KR19990071628A - How to Target Drugs, Therapeutics, and Other Glycozaminoglycan (GAGS) Dosages - Google Patents

Abstract

To treat a disease or condition of a person treatable by a substance capable of transporting a substance to a therapeutic area of the body and of a drug or therapeutic agent to the liver (eg, a carrier capable of binding to a receptor in the liver) In the method;

a) administering an effective amount of a first substance, such as chondroitin sulfate that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver; And

b) administering an amount less than the generally expected effective amount of the drug or therapy and an effective amount of a second substance to transport, wherein the second substance is different from the first substance and the second substance is the site requiring treatment Ability to bind to the liver, and to the liver if the liver is not "down-regulated", and to bind to the liver's phagocytic receptors. This actually decreases and the non-toxic effective amount of the second substance is generally characterized as being less than what is considered effective.

Description

How to Target Drugs, Therapeutics, and Other Glycozaminoglycan (VAS) Dosages

In US Application 07 / 675,908 (Hyal Pharmaceutical Corporation) and PCT Application PCT / CA90 / 00306, Bulletin WO91 / 04058 (Hyal Pharmaceutical Corporation), 10 mg of hyaluronic acid is used to treat areas in need of treatment in the human body. It is detailed that it carries an effective amount of agents and drugs and can penetrate the cells to be treated through all the membranes of the tissue to areas requiring treatment, including scar tissue.

Line 17, page 25 of the PCT application states that there is an additional advantage when using about 200 mg of hyaluronic acid (eg, hyaluronate sodium salt) to reduce their side effects when therapies and drugs are administered. Although side effects of these drugs and therapies, even when administered alone or in excess of their usual acceptable dosages when used alone, such as NSAIDs (non-steroidal anti-inflammatory agents), And associated with common drugs and therapies such as gastrointestinal disorders, neurological disorders, and nervous breakdowns.

The document (p. 25, line 26) elaborates as follows;

"In addition, a combination of NSAIDs (eg, Indocid ^TM ) with hyaluronic acid via the systemic venous route has been shown to result in a better targeted response to diseased tissues. 50-200 mg NSAID-hyaluronic acid (hyaluronate sodium salt) (eg indomethacin-hyaluronic acid) and other chemicals such as ascorbic acid [vitamin C], phloretin and anticancer agents The patient experienced immediate disappearance of the patient's pain with further administration, in the presence of tumors in the lungs or liver, resulting in the dissipation and resorption of renal tissue lesions in a short time, thus improving the function of these organs. And tumor toxins are used in vitro by the action of enhanced macrophages by additional administration of NSAIDs (or steroidal anti-inflammatory drugs) and hyaluronic acid (salts or other forms). Thus, Applicants add that hyaluronic acid to the NSAIDs releases the macrophage function by inhibiting the production of prostaglycan synthase, which blocks the function of the macrophages. Salts and other forms) may enhance the activity of NSAIDs as well as reduce any side effects and toxicity associated with the use of prostaglandin synthase inhibitors. "

U.S. applications 08 / 486,328 and 08 / 520,591 and PCT / CA95 / 00477 (Hyal Pharmaceutical Corporation) use hyaluronic acid to control tissues and cells that can express cell-surface receptors with high affinity for hyaluronic acid. It is detailed. Such cell surface receptors consist of the adsorption molecule ICAM-1, the adsorption molecule CD44 and the adsorption molecule HARLEC (Hyaluronic Acid [Hyaluronan] Receptos Liver Endothelial Cells), and the regulatory molecule Receptor for HA Mediated Motility (RHAMM) for hyaluronan binding. . HARLEC is expressed in hepatic endothelial cells (produced at the cell surface). An effective amount of hyaluronic acid that binds to cell surface receptors regulates cellular activity of tissues and cells that express cell-surface receptors (eg, adsorption or regulatory molecules) that have high affinity for hyaluronic acid in the human body. .

The application (p. 19, line 30) shows that the hepatic hyaluronan-binding activity is high. When the hyaluronan is administered, the liver goes to the liver first because of its greatest binding activity. It circulates systemically and gathers in tumors because the tumors have an excess of hyaluronic acid receptors compared to normal tissues and cells, and they are collected in tumors by the ability of tumor receptors to bind hyaluronic acid (hyaluronan).

Thus, when hyaluronic acid acts as a carrier of drugs and therapeutic agents that deliver drugs to body parts requiring treatment, such complexes are not administered directly to the areas requiring treatment, such as direct injection into a tumor. However, the liver will reach the liver in smaller amounts in areas that require treatment until the ability to bind to hyaluronan is saturated.

In Tovard C. Laurent ets., Biochem J. (1986) 234, 653-538, entitled “Binding of Hyaluronate and Chondroitin Sulfate to Liver Endothelial Cells,” the author states that “circulating HA is a sinusoid of the liver. Are effectively captured and metabolized by endothelial cells) and "chondroitin sulfate (CSA) is also captured and metabolized by liver endothelial cells". The authors also note that "partial inhibition of HA binding by CAS (Smedared et al., 1984) and inhibition of CSA binding by HA (Smedared et al., 1985) indicate that the two polysaccharides are recognized by the same receptor. This hypothesis can be confirmed using the same polymerized oligosaccharides made from the two polymers. "

Hepatic endothelial cells have two different binding proteins, HA (hyaluronan), which include phagocytic receptors that bind to both chondroitin sulfate and hyalonanane (and other glycosaminoglycans (GAGS)). Most of the binding proteins (phagocytic receptors in the liver) are previously bound to chondroitin sulfate, which inhibits the capture of GAGS (glycosaminoglycans), including chondroitin sulfate. It has also been found that sites requiring treatment with metastatic tumors do not have the same dietary receptors that bind to chondroitin sulfate. These sites have receptors that bind hyaluronan. Thus, administration of chondroitin sulfate, which is captured by the phagocytic receptors of the liver first, followed by hyaluronan, will not be captured in the liver.

It is therefore an object of the present invention to provide new methods of treating diseases and disorders in humans by providing more drugs and therapies to the body parts in need of treatment.

Yet another object of the present invention relates to targeting a site that requires treatment to deliver any drug and therapy to a site that requires treatment rather than another site of the body.

It is yet another object of the present invention to provide a novel dosage kit or a combination of substances or chemicals which in turn is targeted to a body part in need of treatment in order to deliver the drug and therapeutic agent to the area in need of treatment.

Another object of the present invention is to reduce the amount of drugs and therapies that are expected to be used to treat a disease or condition. In this regard, when a transport material is used to transport the drug and therapy to a disease or disease site, an object of the present invention is to deliver the drug and therapy to transport the drug and therapy to a site of treatment. The use of a carrier material (eg hyaluronan and its pharmaceutically acceptable salts) is less than the amount normally used. Thus, for example, low-toxic drugs (eg, methotrexate, cisplatin, etc.) are effective drug doses for successful treatment using less than the usual use of hyaluronan as a carrier material. May be necessary.

Those skilled in the art will understand further objects of the present invention in the following summary and examples.

Summary of the Invention

Chondroitin sulfate and hyaluronan bind to hepatic cells, in particular hepatic phagocytic receptors, and chondroitin sulfate does not bind to receptors in tumors (e.g. metastatic tumors), and the cell surface receptors of hyaluronan are regulatory molecules RHAMM (Receptor for HA Mediated Motility) and adsorption molecules ICAM-1, HARLEC, and CD44. This has led to the development of novel methods for treating diseases and disorders, including metastatic tumors. "Down" regulates phagocytic receptors by binding to chondroitin sulfate or other glycosaminoglycans, such as dextran sulfate administered to phagocytic receptors in liver cells, and subsequently to hyaluronic acid (hyaluro) with drugs and therapeutics. Continuous administration of hyaluronan (delivering drugs and therapeutic agents) is not carried out by the liver already filled with binding capacity, but rather by other sites (metastasis tumors) with excessive hyaluronan receptors Is collected by). At the same time, hyaluronan delivers drugs and therapies to sites that require treatment (cytotoxic agents effective for treating tumors). As a result, less pre-treatment with chondroitin sulfate and subsequent administration of carriers such as hyaluronan can be used less than before. It has been found that fewer drugs and therapies are used than previously required to successfully treat a disease or condition.

