MXPA00005274A - Process for obtaining l-dihydroorotic acid and use thereof - Google Patents

Abstract

The invention relates to a process for obtaining L-dihydroorotic acid by chromatography on an anionic exchange material in a base water mixture under a pressure from about 1.1 MPa to about 40 MPa. The process can be used to investigate the in vitro and in vivo activity of N-(4-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide, N-(4-trifluoromethylphenyl)-2-cyano-3-hydroxycrontonamide and similar compounds.

Description

PROCESS FOR OBTAINING L-DIHYDROOROTIC ACID AND ITS USE The invention relates to a process for obtaining L-dihydroorotic acid (hereinafter "L-DHO") by chromatography on an anion exchange material in an aqueous base mixture under a pressure of about 1.1 MPa at about 40 MPa. The method can be used to investigate the in vitro and in vivo activity of N- (4-trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide, N- (4-trifluoromethylphenyl) -2-cyano-3-hydroxyrontonamide and similar compounds. L-DHO can be determined by a chromatographic procedure on silica gel with a subsequent chemical derivation and calorimetric determination (Kesner, L., Aronson, FL, Silverman, M., Chan, PC, Clin.Chem 21/3 (1975) 353). Another method enzymatically converts L-DHO into orotic acid via L-dihydroorotic acid dehydrogenase (hereinafter "DHODH") prepared from rat liver and, after chemical derivation, detects orotate by calorimetric changes (Rogers, LE, Nicolaisen, K., Experientia 28/10 (1972) 1259). The disadvantages of these methods are the interference of other materials in complex physiological solutions. In addition, the aforementioned procedures require a lot of time due to laborious sample preparation and therefore are not applicable for routine analyzes in large clinical studies. In order to provide improved separation and isolation procedures for obtaining L-dihydroorotic acid, it has now been found that it can be obtained by L-DHO chromatography in an aqueous base mixture in an anion exchange material and under a pressure of about 1.1 MPa at about 40 MPa. The procedure can be used for the quantitative determination of L-DHO in cell lysates, of mammalian serum and human serum. This procedure is highly reproducible, sensitive and validated. The invention, as explained in the claims, achieves the object through a chromatography process comprising the steps of: a) obtaining a column comprising a pressure-stable anion exchange material; b) loading the column with a sample solution including L-dihydroorotic acid; c) carry out chromatography; d) eluting the L-dihydroorotic acid with an elution solution containing an aqueous base mixture; said process is carried out under a pressure of about 1.1 MPa at about 40 MPa. The term "pressure stable anion exchange material" refers, for example, to materials such as, for example, divinylbenzene / macroporous ethylvinylbenzene polymer (2,000 ^) or a microporous polyvinylbenzyl ammonium polymer crosslinked with divinylbenzene or mixtures thereof modified with quaternary alkanolammonium.; or macroporous vinylbenzylchloride / divinylbenzene polymer; or else crosslinked polyetheriminopolymer; or silica modified with propyltrimethylammonium; or poly (styrene-divinylbenzene) trimethylammonium. The following products are especially preferred: anion exchange columns Ion Pac AS 11, CarboPac PA1, or CarboPac MA 1 provided by Dionex Corporation, Idstein, Germany, GROM-SIL, Strong Anion, or GROM-SIL, Weak Anion; supplied by Gro P 1000 SAX, Ionospher SA or Chrompack PA; supplied by Chrompack PRP-X100 or RCX-10 supplied by Hamilton. The solution of choice contains an aqueous base mixture. Suitable bases are derived from alkali metals or alkaline earth metals such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, or calcium hydroxide. The concentration of the base is from 1 mmol / L to approximately 200 mmol / L, based on water as solvent, preferably from 2 mmol / L to approximately 120 mmol / L, with 100 mmol / L being particularly preferred. The temperature during the chromatography process is from about 0 ° C to about 50 ° C, preferably from about 15 ° C to about 30 ° C, particularly from about 19 ° C to about 25 ° C. The operating pressure during chromatography is substantially constant. Chromatography can be carried out using different pressures, for example, chromatography can be carried out under a pressure of about 1.1 106 Pa (1.1 MPa) to about 40 10 Pa (40 MPa), particularly 4.1 MPa to 5.5 MPa. The eluent flow rates are from about 0.2 ml / min to about 3 ml / min, preferably 1 ml / min. The loading of the columns, chromatography, and elution of the L-DHO are carried out by known conventional technical methods. A suitable elution is an elution in which the elution has a time gradient of the base, preferably linear, concentration. This concentration gradient can be applied, for example, by a low base concentration (zero in the limit case) present in the elution at the beginning of the elution, and increasing the base concentration during the elution. It is thus possible to achieve a particularly effective separation of L-DHO in samples derived from serum or cell lysates. A preferred base gradient ranges from about 1% NaOH (100 mmol / L) and 99% water (at the start of the elution) to about 60% NaOH and 40% water (at the end of the elution), with the especially preferred range being located from approximately 1% NaOH and 99% water (at the start of the elution) to approximately 15% NaOH and 75% water (at the end of the elution). The base water gradient is changed linearly from 2.5 min to approximately 14 min and from 14 min to approximately 25 min, where the infusion of the gradient is different during these two time periods. A particularly suitable elution can be achieved by using a low base concentration at the beginning of the separation process of about 1% over a period of about 2.5 minutes. The result is the elution of most of the material that interferes from the biological matrix of the column. The analyte separation is achieved by slowly increasing the gradient to approximately 23% base within a time period of a total analysis time of 14 minutes. Then, the base concentration is increased to approximately 60% within 4 minutes to allow elution