Compound for inhibiting virus replication and application of compound in preparation of medicine for resisting feline infectious peritonitis virus

Abstract

The invention belongs to the technical field of medicines, and provides a compound for inhibiting virus replication and application of the compound in preparation of a medicine for resisting feline infectious peritonitis virus (FIPV). The number of the compounds provided by the invention is 10, and the 10 compounds have obvious inhibiting and blocking effects on a 1-bit ribosome frameshift process of the FIPV, and can effectively inhibit the replication and proliferation of the FIPV. The ten compounds can be used for treating and preventing feline infectious peritonitis diseases caused by FIPV infection; the compound disclosed by the invention can be used for preparing anti-FIPV medicines and medicines for treating and preventing feline infectious peritonitis diseases caused by FIPV infection, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to a compound for inhibiting virus replication and application of the compound in preparing a drug for resisting feline infectious peritonitis virus. Background Art

[0002] The number of companion animals continues to grow. By 2024, the total number of pets in China will reach 124 million, including 71.53 million cats (a 6.8% growth rate) and 52.58 million dogs (a 1.1% growth rate). This suggests that the number of pet cats will remain high in the coming years. However, the current status of therapeutic drug research and development for pet cats is not optimistic.

[0003] Feline Infectious Peritonitis Virus (FIPV) infection is one of the biggest threats to pet cats. FIPV infection is known as the "terminal disease of cats" and has a mortality rate of over 95% when untreated. Some studies have shown that the mortality rate of severe cases is close to 100%.

[0004] Feline coronavirus (FCoV) belongs to the order Nidovirales, family Coronaviridae, genus Alphacoronavirus. The FCoV genome is a single-stranded, positive-sense RNA virus, approximately 29 kb in length and containing 11 open reading frames. There are two biotypes of FCoV: feline enteric coronavirus (FeCV) and feline infectious peritonitis virus (FIPV). Over 90% of cats carry the non-pathogenic FeCV throughout their lives, while approximately 5% of infected cats develop feline infectious peritonitis (FIPV) due to viral mutations to FIPV. FIPV infects white blood cells and spreads throughout the body, triggering an immune-mediated vasculitis that leads to organ inflammation and damage. Untreated, the mortality rate in kittens under six months of age is nearly 100%. Feline infectious peritonitis can be categorized into two types based on symptoms: wet and dry. The wet (exudative) type accounts for 70%-80% of cases and is characterized by peritoneal and pleural effusions, abdominal distension, and difficulty breathing, with a rapidly progressive course. The dry (nonexudative) form affects the central nervous system (eg, seizures, ataxia) or the eyes, and symptoms are insidious and more difficult to diagnose and treat.

[0005] In the FIPV genome, the two upstream open reading frames, ORF1a and ORF1b, occupy two-thirds of the genome and contain at least one ribosomal frameshifting site. ORF1a and ORF1b encode two large nonstructural polyproteins, pp1a and pp1ab, the expression of which depends on the -1 ribosomal frameshifting element in the ORF1a / ORF1b overlap region.

[0006] The phenomenon of "-1 frameshifting" during ribosome elongation is very common in RNA viruses. PRRSV, HIV-1, and SARS all have this phenomenon. The specific process is as follows: (1) The mRNA pseudoknot structure forces the ribosome to stall during elongation, and at this time, the anticodon loop of the A-site aminoacyl tRNA and the P-site peptidyl tRNA just bind to the sliding sequence of the mRNA; (2) The sliding sequence causes the tRNAs to shift to the -1 position; (3) The downstream mRNA pseudoknot is opened, and the ribosome continues to move forward, but the reading frame shifts. Previous studies have confirmed that the sliding sequence of FIPV is "UUUAAAC", followed by a three-necked "pseudoknot" structure sequence that mediates ribosome retreat. When the ribosome, in the translation elongation step, moves to the slippery sequence, the tRNA is detached from the ribosome, and the ribosome slides back a step, causing a "-1 frameshift" of the reading frame. The tRNA then re-enters its position. The peptidyl transfer center is intact, and the nascent peptide chain does not detach, allowing the ribosome to enter the translation process of ORF1b. The "-1 frameshifting" phenomenon contributes to the viral genome's streamlined nature, rapid replication, and efficient utilization of genetic material, which facilitates viral growth and reproduction. Inhibiting the -1 frameshifting process of FIPV could block FIPV replication and thereby inhibit viral proliferation. Therefore, research on drugs that can effectively inhibit the -1 frameshifting process of FIPV is of great significance for the development of drugs to combat FIPV and treat diseases caused by FIPV. Summary of the Invention

