serum proteins (6 mg protein / ml gel) to remove 39 doses of non-specific binding.

Frozen sections were fixed in cold methanol for 10 minutes, dried for 10 minutes, and then washed with phosphate-buffered saline (PBS). The peroxidase was blocked with 0.3% hydrogen peroxide in methanol and the sections were washed again in PBS. An avidin / biotin blocking kit (Vector laboratories) was used to block endogenous biotin and biotin binding activity. The sections were then incubated in PBS (Vectastain® Elite ABC) containing 4% goat serum for 30 minutes. HARLEC antiserum was diluted 1: 300 with PBS containing 4% goat serum and incubated for one hour. Sections were washed with PBS and incubated with PBS diluted 1: 200 in goat anti-rabbit antibody (Vectastain Elite ABC) for 30 minutes.

HA staining was performed on frozen sections of cartilage frozen biotinylated hyaluronan binding proteins (b-HABP) treated with methanol and avidin / biotin blocking kit (Vector Laboratories) to block endogenous biotin binding activity.

After incubation with the second antibody or b-HABP, sections were washed and incubated with ABC-Elite Complex (Vectastain Elite ABC). For the development of color, a solution containing 3,3-diarainobenzidine or 3-amino-9-ethylcarbazole • · · ···

40 sets of oxidase substrates (Vector Laboratories) were used and sections were incubated for 5-10 minutes in the mixture. After washing, sections were re-stained in Mayer hematoxylin for 1.5 minutes. The glass slides were placed in Kelser glycerol gelatin (Merck).

Treatment of sections with hyaluronidase

After methanol fixation and washing in PBS, sections were incubated at 37 ° C for 2 hours with 5 U / ml Streptomyces hyaluronidase (Amano Pharmaceutical Co., Ltd, Japan), 1.8 µg / ml pepstatin ( Sigma Chemical Company, St. Louis, Missouri, United States), 1.8 mM EDTA (Merck), 1.8 µg / ml soybean trypsin inhibitor (Sigma), 2.0 mM iodoacetic acid (Sigma), 0, 18 mM 6-amino-n-hexanoic acid (Sigma) and 9.0 mM benzamide (Sigma). Subsequently, we followed the usual staining procedures.

Examination of in vivo recording

14 to 30 days prior to the experiments, under ether anesthesia, CCL 218 human cell line z was found in one hind leg of approximately 200 g of female bare (Rowette Nu / Nu) rats:

(American Tissue Type Collection) approximately 3x10 cells were inoculated subcutaneously [in μΐ s-MEM medium (Gibco)]. The animals were anesthetized and received 2 mg (2-3 x ^{10 6} cpm) of ¹²⁵ IT-HA by intravenous injection. From time to time, unlabeled HA (0.05-1.0 mL of 0.15 M sodium chloride and 10 mM sodium dihydrogen phosphate (pH) was also administered to reduce specific activity and to achieve a higher chemical content of HA). 7.4).

·· * ·· »» «· · · · · · · · · · · · ·

The animals were sacrificed after 18-20 hours and examined for radioactivity as described above (9) for tumors and organs.

RESULTS

To further characterize HA HARLEC / ICAM-1 binding, experiments were performed with isolated rat liver endothelial cells (LEC), radiolabeled HA, and a well-characterized rat

With a monoclonal antibody to ICAM-1 [Ialenti, A. and Di

Rosa, M., Hyaluronic acid modulates acute and chronic infiam1? R matlon, Agents Actions, £ 3, 44-47]. The cell association between I-T-HA and LEC at 37 ° C was inhibited by about 60% (Figure 6). However, approximately 20% of the inhibition was derived from HA in the antibody tissue culture medium, as indicated by the inhibition seen in a control experiment using the same amount of HA but without the antibody (Figure 6).

In order to examine whether HARLEC / ICAM-1 expressed on endothelial cells of tissues other than rat has the same HA binding capacity as was observed for LEC, one of the hind paws of human colon carcinoma had a tissue distribution assay. It was found that 18 to 20 hours after the intravenous injection of 2 mg I-T-HA, most of the radioactivity was as expected in the liver, but only a very small amount of radioactivity in the blood (not shown). The radioactivity in the tumor was more than three times that in the control muscle tissue (wet weight

- 42 • · · »» · · · * * · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·). This increase was statistically significant (p = 0.001, n = 3) (Figure 7).

When the tumor tissue was analyzed by histochemical methods, it was found that HA in the tumor was predominantly in the tumor connective tissue skeleton (stroma) and blood vessels [8. Figure a)], whereas ICAM-1 is predominantly localized in blood vessels in the same areas [Figure 8 Figure b). ICAM-1 staining, although clearly visible, was surprisingly poor [Fig. 8 Figure b). However, staining was dramatically enhanced after treatment with hyaluronidase [8. Figure c).

