EP1207862A2 - Arthritis treatment - Google Patents

Arthritis treatment

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Publication number
EP1207862A2
EP1207862A2 EP00935346A EP00935346A EP1207862A2 EP 1207862 A2 EP1207862 A2 EP 1207862A2 EP 00935346 A EP00935346 A EP 00935346A EP 00935346 A EP00935346 A EP 00935346A EP 1207862 A2 EP1207862 A2 EP 1207862A2
Authority
EP
European Patent Office
Prior art keywords
catechin
arthritis
arthritic
cartilage
agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00935346A
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German (de)
French (fr)
Inventor
David Buttle
Clair Adcocks
Peter Collin
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University of Sheffield
Original Assignee
University of Sheffield
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Filing date
Publication date
Priority claimed from GB0007321A external-priority patent/GB0007321D0/en
Application filed by University of Sheffield filed Critical University of Sheffield
Publication of EP1207862A2 publication Critical patent/EP1207862A2/en
Withdrawn legal-status Critical Current

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the invention relates to the use of catechins, or varients thereof, in the treatment of all forms of arthritis.
  • Green tea is a very common drink in the Far East and its beneficial health properties have been recognised for many hundreds of years. Green tea has therefore been the subject of extensive research to identify the active agents responsible for said beneficial health effects. Green tea is a complex mixture of naturally occurring plant extracts. A group of these is variously described as flavanols, catechins or proanthocyanidins. The commonest of these in green tea are: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG), please see Figure 1.
  • Catechins have been reported to alleviate a number of clinical conditions. These include stroke and cerebral haemorrhage (Sato et al, 1989), cardiovascular and liver diseases (Imai & Nakachi, 1995), bacterial infections (Ikigai et al, 1993) and stomach ulcers (Murakami et al, 1992). Catechins have also been shown to inhibit the release of histamine and leukotriene which indicates that catechins may have benefit with regard to the treatment of various allergic disorders (Matsuo et al, 1997).
  • catechins have also been shown to inhibit the lipopolysaccharide (LPS) induced release of tumour necrosis factor ⁇ (TNF ⁇ ) production at the level of both transcription and release from activated macrophages and therefore may have use in the inhibition of inflammation (Yang et al, 1998).
  • LPS lipopolysaccharide
  • TNF ⁇ tumour necrosis factor ⁇
  • Catechins have been shown to regulate cholesterol levels; to have anti-mutagenic properties; to reduce blood pressure; to inhibit the effects of various agents on the liver and can also protect teeth from decay.
  • Clearly catechins have a number of beneficial health effects which are well known in the art.
  • Catechin is a generic name for a group of compounds based on the structure catechin itself, (3',3',4',5,7-flavanpentol), please see Figure 1.
  • catechins are chondroprotective, that is they inhibit the breakdown of cartilage extracellular matrix.
  • EGCG, ECG and EC at a dose of 20 ⁇ M significantly inhibited proteoglycan loss resulting from treatment of bovine cartilage explants with recombinant human interleukin l ⁇ (rhIL- ⁇ ).
  • rhIL- ⁇ recombinant human interleukin l ⁇
  • EGCG produced a dose-response curve for inhibition of proteoglycan loss, with about 50% inhibition being achieved at a concentration of 2 ⁇ M, please see Figure 2.
  • EGCG showed no toxic effects. At 218 ⁇ M EGCG was without effect on lactate production by the explants.
  • EGCG is reported to inhibit TNF ⁇ synthesis (Yang et al, 1998; Suganuma et al, 1996), which may provide the basis for its anti-inflammatory effects.
  • At a concentration of 20 ⁇ M it inhibited bacterial lipopolysaccharide-stimulated TNF ⁇ synthesis from blood samples from two different volunteers by 66% and 30%o using two different commercially available ELISA kits.
  • this cannot provide the mechanism for inhibition of cartilage breakdown as large amounts of exogenous cytokine are added to the cartilage culture in experiments such as those shown in Figure 2. This leads to the conclusion that catechins have two distinct properties that should be beneficial to arthritis sufferers, an anti-inflammatory and a distinct chondroprotective effect.
  • said catechin is selected from: (+)epicatechin, (+)catechin, (-)epicatechin; (-)catechin, (-)epigallocatechin; (-)gallocatechin; (-)epicatechin gallate; (-)catechin gallate; (-)epigallocatechin gallate; (-)gallocatechin gallate; or varients thereof.
  • said catechin is epigallocatechin gallate.
  • said catechin is epicatechin gallate.
  • said medicament is for the treatment of arthritic conditions selected from: osteoarthritis, rheumatoid arthritis; inflammatory arthritis; osteochondritis; acute pyrophosphate arthritis: reactive arthritis; psoriatic arthritis; juvenile arthritis; lupus erythematosus; Sjogren's syndrome; relapsing polychondritis; ankylosing spondylitis; psoriatic arthritis; MSUM (gout); CPDD (pseudogout, chondrocalcinosis); chondrolysis; bursitis.
  • arthritic conditions selected from: osteoarthritis, rheumatoid arthritis; inflammatory arthritis; osteochondritis; acute pyrophosphate arthritis: reactive arthritis; psoriatic arthritis; juvenile arthritis; lupus erythematosus; Sjogren's syndrome; relapsing polychondritis; ankylosing spondylitis; psoriatic arthritis; MSUM (gout); CP
  • said medicament is for the treatment of osteoarthritis.
  • said medicament is for the treatment of rheumatoid arthritis.
  • said medicament is for the use in prophylactic treatment of arthritis.
  • said prophylactic treatment is for animals with a genetic predisposition to arthritis, preferably osteoarthritis.
  • said prophylactic treatment is to protect animals with an increased probability of developing arthritis due to joint damage (eg. cruciate ligament damage). It is well known in the art that individuals which suffer joint damage have an increased incidence of arthritis (Price et al., 1999).
  • a therapeutic composition comprising at least one catechin and at least one anti-arthritic agent or biopolymer.
  • said composition is for use in the manufacture of a medicament for the treatment of arthritis, ideally osteoarthritis.
  • said anti-arthritic agent is hyaluronic acid, or varients thereof.
  • said anti-arthritic agent is glucosamine, or varients thereof, preferably glucosamine sulphate. It is reported that glucosamine is an effective treatment of arthritic conditions (MacCarty, 1994; MacCarty, 1998). Current opinion suggests that glucosamine stimulates the production of glycosaminoglycans, such as hyaluronic acid in joints.
  • Hyaluronic acid is a polymer of N-acetyl glucosamine and glucuronic acid molecules and is well known to have anti-arthritic properties (Balazs, 1968; Gibbs et al, 1968; Balazs & Gibbs.. 1970; Rydell et al, 1970; Weiss et al, 1981 ; Denlinger, 1982; Balazs,1982; Balazs & Denlinger, 1985; Weiss. & Balazs, 1987; Balazs & Denlinger, 1989; McCain et al, 1989; Adams, 1993; Balazs & Denlinger.
  • Hyaluronic acid can vary in molecular mass from 50kDa to 8 x 10 3 kDa and forms highly viscous solutions. Methods to prepare pure samples, which are non-inflammatory, are well known in the art. For example, EP 0239335 & US 4879375 disclose methods to prepare highly pure fractions of hyaluronic acid which purport to be non-inflammatory. Hyaluronic acid is known to have a variety of therapeutic effects.