The amount of chondroitin sulfate required to down regulate liver cells is at least about 3-5 mg / kg body weight of the patient. However, more amounts may be used, as appropriate, to "stop" the hepatic binding function. It is not a good idea to use less chondroitin sulfate because the liver treats the administered chondroitin sulphate quickly, so after a few hours the liver treats all the chondroitin sulphate. Therefore, it is appropriate to prolong the "blocking" / "downregulation" or "immunization reaction" of liver cells. When chondroitin sulfate is administered (1-2 g in a 70 kg body), administration of chondroitin sulfate may prevent the liver from capturing even the smallest amount of hyaluronan (0.5-1 mg in a 70 kg body) for a significant amount of time. have. Thus, hyaluronan can be used to deliver drugs and therapeutic agents such as methotrexate or cisplatin to a tumor or to the kidney at the site in need of treatment or for other uses set forth in WO 91/04058. Can also be used.

Hyaluronan or its pharmaceutically acceptable salts which are effective for transportation, for example, the required amount of sodium hyaluronate having a molecular weight of 750,000 Daltons or less is actually reduced (e.g., less than 10 mg for a person weighing 70 kg) The amount of drugs and therapies is actually reduced rather than already used. Blocking the liver's binding capacity requires only a small amount of carrier, eg 20 μg / kg, and a drug, eg 10 μg / kg.

Other suitable compounds may be used in place of chondroitin sulfates such as sulfate dextran. Some may be used in the same amount as chondroitin sulfate; Others may be used in larger or smaller amounts. Other GAGSs include Dermatan sulphate or its proteoglycans, Keratan sulphate and the like. Keratan sulfate and the like are technically not glycosaminoglucuronoglycans but are included in GAGS here. Other phagocytic receptor ligands can also be used, such as acylated low density lipoprotein (LDL), acids such as poly-inosinic acid, and the like.

Continuously (e.g. after 3-4 minutes) (e.g. intravenous administration) after administration of chondroitin sulfate, hyaluronan (amounts, forms and molecular weights used are described in US applications 07 / 675,908 and PCT applications WO91 / 04058 and 08 / 468,328 (June 6, 1995), 08 / 50,591 (as of Aug. 30, 1995), drugs and therapeutics [at least 200 mg / 70 kg (weight)] and hyaluronic acid type used When used, the dose is used in excess of the amount normally used or in smaller amounts [(μg / kg) is used], using appropriate means. Because smaller amounts of drugs and therapies and hyaluronan may be used, they may actually be reduced from the amounts specified above and in the references cited therein. If the amount of drugs and therapies is reduced, the side effects are actually reduced. Therefore, the hyaluronic acid type can be reduced to 200 mg in the case of a person weighing 70 kg. In fact, along with “blocking” the liver, it was found that 5 μg / 250 mg (20 μg / 1 kg) hyaluronan was used to target the site in need of treatment in the rat. The same level of dose (μg / 250 mg rat) was found to be useful for the area requiring treatment. Thus, the dosage in WO91 / 04058 may be modified within the scope of the present invention.

In the WO91 / 04058 application it is detailed as follows;

i) In line 3 on page 17 to line 16 on page 18,

"Applicants administer complexes and preparations (injectable formulations) to mammals to treat a disease or condition, and the combination or composition utilizes an effective amount of a drug or therapy to treat the disease or condition, for example , Free radical scavengers (e.g. ascorbic acid (vitamin C), vitamin C (for the treatment of mononucleosis), anticancer agents, chemotherapeutic agents, anti-viral agents e.g. nonionic surfactants e.g. contraceptive creams Delfen ^TM Nonoxynol-9 [nonylphenoxy polyethyl ethanol], and cationic surfactants (eg cetyl pyridinium chloride) and anionic surfactants (eg benzalconium chloride), non-steroidal Anti-inflammatory agents (NSAIDs), for example, indomethacin, naproxine, diclofenac and ketorolac (+/-) tromethamine salt (commercially available as Toadol ^TM ) and steroidal anti-inflammatory drugs, anti-fungal Now, antidote (eg, administered rectally at enema), analgesics, bronchodilators, anti-bacterial agents, antibiotics, drugs for the treatment of vascular ischemia (eg diabetes and Berger's disease), anti-body monoclonal agents, hair growth Topical use minoxidol, diuretics (e.g. purosemide (available as Lasix ^TM )), immunosuppressive agents (e.g. cyclosporin), impokines (interleukin-2 and the like), alpha- and beta-interferon and hyaluronic Lonic acid or salts thereof (e.g. sodium salts) or analogs, verbs, derivatives, complexes, esters, fragments or subunits thereof (preferably hyaluronic acid and salts thereof) are administered together to remove cell membranes of tissues (including scar tissue) Substances can be infiltrated into individual cells to be treated By administering such complexes and preparations to patients with a disease or condition, the disease or condition will be improved more than expected.

The composition may be provided intravenously, intraarterally, in the peritoneum, in the pleura, subcutaneously, into the skin, rectally or orally or by direct injection or as a patch to the patient's skin. Hyaluronic acid or salts and materials thereof may be administered separately but in sufficient quantity continuously or at intervals (as appropriate at the same time), at the same site (eg one intravenously and the other "piggyback"). Is administered in a manner to treat a disease or condition.

ii) Lines 18 to 25 on page 26 to 14

Thus, according to the present invention, NSAIDs, such as indomethacin (dissolved in n-methyl glutamine) or other NSAIDs, can be administered to indomethacin when 200 mg or more of hyaluronic acid is administered to 1-2 mg / kg of NSAID. Even when the amount is high (as needed), no side effects such as gastrointestinal tract, nerve abnormalities, nervous breakdowns, etc. When the hyaluronic acid is used below that amount, the usual side effects recur. For example, the combination of Indocid ^™ with hyaluronic acid was observed to be better, which suggests that it targets diseased tissues even when administered via the systemic venous route. Patients receive 50-200 mg NSAID-hyaluronic acid (hyaluronate sodium salt) (eg indomethacin-hyaluronic acid) and other chemicals such as ascorbic acid [vita. Min C], phloretin, and additional anticancer drugs experienced immediate relief from the patient's pain in the short term when the tumor was in the lung or liver, resulting in the dissipation and resorption of renal tissue lesions in these organs. Thus, dead tumor material and debris and tumor toxins are easily removed in vitro by the action of enhanced macrophages with the addition of NSAIDs (or steroidal anti-inflammatory drugs) and hyaluronic acid (salts or other forms). Therefore, Applicants release the blocking of macrophages by adding hyaluronic acid to the NSAIDs by interfering with the production of prostaglycan synthase, which blocks the function of macrophages. Type not only enhances NSAID activity but also reduces any side effects and toxicity associated with the use of prostaglandin synthase inhibitors. I can see that.