DISCUSSION

The observation that rat ICAM-1 monoclonal antibodies are able to inhibit cellular association of labeled HA with cultured rat liver endothelial cells provides further evidence that ICAM-1 is an essential cell surface receptor for HA. Although inhibition is only about 50%, since cellular association was studied at 37 ° C, inhibition was not complete due to the continuous binding of free receptors from within the cells. High molecular weight HA exhibits an extremely high affinity for receptors on hepatic endothelial cells, and labeled HA is expected to be a major competitor for binding to free receptor sites. It should also be noted that other HA binding sites not affected by 1A29 may be present on ICAM-1.

Monoclonal antibodies used in the assay have previously been shown to inhibit leukocytes in the liver.

- 43 ····························································································································································································· (End (time) to the endothelial cells. Because antibodies cause inhibition of binding between HA and liver endothelial cells, HA binds to a site close to the leukocyte binding site on ICAM-1. Binding of HA to endothelial cells inhibits the adhesion of leukocytes to the same cells. Thus, systemic administration of HA may have a beneficial effect on inflammatory conditions. Similar effects to the anti-inflammatory effects achieved by systemic treatments with ICAM-1 antibodies have been described previously in animal models of acute and chronic inflammation.

Increased uptake of radiolabeled intravenously administered HA into tumor tissues (Figure 7) indicates that the tumor has structures for binding HA. HA staining with the highly specific biotinylated HA binding protein indicated that HA was localized in and around blood vessels. As expected, HARLEC / ICAM-1 staining was also found in blood vessels, although it was significantly weaker [8. Figure b). Application of HA to corneal endothelial cells (CEC) resulted in reduced immunohistochemical staining of CEC receptors, which, in turn, was reversible by treatment with hyaluronidase. In view of this, we have attempted to perform such hyaluronidase treatment on tumor sections. As a result, a

HARLEC / ICAM-1 staining, indicating that HA bound to HARLEC / ICAM-1 caused inhibition of specific antibody binding.

Intravenously administered HA binds predominantly to endothelial cells via ICAM-1. This suggests that HA

as a carrier for delivering cytotoxic drugs to tumors expressing this type of HA receptor.

In the preferred embodiments of the invention described above, many changes can be made within the scope of the invention. It is to be understood that the materials used in the above-described embodiments are illustrative only and are not intended to limit the scope of the invention in any way.

Claims (33)