of a strong binding material. The 60% base should be applied for no more than 6 minutes until a rebalancing is carried out by a 1% aqueous base mixture. The following analysis begins after 45 minutes of total analysis time. The water in the base water gradient must be deionized and degassed water. The separation process according to the present invention is carried out in a column process. The temperature that remains preferably constant during anion exchange chromatography can vary within a wide range. A preferred temperature range is from about -10 ° C to about 50 ° C, particularly, from about 15 ° C to about 25 ° C. The elution of L-DHO is carried out from 10 minutes to 12 minutes after the start of the gradient. The time of the elution process is 1-3 minutes to 25 minutes. The L-DHO is detected by a conductivity detector such as a CD20 model from Dionex Cooperation. In order to minimize baseline displacement and to decrease background conductivity, an anion autoregeneration suppressor can be employed, such as the ASTM-I, 4 mm molding of Dionex Corporation. The process according to the present invention is particularly suitable for analytical chromatography but can also be used for preparation chromatography, especially when the method according to the present invention is carried out with a high pressure liquid chromatography code system ( HPLC) of preparation. The term "preparation chromatography" refers to a purification process in order to obtain, and not only analyze, pure products. The amount of pure products can vary within wide limits, for example from 1 mg to 1000 g, preferably between 50 mg and 500 mg. The method according to the present invention can be used to detect changes in intracellular or extracellular concentrations of L-DHO due to the inhibition of dihydroorotic acid dehydrogenase (DHO-DH). The DHO-DH enzyme is responsible for the conversion of L-dihydroorotic acid during the synthesis of de novo pyrimidine. The inhibition of DHO-DH leads to the accumulation of L-DHO. The method according to the invention can be used for the preparation of a diagnostic assay. The process according to the present invention can be used to determine the activity of DHO-DH inhibitors. The DHO-DH inhibitors are, for example, N- (4-trifluoromethylphenol) -5-? -butylisoxazole-4-carboxylamide, Brequine, N- (4-trifluoromethylphenyl) -2-cyano-3-hydroxyhept-2-en-6-in-carboxamide, -cyano-3-cyclopropyl-3-hydroxy-acrylic acid- (4-cyanophenyl) -amide or N- (4-trifluoromethyl-phenyl) -2-cyano-3-hydroxy-thromonamide. The method according to the present invention can be used to determine the concentrations of L-DHO in plants, cell lines, animals and humans. The determination of L-DHO can be used to monitor the activity of DHO-DH inhibitors in plants, mammals and humans. The method according to the present invention is described in detail in the following examples. Unless otherwise indicated, the percentages refer to weight. Example 1 1.1 Chemicals and reagents Chemicals and reagents were purchased in accordance with the following: NaOH and KOH free of carbonate Baker, Netherlands L-dihydroorotic acid (L-DHO) Sigma, Munich Hcl0 Riedel de Haen, Seelze Eosin, Riedel de Haen chloroform, Seelze RPMI 1640 medium Gibco, Eggenstein Fetal calf serum (FCS) Bio Whitaker, Verviers, Belgium Deionized water must be degassed by helium before use. 1.2 Chromatographic equipment The HPLC system consisted of the following instruments: Item 1.2 Equipment model manufacturer conditioning module SCM 400 Thermo Separation of solvent Products (TSP) (Thermal separation products) Gradient pump P 2000 Binary TSP Autosampler with AS 3000 TSP loop 200 μl Interface SP 4510 TSP Changer AD SN 4000 TSP Duct Detector 20 Dionex ectivity Autogas Suppressor-ASRS-14 mm Anion Dionex neration DS Stabilizers 3-1 Dionex PC Detection 20, Sean Flex, Escon F 563-T Deskjet Jet 550C Escon Programmatic PC 1000 TSP A Peek 1.3 material was used in all the experiments. HPLC conditions The chromatographic separation was carried out with an ion exchange column IonPac AS 11 of 250 x 4 mm Dl (particle size 13 um; P / N 044076, Dionex) equipped with a IonPac AG 11 pre-column of 50 x 4 mm D.l. (particle size 13μm; P / N 044078, Dionex). In addition, an anion trap column ATC-1 (P / N 037151, Dionex) was installed between the gradient pump and the injection valve. In order to minimize baseline displacement and to reduce background conductivity, the ASRS-I suppressor operating at 300 mA was installed. The range of the detector was set at 10 μS. The autosampler was cooled to 14 ° C, but the analysis itself was carried out at room temperature. The mobile phase was composed of 100 mM NaOH (A) and deionized and degassed water (B). With this system the following gradient was produced: Time A B (min) I, 9? -. ' 0 1 99 2.5 1 99 14 23 77 18 60 40 24 60 40 26 1 99 45 1 99 The flow rate was 1 ml / min; the time of the experiment was 45 minutes. 1.4 Standards and quality control samples A standard stock solution was prepared by dissolving 1 mg of L-DHO in 1 ml of water. Aliquots of 400 μl at a temperature of -20 ° C. The stability of these solutions was guaranteed for at least 4 weeks. Definite amounts of the stock solution were added to the cell lysates and human or rat serum and tested to evaluate the linear character between the increase in signal and the concentration of L-DHO sown. The precision and accuracy of the method were investigated by the use of quality control (QC) samples with an L-DHO content in the low, medium and high range of concentrations of the signal / concentration linearity curve. 1.5 Sample preparations 1.5.1. Cells Jurkat cells were obtained in ATCC (TIB 182) and cultured in accordance with what is described in 1.5.1.1. 1.5.1.1 Tissue Culture Conditions Jurkat cells were seeded at 5 x 10 5 / ml and cultured for 24 hours in RPMI 1640 medium and 10% fetal calf serum (FCS). The cells were combined in fresh medium for lysis of cells and the number of cells and percentage of dead cells was calculated by vital microscopy with Eosin. After 24 hours the cell numbers had increased by 1.6-1.8 times with less than 7% of dead cells. The Jurkat cells taken for L-DHO determination showed a proliferation ratio of 1.6-1.8 times in 24 hours and less than 7% of dead cells. 