Examples of materials suitable for use as chemotherapeutic agents include oral administration of novantron (Mitoxantrone), methotrexate, 5-FU (5-fluorouracil), carboplatinum, Methyl CCNU and mitomycin C. "

iii) lines 32 to 37 on page 26;

"Haluronic acid and its salts can be used in a variety of dosages, from -10 to 1000 mg / 70 kg-the appropriate dosage is from 50 to 350 mg / 70 kg. Because of no toxicity, hyaluronic acid can be used in excess (eg For example, 3000 mg / 70 kg). "

iv) lines 27 to 29 on page 29 to 31;

Hyaluronic acid and / or a pharmaceutically acceptable salt (e.g. sodium salt) and one of the verbs, analogs, derivatives, complexes, esters, fragments thereof and subunits, with hyaluronic acid suitable for use in the present invention. Its salts can be obtained from Sterivet Laboratories Limited. One such form is Sodium hyaluronate 20 mg / ml 15 ml vials (300 mg / vial-Lot2F3). Sodium hyaluronate is a 2% solution with an average molecular weight of about 225,000 Daltons. The vial also contains triple distilled sterilized water according to U.S.P. for injectable compositions. Vials of hyaluronic acid and / or salts thereof may be contained in a closed Type 1 borosilicate glass vial with a butyl stopper that does not react with the substance in the vial. "

Hyaluronic acid and / or pharmaceutically acceptable salts (e.g. sodium salts) and segregation / amount in verbs, analogs, derivatives, complexes, esters, fragments and subunits suitably hyaluronic acid and pharmaceutically acceptable salts thereof Consists of hyaluronic acid and / or a salt thereof having the following characteristics;

Iii) a molecular weight range of 150,000-225,000;

Ii) sulfate mucopolysaccharide, based on the total weight, of 1.25% or less;

V) 0.6% or less protein based on total weight;

Iii) less than or equal to 150 ppm iron based on the total weight;

Iii) no more than 15 ppm lead based on total weight;

Viii) no more than 0.0025% glucosamine;

V) glucononic acid is 0.0025% or less;

Iii) no greater than 0.025% N-acetylglucosamine;

Iii) 0.0025% amino acid;

V) a UV absorption coefficient of 0.275 or less at 257 nm;

xi) UV absorption coefficient of 0.275 or less at 280 nm;

xii) pH is 7.3-7.9;

It can be composed of purified, actual pyrogen-free hyaluronic acid powder obtained from a natural source having at least one characteristic in the configuration as described. Suitably hyaluronic acid is mixed with water and the average molecular weight of the hyaluronic acid powder is 150,000 to 225,000.

More suitably, the division of hyaluronic acid has at least one feature selected from the group consisting of:

V) 1% or less sulfate mucopolysaccharide based on total weight;

Ii) 0.4% or less protein based on total weight;

Iii) less than or equal to 100 ppm iron based on the total weight;

Iii) no more than 10 ppm lead based on total weight;

Viii) no greater than 0.00166% glucosamine;

V) glucononic acid is 0.0166% or less;

V) N-acetylglucosamine is 0.0166% or less;

Iii) 0.00166% amino acid;

x) UV absorption coefficient of 0.23 or less at 257 nm;

xi) UV absorption coefficient of 0.19 or less at 280 nm;

xii) pH is 7.5-7.7;

Other forms of hyaluronic acid or salts thereof and verbs, derivatives, complexes, esters, fragments and subunits of hyaluronic acid can be obtained from the suppliers described in the aforementioned literature. In addition, Applicants have shown successful use of hyaluronate supplied and supplied by Life Core ™ Biomedical, Inc. with the following characteristics.

Feature Description

Appearance white-cream particles

Odor No detectable smell

Viscosity average molecular weight <750,000 Daltons

UV / Vis Scan, consistent with 190-820nm reference scan

OD, 260nm <0.25OD units

Hyaluronidase-sensitive positive reaction

Matches IR scan reference

pH, 10mg / g solution 6.2-7.8

Water up to 8%

Protein <0.3mcg / mg NaHy

Acetate <10.0mcg / mg NaHy

Heavy metals, ppm max

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

Microorganisms not observed

Endogenous toxin <0.07EU / mg NaHy

Biological safety test Passed rabbit eye toxicity test

The following references detail hyaluronic acid, its raw materials, manufacturing processes, and their recovery.

U.S. Patent 4,141,973 details hyaluronic acid fragments (including sodium salts) having the following characteristics;

“(a) an average molecular weight of at least about 750,000, suitably at least 1,200,000—ie at least 2000 cm 3 / g with a threshold viscosity of at least about 1400 cm 3 / g;

(b) the protein content is 0.5% or less by weight;

(c) the UV light absorption of the 1% sodium hyaluronate solution at 257 nm is less than 3.0 and less than 2.0 at 280 nm;

(d) the physical viscosity of the 1% sodium hyaluronate solution in physiological buffer is at least 1000 centistokes and preferably at least 10,000 centistokes;

(e) Molar optical rotation of 0.1% -0.2% sodium hyaluronate solution in physiological buffer is -11 x 10 ³ degrees-cm 2 / mol (disaccharide) measured at 220 nm

(f) When sodium hyaluronate 1% solution dissolved in physiological buffer in owl monkeys was replaced with about ½ of the existing liquid vitreous solution, there was no cell infiltration of chondrocytes and whole chambers, no redness in water-soluble fluids, No pathological changes in the cornea, lens, iris, retina and choroid

(g) HUA is free of sterile pyrogen

(h) non-antigenic. "

Canadian Patent 1,205,031 (prior art referred to in US Pat. No. 4,141,937) has a range of 50,000 to 100,000; 250,000 to 350,000; And the hyaluronic acid powder having an average molecular weight of 500,000 to 730,000 and its manufacturing process are described in detail.

If large molecular weight hyaluronic acid is used, it should be diluted for administration and free of intramuscular aggregation.

v) and 37 lines 33 to 35 on page 33;

"Applicants can combine hyaluronic acid (and hyaluronate sodium salt or other forms) with drugs and therapeutics to treat diseases beyond expectations;

For example,

Disease / disease chemical or drug

1. active free radical scavengers of cancer, macrophages,

Increased superoxide dismutase

(superoxide dismutase),

Ascorbic acid (vitamin C) anticancer agent,

NSAID, chemotherapy,

Antidote (cholestyramine).

1A. Dimethyl sulfoxide (DMSO) suffering from brain trauma.

Swollen brain from the recipient

Reducing the climb.

2. Minoxidil-complexes when used topically.

Increases hair growth.

3. Herpes, oral ulcer inflammation, nonionic surfactants eg

Shingles Nonoxynol-9, Anionic Surfactant

(Cetyl pyridinium glolide),

Cationic surfactants (benzaalkonium

Chloride), surfactant

4. Renal dysfunction, heart failure diuretic-furosemide

High blood pressure, edema

5. Infections, acne, mononuclear antibiotics, antimicrobials, antimicrobials, etc.

Ascorbic acid and hyaluronic acid.

6. Transplant cyclosporins.

7. Inflammation, oncolytic non-steroidal anti-inflammatory, NSAID-

Removal of substances (toxins, debris), diclofenac,

Reduced side effects, indomethacin, pyroxicam,

Pain relief (back pain). Ibuprofen, ketorolac tromethamine salt,

Naproxen.

8. Detoxifying enema, antidote, abdominal dialysis.

9. Bronchial expansion. Bronchodilators-Beclomethasone Diproprioonate (Chromoglycate Sodium

Not only used as a bronchodilator),

Thiophylline.

10. Vascular ischemia. Treat limbs for diabetes and Burgus disease using the appropriate drug-Trental.

11.HIV (AIDS) DMSO, Vitamin C, NSAID- (Indomethacin,

Naproxen, ketorolac, tromethamine),

Interferon, vibramycin ^TM (doxcycline),

Tetracycline.

12. Diabetes Insulin

13. Postmenopausal estrogen replacement

14. Local infection prevention anti-metabolites (eg sulfonamides).

15. Swell Prevention DMSO

16. Hypertensive calcium channel blockers, eg heart failure

Nifedipine, β-blockers such as athenol,

Propanolol.