[0007] The present invention aims to provide a compound for inhibiting viral replication and application of the compound in preparing a drug for resisting feline infectious peritonitis virus.

[0008] The present invention provides a compound for inhibiting viral replication, the structural formula of the compound is:

[0009] 、 、 、 、 、 、 、 、 、 .

[0010] Furthermore, the compound can inhibit the replication and proliferation of feline infectious peritonitis virus.

[0011] Furthermore, the compound inhibits the replication and proliferation of feline infectious peritonitis in host cells.

[0012] Furthermore, the compound inhibits the -1 position ribosomal frameshift process of the feline infectious peritonitis virus.

[0013] Furthermore, the IC50 of the compound for the -1 position ribosomal frameshift process of the feline infectious peritonitis virus is less than the CC50 of the compound.

[0014] Furthermore, the compound Sulfatinib has an IC50 of 0.61 μM and a CC50 of 13.94 μM for the -1 position ribosomal frameshift process of the feline infectious peritonitis virus.

[0015] Use of the compound for inhibiting viral replication according to any one of the above items in the preparation of drugs for resisting feline infectious peritonitis virus.

[0016] Furthermore, the medicine is a medicine for treating and / or preventing feline infectious peritonitis.

[0017] Furthermore, the drug is prepared by adding the compound to pharmaceutically acceptable excipients.

[0018] Furthermore, the medicine is an injection preparation or an oral preparation.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. This application discovered 10 compounds (Sulfatinib, Entrectinib, Crizotinib, Regorafenib Hydrochloride, Ensartinib, Gemcitabineelaidate, Lomitapide mesylate, Cobimetinib racemate, Cabozantinib S-malate, and Toceranib) that inhibit viral replication. These 10 compounds have significant inhibitory and blocking effects on the -1 ribosomal frameshift process of FIPV, effectively inhibiting the replication and proliferation of FIPV. These 10 compounds can be used to treat and prevent feline infectious peritonitis caused by FIPV infection; they can be used to prepare anti-FIPV drugs; and they can be used to prepare drugs for treating and preventing feline infectious peritonitis caused by FIPV infection, and they have broad application prospects.

[0021] 2. The IC50 (half inhibitory concentration) of the 10 compounds on the -1 position ribosomal frameshift process (i.e., the replication process) of FIPV is lower than its CC50 (half cytotoxic concentration), further indicating that the 10 compounds have a good inhibitory effect on the replication of FIPV, while having low toxicity to host cells, good antiviral effects and good safety, and have good potential for drug development.

[0022] 3. Among them, the IC50 of the compound Sulfatinib for the -1 position ribosomal frameshift process of FIPV is only 0.61μM, while the CC50 is as high as 13.94μM, indicating that the compound Sulfatinib has a large safety window and good drug development potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0024] Figure 1 Schematic diagram (a), experimental flow diagram (b) and experimental results (c) of 10 compounds inhibiting the FIPV-1 ribosome frameshift process in the luciferase reporter system.

[0025] Figure 2 Schematic diagram (a), experimental flow diagram (b) and experimental results (c) of 10 compounds inhibiting the FIPV-1 ribosome frameshift process in the fluorescent protein reporter system.

[0026] Figure 3 These are the effectiveness and cytotoxicity test results of 10 compounds at different concentrations in inhibiting FIPV strains.

[0027] Figure 4 This is the verification result of the inhibitory effect of the compound Sulfatinib on the CPE of FIPV strain. DETAILED DESCRIPTION

[0028] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and description are to be considered as illustrative in nature and not restrictive.