1.5.1.2 Preparation of cell lysates The cells were suspended in a volume of defined medium and a cell density of samples was determined through vital microscopy using Eosin. Approximately 10 x 10 6 cells were removed which were formed into pellets by centrifugation for 5 minutes at 350 x g, and the supernatant was discarded. Lysis of the cells was carried out by resuspension of the cell pellet in 500 μl of 1.2 M HCL04. The mixture was transferred into 2.0 ml Eppendorf safety capsules and the protein was precipitated by high speed centrifugation for 2 minutes. . The supernatant was completely removed, transferred into glass bottles and after the addition of 500 μl of chloroform completely mixed for 2 minutes by vortexing. The cellular lipids were extracted by chloroform after a 10 minute centrifugation (1502 g) at 10 ° C. The purified supernatant was collected in 2 ml Eppendorf capsules and stored at a temperature of -20 ° C until further use. For HPLC analysis, 100 μl of this supernatant was neutralized with 30 μl of 6 M KOH. After stirring for approximately 5 seconds, the samples were stored on ice for 30 minutes. Subsequently they were centrifuged for 5 minutes at 15,000 rpm. From the clear supernatant, 20 μl was used for HPLC analysis. 1.5.2. Serum In order to reduce the protein content, 200 μl of serum was added to a Microcon filter (10,000 D, model 10, code 42407, A icon) and centrifuged for 30 minutes at 13,000 revolutions per minute (rpm). The resulting flow consisted of approximately 150 μl and contained the analyte L-DHO. From this liquid, 20 μl was used for HPLC analysis. 1.6. Quantification The integrator determined the peak height of the analyte. The calibration curves were obtained by graphing the measured peak heights (y) versus the analyte concentration in the different biological matrices. A weighted linear regression (1 / y) was used to retrocalculate the concentration of L-DHO in standard samples as well as in quality controls. The common correlation coefficient R was provided by PROC GLM based on an analysis of the covariance model using the weighting factor. 1.7. Stability The table shows the analyte stability data at -20 ° C in cell lysates and serum samples after 2 - 3 freeze / thaw cycles of a sample. The L-DHO was stable under the conditions mentioned above in Jurkat cells over a period of at least 4 weeks. The increase detected in the concentration of certain rat serum samples and in a human serum sample after several defrosting cycles of more than 10% can not be explained and shows that the accuracy in these cases can be reduced by up to 15% in general and up to 29% in the worst case. For these reasons, it can only be established that in the serum samples of rat and human L-DHO is definitely stable for at least 1 week. Table 1: Stability data in cell lysates and serum at -20 ° C (n = l) Time concentration residue residue concentration (days) 1 (%) 2 (%) 20 μg / ml 1Q0 μg / ml Jurkat cells 0 19.34 100 100.05 100 7 19.28 99.7 99.09 99.0 14 nd nd 15.32 115.3 29 19.98 103.3 99.35 99.3 Time concentration residue residue concentration (hours) 1 (%) 2 () 5 μg / ml 20 μg / ml rat serum 0 4.30 100 19.94 100 7 4.52 105.1 19.62 98.4 14 4.81 111.9 20.93 105.0 21 5.55 129.1 22.38 112.2 human serum 4. 67 100 20. 22 100 4. 81 103. 0 20 .38 100.8 65 4. 87 104 .3 23 .20 114.7 Residue (%) is a concentration percentage compared to the initial analysis, n.d. not determined In order to simulate the conditions of the samples when they are waiting in the autosampler for the actual analysis, stability was determined for 18 hours at 14 ° C. For this reason, the cell lysates were seeded and then treated with 30 μl of 6 M KOH / 100 μl of lysate before the start of the analysis. Correspondingly, the serum samples were seeded and then deproteinated in accordance with that described in 1.5.2. As can be seen from Table 2, the content of L-DHO is slightly reduced in cells under these conditions to a maximum of about 7%. In serum samples, the analyte is stable under these conditions. For these reasons, only so many HPLC samples were prepared in such a way that the maximum length of stay in the autosampler was less than 18 hours. Table 2: 18 hour stability at 14 ° C (n = l) Time concentration residue concentration residue (hours) 1 (%) 2 20 μg / ml 100 μg / ml Jurkat cells 0 19.96 100 100.02 100 lf 18.60 93.2 97.43 97.4 Time concentration residue concentration (hours) (%) 2 (%) 5 μg / ml 20 μg / ml rat serum 0 4.61 00 19.99 100 lit 4.96 107.6 20.53 102.7 human serum 0 3.34 100 18.78 100 lit 3.37 100.9 22.09 117.6 1. 8. Selectivity Comparison of the lysates of Jurkat cells not seeded with the corresponding chromatograms using cell lysates seeded with 50 μg of L-DHO / ml showed that there is a small peak at 11,983 min, which is the same retention time as L -DHO. It is highly probable that this peak results from the natural content of the analyte in these cells. Furthermore, it can be seen that due to the treatment of cell lysates with KOH, the peak of L-DHO is divided into two peaks. The KOH treatment however is essential to neutralize the acid cell lysate before the HPLC analysis. It can be seen that under the conditions described, the evaluation of the second peak height (retention time (RT) = 11,954 min) can be used to obtain improved results in terms of linearity and reproducibility. Also in the case of rat serum and human being, a white value was found with the same retention time as L-DHO. This is supposed to reflect the natural content of L-DHO in the body. The determination of at least 10 different samples from both species shows that the natural content of L-DHO was lower than the detection limit of 1 μg / ml. 1.9. Linearity The linearity of the determination was evaluated in five calibration curves for the cell line and the different serum samples. Samples were prepared which were treated in five different days with concentrations of L-DHO in the range of 1.5 to 150 μg / ml (cell lysates) and 1-30 μg / ml (serum samples). The results appear in tables 3-5. For the determination of the regression line, peak heights were used. Based on this, the corresponding concentrations of the different standards were retro-calculated according to what is indicated in the different tables.

