Application of Farudodstat in the preparation of drugs for preventing or treating cerebral ischemia-reperfusion injury

Abstract

The present invention discloses the use of farudodstat in preparing a drug for preventing or treating cerebral ischemia-reperfusion injury. By verifying that farudodstat has a significant therapeutic effect on cerebral ischemia-reperfusion injury, the present invention provides a new idea for preventing or treating cerebral ischemia-reperfusion injury and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to the use of Farudodstat in preparing a drug for preventing or treating cerebral ischemia-reperfusion injury. Background Art

[0002] Stroke is the leading cause of death and disability in adults, characterized by high morbidity, disability, mortality, and recurrence rates. Ischemic stroke (IS) refers to ischemic necrosis or softening of localized brain tissue due to cerebral blood circulation disorders, ischemia, and hypoxia, resulting in corresponding neurological impairments. It accounts for approximately 70% of all strokes. Current clinical guidelines for the treatment of ischemic stroke primarily include intravenous thrombolysis, arterial thrombolysis, and interventional thrombectomy, aiming to salvage damaged neurons in the ischemic penumbra through early revascularization. Although reperfusion therapy is the most effective treatment for ischemic stroke, good prognosis is not matched by the restoration of cerebral blood flow, and only half of ischemic stroke survivors achieve functional recovery.

[0003] Cerebral ischemia reperfusion injury (CIRI) is recognized as an important pathological mechanism of poor prognosis. CIRI triggers a series of rapid neuropathological events. Early vascular recanalization can stop ischemic damage, but cannot reduce secondary damage related to the inflammatory response after CIRI. At the same time, these treatments cannot promote nerve regeneration.

[0004] Currently, no effective drugs for ischemic stroke and cerebral ischemia-reperfusion injury have been developed clinically. Summary of the Invention

[0005] Farudodstat (ASLAN003) (CAS No. 1035688-66-4) is an orally active, potent inhibitor of dihydroorotate dehydrogenase (DHODH). Its IC50 for human DHODH is 35 nM. It inhibits protein synthesis by activating the AP-1 transcription factor. It also induces apoptosis and significantly prolongs survival in mice bearing acute myeloid leukemia (AML) xenografts. This study isolated and cultured primary neurons from newborn Sprague-Dawley rats in vitro and induced an in vitro model of cerebral ischemia-reperfusion injury using OGD / R stimulation. Treatment with farudodstat significantly inhibited the OGD / R-induced decrease in activity, inflammatory response, and ferroptosis in these newborn Sprague-Dawley rat primary neurons. This study used C57BL / 6j mice and a transient middle cerebral artery occlusion (t / MCAO) model to simulate cerebral ischemia-reperfusion injury. After treatment with Farudodstat, it was found that Farudodstat could significantly inhibit t / MCAO surgery-induced neurological dysfunction, brain tissue damage, inflammatory response and ferroptosis.

[0006] According to the above results, Farudodstat can inhibit the occurrence of neuroinflammation and ferroptosis during cerebral ischemia-reperfusion injury, thereby exerting an inhibitory effect on cerebral ischemia-reperfusion injury.

[0007] In view of this, the solution of the present invention is:

[0008] The present invention provides use of Farudodstat or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing or treating cerebral ischemia-reperfusion injury.

[0009] Furthermore, the farudodstat or a pharmaceutically acceptable salt thereof is used to inhibit neuronal damage caused by cerebral ischemia-reperfusion injury.

[0010] Furthermore, the farudodstat or a pharmaceutically acceptable salt thereof is used to inhibit neuroinflammatory response caused by cerebral ischemia-reperfusion injury.

[0011] Furthermore, the farudodstat or a pharmaceutically acceptable salt thereof is used to inhibit neuronal ferroptosis caused by cerebral ischemia-reperfusion injury.

[0012] Furthermore, the medicine contains an effective dose of Farudodstat or a pharmaceutically acceptable salt thereof.