[0029] Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the examples of the present invention are commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0030] The names, structural formulas and molecular formulas of the 10 compounds disclosed in this application are shown in Table 1.

[0031] Table 1 Names, structural formulas and molecular formulas of 10 compounds

[0032] Serial number Compound name Compound structure Compound formula 1 Sulfatinib #timg# <![CDATA[C 24 H 28 N6O3S]]> 2 Lomitapide mesylate #timg# <![CDATA[C 40 H 41 F6N3O5S]]> 3 Toceranib #timg# <![CDATA[C 22 H 25 FN4O2]]> 4 Gemcitabine elaidate #timg# <![CDATA[C 27 H 43 F2N3O5]]> 5 Regorafenib Hydrochloride #timg# <![CDATA[C 21 H 16 Cl2F4N4O3]]> 6 Entrectinib #timg# <![CDATA[C 31 H 34 F2N6O2]]> 7 Ensartinib #timg# <![CDATA[C 26 H 27 Cl2FN6O3]]> 8 Crizotinib #timg# <![CDATA[C 21 H 22 Cl2FN5O]]> 9 Cobimetinib racemate #timg# <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 21 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 21 <h2 style=";text-align:left;direction:ltr"> F3IN3O2]]><h2 style=";text-align:left;direction:ltr"> 10 Cabozantinib S-malate #timg# <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 32 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 30 <h2 style=";text-align:left;direction:ltr"> FN3O<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> ]]><h2 style=";text-align:left;direction:ltr">

[0033] Example 1

[0034] Inhibition of FIPV-1 ribosomal frameshifting by 10 compounds (Sulfatinib, Entrectinib, Crizotinib, Regorafenib Hydrochloride, Ensartinib, Gemcitabine elaidate, Lomitapide mesylate, Cobimetinib racemate, Cabozantinib S-malate, Toceranib) (Luciferase reporter system)

[0035] 1. Experimental Methods

[0036] In vitro cell culture was used to investigate whether drugs (10 compounds) could inhibit the -1 ribosomal frameshift process of FIPV. A dual-luciferase reporter gene lentiviral vector containing the FIPV genomic slippery sequence was constructed, and the effects of different drugs were tested in the feline kidney cell line CRFK, with an initial screening concentration of 10 μM.

[0037] Vector construction

[0038] (1) Synthesize the FIPV-1 Frameshift sliding region gene sequence. Double-digest the lentiviral backbone vector with EcoRI and BamHI restriction sites. Digestion conditions: 37°C, 15 minutes. After digestion, the digestion products were recovered by nucleic acid electrophoresis.

[0039] For the synthesized FIPV-1 Frameshift sliding region gene sequence, first denaturation was performed at 95°C for 10 minutes and annealing was performed at 72°C for 30 seconds. Then, the annealing product was mixed with the enzyme digestion product at a mixing ratio of annealing product: enzyme digestion product = 3:1. 10 μl of T4 ligase was added to the above mixture, and ligation was carried out in a constant temperature metal bath at 16°C for 16 hours.

[0040] Transform competent cells with plasmids: Remove competent cells from a -80°C freezer and thaw on ice. Add 10 μL of plasmid (the plasmid is a lentiviral vector containing the FIPV-1 Frameshift region) to 100 μL of competent cells and mix thoroughly. Place on ice for 30 minutes. Heat shock the competent cell and plasmid mixture in 42°C water for 90 seconds. After the heat shock, cool the competent cell and plasmid mixture on ice for 10 seconds. Transfer the cooled mixture to a solid LB plate and spread the liquid evenly over the surface of the solid LB medium using a glass rod. Invert the solid LB medium and incubate at 37°C for 16 hours. After the incubation period, pick a single colony with a pipette tip and inoculate it into liquid LB medium. Incubate at 37°C in a shaker for 8 hours before sending the plasmid to the company for sequencing. Single colonies with positive sequencing results are expanded and plasmid extraction performed.