Table 3: linearity of L-DHO determinations in lysates of Jurkat cells. After sowing the samples were treated with 30μl of 6M KOH and then analyzed once in accordance with that described in 1.3. Day Concentration (μg / ml) 1.5 6 10 50 100 150 1 1.78 5.54 9.09 49.80 102.46 149.09 2 1.90 5.14 9.54 50.77 100.22 150.20 3 1.86 5.24 9.46 51.18 99.91 150.07 4 1.85 5.34 9.39 51.45 100.97 148.71 1.86 5.26 9.53 50.98 100.07 n.d Average 1.85 5.30 9.40 50.80 100.73 149.52 S.D. 0.04 0.15 0.19 0.63 1.05 0.73 C.V. 2.3 2.8 2.0 1.2 1.0 0.5 (%: Day pending intersection Y of y 1 1317.54 -258.17 0.9995 2 1324.84 204.79 0.9995 3 1295.48 302.48 0.9996 4 1306.08 833.08 0.9996 5 1318.67 530.19 0.9993 Medium 1312.52 322.59 SD 11.69 404.93 CV 0.9 125.5 (%) R = 0.9995 Table 4: linearity of L-DHO determinations in rat serum After sowing, the serum was cleaned of proteins according to what is described in 1.5.2 Day Concentration (μg / ml) 1 5 10 20 30 1 1.05 4.48 10.10 22.10 28.61 2 1.16 4.59 8.60 21.07 30.97 3 1.07 4.64 9.81 20.07 30.45 4 1.11 4.62 9.54 19.27 31.65 5 1.06 4.81 9.99 18.89 31.40 Average 1.09 4.63 9.61 20.28 30.62 S., D. 0.05 0.12 0.60 1.32 1.21 C. .V. 4.2 2.6 6.3 6.5 4.0 (%: Day pending intersection Y of and 1 15334 -4719.83 0.9966 2 17559 -4630.68 0.9960 3 17674 -3175.63 0.9995 4 18322 -4296.53 0.9981 5 17738 -829.00 0.9985 Media 17325.4 -3530.33 0 SD 1151.65 1630.62 CV 6.7 46.19 (%) Table 5: linearity of determinations of L-DHO in human serum. After sowing the serum was cleaned of protein according to what is described in 1.5.2. Day Concentration (μg / ml¡ 1 5 10 20 30 1 0.90 5.17 11.30 19.41 39.42 2 0.99 4.87 10.12 21.51 28.68 3 0.94 4.94 11.29 19.41 29.59 4 1.01 4.77 10.32 20.82 29.15 5 1.01 4.81 10.10 20.86 29.28 Average 0.97 4.91 10.62 20.40 29.23 SD 0.05 0.16 0.61 0.95 0.35 CV 5.2 3.2 5.8 4.6 1.2 (%) Day of the intersection and of 1 13498 2930.30 0.9980 2 15663 477.61 0.9982 3 14328 1367.80 0.9982 4 1155777777 11777711..9988 0.9992 5 1155221166 11661122..7777 0.9993 Average 14896.4 1632.09 SD 967.46 881.47 CV 6.5 54.01 (%) R = 0.9986 ^ standard concentration (μg / ml) not determined As can be seen in these tables, the linearity was checked in each case, this is reflected in the individual correlation coefficients? R "that they are in all cases greater than 0.99 The average linear regression line for the concentration curves obtained in 5 different days are described in each table and show that the slope was very well reproducible with a maximum variation 6.7% ma. The retrocalculated standard concentrations presented an average C.V. less than 6.5% which shows the high accuracy of the values. The common correlation value R greater than 0.99 expresses the very high precision and reproducibility of the method. 1.10. Limit of quantification Based on these results, the limit of quantification in Jurkat cells is 1.5 μg / ml. In rat and human serum samples, 1 μg / ml of L-DHO can be detected. Samples planted at these concentrations showed a signal to noise ratio of at least 1: 3. 1. 12. Accuracy and Accuracy The accuracy and precision of replicate determinations of L-DHO in three different concentrations over five different days are summarized in Tables 6-8. Accuracy is expressed as the percentage difference between the amount found and the aggregate amount of L-DHO (Recovery) . The intra-day precision expressed as C.V. (%) was calculated using the two values obtained when a sample was measured twice in a day. Inter-day precision was also expressed as C.V. (%) and was calculated using the mean values found for each control sample in different days Table 6: Accuracy and precision in Jurkat cell lysates after seeding and subsequent neutralization with 30 μl 6M KOH.

