WO2004084886A1

WO2004084886A1 - THE USE OF BLOCKERS FOR THE LONG-TERM INHIBITION OF Na+-K+-ATPase AND/OR Na+/H+ EXCHANGE ACTIVITIES FOR THE PREPARATION OF A MEDICAMENT FOR THE INTESTINAL THERAPY

Info

Publication number: WO2004084886A1
Application number: PCT/PT2004/000005
Authority: WO
Inventors: Patrício Manuel Vieira Araújo Soares da Silva
Original assignee: Universidade Do Porto
Priority date: 2003-03-28
Filing date: 2004-03-26
Publication date: 2004-10-07
Also published as: PT102931B; PT102931A

Abstract

The present invention relates to the use of blockers for the long-term inhibition of Na+-K+-ATPase and/or Na+/H+ exchange activities that act at the level of enzymatic activity inhibition, for the preparation of medicament for the prevention and treatment of changes in intestinal electrolyte absorption associated with inflammatory bowel disease in mammals. The invention is also concerned with a pharmaceutical composition comprising a therapeutically effective amount of the said blocker in combination with a pharmaceutically acceptable carrier.

Description

DESCRIPTION

"THE USE OF BLOCKERS FOR THE LONG-TERM IMBIBITION OF Na⁺- K⁺-ATPase AND/OR Na⁺/H⁺ EXCHANGE ACTIVITIES FOR THE

PREPARATION OF A MEDICAMENT FOR THE INTESTINAL THERAPY"

FIELD OF THE INVENTION

This invention relates to the use of blockers for the long-term inhibition of Na⁺-K⁺-ATPase and/or Na⁺/H⁺ exchange activities that act at the level of enzymatic activity inhibition, the preparation of medicament for the prevention and treatment of changes in intestinal electrolyte absorption associated with inflammatory bowel disease in mammals.

BACKGROUND OF THE INVENTION

There is substantial evidence to suggest that inflammatory bowel disease (IBD) , encompassing Crohn's disease (CD) and ulcerative colitis (UC) , may arise from a disregulated immune response to components of the normal gut flora. An imbalance of T helper cell type 1 (Thl) versus type 2 (Th2) polarisation in favour of Thl cell subsets appears to be a key pathogenic mechanism in chronic IBD (Adorini et al . , XSSI ; Mizoguchi et al . , 1996; Podolsky, 1991) . This concept is supported by studies of mucosal biopsies in patients with IBD, demonstrating an increased expression of proinflammatory cytokines, chemokines, and adhesion molecules (Groux et al . , 1999; Stallmach et al . , 1998; Strober et al . , 1997). Consistent with the hypothesis of a Thl-mediated pathogenesis in IBD, an increase in IFN- γ-secreting cells and IFN-γ mRNA levels has been described in intestinal lesions of patients with CD (Breese et al . , 1993; Niessner et al . , 1995).

Clinically, the dominant symptom in both CD and UC is diarrhoea and recent evidence suggests that this may relate to a reduction in electrolyte absorption rather than increases in electrolyte secretion (Sundaram et al . , 1997a; Sundaram et al . , 1997b). In agreement with this view is the finding that the colonic mucosa of IBD patients responds poorly to secretagogues and that Na⁺ absorption is diminished (Sandle et al . , 1990). Consistent with these findings is the observation that IFN-γ reduces agonist- induced and cAMP-induced Cl^" intestinal secretion (Colgan et al . , 1994). IFN-γ has also been shown to downregulate the cystic fibrosis conductance regulator (CFTR) (Colgan et al . , 1994) , the Na⁺-K⁺-2C1^~ co-transporter (NKCC1) and the Na⁺-K⁺- ATPase (Fish et al . , 1999; Sugi et al . , 2001; Zϋnd et al . , 1996), and the Na⁺/H⁺ exchanger (Rocha et al . , 2001). IFN-γ has direct effects on intestinal epithelial cells, such as changes in cell morphology and decreased levels of proteins involved in transport and barrier function (Adams et al . , 1993; Besanςon et al . , 1994; Colgan et al . , 1994; Fish et al . , 1999; adara et al . , 1989; Sugi et al . , 2001), but the overall cytoskeletal structure appears to be maintained (Madara et al . , 1989). Though the increased production of IFN-γ may have relevant effects on electrolyte and water transport in the inflamed intestine, there is lack of information on the association between IFN-γ and disturbances in intestinal Na⁺-K⁺-ATPase activity in IBD and experimental colitis. Furthermore, the signalling pathways that initiate the IFN- γ-induced downregulation of electrolyte transporters have not been examined.

