Patent 6,706,293

Pharmaceutical composition with adhesion molecule expression regulating activity. The invention relates to a novel pharmaceutical application of a pharmaceutical composition exhibiting regulating activity of the expression of adhesion molecules integrins, selectins and immunoglobulins and its application as anti-inflammatory agent. Said pharmaceutical composition contains Anapsos, a water-soluble extract and a lipo-soluble extract of the rhizomes of Polypodium leucotomos as active substance in addition to acceptable excipients.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to provide a novel therapeutic application of the Anapsos based on the regulation of the different cellular mediators. The regulation of the expression of the adhesion molecules (the decrease of the expression of the chains of integrins and the immunoglobulin superfamily) and the normalization of the lymphocyte CD4+CD29+CD45RA+ population makes the Anapsos a medicament suitable for treatment of diseases which relate to an inflammatory process, in particular in the treatment of multiple sclerosis.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have demonstrated, that the Anapsos (118 mg of a water-soluble extract and 2 mg of a lipid soluble fraction of Polypodium) has regulatory effects in relation to the expression of adhesion molecules, in mononuclear cells of the peripheral blood of healthy humans, both in vivo and in vitro. Further, it has been demonstrated that Anapsos has a capacity similar to azathioprine to recover the normality of the lymphocyte CD4+CD29+CD45RA+ population, present in increased amounts in patients with multiple sclerosis, thereby obtaining a stabilization of the patients.

At a dose from 0 to 5000 .mu.g/ml of Anapsos and using different doses of phytohaemagglutinin, the Anapsos in vitro is capable of inhibiting the increase of the expression of the adhesion molecules (CD54 and CD11b), as induced by the phytohaemagglutinin, in studies realized on mononuclear cells of human peripheral blood. The results are most significant at a dose of 150 .mu.g/ml of Anapsos and 10 .mu.g/ml of phytohaemagglutinin.

After administration of 720 mg of extract per day for 11 days in a human, the Anapsos decreases the percentage of the lymphocyte populations CD11a, CD11b, and CD54. Accordingly, the extract inhibits the expression of certain adhesion molecules of the integrins .beta.2 (CD11a, CD11b) and of the superfamily of immunoglobulins (CD54).

Accordingly, beside the stimulating action on the cellular immunity as performed by the cytokines and its immuno-modulatory action as described in the art, the Anapsos has a strong anti-inflammatory capacity, similar to phenylbutazone, used as a control in anti-inflammatory studies in rats. The anti-inflammatory effects of Anapsos has not been directly related to its capacity of regulating the expression of the adhesion molecules.

A pharmaceutical composition based on water-soluble and lipid soluble extracts of Polypodium has, in clinical studies performed on humans, been demonstrated to be effective against some diseases which involve inflammatory processes such as multiple sclerosis, prostatitis, and pharyngitis. Between them, these diseases have a different aetiological cause.

Below, the invention is described by way of examples. These are not limiting on the scope of the invention.

EXAMPLES

Example 1

Study of the Anti-inflammatory Capacity

                    Materials:
                    Female WISTAR rats of 150 .+-. 15 g
                    Pletismometer LETICA
                    Weight METTLER AJ 100
                    Weight COBOS D 600
                    Phenylbutazone
                    DIFCO
                    CMC and Tween 80

Method:

A study of the anti-inflammatory activity has been made in acute and chronic phase in rats. The method used was as described by Mizushima. The reference medicament (Phenylbutizene, dose 80 mg/Kg) and the products of the study (dose equivalent to 1.25 g extract per kg weight of animal) were dissolved in a solution of 1% of CMC (carboxymethylcellulose) in distillated water (w/v) and Tween 80: distillated water (0.2:3.3-v/v), in order to be administrated orally.

Six days after inoculation, by intradermal route of 0.1 mL of the complete adjuvant Freund (DIFCO) in the basal part of the tail, was injected 0.1 mL of a suspension of carrageenin type IV 2% (w/v) at the aponeurosis of the left back foot.

The volume of the foot of the animal was measured, using a water pletismometer, immediately before the carrageenin injection (basal volume) and afterwards at 3, 5, and 7 hours (acute phase of the inflammation) and at 24, 48, 72, and 96 hours (chronic phase of inflammation).