  • Hyaluronic acid provides visco- supplementation and/or viscolubrication (Balazs & Denlinger, 1993; Peyron, 1993a; Scale et al., 1994; Lussier et al., 1996) to replace fragmented hylauronic acid as a consequence of arthritic disease.
  • said catechin and anti-arthritic agent are administered as a simple admixture.
  • said catechin and anti-arthritic agent are crosslinked, coupled or associated together.
  • Hyaluronic acid has a number of free hydroxyl and carboxyl groups to which catechins may be crosslinked or coupled either directly or via crosslinking agents.
  • hyaluronic acid and a catechin are encapsulated within a liposome preparation as detailed below.
  • said catechin is crosslinked, coupled or associated with hyaluronic acid.
  • a method to crosslink or couple at least one catechin to at least one anti-arthritic agent comprising:
  • said anti-arthritic agent is hyaluronic acid and said catechin is selected from: (+)catechin; (+)epicatechin; (-)catechin; (-)epigallocatechin; (-)gallocatechin; (-)epicatechin gallate; (-)catechin gallate; (-)epigallocatechin gallate; (-)gallocatechin gallate; or varient thereof.
  • a method of treating an arthritic condition comprising administering to an animal a pharmacologically effective amount of the therapeutic composition/medicament according to the invention.
  • said arthritic condition is selected from; osteoarthritis; rheumatoid arthritis; osteochondritis; acute pyrophosphate arthritis; reactive arthritis; psoriatic arthritis; juvenile arthritis; lupus erythematosus: Sj ⁇ gren's syndrome; relapsing polychondritis; ankylosing spondylitis; psoriatic arthritis; MSUM (gout); CPPD (pseudogout, chondrocalcinosis); chondrolysis; bursitis.
  • said arthritic condition is osteoarthritis.
  • said arthritic condition is rheumatoid arthritis.
  • compositions/medicaments can be formulated in a variety of ways to facilitate delivery.
  • liposomal compositions may be usefully employed to deliver said compositions/medicaments.
  • Liposomes are lipid based vesicles which encapsulate a selected therapeutic agent which is then introduced into a patient.
  • the liposome is manufactured either from pure phospholipid or a mixture of phospholipid and phosphoglyceride.
  • liposomes can be manufactured with diameters of less than 200nm, which enables them to be intravenously injected and able to pass through the pulmonary capillary bed.
  • biochemical nature of the liposomes confers permeability across blood vessel membranes to gain access to selected tissues. Liposomes do have a relatively short half-life. So-called STEALTH ® liposomes have been developed which comprise liposomes coated in polyethylene glycol (PEG).
  • the PEG treated liposomes have a significantly increased half-life when administered intravenously to a patient.
  • STEALTH® liposomes show reduced uptake in the reticuloendothelial system and enhanced accumulation in selected tissues.
  • so called immuno-liposomes have been developed which combine lipid based vesicles with an antibody or antibodies, to increase the specificity of the delivery of the therapeutic composition/medicament to a selected cell/tissue.
  • liposomes as a delivery means is described in US 5580575 and US 5542935.
  • compositions/medicaments can be imbided or provided in the form of an oral or nasal spray, an aerosol, suspension, emulsion, and/or eye drop.
  • the medicament may be provided in tablet form.
  • Alternative delivery means include inhalers or nebulisers.
  • the medicament can be delivered by direct injection into a joint. It is envisioned that the compositions/medicaments be delivered intravenously, intramuscularly, subcutaneously or topically. Further still, the medicament may be taken rectally.
  • compositions/medicaments are effective at preventing and/or alleviating arthritic conditions in animals other than humans, for example and not by way or limitation, family pets, livestock, horses.
  • a method to screen for agents with anti-arthritic properties comprises:
  • said inflammatory cytokine is selected from:interleukin-l ⁇ ; interleukin-l ⁇ ; oncostatin M; tumour necrosis factor- ⁇ .
  • said method comprises:
  • vitamin A metabolite is all-/rar ⁇ -retinoic acid.
  • an agent identified by the screening method of the invention is provided.
  • catechins disclosed above can either be isolated from natural plant sources, eg Camellia sinensis, Uncarcia gambir or can be synthesised in the laboratory using methods well known in the art.
  • Table 1 represents the inhibitory effects of catechins on cytokine or vitamin A metabolite-stimulated cartilage breakdown using (i) nasal cartilage explants and (ii) articular cartilage explants;
  • Table 2 represents the inhibitory effects of EGCG and ECG on human osteoarthritic cartilage explants
  • Table 3 represents the inhibitory effects of EGCG and ECG on the degradation of proteoglycan of human rheumatoid knee cartilage
  • Table 4 represents the inhibitory effects of EGCG and ECG on the degradation of proteoglycan of human non-arthritic cartilage
  • Tables 5 and 6 represent the inhibitory effects of EGCG, ECG, EC and EGC on type II collagen degradation in bovine nasal cartilage explants stimulated with rhIL ⁇ ;
  • Table 7 demonstrates lactate production by the explants over the latter part of a 28- day period
  • Table 8 represents the inhibitory effects of EGCG on the synthesis of human TNF ⁇
  • Figure la and lb represents the chemical structures of a selection of catechins and variants thereof;
  • Figure 2 represents the dose-response for EGCG inhibitory activity of TNF ⁇ induced cartilage proteoglycan breakdown
  • Figure 3 represents the macroscopic changes in bovine nasal cartilage explants cultured in the presence or absence of rhIL-l ⁇ , EC and EGC for a 28 day period. Introduction.
  • Cartilage proteoglycan degradation in the bovine system was stimulated with the proinflammatory cytokines interleukinl ⁇ (ILl ⁇ ) or tumour necrosis factor ⁇ (TNF ⁇ ) or with the vitamin A derivative, all-trans retinoic acid (Ret).
  • the degradatory process was initiated in the human cartilage using a combination of interleukinl ⁇ (ILl ⁇ ) and TNF ⁇ .
  • ILl ⁇ interleukinl ⁇
  • TNF ⁇ all-trans retinoic acid
  • Bovine nasal septum and metacarpophalangeal cartilage was prepared as described by Buttle et al, (1992).
  • the nasal septum cartilage was removed using a post-mortem knife and the overlying membrane was discarded.
  • the excised cartilage was wiped with isoprenyl-impregnated Azowipes, placed in a petri dish in the tissue culture hood and washed in sterile phosphate buffered saline (PBS).
  • PBS sterile phosphate buffered saline
  • the cartilage was sliced using a scalpel blade into approximately 2mm x 3mm x 3cm slices, and from these slices discs were cut with the aid of a belt punch. About 200 discs ( ⁇ 3mm diam., 2mm thick) were obtained from each animal.
  • Cartilage explants treated as described above were individually transferred into the wells of a 96-well plate and cultured for up to 9 days (medium change on days 3 and 6 or on day 4) in serum-free DMEM in the presence or absence of recombinant human ILl ⁇ (rhILl ⁇ ), recombinant human ILl ⁇ (rhILl ⁇ ), recombinant human TNF ⁇ (rhTNF ⁇ ) and Ret, either singly or in combination.