[0013] Furthermore, the pharmaceutically acceptable salts include, but are not limited to, pharmaceutically acceptable acid addition salts, such as salts of inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid, and salts of organic acids such as acetic acid, ethanesulfonic acid, benzenesulfonic acid, benzoic acid, citric acid, fumaric acid, gluconic acid, glycolic acid, isethionic acid, lactic acid, lactobionic acid, maleic acid, malic acid, methanesulfonic acid, succinic acid, p-toluenesulfonic acid and tartaric acid; salts of pharmaceutically acceptable bases selected from ammonium salts, alkali metal salts (such as sodium salts, potassium salts) and alkaline earth metal salts (such as magnesium salts, calcium salts) and salts of tromethamine (2-amino-2-hydroxymethyl-1,3-propanediol), diethanolamine, lysine or ethylenediamine.

[0014] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0015] Preferably, the pharmaceutically acceptable excipients include but are not limited to at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a solubilizer, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, a preservative, an antioxidant, an antibacterial agent or a buffer.

[0016] Furthermore, the dosage form of the drug is any one of tablets, capsules, granules, powders or liquid preparations.

[0017] Furthermore, the drug administration route includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, aerosol administration or transdermal administration; preferably oral administration.

[0018] Furthermore, the drug contains a dihydroorotate dehydrogenase inhibitor or a pharmaceutically acceptable salt thereof in a suitable dosage range of 0.001-100 mg / kg body weight, preferably 2-6 mg / kg body weight, and more preferably 4-5 mg / kg body weight; the above dosage can be administered in one dosage unit or divided into several dosage units, and can be based on the doctor's clinical experience and the dosage regimen including the use of other treatment means.

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

[0020] The present invention discovers that farudodstat has the effect of preventing or treating cerebral ischemia-reperfusion injury; by verifying that farudodstat has a significant therapeutic effect on cerebral ischemia-reperfusion injury, it provides a new approach for preventing or treating cerebral ischemia-reperfusion injury and has good application prospects; and farudodstat is an orally active and effective dihydroorotate dehydrogenase inhibitor with extensive toxicity and dosage data. The present invention discovers a new use for an existing known drug and is an important technological breakthrough. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the results of cell activity detection after the OGD / R-treated neurons in Example 1 of the present invention were treated with DMSO or Farudodstat. Indicates p < 0.05.

[0022] Figure 2 These are the results of detecting the mRNA level of Il6 in the neurons treated with OGD / R in Example 1 of the present invention and then treated with DMSO or Farudodstat. Indicates p < 0.01.

[0023] Figure 3 The results (left) and statistical results (right) of protein expression of xCT (Slc7a11), TXNRD1, and GPX4 in the OGD / R-treated neurons in Example 1 of the present invention after treatment with DMSO or Farudodstat are shown. Indicates p < 0.01.

[0024] Figure 4 These are the neurological function score results of C57BL / 6j mice after t / MCAO surgery and DMSO or Farudodstat treatment in Example 2 of the present invention. Indicates p < 0.05.

[0025] Figure 5 These are the TTC staining results (top) and statistical results of infarct volume ratio (bottom) of mouse brain tissue after t / MCAO surgery and DMSO or Farudodstat treatment in Example 2 of the present invention in C57BL / 6j mice. Indicates p < 0.05.

[0026] Figure 6 These are the HE staining results of the mouse brain tissue after t / MCAO surgery and DMSO or Farudodstat treatment in Example 2 of the present invention in C57BL / 6j mice.

[0027] Figure 7 These are the CD11b immunofluorescence staining results (left) and positive cell count results (right) of the brain tissue of C57BL / 6j mice after t / MCAO surgery and DMSO or Farudodstat treatment in Example 2 of the present invention. Indicates p < 0.05.

