A peptide-coupled drug targeting M2 macrophages and its application

Abstract

The present invention discloses a polypeptide-coupled drug targeting M2 macrophages and its application, and belongs to the technical field of polypeptide-coupled drugs. The polypeptide-coupled drug of the present invention is composed of a polypeptide targeting M2 macrophages, a linker and a modified Tasquinimod. The amino acid sequence of the polypeptide targeting M2 macrophages is YEQDPWGVKWWY, and the amino acid sequence of the linker is GGGSKKK. The polypeptide-coupled drug of the present invention can reduce pancreatic fibrosis in CP mice, while reducing the toxic and side effects of Tasquinimod, and inhibiting the polarization of M2 macrophages in pancreatic tissue. The present invention provides a basis for using M2pep‑Tasquinimod as a new drug for chronic inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide-coupled drugs, and in particular relates to a polypeptide-coupled drug targeting M2 macrophages and an application thereof. Background Art

[0002] Chronic pancreatitis (CP) is a multifactorial fibro-inflammatory syndrome in which repeated episodes of pancreatic inflammation lead to extensive fibrotic tissue damage and exocrine and endocrine pancreatic insufficiency. It is estimated that the annual incidence of chronic pancreatitis is 5-12 cases per 100,000 people worldwide, and the prevalence is 50 cases per 100,000 people, which is increasing year by year. Macrophages are differentiated monocytes. According to their activation state, function and secreted factors, macrophages can be divided into classically activated M1 macrophages and selectively activated M2 macrophages; M1 macrophages mainly secrete pro-inflammatory factors and play an important role in the early stage of inflammation, while M2 macrophages express anti-inflammatory factors and play a role in inhibiting inflammatory response and tissue repair. In the progression of CP, macrophages change from the M1 phenotype with pro-inflammatory function in the early stage of CP to the M2 phenotype with fibrotic and anti-inflammatory functions in the late stage. As the severity of pancreatic fibrosis increases, the M2 / M1 cytokine ratio increases. M2 macrophages interact with pancreatic stellate cells (PSCs) by secreting cytokines such as TGF-β, constructing an inflammatory microenvironment that promotes the development of pancreatic fibrosis and aggravates pancreatic damage. Therefore, therapeutic drugs targeting M2 macrophages may be the right direction to cure pancreatic fibrosis in CP.

[0003] S100A9 protein (S100 calcium-binding protein A9) is a calcium-binding protein closely related to inflammation, trauma and tumors. It is mainly expressed in neutrophils, monocytes and keratinocytes. It is an important inflammatory mediator in acute and chronic inflammation and has both anti-inflammatory and pro-inflammatory effects. Studies have found that S100A9 plays an important regulatory role in the occurrence and development of pancreatitis. At the same time, our previous studies have also confirmed that S100A9 inhibitors can significantly reduce the infiltration of M2 macrophages in CP pancreatic tissue. Tasquinimod (4-hydroxy-5-methoxy-N,1-dimethyl-2-oxo-N-[(4-trifluoromethyl)phenyl]-1,2-dihydroquinoline-3-carboxamide, ABR-215050, CAS No: 254964-60-8) is an orally active lead compound. Studies have shown that S100A9 protein is the target of Tasquinimod. Tasquinimod binds to S100A9 protein, inhibits the interaction between S100A9 and its receptors TLR4 and RAGE, and exhibits certain biological activities such as anti-angiogenesis, immunomodulation and inhibition of tumor metastasis. However, phase III clinical trials showed that Tasquinimod has certain toxic and side effects, including loss of appetite, fatigue, weakness, nausea, renal dysfunction, infection and blood lymphatic system dysfunction. These adverse events eventually interrupted the clinical launch of Tasquinimod.