[0041] (2) By homologous recombination, Renilla luciferase was constructed upstream of the -1 Frameshift region, and firefly luciferase was constructed downstream of the -1 Frameshift region.

[0042] The specific method is as follows: Amplify the Renilla luciferase gene sequence by PCR and add homology arms to the upstream and downstream of the sequence. Perform PCR on the vector obtained in step (1) to obtain a linearized vector. After nucleic acid electrophoresis and gel recovery, obtain the Renilla luciferase gel recovery product and the linearized vector gel recovery product. Homologous recombination: Mix the Renilla luciferase gel recovery product and the linearized vector gel recovery product in a mass ratio of 3:1, add 2 μl of homologous recombination enzyme, and react at 37°C for 15 minutes.

[0043] (3) Transform the plasmid into competent cells, select single clones and sequence them, amplify the viral backbone vector with correct sequencing, and extract the plasmid. The transformation of competent cells and extraction steps are the same as step (1).

[0044] 1.2. Establishment of screening cell system

[0045] (1) The viral backbone vector containing dual luciferase and -1 Frameshift region, PMD2.G, and plpax2 packaging vector were mixed with PEI (cationic polymer) at a volume ratio of 4:2:1. 70 μg of PEI was added to every 35 μg of DNA and transfected into 293T cells.

[0046] (2) The supernatant of 293T cell culture was collected 48 h and 72 h after transfection with viral plasmid, and the supernatant was centrifuged at 12000 g for 10 min to remove cell impurities. The lentiviral particles were then collected by cesium chloride gradient centrifugation.

[0047] (3) Add lentiviral particles to CRFK cells. After 7 days of infection, positive cells are screened by Puro. The cells are trypsinized to prepare a single-cell suspension, and single clones are sorted by flow cytometry.

[0048] (4) The genotype of the cultured monoclonal cells was identified, and the positive clones were expanded to obtain CRFK cells containing the -1Frameshift region (CMV-Renilla-framshift-Firefly).

[0049] Drug screening

[0050] (1) CRFK cells containing the -1 Frameshift region were cultured in 96-well plates, and different candidate compounds (i.e., the test compound, DMSO, and 10 compounds) were added thereto, with the drug concentration being 10 μM.

[0051] (2) After 8 h of culture, the cells were lysed, firefly luciferase substrate was added to the lysate, and the luminescence value was detected by a microplate reader.

[0052] (3) Add Renilla luciferase substrate and measure the luminescence value using a microplate reader.

[0053] (4) The ratio of firefly luciferase to Renilla luciferase is used as a reference for -1 Frameshift efficiency. A larger ratio indicates a higher -1 Frameshift efficiency, while a lower ratio indicates a lower -1 Frameshift efficiency. In other words, a lower ratio indicates a higher efficiency of the drug in inhibiting the -1 Frameshift process and a better ability to inhibit the -1 Frameshift process.

[0054] 2. Experimental Results

[0055] Figure 1 Schematic diagram (a), experimental flow diagram (b) and experimental results (c) of 10 compounds inhibiting the FIPV-1 ribosome frameshift process in the luciferase reporter system.

[0056] Among them, such as Figure 1 As shown in (a), when ribosomal frameshifting occurs normally, both reporter genes Renilla and Firefly are expressed. When ribosomal frameshifting is blocked, reporter gene Renilla is expressed, but Firefly is not.

[0057] Figure 1Figure b shows the workflow of the luciferase reporter system. First, the reporter vector is stably integrated into the host cells (feline kidney cells CRFK) via a lentiviral vector. The positive monoclonal cells are treated with candidate compounds, and the inhibitory effect of the compound on the FIPV-1 ribosomal frameshift process is determined using a dual-luciferase reporter gene assay kit and a microplate reader.

[0058] Figure 1 Figure c shows the inhibitory effect of 10 compounds at a concentration of 10 μM on the -1Frameshift process of FIPV in the dual luciferase reporter gene system. Figure 1 As shown in the results in Figure c, the 10 compounds at a concentration of 10 μM all had a very significant inhibitory effect on the -1Frameshift process of FIPV.