Prior art describes polyphenol compounds, some of which are catechin derivatives, and are endowed some therapeutic properties. Among these compounds, epigallocatechin-3-gallate is most probably the best known, even because is the major polyphenol from green tea. Since tea is one of the most widely consumed beverages, second only to water, tea-derived epigallocatechin-3-gallate is, thus, considered safe. Several beneficial effects of epigallocatechin-3-gallate are known, as seen from the following examples. U.S. Pat. Nos. 5,318,986 and 5,670,154 both granted to Hara and Honda (Hara et al . , 1994; Hara et al . , 1997) disclose that tea polyphenols including epigallocatechin-3-gallate inhibit the enzyme activity of alpha amylase and tyrosinase. U.S. Pat. No. 5,391,568 granted to Chung (Chung, 1995) discloses that catechin derivatives inhibit lung cancer in a mammal. U.S. Pat. No. 5,605,929 granted to Liao and Liang (Liao et al . , 1997) discloses that catechin derivatives inhibit the enzyme activity of 5 alpha reductase. U.S. Pat. No. 5,922,756 granted to Chan (Chan, 1999) discloses that catechin derivatives inhibit nitric oxide synthase at the level of gene expression and enzyme activity. U.S. Pat. No. 6,248,341 granted to Anderson and Matsui (Anderson et al . , 2000) discloses that administration of catechin derivative provides an effective method for inhibiting angiogenesis in mammalian tissues. U.S. Pat. No. 6,214,868 granted to Ahn et al . (Ahn et al . , 2001) discloses that administration of catechin derivative provides an effective method for preventing or treating coronary restenosis. U.S. Pat. No. 6,410,061 granted to Morre and Morre (Morre et al . , 2000) discloses that catechin derivatives alone and in combination with other catechins and other anti-cancer therapeutic agents, inhibits the activity of a cancer- specific protein, an isoform of NADH oxidase specific to cancer cells (tNOX) .

Though inhibition of Na⁺-K⁺ ATPase and Na⁺/H⁺ exchanger activities by IFN-γ have been already described (Guzman et al . , 1995; Rocha et al . , 2001; Sugi et al . , 2001; Unno et al . , 1995; Yoo et al . , 2000), it is not known that polyphenol compounds of the catechin group may be endowed with the ability to interfere with these mechanisms attenuating the effect of IFN-γ upon these transporters.

SUMMARY OF THE INVENTION

In particular, this invention relates to the use of a specific blocker of long-term inhibition of Na⁺-K⁺- ATPase and/or Na⁺/H⁺ exchange activities for the preparation of a medicament for the prevention and treatment of changes in intestinal electrolyte absorption associated with intestinal inflammation.

This invention is directed to a pharmacologically acceptable composition for blocking of long-term inhibition of Na⁺-K⁺-ATPase and/or Na⁺/H⁺ exchange activities in a mammal . The composition includes a compound of general formula I :

in which the X groups are the same or different and are selected from H and OH, the Y groups are the same or different and are selected from H and OR groups where R represents H, CH₃ and CH₂-Ph, and Z groups are the same or different and are selected from H and OR groups where R represents H or 0, and a pharmaceutically acceptable carrier, with the active agent present in the composition in an effective amount.