[0004] As a new type of drug conjugate, peptide-drug conjugate (PDC) enters cells through direct penetration or mediated endocytosis of peptides, and then stimulates the cleavable linker to release the drug. Compared with antibody-drug conjugate (ADC), PDC drugs have the advantages of small molecular weight, better tumor penetration, low immunogenicity, large-scale synthesis, and relatively good pharmacokinetics. PDC is expected to become a new generation of targeted drugs after small molecule drugs, monoclonal antibodies, and ADC drugs. However, there is no relevant research on PDC targeting M2 macrophages to treat inflammatory diseases. Summary of the invention

[0005] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide a polypeptide-conjugated drug targeting M2 macrophages and its application. The polypeptide-conjugated drug of the present invention can inhibit the polarization of M2 macrophages in CP pancreatic tissue by targeting them, thereby improving the biological activity of pancreatic fibrosis and reducing the toxic side effects of Tasquinimod.

[0006] The object of the present invention is achieved by the following methods:

[0007] The present invention provides a polypeptide-coupled drug targeting M2 macrophages, wherein the polypeptide-coupled drug consists of a polypeptide targeting M2 macrophages, a linker and a modified Tasquinimod.

[0008] Based on the above technical solution, further, the amino acid sequence of the polypeptide targeting M2 macrophages is YEQDPWGVKWWY.

[0009] Based on the above technical solution, further, the amino acid sequence of the linker is GGGSKKK.

[0010] Based on the above technical solution, further, the structure of the modified Tasquinimod is as follows:

[0011]

[0012] Based on the above technical solution, further, the structure of the peptide-drug conjugate targeting M2 macrophages is as follows:

[0013]

[0014] Another aspect of the present invention provides a pharmaceutical composition, which comprises an effective amount of the above-mentioned polypeptide-coupled drug targeting M2 macrophages and a pharmaceutically acceptable carrier.

[0015] Based on the above technical solution, further, the pharmaceutically acceptable carrier includes a filler, a diluent, a binder, a disintegrant, an emulsifier and a drug-carrying carrier without toxic side effects.

[0016] The present invention also provides the use of the above-mentioned polypeptide-coupled drug and drug composition in the preparation of drugs for treating chronic pancreatitis.

[0017] Based on the above technical solution, further, the dosage form of the drug includes tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.

[0018] Based on the above technical solution, further, the drug can reduce the toxicity of Tasquinimod.

[0019] Based on the above technical solution, further, the drug can inhibit macrophage M2 polarization.

[0020] The beneficial effects of the present invention compared with the prior art are as follows:

[0021] The present invention uses the M2 macrophage targeting peptide-coupled drug M2pep-Tasquinimod to treat CP mice in vivo, and proves that M2pep-Tasquinimod can alleviate pancreatic fibrosis in CP mice, while reducing the toxic and side effects of Tasquinimod and inhibiting M2 macrophage polarization in pancreatic tissue. The present invention provides a basis for using M2pep-Tasquinimod as a new drug for chronic inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0023] Figure 1 is the structural formula of M2pep-Tasquinimod of the present invention.

[0024] Figure 2 These are the targeting detection results of FITC-GGGSKKK-Tasquinimod (FITC-Tasquinimod) and FITC-YEQDPWGVKWWYGGGSKKK-Tasquinimod (FITC-M2pep-Tasquinimod) on M2 macrophages. The data are expressed as mean ± SEM, **P < 0.01 vs FITC-Tasquinimod group.

[0025] Figure 3 HE staining, Masson staining and scoring of pancreas in mice in different experimental groups, including: (A) HE staining and Masson staining; (B) HE score (n=5); (C) Masson score (n=5); (D) detection of pancreatic indexes AMS and LPS (n=5); data are expressed as mean ± SEM. ** P<0.01vs Ctrl group, # P < 0.05 and ## P<0.01vs CP group, && P<0.01vsTasquinimod group.

[0026] Figure 4 HE staining of the intestine, lung, spleen, kidney and heart of mice in different experimental groups.

[0027] Figure 5 The results of liver and kidney toxicity tests of mice in different experimental groups, including (A) renal function ARE and BUN test (n = 5); (B) liver function ARE and BUN test (n = 5); (C) inflammatory marker CRP test (n = 6); data are expressed as mean ± SEM, ** P<0.01vs Ctrl group,# P < 0.05 and ## P<0.01vs CP group, & P < 0.05 and && P<0.01vs Tasquinimod group.

