Use of forskolin and derivatives thereof in the treatment of liver cancer

Abstract

The present application relates to Foskolin induced differentiation treatment of hepatocellular carcinoma. Specifically, the present application relates to a method and use of Forskolin for inducing differentiation of human malignant hepatocellular carcinoma, thereby being applied to the treatment of malignant solid tumors. The research of the present application shows that after treating hepatocellular carcinoma cells with exogenous addition of Forskolin for a period of time, the hepatocellular carcinoma cells can be effectively induced to differentiate, thereby providing a new drug for tumor induced differentiation treatment. The purpose of the present application is to provide the use of Forskolin and its derivatives for anti-hepatoma drugs.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology, cell biology, and medicine. Specifically, it relates to methods and uses of Forskolin-induced differentiation of human hepatocellular carcinoma for the treatment of hepatocellular carcinoma. Background Technology

[0002] Liver cancer (HCC) is one of the most common malignant tumors worldwide, ranking third in cancer-related mortality. In recent years, both the incidence and mortality rates of HCC in my country have been on the rise. Although surgical resection, local treatment, chemotherapy, targeted therapy, and immunotherapy have significantly improved the survival rate of HCC patients, the 5-year survival rate for patients with advanced HCC is less than 20%. Currently, sorafenib and lenvatinib are the main first-line drugs for treating HCC; however, unfortunately, resistance to both sorafenib and lenvatinib is becoming increasingly common among HCC patients. Therefore, research on HCC treatment urgently needs to adjust its approach and find more effective treatment methods.

[0003] Differentiation therapy is a treatment approach that has emerged in recent years, inhibiting tumor growth and spread by promoting the development of tumor cells into a more mature and normal state. The aim of this treatment is to cause tumor cells to lose their malignant characteristics, making them more similar to normal cells and reducing their harm to the body. Since the late 1970s, several signaling molecules and drugs, including retinoic acid (RA), cAMP, sodium butyrate, and cytokines, have been shown to induce terminal differentiation in vitro in acute myeloid leukemia (AML), embryonal carcinoma, or neuroblastoma. These observations pioneered the concept of differentiation therapy, suggesting that terminal differentiation of cancer cells could yield clinical benefits. Its significant success is in the treatment of acute promyelocytic leukemia (APL), a condition that can now be highly curative with a combination of retinoic acid (RA) and arsenic, although both drugs have significant side effects.

[0004] In recent years, the research team of the inventor has made some progress in the field of differentiation therapy for liver cancer. After introducing hepatocyte nuclear factors HNF1α, HNF4α, and HNF3γ (FOXA3) into liver cancer cells for a period of time, tumor cells with malignant phenotypes are transformed into "normal functioning hepatocytes" (multiple gene mutations still exist, but hepatocyte function has been restored) (Cheng Z, Conversion of hepatoma cells to hepatocyte-like cells by defined hepatocyte nuclear factors. Cell Res. 2019 Feb; 29(2):124-135.). Another research result shows that the introduction of HNF3γ alone can also transform poorly differentiated liver cancer cells into highly differentiated, mature cells without malignant phenotypes (Zhou T, m6ARNA methylation-mediated HNF3γ reduction renders hepatocellular carcinoma dedifferentiation and sorafenib resistance. Signal Transduct Target Ther. 2020 Dec 26; 5(1):296.). These research results provide some ideas for differentiation therapy of liver cancer. Professor Zhang Peilin and Academician Wang Hongyang's team discovered that a combination of small molecule drugs, including SB431542 (TGFβ inhibitor), CHIR99021 (GSK3β inhibitor), BIX01294 (H3K9 methyltransferase / G9a inhibitor), and all-trans retinoic acid (ATRA), can induce differentiation of drug-resistant cells, including cell lines, primary cancer cells, and cancer stem cells. The treated cells lost their malignant characteristics and restored their normal hepatocyte phenotype (Zhang X, Small Molecule-Induced Differentiation As a Potential Therapy for Liver Cancer. Adv Sci (Weinh). 2022 May; 9(15):e2103619.). This indicates that differentiation of liver cancer cells can also be induced by small molecule drugs alone.

