Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
  • A61K31/728—Hyaluronic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• This invention relates to the targeting of medicines and therapeutic agents to sites in the body of a mammal in need of treatment and, in one application, finds use in the treatment of malignant tumours in humans.
• W0 1 /04058 also owned by Hyal Pharmaceutical Corporation teaches the use of dosages of at least 10 mg. of forms of hyaluronic acid to transport effective amounts of medicines and /or therapeutic agents to sites in need of treatment in the human body, to penetrate the tissue at the sites in need 1 5 of treatment, including scar tissue, through all membranes into the cells to be treated.
• NSAID non- steroidal anti-inflammatory agent
• hyaluronic acid not only enhance the activity of the NSAID but also reduce any side effects and toxicity that is associated with the use of the prostaglandin synthesis inhibitors.
• Endothelial Cells and regulatory molecule RHAMM (Receptor for HA Mediated Motility), for binding hyaluronan.
• HARLEC is expressed (produced and put on the cell surface) in liver endothelial cells.
• the administration of an effective amount of a form of hyaluronic acid to bind with the cell-surface receptors modulates cellular activity of tissues and /or cells expressing such high affinity cell-surface receptors for hyaluronic acid (for example, an adhesion or regulatory molecule) in the human body.
• the binding capacity of the liver has been found to be so great for hyaluronan that hyaluronan when administered first goes to the liver and if not bound to the liver because the liver has reached its binding capacity for hyaluronan, circulates in the system and collects in for example, a tumour because of the tumour's receptors' ability to bind with hyaluronic acid (hyaluronan) as a result of the tumour having excess receptors for hyaluronic acid (more than normal tissue and cells).
• hyaluronic acid is used as a vehicle for a medicine or therapeutic agent to transport the medicine to a site in the body in need of treatment, unless the combination is administered directly to the site in need of treatment as by injection into a tumour, much of the combination ends up at the liver with lesser amounts at the site in need of treatment, unless and until the liver has reached its binding capacity for hyaluronan.
• hyaluronate and chondroitin sulphate to liver endothelial cells
• liver endothelial cells carry at least two different binding proteins for HA (hyaluronan) including a scavenger receptor that binds to both chondroitin sulphate and hyaluronan, (and other glycosaminoglycan (GAGS)) and, the majority of these different binding proteins (the scavenger receptors on the liver) are inhibited from their take-up of GAGS (Glycosaminoglycans) including chondroitin sulphate (CS) by their being previously bound to, for example, chondroitin sulphate.
• GAGS Glycosaminoglycans
• CS chondroitin sulphate
• a transport agent is used to transport the medicine and /or therapeutic agent to the site of the disease and /or condition
• cytotoxic medicine for example, methotrexate, cisplatin and the like
• cytotoxic medicine for example, methotrexate, cisplatin and the like
• chondroitin sulphate and hyaluronan bind to liver cells and particularly, the scavenger receptors on the liver, chondroitin sulphate does not bind with the receptors on for example, tumours (for example, metastatic tumours) and particularly, the cell surface receptors for hyaluronan comprising, the Regulatory molecule RHAMM (Receptor for HA Mediated Motility), and adhesion molecules ICAM-1, HARLEC and CD44. This has led me to develop my new methods of treatment of disease and conditions including metastatic t mours.
• RHAMM Receptor for HA Mediated Motility
• adhesion molecules ICAM-1, HARLEC and CD44 adhesion molecules
• chondroitin sulphate or other GAGS [Glycosaminoglycan] such as dextran sulphate, other than a form of hyaluronan
• hyaluronan hyaluronic acid
• the subsequently administered amounts of hyaluronan are picked up, not by the liver whose binding capacity has been substantially fulfilled but, by other sites capable of binding with forms of hyaluronan having excess unfilled hyaluronan receptors (such as on metastatic tumours).
• the hyaluronan transports any medicine (or therapeutic agent) to treat the sites in need of treatment (for example, by an effective amount of a cytotoxic agent to treat a tumour).
• a medicine or therapeutic agent
• the amount of the chondroitin sulphate that will "down regulate" the liver cells is preferably in the order of at least about 3-5 mg. of chondroitin sulphate per kilogram of body weight of the patient.