PATENT CLAIMS CLAIMS 1. Tissues and cells expressing a high affinity cell surface receptor for a form of hyaluronic acid, such as an adhesion molecule such as ICAM-1, HARLEC and CD44, and / or a regulatory molecule such as RHAMM. modifying its cellular activity in the human body by administering to the human body a non-toxic effective amount of a human carrier tolerated pharmaceutical carrier such as sterile water, a form of hyaluronic acid such as hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, e.g. sodium hyaluronate having an average molecular weight of less than 750,000 daltons, such as 225,000 daltons, and / or molecular weight fractions of a hyaluronic acid form, such as sodium hyaluronate, homologues, analogs, derivatives, complexes, esters, fragments and / or / or these co and a molecule mimicking a hyaluronic acid form, which mimics the aforementioned hyaluronic acid forms in the sense that it is capable of binding to the same receptors as the hyaluronic acid form, in order to modify the cellular structure of such tissues and / or cells. which express a molecule that functions as a high affinity cell surface receptor for hyaluronic acid, such as an adhesion molecule. 2. The method of claim 1, wherein the method comprises treating a disease and condition. An effective, non-toxic amount of a drug or therapeutic agent for treating the disease is administered. The method of claim 2, wherein the drug and / or therapeutic agent is selected from free radical scavengers such as ascorbic acid, i.e. vitamin C, anticancer agents, chemotherapeutic agents, antiviral agents, e.g. a nonionic surfactant such as nonoxynol-9- (nonylphenoxypolyethoxyethanol) in Delien ™ Contraceptive Cream, anionic surfactants such as cetylpyridinium chloride, cationic surfactants such as benzalkonium chloride, non-steroidal anti-inflammatory drugs active ingredients such as NSAIDs such as indomethacin, naproxen and (+/-) - ketorolac tromethamine salt sold under the trademark Toadol ™, steroidal anti-inflammatory drugs, antifungal agents, such as by enema, rectal administration, analgesics, bronchodilators, antibacterial agents, antibiotics, iron agents for the treatment of cularis ischaemia such as diabetes and Berger disease, antibody monoclonal agents, topical minoxidil for hair growth, diuretics such as furosemide sold under the trademark Lasix ™, immunosuppressants such as cyclosporins, lymphokines such as interleukins and or alpha and interferon beta. 4. A form of hyaluronic acid, such as hyaluronic acid, a pharmaceutically acceptable hyaluronate salt, e.g., an average molecule of less than 750,000 daltons, such as 225,000 daltons. - 47% by weight of sodium hyaluronate, - molecular weight fractions of a hyaluronic acid form, homologues, analogs, derivatives, complexes, esters, fragments and / or subunits of hyaluronic acid and / or combinations thereof and / or a molecule mimicking the hyaluronic acid forms - which imitates the aforementioned forms of hyaluronic acid in the sense that it is capable of binding to the same receptors as the form of hyaluronic acid, to use in the human body to modify the cellular activity of tissues and / or cells which are a they express a high affinity cell surface receptor for hyaluronic acid. The use according to claim 4, wherein an effective non-toxic amount of a drug is also used. The use of claim 5, wherein the drug is selected from free radical scavengers such as ascorbic acid, i.e. vitamin C, anticancer agents, chemotherapeutic agents, antiviral agents such as a nonionic surfactant such as Delien. ™ nonoxynol-9- (nonylphenoxypolyethoxyethanol) contraceptive cream, anionic surfactants such as cetylpyridinium chloride, cationic surfactants such as benzalkonium chloride, non-steroidal anti-inflammatory drugs such as NSAIDs such as indomethacin, naproxen and (+/-) - ketorolac tromethamine salt sold under the trademark Toadol ™, steroidal anti-inflammatory drugs, antifungal agents, detoxifying agents such as enema, rectal, analgesics, bronchodilators, antibiotics, vascular ischemia, e.g. ul for the treatment of diabetes and Berger disease, antibody monoclonal agents, topical minoxidil for hair growth, diuretics such as furosemide under the trademark Lasix ™, immunosuppressants such as cyclosporins, lymphokines such as interleukin-2 and similar lymphokines. and interferon beta. A method of preventing a disease and / or condition comprising modifying the cellular activity of tissues and / or cells expressing a high affinity cell surface receptor for the hyaluronic acid form and thereby preventing the disease and / or condition in a human. a non-toxic effective amount of a hyaluronic acid form, such as hyaluronic acid, a pharmaceutically acceptable salt of hyaluronate, e.g., sodium hyaluronate having an average molecular weight of less than 750,000 daltons, such as 225,000 daltons, a homologous acid of a hyaluronic acid, analogs, derivatives, complexes, esters, fragments and / or subunits thereof and / or combinations thereof and / or a molecule mimicking the above hyaluronic acid forms in the sense that the same it is able to bind to the throat, to which the hyaluronic acid form binds. A process according to claim 7, characterized in that: 49, for the prevention of the disease or condition by co-administration of the active ingredient with the hyaluronic acid form, HA. 9. A dosage amount of a pharmaceutical composition for modifying the cellular activity of a high affinity cell surface receptor for hyaluronic acid, such as ICAM-1, HARLEC, CD44 and RHAMM, wherein the dosage amount is non-toxic. , an effective amount of a hyaluronic acid form such as hyaluronic acid, a pharmaceutically acceptable hyaluronate salt such as sodium hyaluronate having an average molecular weight of less than 750,000 daltons, such as 225,000 daltons, in a human carrier tolerated pharmaceutical carrier such as sterile water, molecular weight fractions of the hyaluronic acid form, homologues, analogs, derivatives, complexes, esters, fragments and / or subunits of the hyaluronic acid and / or combinations thereof and / or a molecule mimicking the hyaluronic acid forms referred to above It mimics uronic acid forms in the sense that it is capable of binding to the same receptors as the hyaluronsa.v form. The dosage amount of claim 9, wherein the dosage amount together with the hyaluronic acid form also comprises an effective non-toxic amount of a drug or therapeutic agent for treating the disease and / or condition. 