[0005] Forskolin, also known as hairy throat extract, is a natural diterpenoid product isolated from the Indian plant *Coleus forskohlii*, with the molecular formula C2. 22 H 34O7, chemically named (3R,4aR,5S,6S,6aS,10S,10aR,10bS)-6,10,10b-Trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f]chromen-5-yl acetate. Forskolin is an adenylate cyclase activator that directly stimulates adenylate cyclase and has been widely used to increase cAMP and induce cAMP-dependent physiological responses. It is currently marketed as a weight-loss dietary supplement, i.e., a health product, and therefore has minimal side effects. Previously, Deng Hongkui's team reported in Science that a combination of five compounds, including Forskolin (5C), treated HBV-infected hepatocytes, restored their normal liver-specific functions, specifically manifested as morphological transformation into primitive hepatocyte-like cells and upregulation of CYP series gene expression of hepatic drug-metabolizing enzymes (Xiang C, Long-term functional maintenance of primary human hepatocytes invitro. Science. 2019 Apr 26; 364(6438):399-402.). However, the authors did not explain the mechanism of this phenomenon. Based on the above research results, we hypothesize that Forskolin may have the potential to induce liver cancer cells to differentiate into cells with "normal hepatocyte-like morphology and function" into drug-grade cells.

[0006] Therefore, the aim of this field is to utilize the small molecule compound Forskolin and its derivatives to induce differentiation of liver cancer cells, thereby providing a new approach for the treatment of liver cancer. Summary of the Invention

[0007] This invention has discovered that Forskolin can induce differentiation of liver cancer cells. Therefore, the purpose of this invention is to provide new pharmaceutical uses for Forskolin and pharmaceutical compositions containing Forskolin. Another purpose is to provide a new method for the treatment of liver cancer.

[0008] A first aspect of the invention provides a novel use of Forskolin, namely, the use of Forskolin or its derivatives in the preparation of medicaments for treating liver cancer.

[0009] Preferably, Forskolin or its derivatives achieve liver cancer treatment by inducing differentiation of hepatocellular carcinoma. Furthermore, by inducing poorly differentiated liver cancer cells to differentiate into mature phenotypes, drugs can inhibit liver cancer cell proliferation and metastasis, and promote apoptosis, thereby achieving the goal of treating liver cancer.

[0010] The Forskolin derivatives described in this invention refer to compounds that can produce Forskolin active ingredients through biochemical reactions in the human body, as well as compounds with similar or better activity than Forskolin produced by chemical modification of Forskolin.

[0011] This invention first demonstrated through in vitro experiments that Forskolin can transform the morphology of Huh7 cells from small and pointed to flat and broad; it can promote the synthesis of fat and glycogen, and the secretion of urea and albumin; it has a significant inhibitory effect on the proliferation of liver cancer cells, significantly inhibiting their invasive ability, and simultaneously arresting the cell cycle at the G0 / G1 phase, thereby inhibiting the progression of the cell cycle. Furthermore, Forskolin can also promote the expression of liver cell-related functional genes and reduce tumor volume.

[0012] The application of Forskolin or its derivatives in the preparation of liver cancer therapeutic drugs according to the present invention also has the following technical features: the therapeutic drug is either Forskolin or its derivatives as the sole active ingredient or a pharmaceutical composition containing Forskolin or its derivatives. It can also be used in combination with other therapeutic drugs, such as with chemotherapy agents.

[0013] In a second aspect, the present invention provides a pharmaceutical composition for treating lung cancer, comprising Forskolin and a pharmaceutically acceptable carrier or excipient.

[0014] The pharmaceutically acceptable carriers or excipients described in this invention refer to additives commonly used in the pharmaceutical field other than the active ingredient, such as diluents (starches, sugars, celluloses, and inorganic salts), excipients, fillers such as starch and sucrose, binders such as water, ethanol, cellulose derivatives, gelatin, and polyvinylpyrrolidone, disintegrants such as dry starch and sodium carboxymethyl starch, solubilizers such as polysorbates and polyoxyethylene fatty acid esters, absorption enhancers, surfactants such as Tween and Span, adsorbent carriers, and lubricants such as magnesium stearate and micronized silica gel. Additionally, other excipients such as flavoring agents and sweeteners may be added to the composition.