• preferably greater amounts are administered to "turn off” the liver. Because the liver processes the administered and “taken up” chondroitin sulphate rapidly, less chondroitin sulphate is not as good as more, as after several hours the liver has processed all the chondroitin sulphate. Thus, prolonged “blockage” / "down regulating" or
• chondroitin sulphate equivalent dose 1-2 grams/70 kg person
• the take-up of even small amounts of hyaluronan by the liver 0.5-1 mg/70 kg/person
• the hyaluronan is available to transport the medicine and /or therapeutic agent to the site in need of treatment (for example, methotrexate or cisplatin to a tumour or furosemide to a kidney or other use proposed by the teachings of WO 91 /04058 which is incorporated herein by reference.
• the amount thereafter required of the transport agent for example, hyaluronan or a pharmaceutically acceptable salt thereof for example, sodium hyaluronate having for example, a molecular weight less than 750,000 daltons may be reduced substantially (for example, to an amount of substantially less than 10 mg/70 kg person such as 0.1 mg/70 kg person) and the amount of medicine and/or therapeutic agent likewise substantially reduced to a mere fraction of what is normally used previously or proposed to be used previously. It may be that with the liver shut down, only micrograms ( ⁇ g)/kg of the body weight, of the transport agent for example, 20 ⁇ g/kg and micrograms ( ⁇ g) of the medicine for example, depending on the medicine 10 ⁇ g/kg of body weight may be only required in the dosage.
• chondroitin sulphate such as dextran sulphate including other GAGS. Some may be used in substantially the same amounts as with chondroitin sulphate; others may be used in higher or lower amounts.
• Other GAGS may include Dermatan sulphate, or their Proteoglycan forms, Keratan sulphate. Keratan sulphate and the like, while not technically a glycosaminoglycuronoglycan, will be considered to be included as a GAG herein.
• Other scavenger receptor ligands may also be used such as acetylated low density lipoproteins (LDL), acids such as poly-inosinic acid and the like.
• LDL acetylated low density lipoproteins
• combinations and formulations for example an injectable formulation
• a mammal for the treatment of a disease or condition
• combinations or formulations employ or incorporate as the case may be a therapeutically effective non-toxic amount of a medicinal and /or therapeutic agent to treat the disease or condition (for example a free radical scavenger (for example ascorbic acid
• Vitamin C for the treatment of mononucleosis
• an anti-cancer agent e.g. chemotherapeutic agent
• anti-viral agents for example a nonionic surfactant, e.g. nonoxynol-9 [nonylphenoxy polyethoxy ethanol] found in DelfenTM contraceptive cream, and anionic surfactants (e.g. cetyl pyridinium chloride) and cationic surfactants (e.g.
• benzalkonium chloride non-steroidal anti-inflammatory drugs (NSAID) for example indomethacin, naproxen and (+/-) tromethamine salt of ketorolac (sold under the trademark ToradolTM) and steroidal anti-inflammatory drugs, anti-fungal agent, detoxifying agents (for example for administration rectally in an enema), analgesic, bronchodilator, anti-bacterial agent, antibiotics, drugs for the treatment of vascular ischemia (for example diabetes and Berger's disease), anti-body monoclonal agent, minoxidil for topical application for hair growth, diuretics (for example furosemide (sold under the trademark LasixTM)), immunosuppressants (for example cyclosporins), lymphokynes (such as interieukin - 2 and the like), alpha- and- ⁇ -interferon and the like) administered with, or carried in, an amount of hyaluronic acid and /or salts thereof (for example the sodium salt) and /
• the formulation can be administered among other methods, intravenously, intra arterially, intraperitoneally, intrapleurally, transdermally, on the skin (topically), rectally, orally or by direct injection (for example into a tumor, into an abscess or similar disease focus) or put on a patch to be secured to the skin of the patient.
• the hyaluronic acid and /or salts thereof and the agent can be administered separately but are administered in sufficient amounts and in an immediate time sequence or interval (preferably concurrently and more preferably simultaneously), preferably at the identical site (e.g. one given intravenously and the other "piggy backed"), to treat the disease or condition.” (ii) at page 25, line 18 to page 26, line 14:
• NSAID for example indomethacin (dissolved in n-methyl glucamine) or other NSAID is administered with greater than 200mg hyaluronic acid for 1 - 2 mg/kg body weight of the NSAID (in one instance indomethacin and NMG), no major toxic side effects occur such as gastro-intestinal distress, neurological abnormalities, depression, etc., even at elevated amounts of indomethacin
• hyaluronic acid is decreased below that amount, the usual side effects may begin to reoccur.