[0059] Example 2

[0060] Inhibition of FIPV-1 ribosomal frameshifting by 10 compounds (Sulfatinib, Entrectinib, Crizotinib, Regorafenib Hydrochloride, Ensartinib, Gemcitabine elaidate, Lomitapide mesylate, Cobimetinib racemate, Cabozantinib S-malate, Toceranib) (fluorescent protein reporter system)

[0061] 1. Experimental Methods

[0062] To further determine the inhibitory activity of the ten compounds, a reporter gene system based on short-half-life fluorescent proteins was constructed. First, ubiquitin was linked to RFP and GFP to obtain UbRFP (ubiquitin-RFP) and UbGFP. Then, Firefly luciferase and Renilla luciferase in the dual-luciferase reporter system were replaced with Ub-RFP and Ub-GFP, respectively. The updated reporter system is: CMV-UbRFP-Frameshift-UbGFP (see Figure 2 (a). For testing, CMV-UbRFP-Frameshift-UbGFP cells were passaged in 96-well plates 24 hours in advance, allowing the cells to reach a confluency of 80%-90% at the time of drug addition (candidate compounds: DMSO and 10 compounds). 0.5 μM of the 10 compounds or DMSO (control group) were added to the culture dish for 1 hour, followed by 5 μM MG-132 (a proteasome inhibitor) for an additional 3 hours. Three replicate wells were set up for each group (see Figure 2Finally, the fluorescence ratio information of GFP and RFP was obtained by fluorescence microscopy (see Figure 2 (as shown in c).

[0063] 2. Experimental Results

[0064] The detection results of the fluorescent protein reporter gene system are as follows Figure 2 As shown in Figure c, in the fluorescent protein reporter gene system, the 10 compounds have a very significant inhibitory effect on the -1 Frameshift process of FIPV.

[0065] Example 3

[0066] Efficacy and safety of 10 compounds (Sulfatinib, Entrectinib, Crizotinib, Regorafenib Hydrochloride, Ensartinib, Gemcitabine elaidate, Lomitapide mesylate, Cobimetinib racemate, Cabozantinib S-malate, Toceranib) in inhibiting FIPV strains

[0067] 1. Experimental Methods

[0068] By culturing CRFK cells in vitro, an in vitro culture system for the FIPV strain was successfully established. FIPV was first added to the CRFK cells, and fresh culture medium was replaced after 6 hours. At the same time, 10 compounds at concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 μM or DMSO (control group) were added. After culturing for 3 days, the viral genome copy number was detected by RT-PCR (upstream primer: 5'-ACAGGTGCGTTACCGCATTG-3'; downstream primer: 5'-ATTGTTGGTTCCACGAGTGCC-3'; product length: 187 bp), and the cell activity was detected by CCK-8.

[0069] 2. Experimental Results

[0070] The results of RT-PCR and CCK-8 were as follows Figure 3 and as shown in Table 2.

[0071] Table 2 Results of the half-inhibitory concentration (IC50) and half-cytotoxic concentration (CC50) of 10 compounds on FIPV replication

[0072] Compound name IC50 (μM) CC50 (μM) Toceranib 3.73 11.07 Cobimetinib racemate 0.89 2.81 Cabozantinib S-malate 9.54 21.44 Lomitapide mesylate 1.49 7.12 Gemcitabine elaidate 0.40 2.47 Ensartinib 1.80 16.78 Regorafenib Hydrochloride 1.51 8.01 Crizotinib 3.91 18.52 Entrectinib 0.95 19.76 Sulfatinib 0.61 13.94

[0073] from Figure 3As shown in Table 2, the IC50 (half-inhibitory concentration) of the 10 compounds for the -1 ribosomal frameshift process (also known as the replication process) of FIPV was less than the CC50 (half-maximal cytotoxic concentration), demonstrating satisfactory antiviral effects. This indicates that the 10 compounds have a good inhibitory effect on FIPV replication while having low toxicity to host cells, exhibiting good antiviral effects and safety, and possessing promising drug development potential. Among them, the IC50 of the compound Sulfatinib for the -1 ribosomal frameshift process of FIPV was only 0.61 μM, while the CC50 was as high as 13.94 μM, indicating that the compound Sulfatinib has a large safety window and good development potential.