  • rhILl ⁇ recombinant human ILl ⁇
  • rhILl ⁇ recombinant human ILl ⁇
  • rhTNF ⁇ recombinant human TNF ⁇
  • Ret Ret
  • sGAGs sulfated glycosaminoglycans
  • Certain catechins were prepared as stock solutions in dimethyl sulfoxide (DMSO) and further diluted in DMSO or DMEM to give the appropriate final concentration in the culture medium and also a
  • sGAGs within the conditioned media and retained in the tissue were either determined by the dimethylmethylene blue assay (Farndale et al., 1986) or by scintillation counting following labelling with SO 4 (Ilic et al, 1995). Data were expressed as the percentage sGAG released from the tissue or as the percentage inhibition of sGAG release. The Mann- Whitney U test for unpaired, non-parametric data was used to determine the statistical significance of the results.
  • Type II collagen breakdown in rhIL-l ⁇ -stimulated bovine nasal cartilage explants
  • bovine nasal cartilage explant cultures were maintained for 28 days, with a twice-weekly medium change, in the presence or absence of rhIL-l ⁇ (4.5nM) and the catechins; EGCG, ECG, EC and EGCG at 20 ⁇ M.
  • type II collagen remaining in the cartilage residues was extracted by digestion with proteinase K (EC 3.4.21.64) at 56°C for 15 hours.
  • the extracts were assayed by- inhibition enzyme-linked immunosorbent assay (ELISA) using a mouse IgG monoclonal antibody to denatured type II collagen, Col2-3/4m as previously described (Hollander et al. 1994).
  • the amount of collagen released at each medium change throughout the culture period was calculated as a percentage of total collagen in each culture well (medium plus tissue residue).
  • Bovine nasal cartilage explants were obtained as described above. The rate of proteoglycan synthesis was assessed by measuring incorporation of 3:> S from 35 SO 4 .
  • Three groups were set up: (a) killed explants (freeze-thawed thrice), (b) explants cultured in serum-free DMEM alone, (c) explants cultured in serum-free DMEM and 2 ⁇ M or 20 ⁇ M EGCG. All three groups were cultured in the presence of 5 ⁇ Ci 33 SO 4 /ml for 18 hours and 3" S incorporation was assessed as previously described by Buttle et al. (1993).
  • Lactate test for assessment of chondrocyte metabolic activity Lactate test for assessment of chondrocyte metabolic activity.
  • a measure of the toxicity of a compound can therefore be made by determining the levels of lactate in the conditioned media by using the lactate oxidase/peroxidase method with a kit from Sigma Chemical Co.
  • Peripheral blood was taken from volunteers and 50 iu/ml heparin was added.
  • the blood was diluted 1 :6 with serum-free DMEM and incubated for 4 hours at 37°C in a water bath in the presence or absence of l ⁇ g/ml lipopolysaccharide (LPS) (E.col ⁇ ) and EGCG (20 ⁇ M).
  • LPS lipopolysaccharide
  • EGCG 20 ⁇ M
  • the blood was freeze-thawed thrice at -40°C and then centrifuged at 1000 rpm for 5 min to remove cell debris.
  • a TNF ⁇ ELISA was then performed on the supernatant following the manufacturers' instructions, either using a kit from R&D systems or from Diaclone Research. RESULTS
  • EGCG significantly inhibited rhTNF ⁇ -stimulated cartilage proteoglycan degradation in a bovine nasal cartilage explant model, but no significant effect was observed for the basal, rhILl ⁇ -, or Ret-stimulated release. Both ECG and EC significantly inhibited ILl ⁇ -stimulated degradation but not rhTNF ⁇ - or Ret- stimulated breakdown.
  • a dose-response curve was constructed (Fig. 2). Inhibition reached statistical significance at 2 ⁇ M (47% inhibition) and increased to 84% and 138% inhibition at 20 ⁇ M and 200 ⁇ M respectively. Where inhibition exceeded 100%) some of the basal breakdown as well as rhTNF ⁇ -stimulated breakdown is indicated.
  • EGCG at 200 ⁇ M was shown not to be toxic over a 5 day period by measurement of lactate levels in the conditioned media of bovine nasal cartilage explants stimulated with rhTNF ⁇ .
  • Explants cultured in DMEM alone produced 896 ⁇ g lactate/explant and in the presence of EGCG, this was 1040 ⁇ g lactate/explant.
  • When explants were cultured in the presence of rhTNF ⁇ a total of 1248 ⁇ g lactate/explant was produced, decreasing slightly to 896 ⁇ g when cultured in the presence of EGCG.
  • ECG (20 ⁇ M) produced significant inhibition of proteoglycan breakdown from human cartilage from osteoarthritic, rheumatoid and non-arthritic joints.
  • EGCG inhibited basal levels of proteoglycan loss from osteoarthritic cartilage (Table 2).
  • EGCG has been reported to inhibit TNF ⁇ synthesis in a human stomach cancer cell line KATO III (Okabe et al, 1999) and in BALB/3T3 cells (Suganuma et al, 1999).
  • KATO III human stomach cancer cell line
  • BALB/3T3 cells BALB/3T3 cells
  • Bovine nasal and articular cartilage explants were cultured for 5 days in serum-free DMEM in the presence or absence of rhIL-l ⁇ (0.3 nM nasal; 3nM articular). rhTNF ⁇ (3nM nasal; 6nM articular) or Ret (l ⁇ M) and in the presence or absence of the catechins EGCG, ECG, EC or EGC at a final concentration of 20 ⁇ M. Medium was changed on day 3. The degradation of cartilage proteoglycan was determined by measuring the sGAG released from the explants as a percentage of total sGAG using the DMB assay. The data are expressed as the mean percentage inhibition of stimulated or basal cartilage proteoglycan degradation ⁇ s.e.m.
  • Human osteoarthritic articular cartilage explants were biosynthetically labelled for 5 days in 5%(v/v) newborn calf serum (NCS)-containing DMEM using 35 SO 4 (5 ⁇ Ci/ml). The explants were washed for 2 days and then cultured for a further 9 days in the presence or absence of a combination of rhlLl ⁇ (3nM) and rhTNF ⁇ (6nM) and also in the presence or absence of the catechins EGCG or ECG at 20 ⁇ M. Medium was changed on day 3 and day 6.
  • the degradation of cartilage proteoglycan was determined by measuring the radiolabel released from the explants as a percentage of total radiolabel by quantifying the 35 S released into the culture medium and that retained in the tissue by use of a scintillation counter. The data are expressed as the mean percentage release ⁇ s.e.m. *p ⁇ 0.05, when the release of sGAG is compared between the groups cultured in the presence of the catechins to those cultured in their absence, as determined by the Mann Whitney U test for 2-tailed, non-parametric data. Table 3
  • Human non-rheumatoid articular cartilage was obtained from a patient suffering from Marfan's Syndrome at surgery, and cultured overnight in 5%(v/v) NCS-containing DMEM. Explants were individually transferred to a 96-well plate and cultured for 9 days in serum-free DMEM in the presence or absence of a combination of rhIL- 1 ⁇ (3nM) and rhTNF ⁇ (6nM) and also in the presence or absence of the catechins EGCG or ECG at 20 ⁇ M. Medium was changed at 3 and 6 days.
  • the degradation of the cartilage proteoglycan was determined by measuring sGAG released from the explants as a percentage of total sGAG using the DMB assay; ** p ⁇ 0.005 when comparing the proteoglycan released from the group cultured with the cytokine combination to the other groups, as determined using the 2-tailed Mann Whitney test for non-parametric data. Table 5
  • Human peripheral blood was diluted 1 :6 with serum-free DMEM and then incubated for 4 hours at 37°C in a water bath in the presence or absence of bacterial lipopolysaccharide (LPS) (l ⁇ g/ml) and EGCG (20 ⁇ M).