• the responses that have been observed are superior when the NSAID (for example IndocidTM) is combined with hyaluronic acid demonstrating clearly that the combination is now "targeting" to the pathological tissue even when administered by the systemic intravenous route.
• NSAID for example IndocidTM
• neoplastic diseases when receiving in addition to other chemicals (for example ascorbic acid [Vitamin C], phloretin and anti-cancer drugs), 50 - 200 mg NSAID - hyaluronic acid (sodium hyaluronate) (for example indomethacin and hyaluronic acid) experience dramatic relief of pain immediately. This is followed within a short period of time by a resolution and resorbtion of neoplastic lesions with an improvement of pulmonary, and liver function if there is tumor present in these organs.
• chemicals for example ascorbic acid [Vitamin C], phloretin and anti-cancer drugs
• 50 - 200 mg NSAID - hyaluronic acid sodium hyaluronate
• indomethacin and hyaluronic acid experience dramatic relief of pain immediately. This is followed within a short period of time by a resolution and resorbtion of neoplastic lesions with an improvement of
• the dead tumor material and the debris and tumor toxins appear to be better eliminated by the body through the action of the macrophages whose activity is enhanced by the addition of the NSAID (or a steroidal anti ⁇ inflammatory drug) administered with hyaluronic acid (or salt or other form thereof).
• the addition of the NSAID for example with hyaluronic acid (sodium hyaluronate) deblocks the macrophages by preventing enzymatic production of prostaglandin synthetase which blocks macrophage functioning.
• the hyaluronic acid (and salt and other forms) not only enhance the activity of the NSAID but also reduce any side effects and toxicity that is associated with the use of the prostaglandin synthesis inhibitors.
• agents suitable for use as chemotherapeutic agents are novantrone (Mitoxantrone),
• the hyaluronic acid and salts thereof may be utilized at varying doses - 10 to 1000 mg/70 kg person with the optimal doses tending to range between 50 and 350 mg/70 kg individual. As there is no toxicity, the hyaluronic acid can obviously be administered in a dose excess (for example 3000 mg/70 kg individual) without any adverse effects.” (iv) at page 29, line 27 to page 33, line 31:
• One form of hyaluronic acid and /or salts thereof (for example sodium salt) and homologues, analogues, derivatives, complexes, esters, fragments, and sub units of hyaluronic acid, preferably hyaluronic acid and salts and thereof suitable for use with Applicant's invention is a fraction supplied by Sterivet Laboratories Limited. One such fraction is a 15 ml vial of Sodium hyaluronate 20mg/ml
• the sodium hyaluronate fraction is a 2% solution with a mean average molecular weight of about 225,000.
• the fraction also contains water q.s. which is triple distilled and sterile in accordance with the U.S.P. for injection formulations.
• the vials of hyaluronic acid and /or salts thereof may be carried in a Type 1 borosilicate glass vial closed by a butyl stopper which does not react with the contents of the vial.”
• the fraction of hyaluronic acid and /or salts thereof may comprise hyaluronic acid and /or salts thereof having the following characteristics: a purified, substantially pyrogen-free fraction of hyaluronic acid obtained from a natural source having at least one characteristic selected from the group consisting of the following: i) a molecular weight within the range of 150,000-225,000; ii) less than about 1.25% sulphated mucopoly- saccharides on a total weight basis; iii) less than about 0.6% protein on a total weight basis; iv) less than about 150 ppm iron on a total weight basis; v) less than about 15 ppm lead on a total weight basis; vi) less than 0.0025% glucosamine; vii) less than 0.
• a purified, substantially pyrogen-free fraction of hyaluronic acid obtained from a natural source having at least one characteristic selected from the group consisting of the following: i) a molecular weight within
• the hyaluronic acid is mixed with water and the fraction of hyaluronic acid fraction has a mean average molecular weight within the range of 150,000-225,000. More preferably the fraction of hyaluronic acid comprises at least one characteristic selected from the group consisting of the following characteristics: i) less than about 1% sulphated mucopolysaccharides on a total weight basis; ii) less than about 0.4% protein on a total weight basis; iii) less than about 100 ppm iron on a total weight basis; iv) less than about 10 ppm lead on a total weight basis; v) less than 0.00166% glucosamine; vi) less than 0.0166% glucuronic acid; vii) less than 0.0166% N-acetylglucosamine; viii) less than 0.00166% amino acids; x) a UV extinction coefficient at 257 nm of less than about 0.23; xi) a UV extinction coefficient at 280
• hyaluronic acid and /or its salts, and homologues, derivatives, complexes, esters, fragments and sub units of hyaluronic acid may be chosen from other suppliers, for example those described in the prior art documents previously referred to.