[0028] Figure 8The results (left) and statistical results (right) of protein expression of xCT (Slc7a11), TXNRD1, and GPX4 in the brain tissue of the infarcted area of ​​C57BL / 6j mice after t / MCAO surgery and DMSO or Farudodstat treatment in Example 2 of the present invention are shown. indicates p<0.05, Indicates p < 0.01. DETAILED DESCRIPTION

[0029] The technical solutions of the present invention will be clearly and completely described below in conjunction with preferred embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] Experimental animals and their breeding

[0031] Cell experiment: One-day-old Sprague-Dawley rats were used as experimental subjects.

[0032] Animal experiment: 8-10 weeks old mice weighing 25-27 g with a C57BL / 6j background were used as experimental subjects.

[0033] Housing environment: SPF-grade mouse feed was purchased from Beijing Huafukang Biotechnology Co., Ltd. Housing conditions: room temperature between 22-24°C, humidity between 40-70%, alternating light and dark lighting for 12 h, and free access to water and food.

[0034] Example 1 Farudodstat inhibits neuronal damage, inflammatory response and ferroptosis induced by OGD / R stimulation

[0035] 1. Primary Neuronal Culture of Newborn Sprague-Dawley Rats

[0036] One-day-old Sprague-Dawley rat pups were euthanized and the cerebral cortex was removed. The cells were minced and digested in 0.125% trypsin (GIBCO, 27250018) at 37°C for 15 minutes. The digestion reaction was then terminated with DMEM-F12 (BioLight, BLCK115) supplemented with 10% fetal bovine serum (Newzeru, FBS-CS500) and DNase (Roche, 10104159001). The cell suspension was filtered through a 40 μm cell strainer (Corning, 352340) to remove clumped cells or incompletely digested tissue. The filtered cell suspension was centrifuged at 1500 rpm for 5 minutes at 4°C to collect the cell pellet. The collected cell pellet was resuspended in DMEM / F-12 complete medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (Biosharp, BL505A). After counting, cells were plated onto culture dishes or flasks coated with poly-L-lysine (10 mg / mL, Sigma) and incubated at 37°C in a 5% CO2 incubator. After 3 hours of incubation, the medium was replaced with Neurobasal-A medium (GIBCO, 10888022) supplemented with 1% L-glutamine (Beyotine, ST083), 1% penicillin / streptomycin, and 2% B27 (GIBCO, 17504044). The medium was changed every 48 hours. Subsequent experiments were performed after 7 days of cell culture.

[0037] 2. Establishment of an oxygen-glucose deprivation / recovery (OGD / R)-induced neuronal brain ischemia model

[0038] To establish the OGD / R model, the neuronal culture medium was first replaced with serum-, glucose-, and sodium pyruvate-free DMEM-F12 medium (11966025, GIBCO), and the cells were cultured at 37°C in an incubator containing 95% N₂ and 5% CO₂ for 3 hours. Following treatment, the culture medium was replaced with normal oxygen, and the cells were transferred to a normal oxygen atmosphere containing 5% CO₂ and cultured at 37°C for an additional 6 hours. Control cells were cultured normally for the same period of time.

[0039] 3. Farudodstat treatment

[0040] Solvent: 10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 300 (PEG300), 5% Tween-80 and 45% saline (Saline, 0.9% NaCl). Vortex and mix before use.

[0041] Neurons were divided into DMSO and Farudodstat treatment groups. After 12 hours of hypoxia, the Farudodstat group was treated with 2 μM Farudodstat, while the DMSO group was treated with the same volume of the solvent. Drug treatment was maintained throughout the OGD / R stimulation period.

[0042] 4. Cell Viability Assay

[0043] Neurons were seeded in 96-well plates (Thermo, 167008) and divided into DMSO and farudodstat-treated groups, with four replicates per group. Three blank wells (no cells were seeded, only an equal volume of culture medium was added) were also set up. After OGD / R stimulation and farudodstat treatment, CCK8 detection reagent was added using a CCK8 detection kit (Dojindo, 44786) according to the manufacturer's instructions. The cells were incubated at 37°C for 2 hours. The absorbance of each group was measured at 450 nm, and relative cell viability was calculated.

