Patent Application
20210393724
The present invention relates to a medicine developed for treatment of psoriasis and production method thereof.
Psoriasis is a systemic inflammatory disease characterized by white colored skin eruption on a red colored base. Psoriasis is mostly seen at the knees and elbows and causes severe itching in the patients. Although it is not clear what causes psoriasis which is a non-infectious disease, it is known that the stress factor has a triggering effect on psoriasis in cases where the body's defense mechanism fails. In 2008, observing that a cream containing Imiquimib (IMQ) active ingredient (Aldara contains 5% Imiquimib, commercial name) used topically to treat genital warts has the side effect of inducing psoriasis-like lesions in humans, and noticing the same effect on mice have attracted attention to this cream [1]. It has been well demonstrated by the studies that IMQ, which binds to Toll-like TLR7 and TLR8 receptors and activates the systemic inflammation pathway as an immune activator, produces a systemic response after topical use in mice leading to formation of psoriasis [2, 3, 4, 5, 6]. Nowadays, it has been decided to show the six main points in mice model histopathologically and immunohistochemically for the efficiency and standardization of the preclinical studies for psoriasis. These are as follows:
Currently, the IMQ-induced mouse model is used as an ideal preclinical model that follows the said six points.
Although there is no treatment that completely cures psoriasis yet, it is tried to be controlled by the dermatologists with various drugs. The subcutaneous immunological agents, which have been started to be used recently, have unfortunately not been a common and favorable option in the treatment of the disease due to their serious side effects (such as immunosuppression, toxicity, etc.), hospitalization and follow-up difficulties, serious costs and inability to prevent recurrence of the disease [7].
The objective of the present invention is to develop a drug formulation for treatment of psoriasis.
“A medicine for treatment of psoriasis and production method thereof” developed to fulfill the objective of the present invention is illustrated in the accompanying figures, in which;
The psoriasis medicine of the present invention comprises 800 to 1000 g of tallow, 250 to 350 g of larch resin, 450 to 550 g of beeswax, 2 to 3 g of gum mastic, 40 to 50 g of propolis, 200 to 250 g of Alkanna tinctoria, 200 to 250 g of alum, 150 to 200 g of Juniper tar. The production method of the psoriasis medicine of the present invention comprises the steps of
In the preferred embodiment of the invention, the obtained elute is applied as an ointment by spreading it on the surface of the skin of the psoriasis patient where the lesion is observed.
The cream named Aldara (Meda Pharma, 3M Health Care) containing 5% Imiquimib (IMQ) was administered once a day for 6 days on the shaved backs and right ears of 8-11 weeks old adult male BALB/c mice such that a total of 62.5 mg cream is applied per day per animal thereby psoriasis inflammation was produced in the animal model. Vaseline was applied to the shaved area of the animals in the control group. The positive control group received 1 mg/kg Mtx (Methotrexate) orally per day. The drug was administered orally at a dose of 5 mg/kg once a day during the experiment. Administration of the drug to the animals in the group where the semi-therapeutic effect of the drug was to be tested was initiated 1 day after the onset of induction of psoriasis with IMQ, and continued until the end of the experiment period. Body weight, and redness, flaking and thickening on the skin were daily measured and scored according to the Psoriasis Area Severity. Index (PASI) to monitor the general health status of the animals and the induction of psoriasis with IMQ. As required by the model, thickening of the right ear skin was also recorded on a regular basis since the day the experiments began.
At the end of the application, animals were anesthetized by isoflurane and then euthanized by cervical dislocation. The size and weight of the spleen tissues were recorded.
The skin samples collected from the IMQ-treated backs of the mice after dissection were fixed in formalin, and paraffin blocks were prepared. Ten sections having a thickness of 10 microns were collected at equal intervals from each animal. The sections were stained with Masson Trichrome and their images were taken by microscope. The epidermis thicknesses were calculated by taking the average of the measurements taken at 5 different points for each section.