• Applicants have successfully employed sodium hyaluronate produced and supplied by LifeCoreTM Biomedical, Inc. having the following specifications
• UV/Vis Scan 190-820nm Matches reference scan
• '(a) an average molecular weight greater than about 750,000, preferably greater than about 1,200,000 - that is, a limiting viscosity number greater than about 1400 c ⁇ vVg., and preferably greater than about 2000 c ⁇ vVg.;
• a kinematic viscosity of a 1% solution of sodium hyaluronate in physiological buffer greater than about 1000 centistokes, preferably greater than 10,000 centistokes;
• Canadian Letters Patent 1,205,031 (which refers to United States Patent 4,141,973 as prior art) refers to hyaluronic acid fractions having average molecular weights of from 50,000 to 100,000; 250,000 to 350,000; and 500,000 to 730,000 and discusses processes of their manufacture.
• Cancer increasing activity free radical scavenger, of macrophages superoxide dismutase, ascorbic acid (Vitamin C) anti-cancer drugs, NSAID, Chemo ⁇ therapeutic Agents, detoxifying Agents (e.g. cholestyramine)
• Hair growth minoxidil combination grow more hair when applied topically
• nonionic surfactants e.g., shingles nonoxynol-9 and anionic, (e.g. cetyl pyridinium chloride) and cationic (e.g. benzalkonium chloride), surfactants
• Bronchodilation bronchodilators e.g. beclo- methasone diproprionate (sodium cromoglycate although not specifically a broncho-dialator), theophylline
• Vascular ischemia treat limbs in respect of diabetes, Berger's disease, etc. with suitable medicine e.g. Trental
• HIV DMSO
• Vitamin C e.g. indomethacin, naproxen, ketorolac tromethamine
• interferon VibramycinTM
• doxcycline e.g. indomethacin, naproxen, ketorolac tromethamine
• a cytotoxic agent, and transport agent for example, hyaluronan may now be required because the hyaluronan together with the medicine and /or therapeutic agent now goes to the site in need of treatment (tumour, for example) and is not taken up by the liver which has now been "down regulated".
• the liver would not be as damaged by the for example, cytotoxic agent as in the post.
• the chondroitin sulphate preferably may have a molecular weight exceeding 20,000 daltons for example, in the order of about between 20,000 and 40,000 daltons although there is a benefit irrespective of the molecular weight of chondroitin sulphate administered.
• higher molecular weight chondroitin sulphate is used so long as it is in a dosage form that can be administered effectively (for example, in sufficient sterile water for intravenous purposes).
• the dextran sulphate or other agents are used so long as it is in a dosage form that can be administered effectively (for example, in sufficient sterile water for intravenous purposes).
• glycosaminoglycans may have a molecular weight for example, in the range between about 20,000 and 500,000 daltons or higher provided the dosages can be effectively administered.
• a method of treating a disease or condition in a human treatable by a medicine and/or therapeutic agent which may be transported by an agent (for example, a form of hyaluronic acid such as sodium hyaluronate) to the site in need of treatment in the body and which agent may also transport the medicine and /or therapeutic agent to the liver (by for example, the transport agent binding to receptors on the liver) comprising:
• the amounts of medicine and /or therapeutic agent that may be effective to treat the site in need of treatment is substantially reduced.
• the amount of the for example, form of hyaluronic acid (transport agent) is substantially reduced so that the effective amount is substantially less than 10 mg/70 kg person (for example, 20 ⁇ g/kg of body weight of the patient being treated).
• a method of protecting the liver from taking up medicines and/or therapeutic agents toxic to the liver when administering the medicine and /or therapeutic agent to a site in need of treatment comprising:
• the amount of the first agent administered under (a) for example, in the order of at least about 3 - 5mg/kg of body weight (for example, 200 - 400 mg/70 kg person) having preferably a molecular weight in the range of 20,000 to 40,000 daltons, may be administered by any suitable manner such as systemically for example, orally, intravenously, subcutaneously or by direct injection proximate, adjacent, or into, the liver (by direct administration into the hepatic artery). Thereafter, (for example, after 3 to
• the amount of the second agent for transport in sub-paragraph (b) for example, sodium hyaluronate having a molecular weight less than 750,000 daltons is administered in an effective amount now found to be substantially less than 10mg/70kg person discussed in WO 91/04058 together with the medicine and/or therapeutic agent.