[0044] The results of cell viability analysis were as follows Figure 1 As shown in the figure, compared with the DMSO group, the cell viability of the Farudodstat-treated group was significantly increased, indicating that Farudodstat can inhibit neuronal damage caused by OGD / R stimulation.

[0045] 5. Cellular Inflammatory Response Detection

[0046] Neurons were seeded in 6-well plates and divided into DMSO and Farudodstat treatment groups, with six replicates per group. After OGD / R stimulation and Farudodstat treatment, cells were harvested and Il6 mRNA levels were measured by RT-PCR. The specific method is as follows:

[0047] Add Trizol reagent (Sigma, 9424) to the culture plate and repeatedly pipette to dislodge cells. Collect the cell suspension into a 1.5 mL centrifuge tube, add chloroform, and vigorously vortex to mix thoroughly. Let the tube rest for 10 minutes before centrifuging at 12,000 rpm in a refrigerated centrifuge for 10 minutes. Collect the upper aqueous phase, add an equal volume of isopropanol, mix thoroughly, let the tube rest for 10 minutes, and centrifuge again at 12,000 rpm in a refrigerated centrifuge for 10 minutes. Discard the supernatant and retain the pellet. Wash the pellet with 75% ethanol three times. Air dry the pellet, dissolve it in DEPC-free water, and measure the RNA concentration. Reverse transcribe 2 µg of RNA using the transcription kit (Vazyme, R323-01) according to the manufacturer's instructions to construct a cDNA library. The reverse-transcribed cDNA is used as a template, and the corresponding primers and qPCR working solution are added to prepare the qPCR system. The resulting cDNA is then transferred to a 96-well or 384-well plate for real-time fluorescence quantitative PCR analysis. The sequences of the gene primers used are shown in Table 1.

[0048] Table 1:

[0049]

[0050] The mRNA expression level of inflammation-related cytokine Il6 was detected as follows Figure 2 Compared with the DMSO group, the mRNA expression of Il6 in the Farudodstat-treated group was significantly decreased, indicating that Farudodstat treatment can significantly inhibit OGD / R-induced neuroinflammation.

[0051] 6. Detection of Cell Ferroptosis

[0052] Neurons were seeded in 6-well plates and divided into DMSO and Farudodstat treatment groups, with triplicate wells per group. After OGD / R stimulation and Farudodstat treatment, cells were harvested and Western blot was performed to analyze activation of the key inhibitory pathway, the xCT-GSH-GPX4 signaling axis. The specific method is as follows:

[0053] Neurons were lysed using SDS lysis buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol). The lysate was incubated at 95°C for 15 minutes, followed by centrifugation, and the supernatant was collected to obtain total protein. Protein concentration was determined using a BCA protein assay kit (Thermo, 23225). Equal amounts of protein were added to loading buffer and separated by 10% SDS-PAGE gel electrophoresis. After electrophoresis, the proteins were transferred to a 0.45 μm PVDF membrane (Millipore, IPVH00010). After transfer, the PVDF membrane was blocked with 5% nonfat dry milk at room temperature for approximately 1 hour. The membrane was then washed three times with TBST for 5 minutes each time and incubated with the primary antibody overnight at 4°C. After washing three times with TBST, the corresponding species secondary antibody (Jackson ImmunoResearch) was incubated for 1 hour at room temperature. The samples were developed with ECL luminescent substrate (Bio-Rad, 1705062), and the signals were collected using a Bio-Rad gel imaging system (ChemiDoc XRS+).

[0054] Information on the primary antibodies used is shown in Table 2.

[0055] Table 2:

[0056]

[0057] The xCT-GSH-GPX4 signaling axis is a key ferroptosis inhibition pathway. Western blot results are shown in Figure 2. Figure 3 As shown in the data, the protein expression levels of xCT (SLC7A11), TXNRD1 and GPX4 in the Farudodstat-treated group were significantly higher than those in the DMSO group, indicating that ferroptosis inhibition was more significant in the Farudodstat-treated group, that is, Farudodstat treatment can inhibit neuronal ferroptosis induced by OGD / R.