[0028] Figure 6 The results show the content of M2 macrophages in the pancreas of mice in different experimental groups. DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0030] Example 1

[0031] 1. Tasquinimod structural modification, the reaction formula is as follows:

[0032]

[0033] The following steps are involved:

[0034] (1) In a single-necked flask, 38% concentrated hydrochloric acid (3 mL) was slowly added to glacial acetic acid (9 mL), followed by the addition of compound 1Cas: 248282-13-5 (1 g, 1 eq) and then heated to 70°C for 18 hours. The product was monitored by LCMS. After the raw material disappeared, the reaction solution was cooled to room temperature. The formed solid was filtered and washed with ethanol (20 mL). Drying gave a white solid compound 2 (0.6 g, 66% yield);

[0035] (2) In a single-necked flask, HATU (1.83 g, 2 eq) and DIPEA (0.94 g, 3 eq) were added to a DMF (10 mL) / THF (10 mL) solution containing compound 2 (0.6 g, 1 eq) in sequence. After stirring at room temperature for 10 min, compound 2a (0.48 g, 1.2 eq) was added and then stirred at 50°C for 2 hours. LCMS monitored the formation of the product and the disappearance of the raw material. Dilute hydrochloric acid was added to adjust the pH to 6-7, concentrated, purified by 4.6*150mm, kromasil C18-5 liquid chromatography column (20%-55% MeCN, 0.1% TFA), concentrated, and freeze-dried to obtain a yellow oil compound 3 (0.5 g, 52.6% yield);

[0036] (3) In a single-necked flask, add 5 ml of KOH (0.21 g, 3 eq) in water, add compound 3 (0.5 g, 1 eq) in THF (10 mL), and stir at 50°C for 4 hours. LCMS monitors the formation of the product, the raw material disappears, dilute hydrochloric acid is added to adjust the pH to 5-6, concentrate, pour out the water, add 5 ml of anhydrous methanol to the yellow viscous substance at the bottom, sonicate, filter, filter and dry to obtain white solid compound 4 (0.2 g, 41.5% yield);

[0037] (4) DCC (0.13 g, 1.2 eq) was added to a suspension of compound 4 (0.2 g, 1 eq) in DMF (5 mL), followed by compound 4a (90 mg, 1.5 eq) and DMAP (6.4 mg, 0.1 eq), and the mixture was stirred at 50°C for 2 hours. The mixture was filtered and the filtrate was purified by a 4.6*150 mm, kromasil C18-5 liquid chromatography column (20%-50% MeCN, 0.1% TFA), concentrated and freeze-dried to obtain a white solid modified tasquinimod (0.12 g, 48% yield, 97.8% purity, M+H+=480.1).