N = 2 means that a sample of cell lysate was seeded at the corresponding concentration and measured twice. Sample of D) ía n Accuracy Control Precedence Medium in Recovery C.V. C.V. (μg / ml) contrada (%) (%) (%) (μg / ml) intra inter day day 1 2 20.10 +0.5 1.4 2 2 19.20 -4.0 1.3 20 3 2 19.40 -2.8 3.9 2.9 4 2 19.51 -2.5 2.2 5 2 18.55 -7.2 1.3 1 2 69.12 -1.3 0.1 2 2 69.13 -1.2 0.8 70 3 2 73.69 +5.3 0.2 2.8 4 2 71.52 +2.2 1.1 5 2 69.64 -0.5 0.9 1 2 123.21 -5.2 2.8 2 2 114.78 -11.7 12.0 130 3 2 135.21 +4.0 0.2 6.0 4 2 126.89 -2.4 1.4 5 2 122.43 -5.8 4.1 Table 7: Accuracy and precision in rat serum after sowing and subsequent deproteinization; n = 2 means that a serum sample was seeded with the corresponding concentration and measured twice. Day Sample n Accuracy control Media in AccuracyRepair (μg / ml) contrada (%) C.V. C.V. (μg / ml) (%) (%) intra inter day day 1 2 7.68 -4.0 0.4 2 2 7.73 -3.4 1.0 8 3 2 7.83 --22..22 22..11 3.0 4 2 7.43 -7.1 1.0 5 2 8.06 +0.7 0.6 1 2 14.53 -3.1 6.8 2 2 14.73 -1.8 1.3 15 3 2 14.26 --55..00 0 0..11 1.8 4 2 14.85 -1.0 0.4 5 2 14.88 -0.8 0.9 1 2 24.98 -0.1 2.5 2 2 25.83 + + 33..33 1 1..33 3.7 3 2 25.43 +1.7 0.6 4 2 24.31 -2.8 2.0 5 2 26.81 +7.2 0.2 Table 8: Accuracy and precision in human serum after sowing and subsequent deproteinization; n = 2 means that a serum sample was seeded with the corresponding concentration and measured twice. Day Sample n Accuracy Accuracy control Average recovery C.V. C.V (μg / ml) contrada (%) (%) (%) (μg / ml) intra inter day day 1 2 7.77 -2.9 0.7 2 2 7.84 -2.0 15.5 3 2 7.43 -7.1 10.9 3.8 4 2 7.17 -10.4 2.1 5 2 7.80 -2.5 1.2 1 2 13.88 -7.5 0.9 2 2 14.00 -6.7 8.5 15 3 2 15.10 + 0.6 1.5 6.0 4 2 15.13 + 0.8 7.2 5 2 16.03 + 6.8 12.8 1 2 26.56 + 6.2 3.4 2 2 23.77 -4.9 0.7 25 3 2 24.80 -0.8 4.7 4.2 4 2 25.46 + 1.8 7.8 5 2 24.54 -1.9 0.2 The results presented here show that in most cases the control values were found with a maximum variation of +/- 10% and that the method is therefore very accurate. Only in one case did the recovery in Jurkat cell determination show a slight difference of this value (-11.7%). This effect can be explained by the high intra-day variation of 12%. The intraday precision in Jurkat cells, rat serum or human serum was less than 5%, 7%, and 10%, respectively. Inter-precision, day in all matrices investigated was very low with a C.V. less than 6.0%. This shows that the results obtained are highly reproducible and accurate.

Example 2 2.1. Tissue culture conditions: - Preparation of serum-free medium: Iscove medium (Biochrom) powder was dissolved in 10 liters of distilled water supplemented with 18.95 g of NaCl, 11.43 g of NaHCO 3, 700 mg of KCl, 10 ml of a solution of 35% NaOH, and 0.5 ml of a 1 M mercaptoethanol solution (Riedel de Haen) and sterile filtered. To a liter of minced Iscove was added 32 mg of human holo-transferin, 1 g of bovine albumin and 1.5 ml of lipids (sigma) before use. - Cell culture: A20.2.J cells were cultured in a serum-free medium (37 ° C, 5% C02) in an expanding culture to logarithmic cell growth. The cells taken for assays had a proliferation rate of 2.2 times in 24 hours. The percentage of dead cells was less than 8% (3). - Cell treatment with N- (4-trifluoromethyl) -2-cyano-3-hydroxy-crotonamide prepared in accordance with that described in EP-0 529 500, then A 77 1726. A77 1726 was dissolved in bidistilled water (10). mM) and was further diluted in serum-free medium. The cells then received the appropriate amount of A77 1726 and were incubated at a temperature of 37 ° C and 5% C02. 2.2. Preparation of cell lysates for determination of DHO: Prepared cells were resuspended in a volume of defined medium and defined cell density. According to the expected content of DHO, between 1 and 50 million cells were removed, they were formed into pellets (5 min, 350 x g) and the supernatant was discarded. The cells were used, by adding 500 μl of 1.2 M HC104. The lysates were transferred into 2 ml Eppendorf safety capsules, and the protein was precipitated by high speed centrifugation for 2 minutes. The acidified lysates were completely removed, transferred into glass jars and, after the addition of 500 μl of chloroform, thoroughly mixed with vortex for 2 minutes. Cellular lipids were extracted after cold centrifugation for 10 minutes (1502 x g, 10 ° C). The purified supernatants were collected in 2 ml Eppendorf capsules for storage at a temperature of -20 ° C until determined by high pressure liquid chromatography (HPLC). 2.3. Determination by HPLC of DHO: The chromatographic separation was carried out in accordance with that described in example 1. The range of the conductive detector was established at 10 μS. The analysis was carried out at room temperature. The mobile phase was composed of 100 mM NaOH (A) and water (B). With this system the gradient system was produced: Time A B (min) (%) (%) 0 1 99 2.5 1 99 14 8 92 22 8 92 28 60 40 32 60 40 34 1 99 49 1 99 The flow rate was 1 ml / min; the experiment time was 49 minutes. 2.4. Results Cells A20.2.J incubated with A77 1726 showed increased amounts of intracellular DHO (tables 9-11).