  • LPS bacterial lipopolysaccharide
  • EGCG EGCG (20 ⁇ M).

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Abstract

The invention relates to the use of catechins in the treatment of various forms of arthritis, including the use of combinations of catechins and other anti-arthritic agents, like hyoluronic acid and glucosamine and glucosamine sulfate, in said treatment; medicaments and compositions for use in said treatment; and methods to identify agents with anti-arthritic properties.

Description

Arthritis Treatment
The invention relates to the use of catechins, or varients thereof, in the treatment of all forms of arthritis.
Green tea is a very common drink in the Far East and its beneficial health properties have been recognised for many hundreds of years. Green tea has therefore been the subject of extensive research to identify the active agents responsible for said beneficial health effects. Green tea is a complex mixture of naturally occurring plant extracts. A group of these is variously described as flavanols, catechins or proanthocyanidins. The commonest of these in green tea are: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG), please see Figure 1.
Catechins have been reported to alleviate a number of clinical conditions. These include stroke and cerebral haemorrhage (Sato et al, 1989), cardiovascular and liver diseases (Imai & Nakachi, 1995), bacterial infections (Ikigai et al, 1993) and stomach ulcers (Murakami et al, 1992). Catechins have also been shown to inhibit the release of histamine and leukotriene which indicates that catechins may have benefit with regard to the treatment of various allergic disorders (Matsuo et al, 1997).
In addition, catechins have also been shown to inhibit the lipopolysaccharide (LPS) induced release of tumour necrosis factor α (TNFα) production at the level of both transcription and release from activated macrophages and therefore may have use in the inhibition of inflammation (Yang et al, 1998).
Catechins have been shown to regulate cholesterol levels; to have anti-mutagenic properties; to reduce blood pressure; to inhibit the effects of various agents on the liver and can also protect teeth from decay. Clearly catechins have a number of beneficial health effects which are well known in the art.
By far the most interest has been in the anti-cancer effects of catechins. Epidemiological studies have suggested that consumption of green tea may help to prevent cancers in humans (Yang et al, 1993), and at least 17 clinical studies have been published, for example Gao et al, 1994.
We have discovered a further beneficial therapeutic effect of catechins which relates to the treatment of arthritis.
Catechin is a generic name for a group of compounds based on the structure catechin itself, (3',3',4',5,7-flavanpentol), please see Figure 1.
We have recently discovered that catechins are chondroprotective, that is they inhibit the breakdown of cartilage extracellular matrix. EGCG, ECG and EC, at a dose of 20μM significantly inhibited proteoglycan loss resulting from treatment of bovine cartilage explants with recombinant human interleukin lα (rhIL-α). When recombinant human TNFα (rhTNFα) provided the catabolic stimulus, EGCG produced a dose-response curve for inhibition of proteoglycan loss, with about 50% inhibition being achieved at a concentration of 2μM, please see Figure 2. In these experiments, EGCG showed no toxic effects. At 218μM EGCG was without effect on lactate production by the explants. Also at an effective concentration range of 2μM and 20μM, 35S incorporation as a measure of proteoglycan synthesis was unaffected. In addition, the inhibitory effect of EGCG on TNF-mediated proteoglycan breakdown was fully reversible following removal of the catechin from the explants (not shown).
EGCG is reported to inhibit TNFα synthesis (Yang et al, 1998; Suganuma et al, 1996), which may provide the basis for its anti-inflammatory effects. We have confirmed that EGCG does indeed have this important property. At a concentration of 20μM it inhibited bacterial lipopolysaccharide-stimulated TNFα synthesis from blood samples from two different volunteers by 66% and 30%o using two different commercially available ELISA kits. However, this cannot provide the mechanism for inhibition of cartilage breakdown as large amounts of exogenous cytokine are added to the cartilage culture in experiments such as those shown in Figure 2. This leads to the conclusion that catechins have two distinct properties that should be beneficial to arthritis sufferers, an anti-inflammatory and a distinct chondroprotective effect.
According to the first aspect of the invention there is provided the use of at least one catechin for the manufacture of a medicament for the treatment of arthritis.
In a preferred embodiment of the invention said catechin is selected from: (+)epicatechin, (+)catechin, (-)epicatechin; (-)catechin, (-)epigallocatechin; (-)gallocatechin; (-)epicatechin gallate; (-)catechin gallate; (-)epigallocatechin gallate; (-)gallocatechin gallate; or varients thereof.
In a further preferred embodiment of the invention said catechin is epigallocatechin gallate.
In yet a further preferred embodiment of the invention said catechin is epicatechin gallate.
In a yet further preferred embodiment of the invention said medicament is for the treatment of arthritic conditions selected from: osteoarthritis, rheumatoid arthritis; inflammatory arthritis; osteochondritis; acute pyrophosphate arthritis: reactive arthritis; psoriatic arthritis; juvenile arthritis; lupus erythematosus; Sjogren's syndrome; relapsing polychondritis; ankylosing spondylitis; psoriatic arthritis; MSUM (gout); CPDD (pseudogout, chondrocalcinosis); chondrolysis; bursitis.
In yet a still further preferred embodiment of the invention said medicament is for the treatment of osteoarthritis.
In yet a still further preferred embodiment of the invention said medicament is for the treatment of rheumatoid arthritis. In yet still a further preferred embodiment of the invention said medicament is for the use in prophylactic treatment of arthritis. Ideally said prophylactic treatment is for animals with a genetic predisposition to arthritis, preferably osteoarthritis.
Alternatively or preferably, said prophylactic treatment is to protect animals with an increased probability of developing arthritis due to joint damage (eg. cruciate ligament damage). It is well known in the art that individuals which suffer joint damage have an increased incidence of arthritis (Price et al., 1999).
According to the second aspect of the invention there is provided a therapeutic composition comprising at least one catechin and at least one anti-arthritic agent or biopolymer. Preferably said composition is for use in the manufacture of a medicament for the treatment of arthritis, ideally osteoarthritis. More ideally still said anti-arthritic agent is hyaluronic acid, or varients thereof.
Alternatively or preferably said anti-arthritic agent is glucosamine, or varients thereof, preferably glucosamine sulphate. It is reported that glucosamine is an effective treatment of arthritic conditions (MacCarty, 1994; MacCarty, 1998). Current opinion suggests that glucosamine stimulates the production of glycosaminoglycans, such as hyaluronic acid in joints.
Hyaluronic acid is a polymer of N-acetyl glucosamine and glucuronic acid molecules and is well known to have anti-arthritic properties (Balazs, 1968; Gibbs et al, 1968; Balazs & Gibbs.. 1970; Rydell et al, 1970; Weiss et al, 1981 ; Denlinger, 1982; Balazs,1982; Balazs & Denlinger, 1985; Weiss. & Balazs, 1987; Balazs & Denlinger, 1989; McCain et al, 1989; Adams, 1993; Balazs & Denlinger. 1993; Moreland et al, 1993; Peyron, 1993a; Peyron, 1993b; Scale et al, 1994; Adams et al, 1995; Band et al, 1995; Baker, 1997 Balazs & Larsen, 1997; Adams, 1998; Denlinger, 1998; Dickson & Hosie, 1998; Estey, 1998; Wobig et al, 1998 and Peyron, 1999.