[0058] Example 2 Study on the Effects of Farudodstat on Neural Damage, Inflammatory Response and Ferroptosis in Cerebral Ischemia-Reperfusion Injury

[0059] C57BL / 6j mice were randomly divided into a DMSO group and a farudodstat-treated group, with 15 mice in each group. A transient middle cerebral artery occlusion (t / MCAO) surgery was used to establish a cerebral ischemia-reperfusion injury model to evaluate the effects of farudodstat on cerebral ischemia-reperfusion injury.

[0060] 1. Transient middle cerebral artery occlusion (t / MCAO) surgery to establish a cerebral ischemia-reperfusion injury model and drug treatment

[0061] Mice were anesthetized with a 2.0% isoflurane and oxygen / nitrous oxide mixture, and cerebral blood flow was continuously monitored using a laser Doppler flowmeter (Perimed, Periflux System 5010). To establish the t / MCAO model, a 6-0 silicone-coated monofilament suture (Doccol Corporation, Cat#602156PK5Re) was inserted into the left external carotid artery, advanced toward the internal carotid artery, and wedged into the cerebral artery circle to occlude the origin of the middle cerebral artery (MCA). After 45 minutes, the suture was removed to restore blood flow and reperfusion. A decrease in cerebral blood flow exceeding 75% during ischemia and a recovery exceeding 70% after reperfusion were considered a successful establishment of the ischemia-reperfusion model. All surgeries were performed under double-blind conditions. Mice in the sham group did not undergo ischemia, but all other procedures were the same as those in the t / MCAO group.

[0062] Dosage regimen: 24 h before modeling, mice in the Farudodstat group were orally gavaged with a suspension of the target compound (50 mg / kg dissolved in the above solvent), and mice in the DMSO group were given an equal volume of solvent control.

[0063] 2. Neurological function score

[0064] After 24 hours of blood flow restoration, neurological function was scored based on the modified Berderson scoring method (9-point system). Higher scores indicate more severe behavioral disorders. The scoring method is as follows:

[0065] 0 points: no symptoms of nerve damage;

[0066] 1 point: when the tail is lifted, the contralateral forelimb is curled up, or the affected forelimb cannot be fully reached;

[0067] 2 points: The opposite shoulder is adducted when the tail is lifted;

[0068] 3 points: Pushing: resistance decreases when pushing to the opposite side;

[0069] 4 points: Can move spontaneously in all directions, but only turns to the opposite side when the tail is removed;

[0070] 5 points: Spinning in circles or only turning in opposite directions during spontaneous movement;

[0071] 6 points: no voluntary movement, movement only when stimulated;

[0072] 7 points: no voluntary movement, no movement even when stimulated;

[0073] 8 points: Death related to cerebral ischemia.

[0074] After scoring, the mice were anesthetized and killed. The brain tissues of the mice in the TTC staining group were isolated for TTC staining and subsequent testing. The brain tissues of the mice in the meristem pathology group were isolated after perfusion and divided into two parts longitudinally along the middle. The front half was fixed in 10% formalin for making paraffin samples, and the brain tissues of the ischemic core area and penumbra of the back half were quickly frozen in liquid nitrogen for subsequent biochemical and molecular biology testing.

[0075] Neurological function score results are shown in Figure 4 After t / MCAO surgery, the score of the Farudodstat-treated group was significantly lower than that of the DMSO group, indicating that Farudodstat can significantly inhibit the neurological dysfunction caused by cerebral ischemia-reperfusion injury.

[0076] 3. Detection of brain tissue infarction and damage

[0077] (1) TTC staining

[0078] After scoring, mice were anesthetized and sacrificed. Brain tissue was removed, frozen at -20°C for 30 minutes, and sliced ​​into seven consecutive 1 mm thick slices using a razor blade. The slices were immediately placed in 2% TTC staining solution and incubated at 37°C for 10 minutes. The slices were rotated occasionally to ensure uniform staining. Normal brain tissue stains bright red, while infarcted areas appear pale. Infarct volume and edema percentage were calculated using Image Pro Plus (version 6.0) software.