17. Prostaglandin Synthesis Inhibition Acetylcyclic Acid

18. Spraying tissue for transplantation

Oxygen of tissue immersed in irrigation

Strengthen offer.

The foregoing description and objects can be modified to reflect the advantages of the invention. Thus, at the time of administration of the hyaluronic acid and the drug or therapy described above, chondroitin sulfate is first administered about 3-5 mg per kg body weight (eg 200-400 mg for a 70 kg human). . As a result, lesser amounts of drugs or therapies, such as cytotoxics, are generally used than those used for treatment because hyaluronan and drugs are used at the site of treatment (eg, tumor). Or because the therapy is provided so that no capture by the liver that has been "down-regulated" occurs. Therefore, the liver is not damaged by cytotoxic substances as in the past.

Suitably the molecular weight of chondroitin sulfate is greater than 20,000 daltons, although irrespective of the molecular weight of the chondroitin sulfate administered, the range is about 20,000 to 40,000. Chondroitin sulfates of high molecular weight are used when appropriately administered in effective dosages (eg, when there is a lack of sterile water for intravenous administration). The molecular weight of dextran sulfate or other substance (such as other glycosaminoglycans) is about 20,000 to 500,000 daltons so that it can be effectively administered.

Thus, according to the present invention, a substance capable of transporting a substance to a therapeutic area of the body (e.g., in the form of sodium hyaluronate) and a substance capable of transporting a drug or therapy to the liver (e.g., a receptor in the liver) Methods of treating a disease or condition of a human treatable by a carrier capable of binding to;

a) administering an effective amount of a first substance, such as chondroitin sulfate that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver;

b) (a) three to four minutes after administration of the substance, a drug or therapeutic agent non-toxic effective amount and a second substance effective amount carrying [drug or therapeutic agent] are administered, wherein the second substance is Different from the first substance (eg hyaluronic acid), the second substance can bind to the site requiring treatment, the liver can bind if the liver is not "down-regulated", and can bind to the phagocytic receptors of the liver. The first substance administered in (a) (e.g., chondroitin sulfate having a molecular weight of 20,000) is captured by the liver, which actually reduces (appropriately eliminates or blocks) the binding ability of the liver to the second substance.

Because the liver is "down regulated", the amount of drug or therapy required to treat the site requiring treatment is reduced. In addition, hyaluronic acid (transport material) is also reduced so that the effective amount is 10 mg or less (20 µg / kg body weight of the person to be treated) for a 70 kg person.

In another aspect of the invention, a method of preventing the liver from receiving a toxic drug or therapy in the liver when administering the drug or therapy to a site in need of treatment

a) administering an effective amount of a first substance, such as chondroitin sulfate that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver;

b) (a) three to four minutes after administration of the substance, a drug or therapeutic agent non-toxic effective amount and a second substance effective amount carrying [drug or therapeutic agent] are administered, wherein the second substance is Different from the first substance (eg hyaluronic acid), the second substance can bind to the site requiring treatment, the liver can bind if the liver is not "down-regulated", and can bind to the phagocytic receptors of the liver. The first substance administered in (a) (eg, chondroitin sulfate with a molecular weight of 20,000) is captured by the liver, which in turn reduces the ability of the liver to bind to the second substance.

The amount of the first substance administered in (a), which has a molecular weight of 20,000 to 40,000 daltons, for example about 3-5 mg per kilogram of body weight (eg 200 to 400 mg for a 70 kg person) in an appropriate manner It may be administered, for example, by oral, intravenous, subcutaneous systemic administration, or by injection directly into the adjacent area of the liver (immediately into the hepatic artery). Thus (after 3-4 minutes), the transporting material of (b), for example, hyaluronate sodium having a molecular weight of 750,000 Daltons or less is 10 mg per 70 kg as detailed in WO 91/04058 together with a drug or therapy. It is administered in the following effective amounts. In order to reduce the side effects of drugs or therapies, at least 200 mg / 70 kg of the sodium hyaluronate provided in WO91 / 04058 is actually reduced because the amount of the drug or therapy currently in effect is reduced from the previous amount. It is. Thus, to reduce the side effects of drugs or therapies, less effective amounts of drug or therapy and less amount of hyaluronic acid are administered. Thus, the amount of drug or therapeutic agent and hyaluronan transporting substance is effective in the amount of μg per kilogram of human body weight.

The first substance is chondroitin sulfate (appropriate) or other suitable substance (eg dextran sulfate or other GAGS [glycosaminoglycan] or proteoglycans thereof that are not hyaluronic acid). Other phagocytic ligands that have an effect can also be used as the first substance. The second substance is suitably hyaluronic acid type such as hyaluronan or sodium hyaluronate.

Thus, according to another aspect of the invention, when a drug or therapy is transported to treat a site other than the liver, the drug or therapy is prevented from being captured by the liver and the drug or therapy is directed to a body part requiring treatment. Dosage kits that deliver the maximum amount of agent are provided;

a) an effective dose of a first substance, such as chondroitin sulfate that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver;

And apart from the dosage of (a),

b) a drug or therapeutic agent non-toxic effective amount and a second substance effective amount to transport; In this case, the second substance may be different from the first substance (eg, hyaluronic acid), the second substance may bind to the site requiring treatment, and may bind to the liver if the liver is not "down-regulated", The first substance administered in (a) capable of binding to the phagocytic receptor (e.g., chondroitin sulfate with a molecular weight of 20,000) binds to the phagocytic receptor in the liver, thereby administering the second substance 3-4 minutes after administration of the substance. When done, the binding capacity of the liver to the second material is actually reduced. The dosage of (b) is the amount of µg / kg body weight, for example 20 µg / kg.

Thus, the method according to another aspect of the present invention

a) administering an effective amount of a first substance, such as chondroitin sulfate that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver; And

b) administering an effective amount of a drug or therapeutic agent (eg, NSAID [non-steroidal anti-inflammatory agent] or a cytotoxic substance (eg, methotrexate and cisplatin and combinations thereof) and a second substance effective amount to transport, wherein the second The substance is different from the first substance (eg hyaluronic acid), the second substance can bind to the site requiring treatment, and can also bind to the liver if the liver is not "down-regulated", serous, lymph and lymphocytes. The binding of the first substance administered in (a), which is capable of transporting to and binding to the liver phagocytic receptor (eg, chondroitin sulfate with a molecular weight of 20,000), is actually reduced by the liver's ability to bind to the second substance.

The first substance is chondroitin sulfate and the second substance is hyaluronic acid type. Each sheep is as described above. For example, the amount of chondroitin sulfate is at least about 3-5 mg / kg, the effective amount of the hyaluronic acid type is 0.1 mg / 70 kg, and its molecular weight is 750,000 daltons or less.

The invention will be described with reference to the following figures and examples.

The present invention relates to the provision of drugs and therapeutic agents to specific parts of the body of a mammal in need of treatment, and to one example, which can be used to treat malignant tumors in humans.

1 shows biodistribution of labeled hyaluronan (HA) after administration of 1 mg of chondroitin sulfate intravenously, 18-20 hours after administration of 1 mg of labeled hyaluronan.

2 illustrates the capture of 1 mg of labeled hyaluronan.

FIG. 3 consists of two diagrams, the upper figure showing MCPM / rat and time (minutes), and the lower figure showing MCPM / organ after 1 mg of chondroitin sulfate at ¹²⁵ I-HA ( ¹²⁵ I-hyaluronan) administration. Is a comparison.

4 illustrates the disruption of the binding of labeled hyaluronan (HA) to NGW cells at 37 ° C. (chondroitin sulfate does not interfere while labeled hyaluronan interferes).

FIG. 5 shows a biometric image showing that labeled hyaluronan (HA) is reduced from being received by the liver after treatment of mice with chondroitin sulfate (CS).