• the at least 200 mg/70kg person of for example, the sodium hyaluronate provided in Application WO91/04058 to reduce the side effects of the medicine and /or therapeutic agent may now be substantially reduced because the amount oi the medicine and /or therapeutic agent that is now effective is substantially less than previously provided.
• the form of hyaluronic acid may now be administered in a dosage together with a lesser amount of what is now an effective amount of medicine and /or therapeutic agent to reduce the side effects of the medicine and /or therapeutic agent.
• the amounts of the medicine and /or therapeutic agent and hyaluronan transport agent may now be ( ⁇ g) microgram amounts per kilogram of body weight to be effective. .
• the first agent may be chondroitin sulphate (preferably) or other su itable agent (such as dextran sulphate or other GAGS [Glycosaminoglycans] and /or their proteoglycan forms which are not a form of hyaluronic acid).
• Other scavenger receptor ligands which are effective may also be used as the first agent.
• the second agent is preferably a form of hyaluronic acid such as hyaluronan or sodium hyaluronate.
• a dosage kit for maximizing the amount of medicine and /or therapeutic agent to be delivered to a site in the body in need of treatment and/or for protecting the liver from taking up medicine and/or therapeutic agent (particularly cytotoxic agents) when medicines and /or therapeutic agents must be delivered to treat sites other than the liver comprising:
• an effective dosage amount comprising an effective non-toxic dosage amount of a medicine and /or therapeutic agent and an effective amount of a second agent which is a transport agent and is a different agent from the first agent (for example, a form of hyaluronic acid) and which second agent will bind to the site in need of treatment and would be capable of binding to the sites of the liver if the liver is not down regulated so that the liver's binding capacity for the second agent has been substantially reduced (preferably eliminated or blocked) by the up-take by the liver of the first agent (for example, chondroitin sulphate Molecular Weight
• the dosage amounts for sub-paragraph (b) may be microgram ( ⁇ g) per kilogram of body weight for example, 20 ⁇ g/kg. Further, according to another aspect of the invention a method is provided, comprising:
• NSAID non-steroidal anti-inflammatory agent
• cytotoxic agent for example, methotrexate and cisplatin and combinations thereof
• an effective amount of a second agent which is a transport agent and is a different agent from the first agent and which second agent is a transport agent which binds to the site in need of treatment and transports to the interstitial fluid, lymph and lymph nodes, and would be capable of binding to the sites of the liver if the liver had not been "down regulated" so that its binding capacity for the second agent has been substantially reduced by the up-take by the liver of the first agent administered under sub-paragraph (a) by binding with the scavenger receptors of the liver.
• the first agent may be chondroitin sulphate and the second agent may be a form of hyaluronic acid.
• the amounts of each may be as previously discussed.
• the amount of chondroitin sulphate may exceed at least about 3-5mg/kg and the effective amount of the form of hyaluronic acid may exceed 0.1 mg/70kg person and may have a molecular weight less than 750,000 daltons.
• FIG 1 illustrates the Biodistribution of Labeled Hyaluronan, 18-20h after Intravenous Injection of 1 mg Chondroitin Sulphate followeded by 1 mg Labeled Hyaluronan;
• FIG 2 illustrates the Uptake of 1 mg Labeled Hyaluronan (HA) With or Without Preinjection of 1 mg Chondroitin Sulphate (CS);
• FIG. 2b illustrates the Uptake of 1 mg Labeled Hyaluronan
• Figure 3 is made up of two drawings, the top drawing comparing MCPM/rat v. Time (min.), and the lower drawing comparing the MCPM/organ when 1 mg of chondroitin sulphate was administered followed by 125 I-HA ( 125 I-Hyaluronan).
• Figure 4 illustrates the inhibition of labeled hyaluronan (HA) binding to NGW cells at 37°C. (The chondroitin sulphate does not interfere whereas the labeled hyaluronan does.)
• Figure 5 shows in vivo images of the reduced liver uptake of labeled hyaluronan (HA) after pre-treatment of rats by chondroitin sulphate (CS).
• HA labeled hyaluronan
• CS chondroitin sulphate
• Figure 6 illustrates the targeting of tumours by trace amount of labeled HA (hyaluronan) after administration of chondroitin sulphate (CS) in an effective amount of 200 - 400 mg/70 kg person of CS.