In the present invention evidence is provided on the decrease in Na⁺-K⁺-ATPase activity in human intestinal cultured epithelial cells after long (24 h) , but not short (0.5 h) , exposure to IFN-γ. On the other hand, short (0.5 h) and long (24 h) exposure to IFN-γ resulted in marked reduction in Na⁺/H⁺ exchange activity. It is an object of the present invention to provide innovative information for the use of compounds to prevent and treat changes in intestinal electrolyte absorption associated with IFN-γ receptor stimulation in a mammal that comprises administering an effective amount of a specific blocker of long-term inhibition of Na⁺-K⁺-ATPase and/or Na⁺/H⁺ exchange activities .

These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing (A) the concentration-dependent and the (B) time-dependent effect of interferon-γ (IFN-γ) on Na⁺-K⁺-ATPase activity in cultured Caco-2 cells. Columns represent means of 5 experiments per group; vertical lines indicate S.E.M. values. Significantly different from control values (* P<0.05) using the Student's "t" test.

Figure 2 is a graph showing (A) the concentration-dependent and the (B) time-dependent effect of interferon-γ (IFN-γ) on Na⁺/H⁺ exchange activity in cultured Caco-2 cells. Columns represent means of 5 experiments per group; vertical lines indicate S.E.M. values. Significantly different from control values ('- P<0.05) using the Student's "t" test.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In particular, this invention relates to the use of a specific blocker of long-term inhibition of Na⁺-K⁺- ATPase and/or Na⁺/H⁺ exchange activities for the preparation of a medicament for the prevention and/or treatment of a mammal afflicted with bowel inflammation disease, specially a chronic one.

Preferably, the mammal is a human subject.

Preferably, the compound is a specific blocker of long-term inhibition of Na⁺-K⁺-ATPase and/or Na⁺/H⁺ exchange activities .

More preferably, the blocker of long-term inhibition of Na⁺-K⁺-ATPase and/or Na⁺/H⁺ exchange activities is a compound of general formula I

in which the X groups are the same or different and are selected from H and OH, the Y groups are the same or different and are selected from H and OR groups where R represents H, CH₃ and CH₂-Ph, and Z groups are the same or different and are selected from H and OR groups where R represents H or 0.

The compounds of general formula I include, but are not limited to epigallocatechin-3-gallate, epicatechin- 3-gallate, catechin-3-gallate, morin-3-gallate, fisetin-3- gallate, myricetin-3-gallate, quercetin-3-gallate, kaempferol-3-gallate and a mixture thereof.

The pharmaceutical employed in the use of the present invention will be an effective blocker of long-term inhibition of Na⁺-K⁺-ATPase activity.

The pharmaceutical employed in the use of the present invention will be an effective blocker of long-term inhibition of Na⁺/H⁺ exchange activity.

The medicament thus obtained is preferentially intended for the systemic administration from 10 to about 100,000 μg/kg of body weight.

Another object of the present invention is a pharmaceutical composition comprising a therapeutically effective amount of a compound according to claims 3 or 4 in combination with a pharmaceutically acceptable carrier. The invention may be more specifically regarded as blocking the long-term inhibition of Na⁺-K⁺-ATPase and Na⁺/H⁺ exchange activities at the level of gene expression and Na⁺ transporter activities. The invention essentially includes compounds of general formula I, both in natural and synthetic forms, and their structural derivatives generated by chemical and molecular biological processes such as via combinatorial library.-

Inflammatory bowel disease refers to idiopathic chronic inflammatory conditions of the intestine, ulcerative colitis and Crohn's disease.

In practising the present invention, a compound of general formula I may be administered in combination with pharmaceutically acceptable carriers. A pharmaceutical formulation suitable for use in the present invention may be prepared in accordance with any of the conventional procedures. In preparing the formulation, the active ingredient is preferably admixed or diluted with a carrier, or enclosed within a carrier that may be in the form of a capsule, sachet or other container. When the carrier serves as a diluent, it may be solid, semi-solid or liquid material acting as a vehicle, excipient or medium for the active ingredient. Thus the formulations may be in the form of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsules, sterile injectable solution sterile packaged powder and the like.

Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavouring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a mammal by employing any of the procedures well known in the art.