Figure 6 illustrates the targeting of tumors to residual amounts of labeled HA (hyaluronan) after administration of CS in an amount of 200-400 mg / 70 kg.

7 is a photographic image illustrating that ¹²⁵ IT-HA is removed.

8-14 illustrate the presence of radioactive HA in the body regardless of pre-administration of other substances.

Targeted to tumors using radiolabeled hyaluronan (HA) administered after pretreatment with chondroitin sulfate (CS) is summarized in Data I and are discussed below.

Data was collected using female Wistar FU rats (average weight of 250 g per mouse) inoculated on the hind limbs of a rat colon sarcoma called NGW. When tumors appear, mice are injected intravenously with 1 mg of chondroitin sulfate (200-400 mg / 70 kg human), followed by 1 mg of HA (hyaluronan) with low radioactivity 30 seconds later. After 18-20 hours, animals are killed and radioactivity in living organs is measured (see FIG. 1).

In the same model, 1 mg of HA (hyaluronan) having similar specific activity was used, wherein the ratio of HA to non-tumor to tumor was 7.79 + 5.00.

Using chondroitin sulfate (CS) pretreatment, the ratio increased to 16.23 + 2.48. The main cause of the increased rate was low capture in tissues other than tumor tissue (mainly healthy leg muscles), but the combined amount increased 27% (see Figure 2).

Four of the five "bases" in each value were found to have a non-tumor to tumor ratio of about 4 (a relatively high ratio, such as 7.79, because one experimental value is high). In an earlier published study of labeled HA alone, a similar but not identical tumor system was used to find that the ratio of HA to non-tumor tissue to tumor was 4 (See "Accessible hyaluronan receptors identical to ICAM-1." in mouse mast cells ", Stefan Gustafson, et al., Glycoconjugate Journal (1995) 12: 350-355). Thus, the use of chondroitin sulfate (CS) for the transport of drugs or therapeutics can be seen to be a real improvement. Administration of chondroitin sulfate (CS) shows about 80% blocking HA (hyaluronan) labeled by the liver about 10-15 minutes (in the absence of chondroitin sulfate (CS), the liver Absorbs about 95% of radioactivity at the time of day; see FIG. 3).

In in vitro experimental data, NGW tumor cells are HA (hyaluronan) that are not blocked by chondroitin sulfate (CS) (or are not blocked / fixed during the time the second substance (eg hyaluronic acid) is received). It can be seen that it has a receptor that accepts.

Figure 4 illustrates three results that clearly show that radiolabeled hyaluronan is not interfered by chondroitin sulfate but by unlabeled hyaluronan.

In FIG. 5, the degree of capturing HA labeled by the liver after CS-pretreatment of mice was reduced in vivo. This figure shows that the collection of HA [0.5-1 mg in the case of 70 kg human body] for a considerable time by CS [1-2 g in the human body of 70 kg body weight] is effectively prevented. Thus, a small amount of active drug (drug or therapeutic agent) (μg / kg) and a small amount of HA (μg per kg body weight of the patient) are used. Therefore, a small amount of HA (10 mg or less for people weighing 70 kg) is used as the transport material. Therefore, treatments have been developed that use small amounts of HA and small amounts of active drugs (or therapeutic agents) that provide maximum targets to the sites (disease sites) that require treatment and also reduce side effects.

In addition, in mice with tumors using CS 1 mg (200-400 mg for 70 kg humans) and small amounts of labeled HA (0.5-1 mg for 70 kg humans), tumors were targeted. Revealed (see FIG. 6). Experimental conditions are the same as described above for NGW tumor rats, but using a small amount of HA. The relatively high radioactivity in the reference muscle is due to the circulating degradation products at this time (about 20 hours after injection). The ratio of HA to tumor in non-tumor tissue was 8.8 (p <0.001, n = 4), indicating that the targeting of HA is good at low doses.

A size extrusion chromatography study was conducted on the molecular weight distribution of radiolabeled HA in urine and serum of rats pretreated with 5 mg of CS and given 5 μg of ¹²⁵ I-HA (hyaluronan) having a molecular weight of 400 kDa (400,000 daltons). In some cases. Blood is collected at 2-4, 10-22, 22-24 and 70 minutes after completion of dosing. Kill animals, collect urine in bladder around 70 minutes, and collect organelles. Looking at the results, it can be seen that over time, the molecular weight of hyaluronan decreases, and by 70 minutes, most of the hyaluronan remaining in the circulation is less than 39 kDa (39,000 daltons). At this time, about 10% of the injected radioactivity was found in urine and its average molecular weight was about 20 kDa (20,000 Daltons). The radioactivity in the liver is only 6-7% and in the kidneys it is about 9% because the substance is filtered and enters the urine. Blood contained about 35%, negligible amounts in other organs, and about 40% was filtered into surrounding tissue. Thus, blocking using chondroitin sulfate (CS) is an ideal way to get some hyaluronan into the tissue. Using chondroitin sulfate pretreatment is an additional factor that increases the binding of intravenously administered hyaluronan (HA) to tumor tissue. It provides a method of transporting drugs or therapeutic agents and hyaluronan (HA) to visceral fluid, lymph and lymphocytes to treat diseases such as cancer or metastasis. Such treatments can be used to prevent metastasis.

Hyaluronan (hyaluronic acid; HA) is a high molecular weight polysaccharide consisting of repeating units of glucuronic acid and N-acetylglucosamine. It is present in high concentrations in connective tissues such as skin and cartilage, in the vitreous and synovial fluid of the eyes (1). Polysaccharides can associate with several proteins in the extracellular matrix and also with some cell-surface HA-binding proteins (2).

Plasma levels of HA are generally very low (10-50 μg / l) but are elevated in diseases such as rheumatoid arthritis, cirrhosis of the liver and various malignancies (3). Circulating hyaluronan comes from surrounding tissue associated with cells and binding proteins, some of which reside in freely movable compartments. Polysaccharides enter the general circulatory system through the lymph (4), 80-90% of which are removed from the lymphocytes before reaching the blood (5). The molecular weight in serum is 1.5X10 ⁵ , and the molecular weight of HA in lymph is about 2X10 ⁶ (6). The main site where HA is removed from the blood stream under normal conditions is due to receptor-mediated endocytosis (1,7) by the liver. The t½ of hyaluronan administered intravenously to experimental animals and humans is a few minutes, and after 15-20 minutes degradation products begin to appear in the circulation (7-9). Collected via coated pits and coated carriers of hepatic endothelial cells (LEC), Kupffer cells and hepatocytes are basically negative for capture both in vivo and in vitro (9-11).

HA captured by LEC is transported to lysosomes, where HA is broken down to monosaccharides and eventually to carbon dioxide, urine and water in hepatocytes (12).

In inhibition studies with LEC in culture, the receptor recognizes ligands other than HA, such as chondroitin sulfate (CS), dextran sulfate (DxS), and sulfate removed CS (13-15). Inhibition studies using polysaccharides that bind to HA receptors in vivo LECs have been shown to indicate that CS and DxS, but not heparin, interfere with HA clearance by the liver. The HA remaining in the circulatory system breaks down into smaller fragments, possibly due to certain saturation mechanisms such as high concentrations of HA interfere with this degradation. This mechanism explains why the circulating HA is smaller than the molecular weight of HA entering the general circulation via lymph. This mechanism of saturation accounts for the significantly higher concentrations of circulating HA that increase plasma viscosity from high molecular weight HA.