• Figure 7 illustrates the photoimaging of clearance of 125j_7_
• Figures 8 to 14 illustrate the presence of ratioactive HA in the body and its characteristics with or without prior administration of other agents.
• a summary of the data I have developed on tumour targeting using radiolabelled hyaluronan (HA) administered after chondroitin sulphate (CS) pretreatment is set out in the Figures and is discussed below.
• NGW a rat colon carcinoma
• the rats received an intravenous injection of 1 mg chondroitin sulphate (200 - 400 mg/70 kg person) followed 30 seconds later by 1 mg HA (hyaluronan) of low specific radioactivity.
• chondroitin sulphate (CS) pretreatment the ratio is increased to 16.23+2.48.
• the increase is mainly due to a lower uptake in the non- tumour tissue (muscle of the healthy leg) but there is also a 27% increase in total amount bound (see Fig. 2).
• tumour to non-tumour ratio of about 4 (the relatively high ratio of 7.79 is due to one single experiment with a high ratio).
• I have seen tumour to non-tumour ratios of about 4 using similar, but not identical tumour systems. See "Accessible hyaluronan receptors identical to ICAM-1 in mouse mast cells", Stefan Gustafson, et al., Glycoconjugate Journal (1995) 12:350-355. Therefore, I have now developed a real improvement using chondroitin sulphate (CS) to the delivery of medicine and /or therapeutic agents.
• CS chondroitin sulphate
• chondroitin sulphate effectively blocks liver uptake of labeled HA (hyaluronan) at 10-15 minutes by about 80% (see Fig. 3) (without chondroitin sulphate (CS) the liver would have absorbed 95% of the radioactivity at this time).
• NGW tumour cells have HA (hyaluronan) take-up receptors that are not inhibited (blocked /immobilized for a period of time from taking up the second agent (for example, hyaluronan)) by chondroitin sulphate (CS).
• HA hyaluronan
• CS chondroitin sulphate
• Figure 4 illustrates three determinations which clearly show that the uptake of radiolabelled hyaluronan is not interfered with by the chondroitin sulphate but is interfered with by the unlabeled hyaluronan.
• Composite Figure 5 shows in vivo images of the reduced liver uptake of labeled HA after CS pre-treatment of rats. This figure shows that also uptake of trace amounts of HA (equivalent to 0.5-1 mg/70 kg person) can be effectively inhibited for an extended time by CS (equivalent dose 1-2 gm/70 kg person).
• CS Equivalent dose 1-2 gm/70 kg person
• HA much less than 10 mg/70 kg person
• tumour rats using 1 mg CS (200 - 400 mg /70 kg person) followed by a trace dose of labeled HA (equivalent to 0.5-1 mg/70kg person) and seen good targeting to the tumour (see Figure 6).
• the experimental conditions are identical to those previously described for NGW tumour rats, except for the low dose of HA.
• chondroitin sulphate (CS) blocking is therefore an ideal way of getting some hyaluronan out into the tissues. This is an additional factor in the increased binding of intravenously administered hyaluronan (HA) to tumour tissue that I found using chondroitin sulphate pretreatment.
• HA intravenously administered hyaluronan
• This provides a further method of delivering a form of hyaluronan (HA) together with a medicine and /or therapeutic agent into interstitial fluid, lymph and lymph nodes for the treatment of disease for example, cancer and metastases. This treatment may also be used to prevent metastases.
• Hyaluronan (hyaluronic acid; HA) is a high molecular weight polysaccharide consisting of repeating units of glucuronic acid and N- acetylglucosamine. It is found in high concentrations in connective tissues such as skin and cartilage, in the vitreous body of the eye and in synovial fluid (1).
• the polysaccharide can associate with several proteins in the extracellular matrix and also with some cell-surface HA-binding proteins (2).
• the serum level of HA is normally very low (10-50 ⁇ g/l), but elevated in certain disease states such as rheumatoid arthritis, liver cirrhosis, and various malignancies (3). Circulating hyaluronan comes from the peripheral tissues where most is associated with cells or binding proteins, but some exists in freely mobilized compartments.
• the polysaccharide enters the general circulation via the lymph (4) after 80- 90% is removed in lymph nodes before reaching the bloodstream (5).
• the Mw in serum is in the order of 1.5xl0 5 while the Mw of HA in lymph is about 2x10 ⁇ (6).