[0038] 2. Chemical Synthesis of M1pep-Tasquinimod

[0039] (1) Swelling of the resin: Weigh the resin and put it into the reaction column, then add DCM to the reaction column, shake for 30 minutes, and activate it for later use. (2) Connect the first amino acid: Filter out the DCM solvent through the sand core and add the first amino acid, then add DIEA 10 times the molar amount of the resin, and finally add a small amount of DMF to dissolve and shake for 1 hour. After the reaction is completed, wash it with DMF and DCM alternately for 6 times. (3) Deprotection: Add 20% piperidine / DMF solution, remove it after 5 minutes, then add 20% piperidine / DMF solution and shake for 15 minutes. (4) Detection: Remove the piperidine solution, take a dozen resins and wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol, heat at 105℃-110℃ for 5 minutes, and turn dark blue for positive, and the reaction can continue to the next amino acid. If it does not change color, it is negative and needs to be deprotected again. (5) First washing: Wash twice with DMF, methanol, and DMF respectively. (6) Condensation: Add amino acids with protective groups and condensing agent HBTU, dissolve with a small amount of DMF, immediately add DIEA, and react for 30 minutes. (7) Second washing: Wash twice with DMF, methanol, and DMF respectively. (8) Detection: Drain the solvent and take a dozen resins, wash three times with ethanol, add one drop of ninhydrin, pyridine, and phenol, and heat at 105℃-110℃ for 5 minutes. If it is colorless, it is a positive reaction. If it is blue, it needs to be condensed again. (9) Extension of the peptide chain: Repeat the above method and connect the remaining amino acids in turn. (10) Connect activated small molecules: Add 2% hydrazine hydrate / DMF solution, drain it after 5 minutes, repeat the operation twice, add the above-mentioned modified Tasquinimod activated molecule, dissolve it with a small amount of DMF, immediately add DIEA, and react for 30 minutes. (11) Detection: Remove the solvent, take a dozen resins, wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol, and heat at 105℃-110℃ for 5 minutes. If it is colorless, it is a positive reaction. If it is blue, it needs to be re-condensed. (12) Contraction of the peptide chain: After the last amino acid is connected, the synthesis of the entire peptide is completed and enters the final contraction stage. Wash the reaction with DMF 3 times, DCM 3 times, and methanol 3 times, and finally drain the peptide resin. (13) Deprotection of amino acid side chains and cutting of resin: Prepare 15mL of cutting solution, in which the volume ratio of each component is: TFA (94.5%), water (2%), EDT (2.5%), TIS (1%). Put the resin into a flask and shake at a constant temperature (30℃) for 2 hours. Blow the lysate as dry as possible with nitrogen, then pour it into a centrifuge tube and slowly pour in ether. Seal the tube and centrifuge it for 5 minutes. Pour out the supernatant and there will be a white solid below. The mixture was then washed with ether for 6 times and evaporated to dryness at room temperature to obtain the crude peptide.

[0040] In this example, a crude peptide sample was obtained, the specific structure of which is as follows Figure 1As shown, it is named M2pep-Tasquinimod, specifically YEQDPWGVKWWYGGGSKKK-[modified Tasquinimod].

[0041] In order to detect the targeting of M2pep-Tasquinimod, M2pep-Tasquinimod was further structurally modified. The specific process is as follows:

[0042] (1) Connecting ACP: Add 3 times the molar amount of fmoc-ACP and 3 times the molar amount of HBTU, dissolve with a small amount of DMF, and immediately add 10 times the molar amount of DIEA, react for 30 minutes. Then remove the solvent, take a dozen resins, wash them three times with ethanol, add one drop of ninhydrin, pyridine, and phenol, heat at 105℃-110℃ for 5 minutes, colorless is a positive reaction, if it is blue, it needs to be condensed again. After confirming that the connection is complete, wash it twice with 15 ml DMF, 15 ml methanol, and 15 ml DMF respectively.

[0043] (2) Deprotection: Add 20 ml of 20% piperidine / DMF solution and remove it after 5 minutes. Add another 20 ml of 20% piperidine / DMF solution and shake for 15 minutes. Then remove the piperidine solution, take a dozen resins, wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol, heat at 105℃-110℃ for 5 minutes, and turn dark blue for a positive reaction, and you can continue to connect FITC. If it does not change color, it is negative and needs to be deprotected again. After confirming that the deprotection is complete, wash it twice with 15 ml of DMF, 15 ml of methanol, and 15 ml of DMF respectively.

[0044] (3) FITC connection: Add 3 times the molar amount of FITC to the resin, dissolve it with a small amount of DMF, and immediately add 10 times the molar amount of DIEA to the resin, and react for 60 minutes. Then remove the solvent, take a dozen resins, wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol, and heat at 105℃-110℃ for 5 minutes. If it is colorless, it is a positive reaction. If it is blue, it needs to be re-coupled. After confirming that FITC is coupled, wash it twice with 15 ml of DMF, 15 ml of methanol, and 15 ml of DMF respectively.

[0045] (4) Peptide shrinkage: After receiving FITC, the final shrinkage stage begins. The reaction is washed with DMF 3 times, DCM 3 times, and methanol 3 times. Finally, the peptide resin is drained.