[0079] (2) HE staining

[0080] After scoring, the mice were anesthetized and killed. The brain tissues of the mice were fixed in 10% neutral formalin, dehydrated, and embedded in paraffin. The tissues were cut into 5 μm serial paraffin sections, dewaxed and hydrated, washed, and stained with hematoxylin (Wuhan Sevier Biotechnology Co., Ltd., G1004) and eosin (Zhuhai Beso Biotechnology Co., Ltd., BA-4024) for routine HE staining. After washing, the sections were dehydrated until transparent, and then mounted to observe brain tissue necrosis.

[0081] The results of TTC staining and HE staining of the above brain tissues are as follows: Figure 5 、 Figure 6 The results showed that after t / MCAO surgery, the pale areas of brain tissue stained by TTC, the cavitation of brain tissue and the nuclear condensation in HE staining in the Farudodstat-treated group were significantly milder than those in the DMSO group, indicating that the cerebral infarction in the Farudodstat-treated group was significantly improved.

[0082] 4. Detection of brain tissue inflammatory response

[0083] Brain tissue from both groups of mice was immunostained for CD11b to assess inflammatory cell infiltration. Paraffin sections were dewaxed and hydrated, blocked with 10% goat serum, and then incubated with a primary antibody (Boster, BM3925, 1:100 dilution) at 4°C overnight. Sections were then washed with PBS and incubated with a secondary antibody (goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Thermo Fisher Scientific, A-11011, Massachusetts, USA)) at 37°C for 1 hour. Nuclei were stained with DAPI.

[0084] CD11b immunofluorescence staining results Figure 7 As shown in the results, the number of CD11b-positive cells in the brain tissue of mice in the Farudodstat-treated group was significantly lower than that in the DMSO group, indicating that Farudodstat administration can significantly inhibit the inflammatory response during cerebral ischemia-reperfusion injury.

[0085] 5. Detection of brain tissue iron death

[0086] Western blot was used to detect the activation of the xCT-GSH-GPX4 signaling axis in the brain tissues of the two groups of mice. Figure 8 Consistent with the results in neurons, after t / MCAO surgery, the expression levels of xCT (SLC7A11), TXNRD1, and GPX4 in the brain tissue of mice in the Farudodstat-treated group were significantly higher than those in the DMSO group, indicating that Farudodstat administration significantly inhibited ferroptosis during cerebral ischemia-reperfusion injury.

[0087] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Use of farudodstat or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating cerebral ischemia-reperfusion injury.

2. The use according to claim 1, characterized in that The farudodstat or a pharmaceutically acceptable salt thereof is used to inhibit neuronal damage caused by cerebral ischemia-reperfusion injury.

3. The use according to claim 1, characterized in that The farudodstat or a pharmaceutically acceptable salt thereof is used to inhibit neuroinflammatory response caused by cerebral ischemia-reperfusion injury.

4. The use according to claim 1, characterized in that The farudodstat or a pharmaceutically acceptable salt thereof is used to inhibit neuronal ferroptosis caused by cerebral ischemia-reperfusion injury.

5. The use according to claim 1, characterized in that The medicament contains an effective dose of Farudodstat or a pharmaceutically acceptable salt thereof.

6. The use according to any one of claims 1 to 5, characterized in that The drug includes pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that The pharmaceutically acceptable excipients include: at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a cosolvent, a solubilizer, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant, an antibacterial agent or a buffer.

8. The use according to any one of claims 1 to 5, characterized in that The dosage form of the drug is any one of tablets, capsules, granules, powders or liquid preparations.

9. The use according to any one of claims 1 to 5, characterized in that: The drug administration route includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, aerosol administration or transdermal administration.

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