[0015] A third aspect of the present invention provides a method for inducing or promoting differentiation of hepatocellular carcinoma in mammals, i.e., a novel treatment method for liver cancer.

[0016] The method includes the following steps: long-term treatment of liver cancer cells with a certain concentration of Forskolin to inhibit the proliferation and metastasis of liver cancer cells and promote their apoptosis.

[0017] The liver cancer described in this invention is preferably human primary hepatocellular carcinoma.

[0018] This invention provides new pharmaceutical applications for Forskolin and a novel method for treating liver cancer by acting on the drug to induce differentiation of liver cancer cells. Attached Figure Description

[0019] Figure 1 This is the chemical structural formula of the small molecule compound Forskolin.

[0020] Figure 2 This shows the changes in cell morphology of Huh7 liver cancer cells after two weeks of Forskolin treatment. A represents cell morphology in the control group, and B represents cell morphology after Forskolin treatment.

[0021] Figure 3 This shows the lipid synthesis status of HCCLM3 liver cancer cells two weeks after Forskolin treatment. A represents the oil red staining of HCCLM3 cells in the control state, and B represents the oil red staining of HCCLM3 cells after Forskolin treatment.

[0022] Figure 4 This shows the glycogen synthesis status of Huh7 liver cancer cells two weeks after treatment with Forskolin. A represents the PAS (periodic acid-Schiff) staining of Huh7 cells in the control state, and B represents the PAS staining of Huh7 cells after Forskolin treatment.

[0023] Figure 5 This is the urea secretion status two weeks after Forskolin treatment of HCCLM3 liver cancer cells.

[0024] Figure 6 This is the albumin secretion status two weeks after Forskolin treatment of HCCLM3 liver cancer cells.

[0025] Figure 7 This refers to the tumor growth inhibition effect of Forskolin treatment on HCCLM3 liver cancer cells after two or three weeks.

[0026] Figure 8 This study describes the inhibitory effect of Forskolin treatment on Huh7 liver cancer cells for two weeks. A represents Huh7 cell migration in the control group, B represents Huh7 cell migration across the membrane after Forskolin treatment, and C represents a quantitative analysis of both cell migration patterns.

[0027] Figure 9 This describes the inhibition of tumor cell colony formation in Huh7 liver cancer cells after two weeks of treatment with Forskolin. A represents Huh7 colony formation in the control state, B represents Huh7 colony formation after Forskolin treatment, and C represents a quantitative analysis of colony formation in both cases.

[0028] Figure 10 This diagram shows the changes in the cell cycle of HCCLM3 liver cancer cells after two weeks of Forskolin treatment. A represents the cell cycle of HCCLM3 cells in the control state, B represents the cell cycle of HCCLM3 cells after Forskolin treatment, and C represents the quantitative analysis of the cell cycle in both cases.

[0029] Figure 11 This is a transcriptomic RNA-Seq analysis of the differences in genes related to liver differentiation and drug metabolism after two weeks of Forskolin treatment of HCCLM3 liver cancer cells. A is a heatmap of the expression profiles of genes related to liver differentiation, and B is a heatmap of the expression profiles of genes related to liver drug metabolism.

[0030] Figure 12 This is a quantitative analysis of the mRNA expression of liver function-related genes after two weeks of Forskolin treatment of HCCLM3 liver cancer cells.

[0031] Figure 13 It is a xenograft formed by subcutaneous inoculation of nude mice with human liver cancer cells treated with Forskolin. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention are merely for illustration and not for limiting the present invention. Simple improvements to the method of the present invention under the premise of the concept of the present invention are all within the scope of protection of the present invention.

[0033] All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions as described in Sambrook et al., *Molecular Cloning: A Laboratory Guide* (New York: Cold Spring Harbor Laboratory Press, 1989), or under standard conditions, or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0034] Example 1: Cell morphology observation of Huh7 liver cancer cells after 3 weeks of Forskolin treatment.