The products of the study, the phenylbutazone, and the carrier were administrated, orally and in portions of 6 animals, 1 hour before the carrageenin injection and at 24, 48 and 72 hours.

Results:

The percentage of inhibition of the inflammation was calculated by comparing the increase of the volume of the animal foot in respect of its basal volume, for each group of animals and in relation to the control group. The control group was given the carrier of phenylbutazone and the products of the study. The statistical significance was evaluated via the T Student test. The results are shown below.

                 % INHIBITION OF THE INFLAMMATION
                   3 h    5 h    7 h   24 h   48 h   72 h   96 h
    Control        --     --     --     --     --     --     --
    Phenylbutazone  41%    27%    21%    36%    60%    56%    40%
    Anapsos        29%    35%    41%    43%    50%    54%    48%

The results show that the Polypodium extract exhibits an inhibitory activity on the inflammation, superior to the control in the acute phase and similar to phenylbutazone in the acute phase.

Example 2

Clinical Studies of the Effect of Anapsos Against Different Pathologies

Multiple sclerosis: Patients with multiple sclerosis were over 1 year provided with a treatment of 720 mg extract/day. The result was that the for the disease most characteristic alterations of the immunological phenotype were corrected giving a clinical stabilization of the patients. Further, the lymphocyte CD4+CD29+CD45RA+ population was normalized.

Prostatitis: Three days treatment with a dose of 240 mg extract, 60 minutes before the principal dinners, gave an improvement of the patients and all symptoms disappeared.

Pharyngitis: A dose of 120 mg extract gave favorable effects in the problems of pharyngitis, in particular for pharyngitis in its subacute period.

Example 3

In Vitro Study of the Adhesion Molecules In Peripheral Blood Mono-nuclear Cells (PBMNc).

Peripheral blood was extracted from 10 healthy individuals and the mononuclear cells was separated via Fycoll-Hypaque density gradient centrifugation. The cells was cultivated in plane bottom microtiter plates, at a titer of 1 million/ml for 48 hours in a CO₂ incubator, with phytohaemagglutinin (PHA) at concentrations of 0, 0.5, 2, 5 and 10 .mu.g/ml and/or with Anapsos at concentrations between 0 to 5000 .mu.g/ml.

Finalized the culturing, the lymphocytes was analyzed by flow cytometry. The expression of determined adhesion molecules (CD11a, CD11b, CD18, CD54) was studied in relation to the different conditions of stimuli. En parallel to the culture as described above, a culture for 5 days was made under the same conditions of stimuli as above. Tritium thymidine was added 16 hours before finalizing the culture. Terminated the culture, the cells was washed with the Harvester and flashing liquid was incorporated into the cells. The cellular incorporation of tritium thymidine was measured via a .beta. counter. During the culturing, the cells were photographed via an inverted microscope. The results are shown below.

Expression, in vitro, of different adhesion molecules of peripheral blood mononuclear cells.

          N = 10        CD11a       CD11b      CD18     CD54
          PHA10          22%         35%        27%      9%
         ANP 150         15%         14%        19%      0%
        PHA + ANP        16%         23%        25%      3%
         CONTROL         17%         20%        20%      2%

Example 4

Expression In Vivo of the Adhesion Molecules In Peripheral Blood Mono-nuclear Cells (PBMNc).

10 voluntary persons took for 11 consecutive days 720 mg/day of Anapsos. Peripheral blood was extracted from all of those persons the day before starting the treatment, the day after, at four days, and after the final administration. The mononuclear cells was separated via Fycoll-Hypaque density gradient centrifugation and the different lymphocyte populations were analyzed with respect of the differentiation markers CD11a and CD11b. The results are shown below.

Expression, in vitro, of different adhesion molecules of peripheral blood mononuclear cells.

        N = 10       PRE       24H     72H     96H     RETIRED
         CD11b       13%       4%      2%      1%        12%
         CD11a       14%       6%      3%      1%        15%

Claim 1 of 4 Claims

What is claimed is:

1. A method for normalization of C4+CD29+CD45RA+ lymphocyte populations comprising administering, to a subject afflicted with a disease wherein said populations are increased, a pharmaceutically effective amount of a composition comprising a water-soluble fraction from rhizomes of Polypodium and a lipid-soluble fraction from rhizomes of Polypodium; and a pharmaceutically acceptable carrier.

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