Materials and methods

Polysaccharides

HA used for labeling, capture- and turnover studies is supplied by Hyal Pharmaceutical Corporation (HPC), Toronto, Canada. The molecular weight distribution of HA is determined by chromatography using 0.25 M NaCl, 0.05% chlorobutanol in a column of Sephacryl HR (Pharmacia, Uppsala, Sweden) labeled 400, 1000, 2000 (16). The HA content in each aliquot is monitored for absorbance at 214 nm. Radioactivity is measured using a Packard auto-gamma counter. Chondroitin sulfate A in bovine organs is described by Sigma Chemical company, St. Louis, U.S.A. (product number 8529). The batch contains 1.9 ng HA / CS μg as determined by the specific radioactivity test of HA (HA-50, Pharmacia, Upssals, Sweden).

Dextran sulfate with a molecular weight of 500,000 Da was obtained from Pharmacia Biotech, Uppsala, Sweden (Code No. 17--340-01).

Repeated purification of heparin with cetylpyridinium chloride (17) in the visceral mucosa was performed by Prof. Provided by Ulf Lindahl, University of Uppsala, Sweden.

Labeling of HA, CS, and Heparin

HA, CS and heparin are labeled with DL-tyrosine (Sigma chemicla company St Louis, USA), as previously described (18) after CNBr-activation of polysaccharides. Briefly, 15 mg HA, CS and heparin were activated at pH 11 with 8 mg CNBr for 5 minutes. Activated polysaccharides are separated from the reaction mixture through a Sephadex G25 (PD 10, Pharmacia, Uppsala, Sweden) small column equalized with 0.2 M borate buffer pH 8.0. Activated polysaccharides were incubated overnight with 1 mg tyrosine (T) (Sigma chemicla company St Louis, USA). Unbound T (tyrosine) in PD 10 column equalized with phosphate buffer salt (pH 7.5) (PBS) containing NaCl (8 g / L), KCl (0.2 g / L) and Na ₂ HPO ₄ (1.15 g / L) T bound to HA (T-HA), CS (T-CS) or heparin (T-Hep).

Portions of T-HA, T-CS or T-Hep were coated with 10 μg 1,3,4,6, -tetrachloro-3a, 6a-diphenylglycouril (Sigma chemical company, St. Louis, USA) film ¹²⁵ I iodo treated by placing in 100 μg T-labeled polysaccharide with 0.5 mCi ¹²⁵ I in a small glass tube.

Unbound ¹²⁵ I was removed using a PD 10 column equalized with PBS and iodinated T-HA ( ¹²⁵ IT-HA), T-CS ( ¹²⁵ IT-CS) or heparin ( ¹²⁵ IT-Hep) was 5 ° C. Store in Specific radioactivity is usually 1500-5000dpm / ng.

In gel filtration chromatography, with an average molecular weight of about 0.5X10 ⁶ Da, ¹²⁵ IT-HA maintained a large molecular weight-profile, and dynamics were found to remove ¹²⁵ IT-HA from the circulation system, biosynthetic high molecular weight labeled HA The organ distribution of was reported. It was possible to capture ¹²⁵ I-labeled T-HA from rat liver endothelial cells isolated in vivo or in vitro, indicating that labeling does not interfere with binding to the specific cell surface receptors found in these cells (1,2). , 9-11).

Gel filtration chromatography on Sephacryl S-1000 and S-3000, calibrated with the HA standard, showed that ¹²⁵ IT-CS and ¹²⁵ IT-Hep unlabeled CS (about 30,000 Da) and heparin (about 20,000 Da) They have the same average molecular weight, which seems to have a similar size distribution pattern.

cell

Single cell suspensions are made from Sprague Sawley rats weighing 200-300 g by collagenase irrigation at 37 ° C. for 10 minutes. Hepatic endothelial cells, Kupffer cells and parenchymal cells were purified by Percoll ^R -centrifugation and selective adsorption described above in Pertoft and Smedsrod (19) showed that about 95% were pure cells (10). , 19). Monolayer cultures were maintained under standard culture conditions in standard medium supplemented with L-glutamine (2 mM) and gentamycin (50 μg / ml) and 10% (v / v) fetal calf serum for parenchymal cells. To supplement. Liver endothelial cells are cultured without serum. All cells are incubated overnight before starting the experiment.

Collection of Cells in Culture

In an unlabeled polysaccharide competition experiment, ¹²⁵ IT-hyaluronan was added to chilled RPMI medium containing L-glutamine (2 mM), gentamicin (50 μg / ml), and fibronectin coated diameter 16 mm plates. Provide 100,000-200,000 liver endothelial cell cultures. Cultures are maintained under standard culture conditions in 3000 ml medium.

After incubation, the medium is removed and the radioactivity is analyzed and in some experiments gel chromatography on a 24 ml Sephacryl 300 column to separate digested from undigested polysaccharide. Cells were washed three times in phosphate buffer salt (pH 7.5) (PBS) containing NaCl (8 g / L), KCl (0.2 g / L) Na ₂ HPO ₄ (1.15 g / L) (8). ) Radioactivity or homogenized or aliquots were analyzed. Nonspecific binding is corrected by measuring radioactivity in cell-free dishes, which are generally below the background level.

Biometric research

Sprague Dawley rats weighing 200-250 g are anesthetized using pentobarbital (45 mg / kg body weight). ₅ μg of ¹²⁵ IT-hyaluronan or ¹²⁵ IT-Hep (8-15 × 10 ⁶ cpm) is injected into the tail vein in 0.8-1.0 ml of 0.15 M NaCl, 10 mM Na ₂ HPO ₄ pH 7.4. In some studies, 1-5 mg unlabeled polysaccharide is administered 30 seconds before the labeled polysaccharide. Blood is taken periodically at the end of the tail during the cycle. In some cases the serum was subjected to size extrusion chromatography on Sephacryl S 300 and analyzed for radioactivity of the eluate.

Kill rats after 10-22 minutes. Radioactivity is tested in the liver, lungs, kidneys, heart, pancreas, and in some cases, urine. Processing is performed using a Macintosh SE / 30 ^R , Macintosh IIsi ^R or Macintosh 7200 computer (Apple Computer Inc. Cupertino, CA, USA). Run using Cricket Graph ^R program (version 1.3, Criket software, Malvern, PA, USA) and Canvas (version 3.0.2, Deneba Systems Inc., Miami, FI, USA). Statistical analysis was performed using Statworks ^R (version 1.1, Cricket Software, Malvern, PA, USA).

Scintillation research

Mice were anesthetized and injected as described above. In kinetic studies, the mice are placed and injected into a Fuji phosphoimager screen with a high resolution metal collimator located between the mouse and the screen. The screen is exposed for 10 minutes, the image is developed and analyzed on a Fuji 2000 phospho imager. Some phase analytical studies are performed using NIH Image software.

result

Intravenously administered ¹²⁵ IT-HA traces can be seen to be rapidly removed by rat liver via phosphoimaging (FIG. 7). Small amounts of ¹²⁵ IT-HA lost from labeled polysaccharides can be seen in the urine bladder (FIG. 7). For 22 hours, about 20% of radioactivity disappeared from the liver and was found in urine (planted in the intestine).

^When CS was administered at a dose of about 20 mg / kg body weight prior to ¹²⁵ IT-HA administration, it could be seen that rapid removal disappeared and most of the radioactivity was dispersed throughout the animal for several hours (FIG. 7). After CS blocking, radioactivity is mainly seen in the blood and some are collected in the liver, spleen, kidney (Fig. 8), and it can be seen that the molecular weight is sharply reduced (Fig. 9). Some labeled polysaccharides with a molecular weight of 10,000-40,000 can be seen in urine (FIGS. 8 and 9). The capture of some labeled HA by the liver was found in urine when blocked by unlabeled HA (1 mg / kg bw) (FIG. 8). After CS blocking, the rapid decrease in molecular weight of circulating ^125- IT-HYA disappeared, and more radioactivity was maintained in the general circulation after HA blocking than after CS blocking (FIGS. 8 and 10). However, trace amounts of radioactivity can be found in urine even after 70 minutes, and these substances have the same molecular weight when radioactivity is found in urine after CS block (Figures 8, 9, 10).