11. A dosage amount of a pharmaceutical composition - 50, characterized in that the dosage amount contains the following components: i) a non-toxic, therapeutically effective amount of a pharmaceutical and / or therapeutic agent for treating a disease or condition; ii) non-toxic effective amounts of a hyaluronic acid form, e.g., hyaluronic acid, a pharmaceutically acceptable hyaluronate salt, e.g., sodium hyaluronate having an average molecular weight of less than 750,000 daltons, such as 225,000 daltons, homologues of hyaluronic acid, , derivatives, complexes, esters, fragments and / or subunits thereof and / or combinations thereof and / or a molecule mimicking the hyaluronic acid forms - which mimics the aforementioned hyaluronic acid forms in the sense that it is capable of binding to the same receptors as the hyaluronic acid form binds, a component whose function is to modify the cellular activity of tissues and / or cells in the human body that express a molecule that functions as a high affinity cell surface receptor for hyaluronic acid; and iii) a pharmaceutically acceptable carrier such as sterile water; wherein component (ii) is in such a form that, when used in a dosage amount of the pharmaceutical composition, component (ii) • ·· • · ···. · · ······ · · · · · · · · · 51 is capable of modifying the cellular activity of tissues and cells expressing in the human body a high affinity cell surface receptor for the hyaluronic acid form, such as ICAM-1, HARLEC, CD44 and RHAMM, wherein modifying cellular activity, for example, is based on the fact that component (ii) binds to a receptor for the hyaluronic acid form and / or the HA mimicking molecules, and component (ii) is immediately capable of transporting component (i) to the body or skin, if component ii) is a form of hyaluronic acid. 12. The dosage amount of claim 11, wherein the suitable pharmaceutical and therapeutic agents are selected from the group consisting of free radical scavengers such as ascorbic acid, i.e. vitamin C, anticancer agents, chemotherapeutic agents, antiviral agents such as a nonionic agent. surfactants such as nonoxynol-9- (nonylphenoxypolyethoxyethanol) in the Delien ™ contraceptive cream, anionic surfactants such as cetylpyridinium chloride, cationic surfactants such as benzalkonium chloride, non-steroidal anti-inflammatory drugs such as NSA such as indomethacin, naproxen and (+/-) - ketorolac tromet'hamine salt sold under the trademark Toadol ™, steroidal anti-inflammatory drugs, antifungal agents, detoxifying agents such as by enema, rectal administration, analgesics, bronchodilators, Wed ant, antibiotics, for treating vascular ischemia such as diabetes and Berger disease 52 active substances, antibody monoclonal agents, topical minoxidil for hair growth, diuretics such as furosemide sold under the trademark Lasix ™, immunosuppressants such as cyclosporins, lymphokines such as interleukin-2 and similar lymphokines or interferon alpha and beta. 13. One of Figs. A container containing multiple doses of a dose amount as claimed in any one of claims 1 to 5, from which the individual dose quantities may be withdrawn. Use of the following components in the manufacture of a medicament for modifying the cellular activity of a high affinity cell surface receptor for a form of hyaluronic acid: i) a non-toxic, therapeutically effective amount of a pharmaceutical and / or therapeutic agent for treating a disease or condition; ii) non-toxic, effective amounts of a hyaluronic acid form, e.g., hyaluronic acid, a pharmaceutically acceptable hyaluronate salt, e.g., sodium hyaluronate having an average molecular weight of less than 750,000 daltons, such as 225,000 daltons, homologues of hyaluronic acid, , derivatives, complexes, esters, fragments and / or subunits thereof and / or combinations thereof and / or a molecule mimicking the hyaluronic acid forms, which mimics the aforementioned forms of hyaluronic acid in the sense that it is capable of binding to the same receptors as hyaluronic band-for • · · Β ♦ · ··· Β »· ···· 53 today, which is responsible for modifying the cellular activity of tissues and / or cells in the human body that express a molecule that functions as a high affinity cell surface receptor for hyaluronic acid; and iii) a pharmaceutically acceptable carrier such as sterile water; wherein component (ii) is in such a form that when used in a dosage amount of the pharmaceutical composition, component (ii) is capable of modifying the cellular activity of tissues and cells that have a high affinity cell surface receptor for the hyaluronic acid form in the human body, such as ICAM-1. et al., HARLEC, CD44, and RHAMM, wherein the modification of cellular activity is based, for example, on component (ii) binding to a receptor for the hyaluronic acid form and / or HA mimicking molecules, and component (ii) it is capable of transporting component (i) to the body or skin immediately if component (ii) is a form of hyaluronic acid. The use of claim 14, wherein modifying the cellular activity of the tissue and cells is used to treat or prevent one or more of the following diseases or conditions requiring treatment or prevention: (i) colds; (ii) stroke; ······································································ ··· - 54 (iii) inflammatory processes; (iv) fibrosis; and (v) oncogenic control. 