[0046] (5) Deprotection of amino acid side chains and cleavage of resin: Prepare 15 ml of cleavage solution, in which the volume ratio of each component is: TFA (94.5%), water (2%), EDT (2.5%), TIS (1%). Put the resin into a flask and shake at a constant temperature (30°C) for 2 hours. Blow the lysate as dry as possible with nitrogen, then pour it into a centrifuge tube and slowly pour in ether. Seal the tube and centrifuge it in a centrifuge for 5 minutes. Pour off the supernatant and there will be a white solid below. Wash it with ether for 6 times, then evaporate it at room temperature to obtain the crude peptide (FITC-YEQDPWGVKWWYGGGSKKK-Tasquinimod). In addition, the same operation was performed on Tasquinimod to obtain FITC-Tasquinimod (FITC-GGGSKKK-Tasquinimod). In the in vitro experiment, RAW264.7 cells were inoculated in 6-well plates and cultured for 48 hours. The culture medium or IL-4 (20 ng / ml) was added to the Taquinimod group and the M2pep-Tasquinimod group for 24 hours. The culture medium was discarded, and 10 μM FITC-Taquinimod and FITC-M2pep-Tasquinimod were added to the two groups for 24 hours. The cell pellet was collected for subsequent flow cytometry experiments. The cell flow cytometry results are shown in Figure 2. Figure 2 As shown, the fluorescence intensity of the FITC-M2pep-Tasquinimod group was significantly higher than that of the FITC-Tasquinimod group, that is, FITC-YEQDPWGVKWWYGGGSKKK-Tasquinimod had better targeting ability to IL-4-induced M2 macrophages.

[0047] Example 2

[0048] This example is an in vivo experiment. According to the method reported in the literature, a mouse model of chronic pancreatitis was induced by caerulein: the experimental animals were raised in an SPF animal room, alternating day and night for 12 hours, with an ambient temperature of 25±2°C and a relative humidity of 65-70%; 32 C57BL / 6 mice were randomly divided into a control group, a model group, a Tasquinimod treatment group and an M2pep-Tasquinimod treatment group, with 8 mice in each group. The mice in the model group were intraperitoneally injected with caerulein (50 μg / kg) on ​​Mondays, Wednesdays and Fridays, once every hour, 5 times a day, for 8 weeks. The mice in the Tasquinimod treatment group and the M2pep-Tasquinimod treatment group were injected with Tasquinimod (2.5 mg / kg, 0.154 μmol / mouse) and M2pep-Tasquinimod (0.154 μmol / mouse) respectively on the second day of the injection of caerulein, and the injections were continued for four weeks. The control group mice were intraperitoneally injected with an equal amount of 0.9% NaCl at the same dosing cycle. During the modeling process, a 1 ml syringe was used for slow push injection. After the injection was completed, the syringe was rotated and the needle was pulled out to prevent the caerulein from leaking out. If necessary, local gentle pressure was applied for a few seconds to prevent bleeding.

[0049] The experimental mice were sampled 24 hours after the last injection of the drug in the 8th week. After the mice were anesthetized by inhalation of isoflurane, their abdomens were facing up, their heads and limbs were fixed on the operating table, their backs were raised to fully expose the abdominal aorta, and blood was collected by needle. The extracted blood was placed at 4℃ for 2h, 4000rpm, centrifuged for 10min, and the upper serum was taken and stored in a -80℃ refrigerator; the pancreas was quickly separated, and a part of the mouse pancreas, heart, liver, spleen, lung, kidney and part of the intestinal tissue were fixed in 4% paraformaldehyde solution for subsequent preparation of HE staining and Masson staining to observe tissue pathological changes. Part of the pancreatic tissue was digested with collagenase and macrophages were isolated and analyzed for M2 polarization by cell flow cytometry. The remaining mouse pancreatic tissue was washed with saline, quickly placed in liquid nitrogen, and then transferred to a -80℃ refrigerator for storage for inspection. Amylase (AMS) and lipase (LPS) assay kits were used to detect serum AMS and LPS levels, and C-reactive protein (CRP), creatinine (CRE), blood urea nitrogen (BUN), aspartate aminotransferase (ALT) and alanine aminotransferase (AST) assay kits were used to detect serum CRP, CRE, BUN, ALT and AST levels.