The results presented in Table 9 demonstrate that DHO levels correlate directly with the number of cells extracted. Table 9: Correlation of intracellular DHO concentrations and number of cells cultured with A77 1726 Cells DHO concentration (μg / ml ± Standard Deviation) (x 106) Experiment 1 '(E10 B) Experiment 2 (E14) 1 14.08 ± 2.40 11.75 ± 0.23 2 19.51 ± 6.47 28.27 ± 0.79 4 53.58 ± 3.50 57.20 ± 2.76 73.01 ± 1.49 85.38 ± 2.33 99.89 ± 5.96 107.02 ± 3.47 10 113.37 + 4.24 128.73 ± 1.95 Cells A20.2.J were treated with 5 μM of A77 1726 and cultured for 24 hours (37 ° C, 5% C02) and then prepared for DHO extraction (n = 3). To optimize cell culture methods and to determine the best cell / A77 1726 molar ratio, various densities of A20.2.J cells were incubated together with A77 1726 (5 μM). The samples were removed at different times and the DHO concentrations were determined (table 10). Due to the fact that the DHO concentrations correlate directly with the amount of cells extracted (see table 9), for the following experiments the DHO concentrations were extrapolated to 10 x 106 cells in μg / ml. The best linear increase in DHO level was found at a density of 1 x 106 cells / ml. Using this cell density, the time-dependent increase of intracellular levels of DHO in cells incubated with A77 1726 was studied. Detectable amounts of DHO could be determined after 1 hour of incubation, irrespective of the drug concentration (Table 11). A linear increase was observed with the highest amount of DHO determined after 6 hours (table 11). After this period of time saturation was observed without further increase in DHO.

Table 10: Cell density and time-dependent increase of intracellular DHO concentrations DHO concentration time μg / ml mean ± incubation 1 x 10 £ standard deviation (h) cells / ml 2 x 105 3 X 10 cells / ml cells / ml 1 6.25 ± 0.42 5.47 + 0.10 5.63 ± 0.19 4 33.06 ± 2.26 26.36 ± 0.42 19.39 ± 1.87 7 60.07 ± 0.01 42.31 ± 1.07 28.79 ± 0.34 Several quantities of A20.2.J cells were incubated together with A77 1726 (5 μM) during the time periods provided above, and their intracellular DHO concentrations were determined for each sample point (* all extrapolated values for 10 x 106 cells) (n = 2) Table 11: Time-dependent increase of intracellular L-DHO concentrations Concentration Time DHO μg / ml mean ± incubation Standard Deviation (h) A77 1726 (5μM) A77 1726 (lOμM) A77 1726 (25μM) 0 2 2..7733 ±± 00..1100 2.73 ± 0.10 2.73 ± 0.0Í 1 9.20 + 0.09 8.5 + 0.39 15.05 ± 0.26 2 24.03 ± 1.15 20.74 ± 1.43 26.06 ± 0.20 4 59.48 ± 0.72 58.65 ± 2.06 50.21 ± 1.54 7 87.54 ± 1.58 82.65 ± 4.50 114.89 ± 3.61 One million cells A20.2.J / ml were incubated together with various concentrations of A77 1726 and their intracellular DHO concentrations is were determined at different time points. The data appear as one μg / ml of DHO ± standard deviation extrapolated to ten million cells (0 - 4h = n = 2, 7h = n = 4). Incubation of tumor cells A20.2.J with A77 1726 resulted in a rapid accumulation of L-DHO due to the inhibition of DHO-DH. The intracellular concentrations of L-DHO correlated with the number of cells and were time dependent. L-DHO monitoring is a surrogate marker for the immunomodulation activity of A77 1726 in patients. Example 3 Animals: Male Wistar-Lewis rats (Mollegaard Breading Center Ltd., Ejby, DK) with a body weight of 160 to 200 g. Arthritis by adjuvant: the disease was induced by the injection of 0.1 ml of Freund's adjuvant (6 mg of Mycobacterium smegmatis suspended in 1 ml of heavy white paraffin oil (Merck, Darmstadt) in the root of Wistar-Lewis rat tails. The pathological symptoms generally appeared between 10 and 14 days after the induction of the disease. Pharmacological treatment: drugs were suspended in 1% carboxymethylcellulose (COMC). Healthy animals (n = 18) and sick rats due to the adjuvant (n = 18) (day 9 of the disorder), received 10 mg / kg, p.o. of N-4- (trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide, then leflunomide twice a day (at 7:30 am and at 1:30 p.m.) for 5 days, that is, at the time points Oh, 6h, 24h, 30h, 48h, 54h, 72h, 78h, 96h (see table 12). At the time point Oh, 3 animals from both the group of sick animals and the group of non-diseased animals were sacrificed to determine the baseline levels. 3 healthy animals and 3 additional sick animals were treated with placebo (COMC alone) for 5 days. Sampling: three animals per group were slaughtered at each sampling time point. Serum and splenocytes were taken at 3h, 7h, 27h, 51h, 75h, and 99h (see table 12). Except for the 7 h value, the samples were taken three hours after the last drug application. The value of 7 h was taken one hour after the second administration. Samples of animals treated with placebo were taken at 0 h (n = 3) and at 99 h (n = 3). Table 12: administration of leflunomide and tissue sampling times Time: 0 3 6 7 24 27 30 48 51 54 72 75 78 96 99 Drug ttttttttt (po) Sampling ttttttt Sample preparation: The blood collected by cardiac puncture was stored for 30 days. minutes at a temperature of 4 ° C and then centrifuged for 10 minutes at 3000 rpm. Serum was separated and stored in Eppendorf capsules at a temperature of -20 ° C (3). Prior to HPLC analysis, the frozen serum was thawed and, in order to remove the proteins, 200 μl of serum was added to a Microcon filter (model 10, code 42407, Amicon) and centrifuged for 30 minutes at 13,000 rpm. - Spleens were taken (n = 3) and combined for L-DHO analysis. The cells separated by passage through a stainless steel strainer were treated with 0.17 M NHC1 to lyse the erythrocytes. Aliquots of 50 million spleen cells per group were prepared, placed in capsules for centrifugation and the supernatant was discarded. The cell pellet, under permanent mixing, received 500 μl of a 1.2 M HC104 solution to lyse the cells and centrifuged for 2 minutes. The acid cell lysate was completely transferred to glass bottles, 500 μl of chloroform was added, and mixed for 2 minutes with a Vortex mixer. Cell lipids were precipitated by centrifugation (10 min 1502 x g and 10 ° C). The supernatant was placed in 2 ml capsules and stored at a temperature of -20 ° C.