The pharmaceutical formulation of the present invention can be administered via various routes including oral, enema, subcutaneous, intravenous and intramuscular introduction. In case of human, a typical daily dose of a compound of general formula I may range from 10 to about 100,000 μg/kg of body weight and can be administered in a single or in divided doses.

However, it should be understood that the amount of the active ingredient actually administered ought to be determined in light of various relevant factors, including the condition to be treated, the chosen route of administration, the age, the sex and body weight of the individual patient, and the severity of the patient's symptom; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.

The active ingredient may also be used in combination with other therapeutic agents, for example, anti-inflammatory agents, particularly corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs) , and immunosuppressants .

The following examples are intended to illustrate the present invention without limiting its scope.

REFERENCE EXAMPLE 1

Inhibi tion of Na^-K* -ATPase activi ty in Caco-2 cells after long- term exposure to INF-χ

Caco-2 cells (ATCC 37-HTB; passages 39-49) were obtained from the American Type Culture Collection (Rockville, MD) and maintained in a humidified atmosphere of 5% C0₂-95% air at 37°C. Cells were grown in Minimal Essential Medium (Sigma Chemical Company, St. Louis, Mo, USA) supplemented with 10^s U/L penicillin G, 250 μg/L amphotericin B, 100 ng/L streptomycin (Sigma) , 20% foetal bovine serum (Sigma) and 25 mmol/L N-2 - hydroxyethylpiperazine-N' -2-ethanosulfonic acid (HEPES; Sigma) . For subculturing, the cells were dissociated with 0.05% trypsin-EDTA, split 1:3 and subcultured in Costar Petri dishes with 21 cm² growth area (Costar, Badhoevedorp , The Netherlands) . For studies on Na⁺-K⁺-ATPase activity, the cells were seeded in 21 cm² plastic culture dishes at a density of 2.0 x 10⁴ cells/cm². The cell medium was changed every 2 days, and the cells reached confluence after 7 days of initial seeding. For 24 h prior to each experiment, the cell medium was free of foetal bovine serum. Experiments were generally performed 5 days after cells reached confluency, usually 12 days after the initial seeding; each cm² contained about 100 μg of cell protein.

On the day of the experiment, Caco-2 cells cultured in 21 cm² plastic culture dishes were dissociated with 0.05% trypsin-EDTA and suspended in Hanks' medium. Na⁺-K⁺-ATPase activity in isolated intestinal epithelial cells was measured by the method of Quigley and Gotter (Quigley et al . , 1969) and adapted in our laboratory with slight modifications (Vieira-Coelho et al . , 2000). Na ⁺-K⁺- ATPase activity is expressed as nanomoles Pi per miligram protein per minute and determined as the difference between total and ouabain-sensitive ATPase. Protein concentration was measured using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA) and bovine serum albumin as standard.

When Caco-2 cells were treated for 24 h with increasing concentrations of human IFN-γ (100, 300 and- 1,000 U/ml) the decrease in Na⁺-K⁺-ATPase activity was a concentration effect (figure 1A) . As indicated in figure IB the maximal inhibitory effect of IFN-γ (1000 U/ml) upon Na⁺-K⁺-ATPase activity was obtained at 24 h with less marked inhibition at 48 h. Na⁺-K⁺-ATPase activity in Caco- 2 cells was not affected after 0.5 h exposure to IFN-γ (1000 U/ml) (figure IB) .

REFERENCE EXAMPLE 2

Inhibi tion of Na⁺/ϊ exchange activi ty in Caco-2 cells after long- term exposure to INF-γ