[0035] Huh7 / Control and Huh7 / Treated cells, treated with DMSO and Forskolin for 3 weeks, were seeded at a rate of 2000 cells per well in 6-well plates. Seven days later, the morphology of live cells was observed under an inverted phase-contrast microscope (×200x). Under light microscopy, Huh7 / Control cells were observed to be small and sharp (e.g., ...). Figure 2 As shown in A), while Huh7 / Treated cells are flat and broad (as shown in A). Figure 2 (as shown in B).

[0036] Example 2: Effect of Forskolin treatment on adipogenesis in HCCLM3 liver cancer cells for 2 weeks

[0037] HCCLM3 / Control and HCCLM3 / Treated cells were seeded into 12-well plates. After one day of adhesion, the culture medium was removed, the cells were washed twice with PBS, fixed with 10% neutral formaldehyde for 30 min, washed with 70% ethanol for 1 min, stained with oil red dye prepared with 70% ethanol for 15 min, washed with 70% ethanol for 1 min, stained with hematoxylin for 90 sec, and then rinsed with water for 15 sec. The staining was observed under an optical microscope.

[0038] The results showed that HCCLM3 / Treated cells had a higher lipid droplet content than HCCLM3 / Control cells. Figure 3 ).

[0039] Example 3: Effect of Forskolin treatment on glycogen synthesis in Huh7 liver cancer cells for 2 weeks

[0040] Huh7 / Control and Huh7 / Treated cells were seeded into 12-well plates. After one day of adhesion, the culture medium was removed, the cells were washed twice with PBS, fixed with 10% neutral formaldehyde for 10 min, rinsed with water for 1 min, 1% periodic acid for 5 min at room temperature, washed with distilled water for 5 min, Schiff's solution for 15 min at room temperature, washed with distilled water for 5 min, and the staining was observed under an optical microscope.

[0041] The results showed that Huh7 / Treated cells were redder and produced more glycogen than Huh7 / Control cells. Figure 4 ).

[0042] Example 4: Effect of Forskolin treatment on urea secretion in HCCLM3 liver cancer cells for 2 weeks

[0043] HCCLM3 / Control and HCCLM3 / Treated cells were cultured at 10,000 cells per well, with 3 replicates per cell type. After cell adhesion, each well was replaced with 100 μL of serum-free culture medium and incubated at 37°C in a 5% CO2 incubator for 24 h. The supernatant was then collected, and urea levels were determined according to the instructions of the Human Urea ELISA Kit (purchased from Shanghai Yanjin Biotechnology Co., Ltd.).

[0044] The results showed that HCCLM3 / Treated cells had higher urea secretion compared to HCCLM3 / Control cells. Figure 5 ).

[0045] Example 5: Effect of Forskolin treatment on albumin secretion in HCCLM3 liver cancer cells for 2 weeks

[0046] HCCLM3 / Control and HCCLM3 / Treated cells were cultured at 10,000 cells per well, with 3 replicates per cell type. After cell adhesion, each well was replaced with 100 μL of serum-free medium and incubated at 37°C in a 5% CO2 incubator for 24 h. The supernatant was then collected, and albumin was measured according to the instructions of the HumanALB ELISA Kit (purchased from Shanghai Yanjin Biotechnology Co., Ltd.).

[0047] The results showed that HCCLM3 / Treated cells secreted more albumin than HCCLM3 / Control cells. Figure 6 ).

[0048] Example 6: Effect of Forskolin treatment on HCCLM3 liver cancer cells for 1-2 weeks on cell growth inhibition

[0049] HCCLM3 cells in logarithmic growth phase were seeded into three 6-well plates and treated with DMSO and Forskolin for a period of time (T1W: 1 week of treatment; T2W: 2 weeks of treatment). Then, they were seeded into 96-well plates at a density of 5000 cells per well with 3 replicates. 200 μL of culture medium was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator for 0d, 1d, 3d, and 5d (d: days). At each time point, the culture medium was removed, and 100 μL of CCK8 working solution was added to each well. After incubation for 1 hour, the plates were read at a wavelength of 450 nm using a microplate reader. The OD value of each well was read and the average value was calculated. A growth curve was plotted with time on the x-axis and the average OD value on the y-axis.