It is naturally occurring in all mammals in a variety of tissues (eg. synovial fluid, vitreous humour) and some bacterial species. Hyaluronic acid can vary in molecular mass from 50kDa to 8 x 103 kDa and forms highly viscous solutions. Methods to prepare pure samples, which are non-inflammatory, are well known in the art. For example, EP 0239335 & US 4879375 disclose methods to prepare highly pure fractions of hyaluronic acid which purport to be non-inflammatory. Hyaluronic acid is known to have a variety of therapeutic effects. For example, and not by the way of limitation, the treatment of various skin disorders, (described in US 5914322) and the treatment of articular degeneration as a consequence of corticosteroid treatment, (described in US 4801619). Hyaluronic acid, and the like, provide visco- supplementation and/or viscolubrication (Balazs & Denlinger, 1993; Peyron, 1993a; Scale et al., 1994; Lussier et al., 1996) to replace fragmented hylauronic acid as a consequence of arthritic disease.
In a preferred embodiment of the invention said catechin and anti-arthritic agent are administered as a simple admixture. Alternatively, said catechin and anti-arthritic agent are crosslinked, coupled or associated together.
It is possible to crosslink or conjugate hyaluronic acid to various therapeutic molecules. For example, EP 0296740 describes the production of hyaluronic acid conjugates. Hyaluronic acid has a number of free hydroxyl and carboxyl groups to which catechins may be crosslinked or coupled either directly or via crosslinking agents. Alternatively, hyaluronic acid and a catechin are encapsulated within a liposome preparation as detailed below.
In an alternative embodiment of the invention said catechin is crosslinked, coupled or associated with hyaluronic acid.
According to a third aspect of the invention there is provided a method to crosslink or couple at least one catechin to at least one anti-arthritic agent comprising:
i) providing at least one catechin and at least one anti-arthritic agent;
ii) providing conditions conducive to the crosslinking or coupling of said catechin to said agent; and, optimally iii) purifying the crosslinked or coupled complex from the reaction mixture.
In a preferred method of the invention said anti-arthritic agent is hyaluronic acid and said catechin is selected from: (+)catechin; (+)epicatechin; (-)catechin; (-)epigallocatechin; (-)gallocatechin; (-)epicatechin gallate; (-)catechin gallate; (-)epigallocatechin gallate; (-)gallocatechin gallate; or varient thereof.
According to a yet further aspect of the invention there is provided a method of treating an arthritic condition comprising administering to an animal a pharmacologically effective amount of the therapeutic composition/medicament according to the invention.
In a preferred method of the invention said arthritic condition is selected from; osteoarthritis; rheumatoid arthritis; osteochondritis; acute pyrophosphate arthritis; reactive arthritis; psoriatic arthritis; juvenile arthritis; lupus erythematosus: Sjόgren's syndrome; relapsing polychondritis; ankylosing spondylitis; psoriatic arthritis; MSUM (gout); CPPD (pseudogout, chondrocalcinosis); chondrolysis; bursitis.
In a still preferred method of the invention said arthritic condition is osteoarthritis.
In a yet still further preferred method of the invention said arthritic condition is rheumatoid arthritis.
It will be apparent to one skilled in the art that the therapeutic compositions/medicaments can be formulated in a variety of ways to facilitate delivery. For example, liposomal compositions may be usefully employed to deliver said compositions/medicaments.
Liposomes are lipid based vesicles which encapsulate a selected therapeutic agent which is then introduced into a patient. Typically, the liposome is manufactured either from pure phospholipid or a mixture of phospholipid and phosphoglyceride. Typically liposomes can be manufactured with diameters of less than 200nm, which enables them to be intravenously injected and able to pass through the pulmonary capillary bed. Furthermore the biochemical nature of the liposomes confers permeability across blood vessel membranes to gain access to selected tissues. Liposomes do have a relatively short half-life. So-called STEALTH® liposomes have been developed which comprise liposomes coated in polyethylene glycol (PEG). The PEG treated liposomes have a significantly increased half-life when administered intravenously to a patient. In addition STEALTH® liposomes show reduced uptake in the reticuloendothelial system and enhanced accumulation in selected tissues. In addition, so called immuno-liposomes have been developed which combine lipid based vesicles with an antibody or antibodies, to increase the specificity of the delivery of the therapeutic composition/medicament to a selected cell/tissue.
The use of liposomes as a delivery means is described in US 5580575 and US 5542935.
It will be apparent to one skilled in the art that the compositions/medicaments can be imbided or provided in the form of an oral or nasal spray, an aerosol, suspension, emulsion, and/or eye drop. Alternatively the medicament may be provided in tablet form. Alternative delivery means include inhalers or nebulisers.
Alternatively or preferably the medicament can be delivered by direct injection into a joint. It is envisioned that the compositions/medicaments be delivered intravenously, intramuscularly, subcutaneously or topically. Further still, the medicament may be taken rectally.
It will also be apparent that compositions/medicaments are effective at preventing and/or alleviating arthritic conditions in animals other than humans, for example and not by way or limitation, family pets, livestock, horses.
According to a fourth aspect of the invention there is provided a method to screen for agents with anti-arthritic properties. In a preferred method of the invention said method comprises:
i) providing a cartilage sample.
ii) addition of an effective amount of at least one agent to be tested.
iii) addition of at least one pro-inflammatory cytokine; and
iv) monitoring at least one molecule indicative of cartilage breakdown.
In a further preferred embodiment of the invention said inflammatory cytokine is selected from:interleukin-lα; interleukin-l β; oncostatin M; tumour necrosis factor-α. In a preferred method of the invention said method comprises:
i) providing a cartilage sample; ii) addition of an effective amount of at least one agent to be tested; iii) addition of one vitamin A metabolite; and iv) monitoring at least one molecule indicative of cartilage breakdown.
Preferably said vitamin A metabolite is all-/rarø-retinoic acid.
It will be apparent to one skilled in the art that methods to monitor cartilage degradation are well known and are herein described.
According to a further aspect of the invention there is provided an agent identified by the screening method of the invention.
It will also be apparent to one skilled in the art that catechins disclosed above can either be isolated from natural plant sources, eg Camellia sinensis, Uncarcia gambir or can be synthesised in the laboratory using methods well known in the art.
An embodiment of the invention will now be described, by example only, and or with reference to the following Tables and Figures: Table 1 represents the inhibitory effects of catechins on cytokine or vitamin A metabolite-stimulated cartilage breakdown using (i) nasal cartilage explants and (ii) articular cartilage explants;
Table 2 represents the inhibitory effects of EGCG and ECG on human osteoarthritic cartilage explants;
Table 3 represents the inhibitory effects of EGCG and ECG on the degradation of proteoglycan of human rheumatoid knee cartilage;
Table 4 represents the inhibitory effects of EGCG and ECG on the degradation of proteoglycan of human non-arthritic cartilage;
Tables 5 and 6 represent the inhibitory effects of EGCG, ECG, EC and EGC on type II collagen degradation in bovine nasal cartilage explants stimulated with rhILα;
Table 7 demonstrates lactate production by the explants over the latter part of a 28- day period;
Table 8 represents the inhibitory effects of EGCG on the synthesis of human TNFα;
Figure la and lb represents the chemical structures of a selection of catechins and variants thereof;
Figure 2 represents the dose-response for EGCG inhibitory activity of TNFα induced cartilage proteoglycan breakdown; and
Figure 3 represents the macroscopic changes in bovine nasal cartilage explants cultured in the presence or absence of rhIL-lα, EC and EGC for a 28 day period. Introduction.