The determination of the serum concentrations of A77 1726 was carried out in the following manner: the serum samples were brought to room temperature and thoroughly mixed using a vortex mixer. The serum was placed with pipettes (200 μl) in Eppendorf capsules and the internal standard was added (A77 1726, 2 μg in 400 μl of acetonitrile). The tubes were then mixed in a vortex mixer and centrifuged at 2500 rpm (at room temperature) for 10 minutes. For HPLC analysis, the supernatant (400 μl) was transferred to a flask and water (400 μl) was added and mixed. The HPLC conditions were as follows: the equipment consisted of a TSP P2000 pump, a TSP AS1000 autosampler, a TSP SP4270 integrator, and a TSP UV100 UV detector. The detection was at a wavelength of 292 nm. The mobile phase consisted of 650 ml of methanol (CHROMASOLV), 2.42 g of tetrabutylammonium bromide, and 350 ml of 0.05 M ammonium acetate. The flow rate was 0.5 ml / min. A column of 10 cm CHROMPACK Spherisorb ODS-2 was used with a 1 cm reverse phase protection column (R2). 100 μl was injected into the column and the experiment time was 7 minutes. HPLC determination of L-DHO concentrations: the chromatographic separation was carried out in accordance with that described in example 1. The range of conductivity detector was set at 10 μS; the analysis was carried out at room temperature. The mobile phase consisted of 100 mM NaOH (A) and water (B) With this system, the following gradients were produced: Serum Time AB (min) (%) (%) 0 1 99 2.5 1 99 14 23 77 18 60 40 24 60 40 26 1 99 45 1 99 Splenocytes Time AB (min) (%) (%) 0 1 99 2.5 1 99 14 8 92 22 8 92 28 60 40 32 60 40 34 1 99 49 1 99 The flow rate it was 1 ml / min.

Both healthy rats and diseased rats showed concentrations of cellular L-DHO (table 13) and serum levels (table 14) increased after oral administration of leflunomide. This increase was correlated with the serum concentrations of A77 1727 determined in these animals (Table 15). Initially, 3 hours after the oral administration of drug, A77 1726 reached a concentration of approximately 26 μg / ml in rats diseased by the adjuvant and 31 μg / ml in non-diseased rats. These values peaked one hour after the second administration (7 hours), but dropped to values between 7 and 12 μg / ml during the experiment in both diseased rats and healthy rats. It took 51 hours for sick animals to reach these concentrations, while healthy animals had reached that concentration after 27 hours (Table 15). The serum concentrations of A77 1726 were correlated with the concentrations of L-DHO in the serum of rodents diseased by adjuvant and healthy rodents. In contrast to the serum concentrations of L-DHO, which were equilibrated to approximately 5 μg / ml during the experiment, the amount of L-DHO found in the splenocytes fell below the limit of detection (1.5 μg / ml) after 99 hours. Table 13: L-DHO Concentrations in splenocytes [50 x 10d cells] treated with Leflunomide Time Sick rats per adjuvant (hours) Mean Value Deviation Single deviation [μg / ml] standard standard [μg / ml] [%] 0 < D.L. (Pla < D.L. < D.L. bait) 3 5.81 4.60 5.21 0.60 11.52 7 13.00 11.22 12.11 0.89 7.35 27 16.28 16.84 16.56 0.28 1.69 51 3.63 3.97 3.80 0.17 4.47 75 2.58 2.71 2.65 0.06 2.26 99 < D.L. < D.L. < D.L. Placebo < D.L. (99) < D.L. < D.L. Time Non-diseased rats (hours) Mean value Deviation Unique deviation [μg / ml] standard standard [μg / ml] [%] 0 < D.L (pla < D.L < D.L bait) 3 5.09 6.31 5.70 0.61 10.70 7 16.77 14.87 15.82 0.95 6.01 27 8.69 10.25 9.47 0.78 8.24 51 1.84 2.34 2.09 0.25 11.96 75 1.92 2.24 2.08 0.16 7.69 99 < D.L. < D.L. <; D.L. Placebo < D.L. (99) < D.L. < D.L. Animals were treated with Leflunomide or placebo, vessels were removed (n = 3) and combined in accordance with what was described. The combined splenocytes were assayed in duplicate. DL = limit of detection (1.5 μg / ml); Table 14: L-DHO concentrations in serum of rats treated with Leflunomide Diseased rats by adjuvant Time rat mean value Deviation Deviation (hours) only [μg / ml] standard standard [μg / ml] [%] 1 < D.L. 0 2 < D.L. < D.L. (pla3 < DL bait) 1 7.85 3 2 9.66 8.52 0.99 11.70 3 8.05 1 19.55 7 2 17.00 17.54 1.81 10.30 3 16.06 1 19.73 27 2 15.95 16.45 3.06 18.60 3 13.67 1 3.06 51 2 3.68 3.27 0.36 11.00 3 3.06 1 5.38 75 2 4.45 5.14 0.61 11.90 3 5.60 1 5.48 99 2 4.89 5.02 0.41 8.30 3 4.68 placebo 1 < D.L. 99 2 < D.L. < D.L. 3 < D.L. Non-sick rats Time rat mean value Deviation Deviation (hours) single [μg / ml] standard standard [μg / ml] [%] 1 < D.L. 0 2 < D.L. < D.L. (pla3 < DL bait) 1 11.92 3 2 10.88 11.13 0.67 6.30 4 10.60 1 20.29 7 2 18.14 18.66 1.45 7.80 4 17.54 1 8.22 27 2 8.42 8.43 0.21 2.50 3 8.64 1 4.87 51 2 4.77 4.55 0.46 10.20 3 4.02 1 4.60 75 2 4.17 4.33 0.23 5.40 3 4.23 1 5.67 99 2 6.64 5.75 0.85 14.80 3 4.95 placebo 1 < D.L. (99) 2 < D.L. < D.L. 3 < D.L. The animals were treated with Leflunomide or placebo and the bleeding was extracted, prepared and tested in accordance with what was described. The serum concentrations of L-DHO were determined for each animal individually. DL = limit of detection (0.5 μg / ml); Table 15: Concentrations A77 1726 in rats serum treated with leflunomide Diseased rats by adjuvant Time rat average value Deviation Deviation (hours) only [μg / ml] standard standard [μg / ml] [] 1 0.0 0 2 0.0 0.0 (pla3 0.0 bait) 1 26.8 3 2 27.2 26.33 1.17 4.45 3 25.0 1 44.1 7 2 45.2 42.3 4.11 9.71 3 37.6 1 21.3 27 2 22.0 19.7 3.34 16.92 3 15.9 1 9.1 51 2 10.2 9.2 1.00 10.93 3 8.2 1 9.0 75 2 7.3 9.9 3.09 31.34 3 13.3 1 12.0 99 2 11.9 12.2 0.38 3.11 3 12.6 placebo 1 0.0 99 2 0.0 0.0 3 0.0 Non-sick rats Time rat mean value Deviation Deviation (hours) single [μg / ml] standard standard [μg / ml] [%] 1 0.0 0 2 0.0 0.0 (pla3 0.0 bait) 1 32.0 3 2 30.6 30.7 1.21 3.92 3 29.6 1 47.7 7 2 49.4 47.9 1.46 3.04 3 46.5 1 10.2 27 2 9.9 9.6 0.74 7.65 3 8.8 1 7.8 51 2 8.7 7.4 1.48 19.97 3 5.8 1 6.7 75 2 9.4 1.35 16.70 3 8.2 1 14.6 99 2 11.4 12.7 1.67 13.08 3 12.2 placebo 1 0.0 (99) 2 0.0 0.0 3 0.0 The animals were treated with leflunomide or placebo and the blood was drawn, prepared and tested in accordance with what was described. Serum concentrations A77 1726 were determined for each animal individually.

The orally applied leflunomide is converted very rapidly to A77 1726 in vivo. A77 1726 is the active metabolite of leflunomide (US 5,679,709). Incubation of sick rats by the adjuvant and healthy rats with leflunomide resulted in a rapid accumulation of L-DHO in their serum and splenocytes. The concentration of L-DHO correlated with the serum concentrations of A77 1726, thus demonstrating the active suppression of DHO-DH through this molecule in vivo. In human clinical studies, monitoring of L-DHO may be a surrogate marker for the immunomodulation activity of leflunomide in patients.

Claims (5)

1. CLAIMS 1. A process for obtaining L-dihydroorotic acid by chromatography comprising the steps of: a) obtaining a column comprising a pressure-stable anion exchange material; b) loading the column with a sample solution including L-dihydroorotic acid; c) chromatography; d) eluting the L-dihydroorotic acid with an elution solution containing an aqueous base mixture; said process is carried out under a pressure comprised within a range of about 1.1 MPa to about 40 MPa.
2. 2. A process according to claim 1, wherein the base is sodium hydroxide.
3. 3. A process according to claim 1, wherein the pressure-stable anion exchange material is a divinylbenzene / ethylvinylbenzene polymer or a polyvinylbenzylammonium polymer crosslinked with divinylbenzene or mixtures thereof modified with quaternary alkanolammonium. 4. A process according to claim 1, wherein the pressure is from about
4. 4.1 MPa to about 5.5 MPa.
5. 5. A process according to claim 1, wherein the elution of the water-based mixture has a time gradient of the base concentration, preferably with a linear development. A process according to any of claims 1 to 5, wherein the L-dihydroorotic acid is detected through a conductivity detector. The use of the method according to any of claims 1 to 6 for the preparation of a diagnostic assay. The use of the method according to any of claims 1 to 7 for determining the inhibitors of dihydroorotic acid dehydrogenase. The use of the method according to claim 8, wherein the inhibitor of dihydroorotic acid dehydrogenase is N-4 (trifluoromethylphenyl) -5-methylisoxazole-4-carboxamide, Brequine, N- (4-tri-fluoromethylphenyl) -2-cyano -3-hydroxyhept-2-en-6-incarboxamide, 2-cyano-3-cyclopropyl-3-hydroxy-acrylic acid (4-cyanophenyl) -amide and / or N- (4-trifluoromethylphenyl) -2-cyano-3 -hydroxy-romonamide. . The use of the determination of L-dihydroorotic acid to monitor the activity of dihydroorotic acid dehydrogenase inhibitors.