16. The method of claim 1 or 2, wherein the modification of the cellular activity of the tissue and cells, as the case may be, is for the treatment or prevention of one or more of the following diseases or conditions requiring treatment or prevention: (i) colds; (ii) stroke; (iii) inflammatory processes; (iv) fibrosis; and (v) oncogenic control. The use according to claim 4 or 5, wherein the modification of the cellular activity of the tissue and cells is used to treat or prevent one or more of the following diseases or conditions requiring treatment or prevention, as appropriate: (i) colds; (ii) stroke; (iii) inflammatory processes; (iv) fibrosis; and (v) oncogenic control. 18. The method of claim 7 or 8, wherein the disease and / or condition to be prevented is one of the following: (i) colds; • · · · * · · · · · · · · · · · · · · - 55 (ii) stroke; (iii) inflammatory processes; (iv) fibrosis; (v) cancer and metastases; (vi) cancer and metastases by oncogenic control. Use of a hyaluronic acid form - HA - and a suitable diluent for the manufacture of a medicament for the prevention of stroke, cold or viral disease or condition in a human, wherein the hyaluronic acid form is selected from pharmaceutically acceptable salts of hyaluronic acid and / or combinations thereof, and wherein the effective amount of the hyaluronic acid form administered by attaching to the surface receptors expressed by tissues and cells prevents the disease or condition from entering the tissue and cells, so that the same sites on the surface expressed surface receptors for binding the disease or condition now. form, and thus the hyaluronic acid form prevents the disease or condition. 20. The use according to claim 19, further comprising administering an effective non-toxic amount of a drug. The use of claim 20, wherein the drug is selected from the group consisting of free radical scavengers, non-steroidal anti-inflammatory drugs, i.e. NSAIDs, acetylsalicylic acid, antiviral agents such as nonionic surfactants, anionic surfactants and cationic surfactants. materials, antifungal ··· ··· - 56 active ingredients, detoxifying agents and antibacterial agents. Use of a form of hyaluronic acid and a suitable diluent for the preparation of a dosage amount of a pharmaceutical composition for preventing a disease and / or condition, wherein the dosage amount is in a non-toxic effective amount of a hyaluronic acid form, a hyaluronic acid, an acceptable salt thereof and / or combinations thereof to bind the hyaluronic acid form to the expressed surface receptors of tissues and / or cells and thus prevent the disease or condition from binding to the same sites on the expressed surface receptors as the hyaluronic acid. form, and wherein the disease or condition is one of: (i) colds; (ii) stroke; (iii) a viral disease. The use of claim 25, wherein the dosage amount further comprises, in addition to the hyaluronic acid form, an effective, non-toxic amount of a drug or therapeutic agent for the prevention of the disease and / or condition. 27. The use of claim 26, wherein the therapeutic and therapeutic agents are selected from the group consisting of free radical scavengers, non-steroidal anti-inflammatory drugs (NSAIDs), acetylsalicylic acid, antiviral agents such as nonionic surfactants. 9 · • · · · · · · · · · · · · · · · · · · · · · · · · · · · - 57 substances, anionic surfactants and cationic surfactants, antifungal agents, detoxifying agents and antibacterial agents. The use of claim 22 or 23, wherein the disease or condition requiring prevention is one of the following: (i) colds; (ii) stroke; (iii) a viral disease. 29. Use of a hyaluronic acid form selected from the group consisting of hyaluronic acid, a pharmaceutically acceptable salt of hyaluronic acid, and combinations thereof, for the manufacture of a medicament for binding tissue and / or cell surface receptors expressing a high affinity cell surface receptor for the hyaluronic acid form, for the prevention of a disease or condition caused by the attachment of a virus to receptors that have a high affinity for the hyaluronic acid form, and thus the pharmaceutical composition prevents the virus from attaching to these receptors. 30. Use of hyaluronic acid in the human body for binding to tissue and / or cell surface receptors expressing high affinity cell surface receptors of the hyaluronic acid form and thus for the manufacture of a medicament for the prevention of a viral disease. 31. The use of hyaluronic acid is a disease ···· ·· · · 58, wherein the hyaluronic acid, by binding to the HA receptor of an adhesion molecule, prevents the organism carrying the disease or condition from binding to the cells and tissues of the body by attaching it to the adhesive molecule. 32. The use of claim 31, wherein the effective non-toxic amount is a pharmaceutical agent or therapeutic agent for controlling the disease. 33. The use of claim 20, wherein the drug is an effective amount of acetylsalicylic acid, i.e. aspirin. The use of claim 33, wherein the condition is stroke. 35. 4-6, 14, 15, 17 and 19-34. The use according to any one of claims 1 to 4, wherein the hyaluronic acid form has a molecular weight of less than 750,000 daltons. 36. Figures 4-6, 14, 15, 17 and 19-35. The use according to any one of claims 1 to 3, wherein the hyaluronic acid form has a molecular weight of greater than about 150,000 daltons.