Cartilage proteoglycan degradation in the bovine system was stimulated with the proinflammatory cytokines interleukinlα (ILlα) or tumour necrosis factor α (TNFα) or with the vitamin A derivative, all-trans retinoic acid (Ret). The degradatory process was initiated in the human cartilage using a combination of interleukinl β (ILl β) and TNFα. These are standard in vitro models for the breakdown of cartilage proteoglycan components that occurs in arthritis (Bryson et al, 1998; Ilic et al, 1992). Methods
The inhibition of cartilage proteoglycan degradation.
Bovine nasal septum and metacarpophalangeal cartilage was prepared as described by Buttle et al, (1992). The nasal septum cartilage was removed using a post-mortem knife and the overlying membrane was discarded. The excised cartilage was wiped with isoprenyl-impregnated Azowipes, placed in a petri dish in the tissue culture hood and washed in sterile phosphate buffered saline (PBS). The cartilage was sliced using a scalpel blade into approximately 2mm x 3mm x 3cm slices, and from these slices discs were cut with the aid of a belt punch. About 200 discs (~3mm diam., 2mm thick) were obtained from each animal.
Slices of cartilage from the bovine metacarpophalangeal joint were dissected and cut into small pieces similar in size to the nasal explants. All bovine cartilage explants were cultured overnight in DMEM (Dulbecco's Modified Eagles Medium) containing newborn calf serum (NCS) (5%) and hydrocortisone (0.1 μg/ml) prior to the start of an experiment.
In different experiments human articular cartilage was obtained following various surgical procedures. The cartilage was dissected into slices and in some experiments the proteoglycan was biosynthetically labelled with 5 μCi/ml 3: SO4 in 5% NCS- containing DMEM for 5 days. The cartilage was then washed in 5%> NCS-containing DMEM without radiolabel for 2 days prior to the start of an experiment. The effect of EGCG and ECG on the degradation of the proteoglycan component of bovine and human cartilage.
Cartilage explants treated as described above were individually transferred into the wells of a 96-well plate and cultured for up to 9 days (medium change on days 3 and 6 or on day 4) in serum-free DMEM in the presence or absence of recombinant human ILlα (rhILl α), recombinant human ILlβ (rhILl β), recombinant human TNFα (rhTNFα) and Ret, either singly or in combination. On average greater than 50% of the sulfated glycosaminoglycans (sGAGs) in the explants was released following stimulation, which was twice the basal release. Certain catechins were prepared as stock solutions in dimethyl sulfoxide (DMSO) and further diluted in DMSO or DMEM to give the appropriate final concentration in the culture medium and also a constant 1% (v/v) DMSO concentration.
sGAGs within the conditioned media and retained in the tissue (measured following papain digestion) were either determined by the dimethylmethylene blue assay (Farndale et al., 1986) or by scintillation counting following labelling with SO4 (Ilic et al, 1995). Data were expressed as the percentage sGAG released from the tissue or as the percentage inhibition of sGAG release. The Mann- Whitney U test for unpaired, non-parametric data was used to determine the statistical significance of the results.
Type II collagen breakdown in rhIL-lα-stimulated bovine nasal cartilage explants.
In order to investigate the effect of catechins on type II collagen degradation, bovine nasal cartilage explant cultures were maintained for 28 days, with a twice-weekly medium change, in the presence or absence of rhIL-lα (4.5nM) and the catechins; EGCG, ECG, EC and EGCG at 20μM. At the completion of the experiment the type II collagen remaining in the cartilage residues was extracted by digestion with proteinase K (EC 3.4.21.64) at 56°C for 15 hours. The extracts were assayed by- inhibition enzyme-linked immunosorbent assay (ELISA) using a mouse IgG monoclonal antibody to denatured type II collagen, Col2-3/4m as previously described (Hollander et al. 1994). The amount of collagen released at each medium change throughout the culture period was calculated as a percentage of total collagen in each culture well (medium plus tissue residue).
Effect of catechins on proteoglycan synthesis.
Bovine nasal cartilage explants were obtained as described above. The rate of proteoglycan synthesis was assessed by measuring incorporation of 3:>S from 35SO4. Three groups were set up: (a) killed explants (freeze-thawed thrice), (b) explants cultured in serum-free DMEM alone, (c) explants cultured in serum-free DMEM and 2μM or 20 μM EGCG. All three groups were cultured in the presence of 5μCi 33SO4/ml for 18 hours and 3"S incorporation was assessed as previously described by Buttle et al. (1993).
Lactate test for assessment of chondrocyte metabolic activity.
Chondrocytes respire anaerobically (Stefanovic-Racic et al, 1994). A measure of the toxicity of a compound can therefore be made by determining the levels of lactate in the conditioned media by using the lactate oxidase/peroxidase method with a kit from Sigma Chemical Co.
The effect of catechins on TNFα production by human peripheral blood cells.
Peripheral blood was taken from volunteers and 50 iu/ml heparin was added. The blood was diluted 1 :6 with serum-free DMEM and incubated for 4 hours at 37°C in a water bath in the presence or absence of lμg/ml lipopolysaccharide (LPS) (E.colϊ) and EGCG (20 μM). The blood was freeze-thawed thrice at -40°C and then centrifuged at 1000 rpm for 5 min to remove cell debris. A TNFα ELISA was then performed on the supernatant following the manufacturers' instructions, either using a kit from R&D systems or from Diaclone Research. RESULTS
Inhibition of basal and stimulated levels of bovine nasal and articular cartilage proteoglycan degradation by the catechins at 20 uM.
As shown in Table l(i). EGCG significantly inhibited rhTNFα-stimulated cartilage proteoglycan degradation in a bovine nasal cartilage explant model, but no significant effect was observed for the basal, rhILlα-, or Ret-stimulated release. Both ECG and EC significantly inhibited ILlα-stimulated degradation but not rhTNFα- or Ret- stimulated breakdown.
In the bovine articular explant model (Table 1 ii), EGCG again potently inhibited the rhTNFα-stimulated response, whilst also inhibiting, but to a lesser degree the basal, rhIL-lα and Ret response. Both the rhIL-α and Ret responses were more potently inhibited by ECG.
Dose-response for the inhibition of rhTNFα-stimulated bovine cartilage proteoglycan degradation by EGCG.
In view of the potent inhibition of rhTNFα-stimulated cartilage proteoglycan degradation by 20μM EGCG, a dose-response curve was constructed (Fig. 2). Inhibition reached statistical significance at 2 μM (47% inhibition) and increased to 84% and 138% inhibition at 20μM and 200μM respectively. Where inhibition exceeded 100%) some of the basal breakdown as well as rhTNFα-stimulated breakdown is indicated.
Influence of catechins on the lactate output of chondrocytes.
EGCG at 200 μM was shown not to be toxic over a 5 day period by measurement of lactate levels in the conditioned media of bovine nasal cartilage explants stimulated with rhTNFα. Explants cultured in DMEM alone produced 896 μg lactate/explant and in the presence of EGCG, this was 1040 μg lactate/explant. When explants were cultured in the presence of rhTNFα a total of 1248 μg lactate/explant was produced, decreasing slightly to 896 μg when cultured in the presence of EGCG. These results demonstrate that over a 5-day period EGCG did not have a marked effect on lactate output by chondrocytes.