[0050] The results of HE staining and Masson staining are as follows Figure 3As shown in the figure, HE staining results showed that compared with the model group, the pancreatic tissue edema, hemorrhage, acinar cell necrosis and inflammatory cell infiltration of the mice in the Tasquinimod group were significantly reduced, and the pancreatic tissue damage of the mice was improved to a certain extent. The above results indicate that Tasquinimod has a certain therapeutic effect on mice with chronic pancreatitis. Compared with the use of Tasquinimod alone, the pancreatic tissue edema, hemorrhage, acinar cell necrosis and inflammatory cell infiltration of the M2pep-Tasquinimod experimental group were significantly reduced, indicating that M2pep-Tasquinimod has a better therapeutic effect on chronic pancreatitis.

[0051] The results of Masson staining also revealed that the degree of pancreatic fibrosis in the Tasquinimod group mice was significantly reduced compared with the model group, and the degree of fibrosis in the M2pep-Tasquinimod group mice was lower than that in the Tasquinimod group. In addition, compared with the Tasquinimod group, the levels of AMS and LPS in the serum of the M2pep-Tasquinimod group mice were significantly decreased. These results confirmed that M2pep-Tasquinimod had a better therapeutic effect on pancreatic damage and fibrosis caused by CP, and significantly improved the biological activity of Tasquinimod.

[0052] HE staining of the intestine, lung, spleen, kidney and heart of mice in different experimental groups Figure 4 As shown, the results showed that the Tasquinimod group showed certain shrinkage and structural damage of the glomeruli. At the same time, the Tasquinimod group showed an increase in immune cells in the spleen and hyperimmunity. The intestines, lungs, spleen, kidneys and hearts of mice in the M2pep-Tasquinimod group were significantly improved, with no significant difference from the control group.

[0053] The results of liver and kidney toxicity experiments in different experimental groups of mice are as follows Figure 5 As shown in the results, the Tasquinimod group showed glomerular shrinkage and structural damage, suggesting that long-term use of Tasquinimod has certain nephrotoxicity. At the same time, the Tasquinimod group showed an increase in spleen immune cells and hyperimmunity ( Figure 4 ). The CRE content in the Tasquinimod group was significantly higher than that in the model group and M2pep-Tasquinimod, which also indicates that Tasquinimod can cause certain damage to the kidneys ( Figure 5 ), all indicators of the M2pep-Tasquinimod group were significantly improved compared with those of the Tasquinimod group, and M2pep-Tasquinimod significantly reduced the toxic and side effects of Tasquinimod.

[0054] Flow cytometry was used to detect the content of M2 macrophages in the pancreas of mice in different experimental groups. Figure 6 The results showed that compared with the CP group, the expression of CD206 decreased in the Tasquinimod group and the M2pep-Tasquinimod group, indicating that both groups could inhibit the M2 polarization of pancreatic macrophages in mice. Compared with the Tasquinimod group, the M2pep-Tasquinimod group had a decrease in M2 macrophages (CD68 + -CD206 + The content of M2pep-Tasquinimod in macrophage M2 polarization was lower.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A peptide-conjugated drug targeting M2 macrophages, characterized in that: The structure of the peptide-coupled drug targeting M2 macrophages is shown below:

2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective amount of the polypeptide-coupled drug targeting M2 macrophages according to claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutically acceptable carriers include fillers, diluents, binders, disintegrants, and emulsifiers.

4. Use of the polypeptide-coupled drug targeting M2 macrophages according to claim 1 or the pharmaceutical composition according to claim 2 or 3 in the preparation of a drug for treating chronic pancreatitis.

5. The use according to claim 4, characterized in that: The dosage forms of the medicine include tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.

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