It can be seen that at 200 mg / kgbw DxS (dextran sulfate) effectively blocks the capture by the liver, and when the capture by the liver is low, the outflow of labeled HA in the general circulation is increased and the recovery of injected HA is increased. It can be seen that it is low (Fig. 11). 1 mg / kgb.w. When testing the dose, it can be seen that 30-40% inhibition of capture by the liver (FIG. 11).

Heparin did not affect the removal of ¹²⁵ IT-HYA at 20 mg / kgbw, and HA did not interfere with the removal of ¹²⁵ IT-Hep in trace amounts at 20 mg / kgbw (FIG. 12). CS partially inhibited liver capture of ¹²⁵ IT-Hep (FIG. 12).

Binding of ¹²⁵ IT-HYA to LEC in culture was effectively inhibited by HA, CS, DxS (FIG. 13).

Studying the distribution of ¹²⁵ IT-CS intravenously, when recovering total radioactivity, liver capture was lower than for ¹²⁵ IT-HYA and urine excretion was high (FIG. 14). Entrapment by the liver is effectively inhibited by unlabeled CS and HA, increasing clearance to urine (FIG. 14).

discussion

It has been shown by experiments with LEC isolated from culture that HA receptor mediated endocytosis by LEC is not specific for HA (13-15). In such studies, receptors recognize ligands such as CS and DxS. This study was conducted to determine if some negatively charged polysaccharides influence the movement of circulating HA in living organisms. Labeling techniques that do not affect the molecular weight and do not interfere with the cell binding capacity of polysaccharides were induced with high specific activity g-spinning (18, 20-23). Such polysaccharides have advantages in many ways, they can be detected in small amounts, and the distribution of them in living animals can be recorded using scintogram or phosphoimaging techniques. Mice were chosen as experimental animals in this study, and these species were used early in many migration and capture studies, and normal blood levels and measured migration rates were similar to those found in humans (1,3,9,10,24). .

In our study, CS and DxS, except for heparin, inhibited receptor mediated endocytosis by the liver, preventing the removal of HA from the bloodstream (FIGS. 7 and 11). Heparin removed by the mechanism appears to be unaffected by HA, and CS appears to partially reduce the capture of the labeled heparin by the liver (FIG. 12), but inhibition occurs partially, as is the blocking of labeled HA. It is not effective. As a result of blocking the HA receptors in the liver by CS or DxS, HA remaining in the circulatory system is broken down into smaller fragments by a mechanism that appears to be a specific saturation mechanism and high concentrations of HA interfere with this degradation (FIGS. 9 and 10). . Fragmentation lowers the number of injections injected, and low molecular weight HA is filtered through tissue and goes to the urine via kidneys (FIGS. 7, 9 and 10). Size of the serum or plasma in the Sephacryl S 300 column is determined by size of the circulating material by extrusion chromatography. Such gels are not able to separate HA above 50 kDa well, and in order to provide unlabeled HA to reduce capture by the liver, the injection material having a molecular weight of about 400 kDa is degraded by shear force to a molecular weight of about 50 kDa. However, by breaking down into smaller fragments, the appearance of the chromatography of the material at the included sites occurs immediately after injection only in the case of CS blocking, and not so large when unlabeled HA is used as the blocking material. 9 and 10).

Recognition by the same receptor in the liver can be seen as the fact that unlabeled CS and HA interfere with the capture of ¹²⁵ IT-CS by the liver (FIG. 14). The capture of CS by the liver is not as great as the capture of HA by the liver depends on the molecular weight of about 300 kDa compared to about 400 kDa for HA. Thus, in some cases, it is quickly removed from the circulation system by filtration and some of the injected material remains in the general circulation for a long time and is collected by the liver.

DxS binds to the same receptors as CS and HA but has a low affinity, so when using DxS instead of CS or HA, more amount is required to interfere with liver capture of ¹²⁵ IT-HAs (FIG. 11).

In our results, the migration of naturally occurring polysaccharides HA and CS appears to have some effect on the liver's effective capture of circulating polysaccharides via the usual receptors in the LEC. Thus, under some conditions, high levels of circulating HA are comparable to increased outflow of CS from the tissue to the circulation and vice versa. The results are divided due to the presence of a specific mechanism of degradation of HA in the circulation. If LEC is blocked by CS, urinary excretion of HA will increase significantly in relation to its effect at the serum level, and HA entering the circulation. Since the outflow is increased or liver clearance is reduced, the serum levels of HA are increased, resulting in a slight increase in urine output. The weak correlation between such serum levels and urine excretion has already been mentioned in rheumatoid arthritis (RA), primary biliary sclerosis (PBC) and Werners syndrome (25). All three diseases have elevated serum levels of HA. However, urinary excretion was slightly increased in RA and PBC, whereas in Werners syndrome, although serum levels were lower than in PBC and RA, urinary excretion was increased several times.

The presence of a saturation mechanism of saturation of HA in the circulatory system explains why the circulating HA has a much lower molecular weight than HA entering the general circulatory system via lymph (6, 26). The reduction of polymer size by mechanical shear has been proposed by the mechanism proposed by Fraser (26). However, the molecular weight and concentration of the unlabeled HA used to interfere with liver collection in the present invention does not seem to be affected to the extent that viscosity is inhibited by shearing. According to Fraser, degradation by serum hyaluronidase does not occur under physiological conditions, but the amount of free radicals in the serum is too small to reduce its size, and the breakdown of large molecular weight HA is high. It does not interfere with. The study also refutes that free radicals attack when degradation occurs in the presence of large amounts of CS that removes any active free radicals. Molecular weight dependent binding of HA required for activity results in degradation sites by hyaluronidase immobilized on the cell surface. Research also needs to characterize this mechanism, which is an important part of HA metabolism.

Many changes may be made without departing from the scope of the invention and all materials used herein are for the purpose of illustrating the invention and are not intended to be limiting thereof.

Claims (45)