The effect of EGCG on the synthesis of bovine nasal cartilage proteoglycan.
EGCG at 2 μM had no significant effect on proteoglycan synthesis in bovine nasal cartilage explants, giving a non-significant stimulation (12% ± 15%), whilst at 20 μM it gave a non-significant inhibition of 32%o + 8%> (2 animals, n=8 explants/animal).
The effect of catechins on human cartilage proteoglycan degradation.
As shown in Tables 2, 3 and 4, ECG (20 μM) produced significant inhibition of proteoglycan breakdown from human cartilage from osteoarthritic, rheumatoid and non-arthritic joints. EGCG inhibited basal levels of proteoglycan loss from osteoarthritic cartilage (Table 2).
The effect of catechins on collagen degradation in rhIL-lα-stimulated cartilage proteoglycan degradation.
As Tables 5 and 6 demonstrate, the culture of bovine nasal cartilage explants for 28 days in the presence of rhILlα resulted in the almost complete degradation of the explants (shown visually in Fig. 3), with almost total release of type II collagen by ILlα. EGCG, ECG and EGC (20 μM) significantly reduced this degradation (Table 6) with the percentage type II collagen released from the explants decreasing by more than 50%) in all three cases. As Table 7 shows, the culture of bovine nasal cartilage explants for a 28-day period in the presence of rhILlα and EGCG or ECG was not associated with any toxic effects, as determined by the levels of lactate in the conditioned media over the 17-20 day and 24-28 day culture periods. The effect of the catechins on TNFα synthesis by human peripheral blood cells.
EGCG has been reported to inhibit TNFα synthesis in a human stomach cancer cell line KATO III (Okabe et al, 1999) and in BALB/3T3 cells (Suganuma et al, 1999). We tested the effects of EGCG (20 μM) on LPS-stimulated TNFα synthesis by whole blood. In separate experiments using blood samples from different volunteers and ELISA kits from different sources, we confirmed the inhibition of TNFα synthesis by EGCG (Table 8).
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Table 1
The inhibition of cartilage proteoglycan degradation in bovine nasal and articular cartilage explants by the catechins (20uM).
(i) Nasal cartilage explants
% Inhibition of stimulated sGAG release from cartilage
(ii) Articular cartilage explants
% Inhibition of stimulated sGAG release from cartilage
Table 1
Bovine nasal and articular cartilage explants were cultured for 5 days in serum-free DMEM in the presence or absence of rhIL-lα (0.3 nM nasal; 3nM articular). rhTNFα (3nM nasal; 6nM articular) or Ret (lμM) and in the presence or absence of the catechins EGCG, ECG, EC or EGC at a final concentration of 20 μM. Medium was changed on day 3. The degradation of cartilage proteoglycan was determined by measuring the sGAG released from the explants as a percentage of total sGAG using the DMB assay. The data are expressed as the mean percentage inhibition of stimulated or basal cartilage proteoglycan degradation ± s.e.m. (a) 4 animals, n=32; 6 animals, n= 48; (c) 2 animals, n=16; (d) 7 animals, n=56. * p<0.05, ** p<0.005 and *** p<0.0005 as determined using the 2-tailed Mann- Whitney test for non-parametric data.
Table 2
The inhibition of cartilage proteoglycan degradation in human osteoarthritic cartilage explants by EGCG and ECG (20uM)
Human osteoarthritic articular cartilage explants were biosynthetically labelled for 5 days in 5%(v/v) newborn calf serum (NCS)-containing DMEM using 35SO4 (5μCi/ml). The explants were washed for 2 days and then cultured for a further 9 days in the presence or absence of a combination of rhlLl β (3nM) and rhTNFα (6nM) and also in the presence or absence of the catechins EGCG or ECG at 20μM. Medium was changed on day 3 and day 6. The degradation of cartilage proteoglycan was determined by measuring the radiolabel released from the explants as a percentage of total radiolabel by quantifying the 35S released into the culture medium and that retained in the tissue by use of a scintillation counter. The data are expressed as the mean percentage release ± s.e.m. *p<0.05, when the release of sGAG is compared between the groups cultured in the presence of the catechins to those cultured in their absence, as determined by the Mann Whitney U test for 2-tailed, non-parametric data. Table 3
The inhibition of cartilage proteoglycan degradation in human rheumatoid cartilage explants by EGCG and ECG (20LIM)
Human rheumatoid articular cartilage was obtained at surgery and cultured overnight in 5%(v/v) NCS-containing DMEM. Explants were individually transferred to a 96- well plate and cultured for 9 days in serum-free DMEM in the presence or absence of a combination of rhIL-l β (3nM) and rhTNFα (6nM) and also in the presence or absence of the catechins EGCG or ECG at 20 μM. Medium was changed at 3 and 6 days. The degradation of the cartilage proteoglycan was determined by measuring the sGAG released from the explants as a percentage of total sGAG using the DMB assay. * p<0.05 when comparing the proteoglycan released from the group cultured with the cytokine combination to the other groups, as determined using the 2-tailed Mann Whitney test for non-parametric data. Table 4
The inhibition of cartilage proteoglycan degradation in human non- arthritic cartilage explants by EGCG and ECG (20uM)
Human non-rheumatoid articular cartilage was obtained from a patient suffering from Marfan's Syndrome at surgery, and cultured overnight in 5%(v/v) NCS-containing DMEM. Explants were individually transferred to a 96-well plate and cultured for 9 days in serum-free DMEM in the presence or absence of a combination of rhIL- 1 β (3nM) and rhTNFα (6nM) and also in the presence or absence of the catechins EGCG or ECG at 20 μM. Medium was changed at 3 and 6 days. The degradation of the cartilage proteoglycan was determined by measuring sGAG released from the explants as a percentage of total sGAG using the DMB assay; ** p<0.005 when comparing the proteoglycan released from the group cultured with the cytokine combination to the other groups, as determined using the 2-tailed Mann Whitney test for non-parametric data. Table 5
Time-course for type II collagen degradation in rhILlα-stimulated bovine nasal cartilage explants treated with or without ILlα and EGCG or ECG.
Days
0-3 0-7 0-10 0-14 0-17 0-21 0-24 0-28
Treatment % Release of type II collagen
CONTROL l±f 0±8 0±9 0+7* 0+5** 0±6*** 0±6*** 0±5***
IL-1 3±1 4+1 5+1 9+3 14±6 34±8 47±8 90±6
IL-1 + EGCG (20μM) ι± 2±1 2±1* 3+1* 4± 12±5" 19±7* 29±10***
IL-1 + ECG (20nM) 4±2 6+2 16+4* 26+8 9±10 32±10 37±12 41+1 1
Bovine nasal cartilage explants were cultured for 28 days in the presence or absence of rhILlα (4.5nM) and the catechins EGCG or ECG (20μM). Medium was changed twice a week. The degradation of type II collagen was measured using the CB11B inhibition ELISA and the data were expressed as the cumulative release of type II collagen. * p<0.05, ** p<0.05 and *** p<0.005 as determined by the Mann Whitney U test for 2-tailed, non-parametric data and compared to the group cultured in the presence of ILlα alone. Data relate to 2 animals, n=6/group/animal. Table 6
The inhibition at day 28 of type II collagen degradation in rhILlα- stimulated bovine nasal cartilage explants by EGCG, ECG, EC and EGC.