Na⁺/H⁺ exchanger activity was assayed as the initial rate of pHi recovery after an acid load imposed by 10 mM NH₄C1 followed by removal of Na⁺ from the Krebs ' modified buffer solution (in mM: NaCl 140, KC1 5.4, CaCl₂ 2.8, MgS0₄ 1.2, NaH₂P0₄ 0.3, KH₂P0₄ 0.3, HEPES 10, glucose 5, pH = 7.4, adjusted with Tris base), in the absence of C0₂/HC0₃ (Gomes et al . , 2002a). Intracellular pH measurements were performed in Caco-2 cells cultured in 96 well plates. After loading the cells with 5 μM of the acetoxymethyl ester of 2 ' , 7 ' -bis (carboxyethyl) -5 (6) - carboxyfluorescein (BCECF-AM) at 37 °C for 40 min, test compounds were added to the extracellular fluid 15 min before starting the sodium-dependent pHi recovery period. Cells were placed in the sample compartment of a dual- scanning microplate spectrofluorometer (Spectramax Gemini, Molecular Devices, Sunnyvale, USA) , and fluorescence was measured every 19 s alternating between 440 and 490 nm excitation at 535 nm emission, with a cutoff filter of 530 nm. In both types of experiments, the ratio of mtracellular BCECF fluorescence at 490 and 440 nm was converted to mtracellular pH values by comparison with values from an mtracellular calibration curve using the nigericin (10 μM) and high-K⁺ method (Gomes et al . , 2002b) .

When Caco-2 cells were treated for 24 h with increasing concentrations of human IFN-γ (100, 300 and 1,000 U/ml) the decrease in Na⁺/H⁺ exchanger activity was a concentration effect (figure 2A) . As indicated in figure 2B the maximal inhibitory effect of IFN-γ (1000 U/ml) upon Na⁺/H⁺ exchanger activity was obtained at 24 h with less marked inhibition at 48 h. In contrast to that observed for Na⁺-K⁺-ATPase (see above) , Na⁺/H⁺ exchanger activity in Caco-2 cells was markedly reduced after 30 min or 3 h exposure to IFN-γ (1000 U/ml) (figure 2B) .

REFERENCE EXAMPLE 3

Compounds of general formula I prevent inhibi tion of Na⁺- FX -ATPase and Na* /it exchange activi ties in Caco-2 cells after long- term exposure to INF-γ

As shown in Table 1 , compounds of general formula I , but not their non-gallate derivatives , markedly attenuated the inhibition of Νa⁺-K⁺-ATPase and Na⁺/H⁺ exchange activities induced after 24 h exposure to IFN-γ ( 1 , 000 U/ml) . By contrast, inhibition of Na⁺/H⁺ exchanger induced after 3 h exposure to IFN-γ (1,000 U/ml) was not antagonised by compounds of general formula I.

REFERENCE EXAMPLE 4

This example illustrates a clinical trial and therapy by oral administration of compounds of general formula I

A patient having inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis) is selected for therapy. The patient weighs 80 kilograms. For infants or children the doses suggested are lowered in a linear fashion based on body weight or surface area. The female patient of child-bearing potential is given a pregnancy test to confirm that the patient is not pregnant. Provided that the patient is not pregnant and does not plan to become pregnant during treatment, a compound of general formula I that modifies intestinal Na⁺-K⁺-ATPase and Na⁺/H⁺ exchange activities is orally administered in a dosage of 10 to about 100,000 μg/kg of body weight and can be administered in a single or in divided doses. The patient is monitored for improvement in the manifestations of the index disease. Additionally, a complete blood count, including white cell count and differential, a platelet count, and liver function tests (such as levels of alkaline phosphatase, lactose dehydrogenase, and transaminases) are checked prior to treatment and periodically thereafter. The dosage is tapered when the manifestations of the disease subside, or discontinued if indicated. While the invention has been described with respect to the above specific embodiments, it should be recognised that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