After dissection, the spleen tissues were divided into smaller parts by a scalpel a petri dish containing RPMI medium, then they were thoroughly ground with a pasteur pipette and washed without being allowed to disintegrate. After the cell suspension was passed through a 70 μm filter, it was taken up in PBS and centrifuged for 10 minutes at 2000 rpm. The precipitated cells were dissolved in sterile lysis solution containing 0.037 g/L EDTA, 1 g/L potassium bicarbonate and 8.29 g/L ammonium chloride, and incubated at room temperature for 10 minutes. The supernatant obtained after centrifugation at 800 rpm for 10 minutes was dissolved in PBS and centrifuged at 2000 rpm for 10 minutes. The precipitated cells were dissolved in 10 ml of cRPMI medium and the number of cells was determined.
CFSE was placed on the isolated cells for staining purposes. This process and the rest were carried out in dark environment. Cells incubated at +4° C. for 6 minutes were then placed in cRPMI and centrifuged at 2000 rpm for 5 minutes. cRPMI was added to the precipitated cells and they were again centrifuged at 1200 rpm for 5 minutes. The precipitated cells were resuspended in cRPMI medium and the number of cells was determined. 55×105 cells were seeded in each well. The lymphocytes were stimulated with CD3 and CD28. The cultures were placed in a 37° C. incubator for 3 days.
The isolated lymphocytes were spread into each well of 48-well plates at a concentration of 5×105 cells per well. Lymphocytes stained with CFSE were cultured for 3 days for proliferation analysis. Non-stimulatory and anti-CD3+CD28 (CDmix) stimulated cultures of lymphocyte cells in each group were performed. On the third day, proliferation analyses of the lymphocyte cells were examined by flow cytometry. CFSE fluorescence staining can be displayed in FL-1 in flow cytometry. After the analyses, proliferation of the T cells was compared in the presence or absence of stimuli.
After 3 days of culturing of the isolated lymphocyte cells, CD4+CD25+FoxP3+ cell numbers were examined. After enabling the cells to be homogenously dissociated by pipetting in the wells, they were taken out of this homogenous solution and put into flow tubes. The cells, to which PBS was added, were centrifuged at 1200 rpm for 5 minutes. CD4 and. CD25 were added to the tubes and vortexed and were incubated at room temperature in the dark for 20 minutes. Then the staining buffer solution was added and centrifuged at 250 g for 10 minutes. FoxP3 buffer solution was added to the precipitated cells and they were vortexed and incubated at room temperature in the dark for 10 minutes. They were then centrifuged at 500 g for 5 minutes. Staining buffer solution was added to the precipitated cells and they were vortexed and centrifuged at 500 g for 5 minutes. FoxP3 buffer solution C was added to the precipitated cells and they were vortexed and incubated at room temperature in the dark for 30 minutes. Then the staining buffer solution was added and centrifuged at 500 g for 5 minutes. This process was repeated once more for the precipitated cells. Then the FoxP3 antibody was added and the vortex process was carried out slowly. They were incubated at room temperature in dark for 30 minutes and then the staining buffer solution was added and centrifuged at 500 g for 5 minutes, The staining buffer solution was added to the precipitated cells and they were analyzed in the flow cytometry device.
In the literature, the use of drugs prepared with traditional methods in psoriasis is frequently seen [3]. Especially in Chinese and Indian medicine, certain herbal mixtures have been used in this field for centuries. Recently, it has been observed that these traditional drugs are being tested in controlled preclinical studies. An important reason for this is the ease of use of the standardized IMQ-mouse model which has become widely preferred for evidence-based medicine practices in psoriasis. When we study the chemical behavior of these herbal compounds that are traditionally used in psoriasis and similar skin diseases, the strong anti-inflammatory properties of these mixtures stand out. These anti-inflammatory systemic features exhibit promising although limited number of positive data in both preclinical and clinical histopathology [3]. We demonstrated that the drug, which is developed for the treatment of psoriasis and prepared in the above-mentioned formulation, is effective in psoriasis pathophysiology by testing it on the IMQ-mouse model in a similar manner to the samples in the literature.
The activity of the product can be demonstrated by the graphical representations generated during the development of the invention as follows:
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018/16367 | Nov 2018 | TR | national |
This application is the national phase entry of International Application No. PCT/TR2019/050019, filed on Jan. 9, 2019, which is based upon and claims priority to Turkish Patent Application No. 2018/16367, filed on Nov. 1, 2018, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/TR2019/050019 | 1/9/2019 | WO | 00 |