(i) The % release of type II collagen after 28 days of culture from explants cultured in the presence of 20uM EGCG or ECG.
% release of type II collagen
control -l± 4 ** * rhIL-lα 95± 6 rhIL-lα + 20μM EGCG 31±10 ** * rhIL-lα + 20μM ECG 35±10 ***
(ii) The % release of type II collagen after 28 days in culture from explants cultured in the presence of 20uM EC or EGC.
Table 6: Bovine nasal cartilage explants were cultured for 28 days in the presence or absence of rhIL-lα (4.5nM) and the catechins EGCG, ECG, EC or ECG (20μM). Medium was changed twice a week. The degradation of cartilage type II collagen was measured using the CBl IB assay and the data are expressed as the cumulative release of type II collagen. * p<0.05, ** p<0.05 and *** p< 0.005 as determined by the Mann Whitney U test for 2-tailed, non-parametric data and compared to the group cultured in the presence of rhIL-lα alone. Data in each table relates to the results obtained from 4 animals, n=14/group in total.
Table 7
Lactate levels in the conditioned media of bovine nasal explants cultured with or without rhILlα and EGCG or ECG for 28 days.
Bovine nasal cartilage explants were cultured for a 28 day period in the presence or absence of rhILlα and the catechins EGCG and ECG at 20μM. Medium was changed twice a week and stored at -20°C awaiting assay. Lactate levels in the 17-20 day and 24-28 day medium was determined using a kit from Sigma. The data relate to two animals, n=6/animal. The Mann Whitney U test for 2-tailed, non-parametric data was performed, with all groups being compared to the group cultured in the presence of IL-lα. No significant differences were observed.
Table 8
The inhibition by EGCG of TNFα production by human peripheral blood cells.
(ϊ)R&D Systems Kit
(ii) Diaclone Research Kit
Human peripheral blood was diluted 1 :6 with serum-free DMEM and then incubated for 4 hours at 37°C in a water bath in the presence or absence of bacterial lipopolysaccharide (LPS) (lμg/ml) and EGCG (20μM). The blood was freeze-thawed thrice and the levels of TNFα were quantified using an ELISA kit. ***p< 0.0005, nd = not detectable.

Claims

1. The use of at least one catechin in the manufacture of a medicament for the treatment of arthritis.
2. The use according to Claim 1 wherein said catechin is selected from:(+)epicatechin; (+) catechin; (-)epicatechin;(-) catechin; (-) epigallocatechin;(-) gallocatechin; (-)epicatechin gallate; (-) catechin gallate; (-) epigallocatechin gallate; (-) gallocatechin gallate.
3. The use of a catechin according to claim 1 or 2 wherein said catechin is epigallocatechin gallate.
4. The use of a catechin according to claim 1 or 2 wherein said catechin is epicatechin.
5. The use according to any of claims 1-4 wherein said arthritic condition is selected from: osteoarthritis; rheumatoid arthritis; inflammatory arthritis; osteochondritis; acute pyrophosphate arthritis; reactive arthritis; psoriatic arthritis; juvenile arthritis; lupus erythematosus; Sjogren's syndrome; relapsing polychondritis; ankylosing spondylitis; psoriatic arthritis; MSUM (gout); CPPD (psuedogout, chondrocalcinosis); chondrolysis; bursitis.
6. The use according to claim 5 wherein said medicament is for the treatment of osteoarthritis.
7. The use according to claim 5 wherein said medicament is for the treatment of rheumatoid arthritis.
8. The use according to any of claims 1-7 wherein said medicament is for the prophylactic treatment of arthritis.
9. The use according to claim 8 wherein said prophylactic treatment is for animals with a genetic predisposition to arthritis.
10. The use according to claim 8 wherein said prophylactic treatment is to protect animals with an increased probability of developing arthritis due to joint damage.
1 1. A therapeutic composition comprising at least one catechin and at least one anti-arthritic agent or biopolymer.
12. A therapeutic composition according to claim 11 wherein said anti-arthritic agent is hyaluronic acid.
13. A therapeutic composition according to Claim 1 1 wherein said anti-arthritic agent is glucosamine.
14. A therapeutic composition according to claim 13 wherein glucosamine is g glucosamine sulphate.
15. A therapeutic composition according to any of Claims 1 1 - 14 wherein said c composition is immune silent.
16. A therapeutic composition according to any of claims 1 1-15 wherein said catechin and said anti-arthritic agent are administered as an admixture.
17. A therapeutic composition according to any of claims 1 1-15 wherein said catechin and said anti-arthritic agent are cross-linked, coupled or associated together.
18. A therapeutic composition according to claim 17 wherein said catechin is cross-linked, coupled or associated with hyaluronic acid.
19. A method to cross-link or couple at least one catechin to at least one anti- arthritic agent comprising:
i) providing at least one catechin and at least one anti-arthritic agent; ii) providing conditions conducive to the cross-linking or coupling of said catechin to said agent; and optionally iii) purifying the cross-linked or coupled complex from the reaction mixture.
20. A method according to claim 19 wherein said anti-arthritic agent is hyaluronic acid and said catechin is selected from: (+)epicatechin; (+) catechin;(-) epicatechin;(-)catechin;(-)epigallocatechin;(-)gallocatechin;(-)epicatechin gallate; (-) catechin gallate; (-) epigallocatechin gallate;(-) gallocatechin gallate.
21. A method for treating an arthritic condition comprising administering to an animal a pharmacologically effective amount of the medicament according to any of Claims 1- 4 or a therapeutic composition according to any of Claims 1 1- 18.
22 A method according to Claim 21 wherein said arthritic condition is selected from: osteoarthritis; rheumatoid arthritis; inflammatory arthritis; osteochondritis; acute pyrophosphate arthritis; reactive arthritis; psoriatic arthritis; juvenile arthritis; lupus erythematosus; Siogren's syndrome; relapsing polychondritis; ankylosing spondylitis; psoriatic arthritis; MSUM (gout); CPPD (psuedogout, chondrocalcinosis); chondrolysis; bursitis.
22. A method of treating an arthritic condition according to claims 21 or 22 wherein said arthritic condition is osteoarthritis.
23. A method of treating an arthritic condition according to claims 21 or 22 wherein said arthritic condition is rheumatoid arthritis.
24. A method to screen for agents with anti-arthritic properties.
25. A method according to claim 24 wherein said method comprises:
i) providing a cartilage sample; ii) addition of an effective amount of at least one agent to be tested; iii) addition of at least one pro-inflammatory cytokine; and iv) monitoring at least one molecule indicative of cartilage breakdown.
26. A method according to claim 25 wherein said pro-inflammatory cytokine is selected from: interleukin- lα; interleukin- l β; oncostatin M; tumour necrosis factor α.
27. A method according to claim 24 wherein said method comprises:
i) providing a cartilage sample; ii) addition of an effective amount of at least one agent to be tested; iii) addition of at least one vitamin A metabolite; and iv) monitoring at least one molecule indicative of cartilage breakdown.
28. The method according to claim 27 wherein said vitamin A metabolite is all trans-retinoic acid.
29. An agent identified by the screening methods of claims 24-28.
EP00935346A 1999-06-07 2000-06-06 Arthritis treatment Withdrawn EP1207862A2 (en)

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