[0074] Example 4

[0075] Inhibitory effect of the compound Sulfatinib on the cytopathic effect (CPE) of FIPV strains

[0076] 1. Experimental Methods

[0077] To verify the CPE inhibitory ability of Sulfatinib against FIPV strains, FIPV was first added to CRFK cells. Fresh culture medium was replaced after 6 hours, and 0.5 μM of the compound Sulfatinib or DMSO (control group) was added at the same time. 0.5 μM GS-441524 (a competitive inhibitor of nucleoside triphosphate, which has strong antiviral activity against FIPV and low cytotoxicity) was added to the head-to-head control group. After culturing for 3 days, the cell pathological changes were observed under bright field microscopy.

[0078] 2. Experimental Results

[0079] Bright field microscope observation results (photos) are as follows Figure 4 As shown in the results, the sulfatinib-treated group showed no obvious lesions; the DMSO control group showed significant lesions; and the head-to-head control group, GS-441524, showed some lesions, with results intermediate between the sulfatinib-treated and DMSO groups. These results suggest that sulfatinib has a significant inhibitory effect on FIPV cultured in vitro, and is superior to GS-441524 in this regard.

[0080] In summary, the present invention provides 10 compounds (Sulfatinib, Entrectinib, Crizotinib, Regorafenib Hydrochloride, Ensartinib, Gemcitabine elaidate, Lomitapide mesylate, Cobimetinib racemate, Cabozantinib S-malate, Toceranib) that inhibit viral replication and their use in the preparation of anti-FIPV drugs. The 10 compounds of the present invention have significant inhibitory and blocking effects on the -1 Frameshift process of FIPV and can effectively inhibit the replication and proliferation of FIPV; the 10 compounds can be used to treat and prevent feline infectious peritonitis caused by FIPV infection; can be used to prepare anti-FIPV drugs, and can be used to prepare drugs for treating and preventing feline infectious peritonitis caused by FIPV infection, and have broad application prospects.

Claims

1. A compound for inhibiting viral replication, characterized in that The structural formula of the compound is: 、 、 、 、 、 、 、 、 、 。 2. The compound for inhibiting viral replication according to claim 1, characterized in that The compound can inhibit the replication and proliferation of feline infectious peritonitis virus.

3. The compound for inhibiting viral replication according to claim 2, characterized in that The compound inhibits the replication and proliferation of feline infectious peritonitis in host cells.

4. The compound for inhibiting viral replication according to any one of claims 1 to 3, characterized in that The compound inhibits the -1 ribosomal frameshift process of the feline infectious peritonitis virus.

5. The compound for inhibiting viral replication according to claim 4, characterized in that The IC50 of the compound against the -1 position ribosomal frameshift process of the feline infectious peritonitis virus is less than the CC50 of the compound.

6. The compound for inhibiting viral replication according to claim 5, characterized in that The compound Sulfatinib has an IC50 of 0.61 μM and a CC50 of 13.94 μM for the -1 position ribosomal frameshift process of the feline infectious peritonitis virus.

7. Use of the compound that inhibits viral replication according to any one of claims 1 to 6 in the preparation of a medicament for resisting feline infectious peritonitis virus.

8. Use of the compound for inhibiting viral replication according to claim 7 in the preparation of a medicament for resisting feline infectious peritonitis virus, characterized in that: The drug is used for treating and / or preventing feline infectious peritonitis.

9. Use of the compound for inhibiting viral replication according to claim 8 in the preparation of a medicament for resisting feline infectious peritonitis virus, characterized in that: The medicine is prepared by adding the compound and pharmaceutically acceptable auxiliary materials.

10. Use of the compound for inhibiting viral replication according to claim 8 in the preparation of a medicament for resisting feline infectious peritonitis virus, characterized in that: The medicine is an injection preparation or an oral preparation.

Images