Substances that can transport substances (such as sodium hyaluronate) to the therapeutic area of the body and those that can transport drugs or therapeutics to the liver (eg carriers that bind to receptors in the liver) A method of treating a disease or condition of a human treatable by; a) administering an effective amount of a first substance, such as chondroitin sulfate that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver; b) administering a drug or therapeutic agent non-toxic effective amount and a second substance effective amount that plays a role of transport, wherein the second substance is different from the first substance (eg hyaluronic acid) and the second substance is treated A first substance administered in (a) (eg, chondroitin sulfate having a molecular weight of 20,000) that can bind to a site requiring and can bind to the liver if the liver is not "down-regulated." ) Is collected by the liver, the ability of the liver to bind to the second substance is actually reduced. The method of claim 1, wherein the first substance is glycosaminoglycan but not hyaluronic acid type. The method of claim 1, wherein the first substance is chondroitin sulfate. The method of claim 1, 2 or 3, wherein the second substance (transport substance) is selected from the hyaluronic acid type. The method of claim 1, 2, 3 or 4, wherein the hyaluronic acid type is selected from hyaluronic acid (hyaluronan) and a pharmaceutically acceptable salt thereof. 5. The method of claim 3 or 4, wherein the amount of chondroitin sulfate is at least about 3-5 mg / kg body weight of the person. 7. The method of claim 6, wherein the second substance is an effective amount of a hyaluronic acid type having a molecular weight of 750,000 Daltons or less. A method of preventing the liver from capturing drugs or therapeutics that are toxic to the liver; a) administering an effective amount of a first substance that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver; b) administering a non-toxic effective amount of a drug or therapeutic agent and an effective amount of a second substance which is a transport agent, wherein the second substance is different from the first substance and the second substance can bind to the site requiring treatment In which the first substance administered in (a), which is capable of binding to the liver's phagocytic receptors, is captured by the liver, thereby actually reducing the ability of the liver to bind to the second substance. Characterized in that the method. 9. The method of claim 8 wherein the first substance is chondroitin sulfate and the second substance is selected from the hyaluronic acid type. 10. The method of claim 9, wherein the hyaluronic acid type is selected from hyaluronic acid and its pharmaceutically acceptable salts. The method of claim 10, wherein the amount of chondroitin sulfate is at least about 3-5 mg / kg human body weight. The method of claim 11, wherein the amount of hyaluronic acid type is about 0.1 mg or more per 70 kg of human body weight. The method of claim 11 or 12, wherein the hyaluronic acid type has a molecular weight of 750,000 Daltons or less. When a drug or therapy is transported to treat a site other than the liver, the drug or therapy prevents the drug or therapy from being captured by the liver, and in a dosage kit that maximizes the transport of the drug or therapy to the body site in need of treatment. ; a) an effective dose of a first substance, such as chondroitin sulfate that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver; b) a drug or therapeutic agent non-toxic effective amount and a second substance effective amount to transport; In this case, the second substance is different from the first substance, the second substance may bind to a site requiring treatment, the liver may bind to the liver when the liver is not down-regulated, and may bind to the phagocytic receptor of the liver ( The first substance administered in a) binds to the phagocytic receptors in the liver. (a) When the second substance is administered after administration of the substance, the kit's ability to bind liver to the second substance is actually reduced. 15. The kit of claim 14, wherein the first substance is chondroitin sulfate and the second substance is selected from the hyaluronic acid type. 16. The kit of claim 15, wherein the hyaluronic acid type is selected from hyaluronic acid and its pharmaceutically acceptable salts. 17. The kit of claim 16, wherein the amount of chondroitin sulfate is at least about 3-5 mg / kg body weight of the person. 18. The kit according to claim 17, wherein the amount of hyaluronic acid type is about 0.1 mg or more per 70 kg of human body weight. 19. The kit according to claim 17 or 18, wherein the hyaluronic acid type has a molecular weight of 750,000 Daltons or less. To treat a disease or condition of a person treatable by a substance capable of transporting a substance to a therapeutic area of the body and of a drug or therapeutic agent to the liver (eg, a carrier capable of binding to a receptor in the liver) In the method; a) administering an effective amount of a first substance, such as chondroitin sulfate that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver; And b) administering an amount less than the generally expected effective amount of the drug or therapy and an effective amount of a second substance to transport, wherein the second substance is different from the first substance and the second substance is the site requiring treatment Ability to bind to the liver, and to the liver if the liver is not "down-regulated", and to bind to the liver's phagocytic receptors. This actual reduction and the non-toxic effective amount of the second substance is generally less than the amount that is considered effective. 21. The method of claim 20, wherein the first substance is glycosaminoglycan but not hyaluronic acid type. The method of claim 20 or 21, wherein the first substance is chondroitin sulfate. 23. The method of claim 20, 21 or 22, wherein the second substance (transport substance) is selected from the hyaluronic acid type. The method of claim 20, 21, 22 or 23, wherein the hyaluronic acid type is selected from hyaluronic acid (hyaluronan) and a pharmaceutically acceptable salt thereof. 24. The method of claim 22 or 23, wherein the amount of chondroitin sulfate is at least about 3-5 mg / kg body weight of the person. The method of claim 25, wherein the second agent is in the form of hyaluronic acid having a molecular weight of 750,000 Daltons or less in an amount of about 20 μg / kg body weight of the patient. A method of preventing the liver from capturing drugs or therapeutics that are toxic to the liver; a) administering an effective amount of a first substance that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver; b) The drug or therapeutic agent is administered with an effective amount of the second substance which is actually less than the amount normally used and the transport agent, where the second substance is different from the first substance and the second substance is in need of treatment. Binding of the liver to the second substance by being captured by the liver, the first substance administered in (a) capable of binding to the site and capable of binding the liver if the liver is not "down-regulated" and capable of binding to the liver's phagocytic receptor The capacity is actually reduced, wherein the non-toxic effective amount of the second substance is generally less than the amount that is considered effective. 28. The method of claim 27, wherein the first substance is chondroitin sulfate and the second substance is selected from the hyaluronic acid type. 29. The method of claim 28, wherein the hyaluronic acid type is selected from hyaluronic acid and its pharmaceutically acceptable salts. The method of claim 29, wherein the amount of chondroitin sulfate is at least about 3-5 mg per kg. The method of claim 11, wherein the amount of hyaluronic acid type is about 0.1 mg or more per 70 kg of human body weight. 31. The method of claim 30, wherein the second substance is in the form of hyaluronic acid having a molecular weight of 750,000 Daltons or less in an amount of about 20 μg / kg body weight of the patient. When a drug or therapy is transported to treat a site other than the liver, the drug or therapy prevents the drug or therapy from being captured by the liver, and in a dosage kit that maximizes the transport of the drug or therapy to the body site in need of treatment. ; a) an effective dose of a first substance, such as chondroitin sulfate that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver; b) the drug or therapeutic agent is in an amount less than the normally effective amount and the second substance effective amount to transport; In this case, the second substance is different from the first substance, the second substance may bind to a site requiring treatment, the liver may bind to the liver when the liver is not down-regulated, and may bind to the phagocytic receptor of the liver ( The first substance administered in a) binds to the phagocytic receptors in the liver. Kits, characterized in that the effective amount is generally less than the amount that is said to be effective. 34. The kit of claim 33, wherein the first substance is chondroitin sulfate and the second substance is selected from the hyaluronic acid type. 35. The kit of claim 34, wherein the hyaluronic acid type is selected from hyaluronic acid and its pharmaceutically acceptable salts. 36. The kit of claim 35, wherein the amount of chondroitin sulfate is at least about 3-5 mg / kg body weight of the person. 37. The kit according to claim 36, wherein the amount of hyaluronic acid type is about 0.1 mg or more per 70 kg of human body weight. 37. The kit of claim 36, wherein the second material is in the form of hyaluronic acid having a molecular weight of 750,000 Daltons or less in an amount of about 20 µg / kg body weight of the patient. A method of preventing metastasis in a cancer patient; a) administering an effective amount of a first substance that does not bind to the site requiring treatment but binds to a receptor in the liver for "downregulation" of the liver; And b) administering an effective amount of a drug or therapeutic agent and an effective amount of a second substance to be transported, wherein the second substance is different from the first substance, and the second substance may bind to the site requiring treatment, and The first substance administered in (a) (eg, chondroitin sulfate with a molecular weight of 20,000), which can bind to the liver when not "downregulated" and which can transport to serous, lymph and lymphocytes and bind to the liver's phagocytic receptors Being collected by the liver, wherein the ability of the liver to bind to the second material is actually reduced. 40. The method of claim 39, wherein the first substance is chondroitin sulfate and the second substance is selected from the hyaluronic acid type. 41. The method of claim 40, wherein the hyaluronic acid type is selected from hyaluronic acid and its pharmaceutically acceptable salts. 42. The method of claim 41, wherein the amount of chondroitin sulfate is at least about 3-5 mg / kg human body weight. 43. The method of claim 42, wherein the amount of hyaluronic acid type is about 0.1 mg or more per 70 kg of human body weight. 44. The method of claim 42 or 43, wherein the second material is in the form of hyaluronic acid having a molecular weight of 750,000 Daltons or less in an amount of about 20 μg / kg body weight of the patient. 45. The method of claim 39, 40, 41, 42, 43 or 44, wherein the drug or therapy is selected from NSAIDs and cytotoxic substances or combinations thereof.