[0050] After several days of continuous measurement of three-cell growth curves, it was found that Forskolin had a significant inhibitory effect on the proliferation of liver cancer cells. The control group cells proliferated significantly over time, while the treatment group cells proliferated at a slower pace. Figure 7 ).

[0051] Example 7: Effect of Forskolin treatment on HCCLM3 liver cancer cells for 2 weeks on cell invasion inhibition

[0052] HCCLM3 cells in logarithmic growth phase were seeded into two 6-well plates and treated with DMSO and Forskolin, respectively. After two weeks, the cells were seeded at a density of 2 × 10⁶ cells per well. 5Seeds were seeded at a density of 200 μL of culture medium per well in the upper chamber of a transwell plate and placed in a 24-well plate. Culture medium containing 20% ​​bovine serum was injected into the lower 24-well plate and the plate was incubated at 37°C in a 5% CO2 incubator. After 20 h, the transwells were removed, the culture medium in the upper chamber was aspirated, the plates were washed twice with PBS, fixed with 10% neutral formaldehyde for 10 min, washed twice with PBS, stained with 0.1% crystal violet for 15 min, washed twice with PBS, and the stained cells in the upper chamber were carefully wiped away with a cotton swab and observed under an inverted phase-contrast microscope (×100x).

[0053] The results showed that Forskolin could significantly inhibit the invasive ability of liver cancer cells. Figure 8 ).

[0054] Example 8: Effect of Forskolin treatment on HCCLM3 liver cancer cells for 2 weeks on cell clones

[0055] HCCLM3 cells in logarithmic growth phase were harvested and processed at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of / well in 6-well plates. After treatment with DMSO and Forskolin for two weeks, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and then photographed in each well to count the cell clones.

[0056] The results showed that the number of cell clones in the HCCLM3 / Treated group was significantly lower than that in the HCCLM3 / Control group. Figure 9 ).

[0057] Example 9: Effect of Forskolin treatment on the cell cycle of HCCLM3 cells for 2 weeks

[0058] HCCLM3 cells were treated with Forskolin for one week, then the culture medium was removed, and the cells were washed twice with PBS. The cells were then fixed overnight at 4°C with cold 70% ethanol. After removing the fixative, 500 μL of PI / RNase A staining solution was added, and the cells were incubated at room temperature in the dark for 60 min. Cell apoptosis was detected using flow cytometry, and the data were analyzed using FlowJO software.

[0059] Forskolin primarily arrests the cell cycle at the G0 / G1 phase, thereby inhibiting the progression of the cell cycle. Figure 10 ).

[0060] Example 10: Effects of Forskolin treatment on the expression of genes related to liver differentiation and drug metabolism in HCCLM3 cells after 2 weeks.

[0061] After treating HCCLM3 cells with Forskolin for two weeks, the cells were washed twice with PBS, and then lysed in six-well plates with Trizol. The samples were numbered: the control group had three replicates (Control-1, Control-2, Control-3); the Forskolin-treated group had three replicates (Treated-1, Treated-2, Treated-3). RNA-seq was used for transcriptome sequencing, and Z-cores were applied to the expression values ​​of genes related to differentiation and drug metabolism.

[0062] Heatmap results showed that the expression levels of differentiation and drug metabolism-related genes were higher in the Forskolin-treated group than in the control group. Figure 11 ).

[0063] Example 11: Real-time quantitative PCR was used to detect the expression of hepatocyte-related functional genes after treating HCCLM3 liver cancer cells with Forskolin for 2 weeks.