• the major site for elimination of HA from the bloodstream, under normal circumstances, is via receptor mediated endocytosis by the liver (1,7).
• the t ⁇ 2 of intravenously administered hyaluronan to experimental animals and man is in the order of a few minutes, and already after 15-20 min the degradation products start to appear in the circulation (7-9).
• the uptake is via coated pits and coated vesicles in liver endothelial cells (LEC), while Kupffer cells and hepatocytes are essentially negative for uptake both in vivo and in vitro (9-11).
• the HA taken up by LEC is transported to lysosomes where it is degraded to monosaccharides that ultimately are broken down to carbon dioxide, urea and water in the hepatocytes (12).
• HA used for labelling and uptake- and turnover- studies was supplied by Hyal Pharmaceutical Corporation (HPC), Toronto, Canada.
• the molecular weight distribution of the HA was determined by chromatography on a calibrated column of Sephacryl HR with porosities noted as 400, 1000 and 2000 (Pharmacia, Uppsala, Sweden) in 0.25M NaCl, 0.05% chlorbutanol (16).
• the HA content in each fraction was monitored by determination of the absorbance at 214 nm. Radioactivity was measured by gamma-counting on a Packard auto-gamma gamma-counter.
• Chondroitin sulphate A from bovine trachea was from Sigma chemical company, St. Louis, U.S.A. (product number 8529). This batch contained 1.9 ng HA/ ⁇ g CS as determined by a specific radioassay for HA (HA-50, Pharmacia, Upssala, Sweden).
• Dextran sulphate with a Mw of approximately 500,000 Da was from Pharmacia Biotech, Uppsala, Sweden (Code No. 17-0340-01).
• Heparin from intestinal mucosa and purified by repeated precipitation with cetylpyridinium chloride (17) was a kind gift from professor Ulf Lindahl, University of Uppsala, Sweden.
• HA, CS and heparin were labelled with DL-tyrosine (Sigma chemical company St. Louis, U.S.A.) as previously described (18), after CNBr- activation of the polysaccharide. Briefly, 15mg HA, CS or heparin was activated by pH 11 by 8mg CNBr for 5 min. The activated polysaccharide was separated from the reaction mixture on a small column of Sephadex G25 (PD 10, Pharmacia, Uppsala, Sweden) equilibrated with 0.2M borate buffer pH 8.0.
• DL-tyrosine Sigma chemical company St. Louis, U.S.A.
• T tyrosine
• T-HA tyrosine
• CS T- CS
• T-Hep heparin
• T-HA, T-CS or T-Hep was iodinated with 125 ⁇ by placing lOO ⁇ g xof T-labelled polysaccharide together with 0.5 mCi 125 ⁇ in a small glass tube covered with a film of lO ⁇ g l ,3,4,6-tetrachloro-3a,6a- diphenylglycouril (Sigma chemical company, St. Louis, U.S.A.). Unincorporated 125j was removed on a PD 10 column equilibrated with
• T-HA 12 5l-T-HA
• T-CS 125 I-T-CS
• the l 5l-T-HA kept a high molecular weight-profile upon gel filtration chromatography with a mean Mw of around 0.5x10 ⁇ Da, and was found to be cleared from the circulation with the kinetics and organ distribution reported for biosynthetically labelled HA of high Mw.
• the 125j_ labelled T-HA was also taken up by isolated rat liver endothelial cells both in vivo and in vitro, indicating that the labelling does not interfere with the binding to specific cell-surface receptors found on these cells (1, 2, 9-11).
• the 125l-T-CS anc j 125_ ⁇ -Hep was, by gel filtration chromatography on Sephacryl S-1000 and S-300 calibrated with HA standards, found to have the same mean Mw as the unlabelled CS (approximately 30,000 Da) and heparin (approximately 20,000 Da) and showed similar size distribution patterns.
• a single cell suspension was prepared from the liver of Sprague Dawley rats, weighing 200-300 g, by collagenase perfusion for 10 minutes at 37°C. Liver endothelial cells, Kupffer cells and parenchymal cells were purified by Percoll®-centrifugation and selective adherance as described by Pertoft and Smedsr ⁇ d (19), giving approximately 95% pure cells (10, 19). Monolayer cultures were maintained under standard culturing conditions in RPMI medium supplemented with L-glutamine (2 mM), gentamicin (50 ⁇ g/ml) and, in the case of parenchymal cells, 10% (v/v) fetal calf serum.