DISCUSSION

An important aspect of the present invention is related with the fact that compounds of general formula I were particularly efficacious in reversing the inhibition of Na⁺-K⁺ ATPase and/or Na⁺/H⁺ exchanger activities after prolonged, but not acute, exposure to IFN-γ. It should be stressed the finding that acute inhibition of Na⁺/H⁺ exchange activity by IFN-γ was insensitive to compounds of general formula I . On the other hand, it was observed that IFN-γ failed to inhibit Na⁺-K⁺ ATPase activity. Thus, it is suggested that that compounds of general formula I act specifically upon mechanisms of long-term regulation of the Na⁺ transporters, namely the Na⁺-K⁺ ATPase and/or Na⁺/H⁺ exchanger, though the nature of the intrinsic events is not known at present. According to Yoo et al . (Yoo et al . , 2000) , inhibition of Na⁺-K⁺ ATPase activity during the prolonged exposure to IFN-γ is likely not to be related to the stimulation of nitric oxide synthase, for which the information mentioned in U.S. patent No. 5,922,756 granted to Chan (Chan, 1999) should not be considered of relevance for the purpose of the present invention. In addition, only compounds of general formula I, but not the correspondent non-gallate derivatives, were effective in reversing the inhibition of Na⁺-K⁺ ATPase and/or Na⁺/H⁺ exchanger activities after prolonged exposure to IFN-γ, which contrast with that reported above for the catechin derivatives .

Tables

Table 1. Effect of compounds of general formula I (20 μM) , and their non-gallate derivatives (20 μM) , upon inhibition of Na⁺-K⁺-ATPase and Na⁺/H⁺ exchange activities induced after 0.5 h and 24 h exposure to IFN-γ (1,000 U/ml) .

Values are means + S.E.M. of 5 experiments per group. Significantly- different from corresponding control values (* P<0.05) or values for IFN-γ using the Newman-Keuls test.

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Claims

1. The use of a specific blocker of long-term inhibition of Na⁺-K⁺-ATPase and/or Na⁺/H⁺ exchange activities for the preparation of a medicament for preventing and/or treating changes in intestinal electrolyte absorption associated with inflammatory bowel disease in a mammal.

2. The use according to claim 1, wherein the mammal is human.

3. The use according to claim 1, wherein the blocker of long-term inhibition of Na⁺-K⁺-ATPase and/or Na⁺/H⁺ exchange activities is a compound of the general formula I :

in which the X groups are the same or different and are selected from H and OH, the Y groups are the same or different and are selected from H and OR groups where R represents H, CH₃ and CH₂-Ph, and Z group is selected from H and OR groups where R represents H or 0.

4. The use according claim 3, wherein the compound of general formula I is selected from the group consisting of epigallocatechin-3-gallate, epicatechin-3- gallate, catechin-3-gallate, morin-3-gallate, fisetin-3- gallate, myricetin-3-gallate, quercetin-3-gallate, kaempferol-3-gallate and a mixture thereof.

5. The use according to claims 1 to 4, wherein said blocker is a specific blocker of long-term inhibition of Na⁺-K⁺-ATPase activity.

6. The use according to claims 1 to 4 , wherein said blocker is a specific blocker of long-term inhibition of Na⁺/H⁺ exchange activity.

7. The use according to claims 1 to 6 , wherein the medicament is administered systemically in an amount from about 10 to about 100,000 μg/kg/treatment .

8. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claims 3 and 4 in combination with a pharmaceutically acceptable carrier.

9. A method for preventing and/or treating changes in intestinal electrolyte absorption associated with inflammatory bowel disease in a mammal that comprises administering an effective amount of a specific blocker of long-term inhibition of Na⁺-K⁺-ATPase and/or Na⁺/H⁺ exchange activities .

10. The method according to claim 9, wherein the mammal is human.

11. The method according to claim 9, wherein the blocker of long-term inhibition of Na⁺-K⁺-ATPase and/or Na⁺/H⁺ exchange activities is a compound of the general formula I :

12. The method according claim 11, wherein the compound of general formula I is selected from the group consisting of epigallocatechin-3-gallate, epicatechin-3- gallate, catechin-3-gallate, morin-3-gallate, fisetin-3- gallate, myricetin-3-gallate, quercetin-3-gallate, kaempferol-3-gallate and a mixture thereof.

13. The method according claims 9 to 12, wherein said blocker is a specific blocker of long-term inhibition of Na⁺-K⁺-ATPase activity.

14. The method according claims 9 to 12, wherein said blocker is a specific blocker of long-term inhibition of Na⁺/H⁺ exchange activity.

15. The method according claims 9 to 14, wherein the medicament is administered systemically in an amount from about 10 to about 100,000 μg/kg/treatment .