[0064] (1) Treat HCCLM3 cells with Forskolin in a 6-well plate for two weeks; (2) After two weeks, remove the culture medium, wash twice with PBS, add 1 ml of TRIZOL, and let stand at room temperature for 3 min; (3) Add 200 μl of chloroform and shake vigorously for 30 sec, and let stand at room temperature for 3 min; (4) Centrifuge at 4℃, 12,000 rpm for 15 min; (5) Transfer the upper aqueous phase containing RNA to a new 1.5 ml Eppendorf tube, add 500 μl of isopropanol to precipitate RNA, shake vigorously for 30 sec, and let stand on ice for 10 min; (6) Centrifuge at 4℃, 12,000 rpm for 15 min; (7) Discard the supernatant, add 0.5 ml of 75% ethanol to wash the RNA precipitate, and centrifuge at 4℃, 12,000 rpm for 5 min; (8) Remove the ethanol, dry the RNA with a hairdryer, and when a transparent precipitate appears, dissolve the RNA in 30 μl of DEPC water; (9) Measure the RNA content using a UV spectrophotometer and store at -20℃;

[0065] (10) RNA reverse transcription: After determining the RNA concentration, take 2 μg of RNA and add it to a PCR tube. Add 1 μl of Random primer N6 and make up the volume to 17.5 μl with RNase-free water. Pre-treat at 70℃ for 5 min, then immediately incubate on ice for at least 2 min. Then add the pre-prepared reverse transcription mixture: 5 μl 5×RTbuffer, 1 μl dNTPs, 1 μl mouse reverse transcriptase, and 0.5 μl RNase inhibitor. Perform reverse transcription on a regular PCR instrument at 37℃ for 60 min and 70℃ for 10 min to obtain cDNA, which is then stored at -20℃.

[0066] (11) Preparation of Real-time PCR system: 1 μl cDNA, 1 μl upstream / downstream primer mixture (5 μM), 5 μl 2×SYRB Green Master, 3 μl MilliQ water, centrifuge and mix to a total volume of 10 μl. (12) Real-time PCR steps: Detection was performed using a Roche LightCycler 480 fluorescence quantitative analyzer. Pre-denaturation was performed at 95℃ for 10 min, followed by amplification cycles: 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 35 s, for 40 cycles, with a final extension at 72℃ for 10 min. See Table 1 for details of the Real-time PCR gene primers used.

[0067] Table 1 Primer sequences for human liver function

[0068]

[0069] Albumin (ALB); Cytochrome P450 family 1A2 (CYP1A2); Glutamine synthetase (GS); Multidrug resistance-associated proteins 2 (MRP2); Asialoglycoprotein receptor 1 (Asgpr1); Alpha-fetoprotein (AFP).

[0070] The results showed that, compared with the control group, the mRNA expression of hepatocyte-related functional genes ALB, CYP1A2, GS, MRP2, and Asgpr1 was upregulated in the treatment group, while AFP was downregulated. Figure 12 ).

[0071] Example 12: Forskolin treatment of HCCLM3 cell subcutaneous tumor-bearing nude mice

[0072] HCCLM3 liver cancer cells were treated at a rate of 5 × 10 6 A certain amount was injected subcutaneously into the left axilla of nude mice, and the subcutaneous tumor was allowed to grow to approximately 260 mm². 3 At the time of administration, 10 mg / kg of Forskolin was injected intraperitoneally, while the control group was injected with an equal volume of a solvent composed of 5% DMSO, 40% PEG300, 5% Tween 80, and 50% ddH2O. Injections were administered every other day, and changes in tumor size were recorded.

[0073] The results showed that after 20 days of treatment, the tumor volume in the experimental group of nude mice was significantly smaller than that in the control group. Figure 13 ).

[0074] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of Forskolin or its derivatives in the preparation of drugs for treating liver cancer.

2. The application according to claim 1, characterized in that, The liver cancer treatment drug mentioned is a drug that induces liver cancer differentiation.

3. The application according to claim 1, characterized in that, The drug that induces liver cancer differentiation is a drug that induces poorly differentiated liver cancer cells to differentiate into a mature phenotype.

4. The application according to any one of claims 1 to 3, characterized in that: Forskolin or its derivatives in combination with other therapeutic agents.

5. A pharmaceutical composition for treating liver cancer, characterized in that, Contains Forskolin or pharmaceutically acceptable salts, esters, hydrates, or combinations thereof, as well as excipients.

6. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition is selected from tablets, capsules, oral liquid preparations, granules, and injections.

7. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition is prepared using conventional or special formulation processes.

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