• Liver endothelial cells were cultured entirely without serum. All cells were cultivated overnight before the start of the experiments. Uptake studies with cells in culture: l 5l-T-hyaluronan, and in competition experiments unlabelled polysaccharides, were added to cold
• RPMI medium containing L-glutamine (2 mM) and gentamicin ⁇ 50 ⁇ g/ml
• RPMI medium containing L-glutamine (2 mM) and gentamicin ⁇ 50 ⁇ g/ml
• the cultures were kept under standard culturing conditions in 300ml medium. After the termination of the incubations, the medium was removed and analyzed for radioactivity, thereafter it was in some experiments subjected to gel cromatography on a 24ml Sephacryl 300 column to separate degraded from undegraded polysaccharide.
• the cells washed three times in phosphate buffered saline (pH 7,5)(PBS), containing NaCl (8g/l), KCl (0,2 g/l), KH2PO4 (0,2 g/l) and Na2HP ⁇ 4 (1,15 g/l), analyzed for radioactivity, or homogenized and fractionated as described earlier (18). Unspecific binding was corrected for by measurement of radioactivity associated to dishes without cells, which generally was just above background levels. In vivo studies: Sprague Dawley rats, weighing 200-300g, were ⁇ mesthetized with pentobarbital (45mg/kg body weight).
• the rat was killed. Liver, lungs, kidneys, heart, spleen and in some instances urine were assayed for radioactivity.
• the data were processed using a Macintosh SE/30®, Macintosh Ilsi® or Macintosh 7200 computer (Apple computer Inc. Cupertino, CA, U.S.A.).
• the graphs were constructed using the Cricket Graph® program (version 1.3, Cricket software, Malvern, PA, U.S.A.) and Canvas (verson 3.0.2, Deneba Systems Inc., Miami, Fl, U.S.A.).
• Statistical analysis was performed using Statworks® (version 1.1, Cricket Software, Malvern, PA, U.S.A.).
• the radioactivity after CS blocking was mainly found in the blood with some uptake in liver, spleen and kidney (Fig. 8) and was found to rapidly decrease in Mw (Fig. 9).
• Some labelled polysaccharides with Mw of about 10,000-40,000 Da were found in urine (Fig. 8 and 9). Only minute amounts of labelled HA could be found in the urine when liver uptake was blocked by unlabelled HA (1 mg/kg b.w.) (Fig. 8).
• the rapid decrease in Mw of circulating l 5l-T-HYA seen after CS blocking was not seen with HA blocking and more radioactivity stayed within the general circulation after HA blocking than after CS blocking (Fig. 8 and Fig. 10).
• trace amounts of radioactivity could be found in the urine after 70 min., this material had the same Mw as the radioactivity found in urine after CS blocking (Figs. 8, 9 and 10).
• DxS Extran Sulphate
• a dose of lmg/kg b.w. was tested it was found that the liver uptake was inhibited by 30-40% (Fig. 11).
• Heparin at a dose of 20mg/kg b.w. did not affect the clearance of 125j/r- HYA (Fig. 8), nor could HA at a dose of 20mg/kg b.w. inhibit the clearance of 125 ⁇ _ ⁇ _Hep at a tracer dose (Fig. 12).
• CS could partially inhibit the liver uptake of l 2 5l-T-Hep (Fig. 12).
• liver uptake could effectively be inhibited by unlabelled CS and HA, resulting in increased urinary clearance (Fig. 14).
• the fragmentation results in low recovery of injected dose and the low Mw HA is filtered out into the tissues and via the kidneys out into the urine (Fig. 7, 9 and 10).
• liver uptake of CS is not as high as that of HA probably depends on the fact that the CS used only has a Mw of around 30 kDa compared to about 400 kDa for the HA, and some is therefore rapidly removed from the circulation by filtration so that only a fraction of that injected remains long enough in the general circulation to be taken up by the liver.
• DxS seems also to bind to the same receptors as CS and HA but with lower affinity as a higher dose is needed to inhibit liver uptake of ⁇ ⁇ I-T-HA using DxS than using CS or HA (Fig. 11).
• Our results indicate that the turnovers of the naturally occuring polysaccharides HA and CS are partially an effect of liver uptake of the circulating polysaccharides via a common receptor on LEC. It is therefore possible, that high levels of circulating HA in some conditions can be secondary to increased outflow of CS into the general circulation from the tissues, and vice versa.

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