Introduction

Top Abstract Introduction Methods Results Discussion References

Decreased synthesis of eicosanoids by reduction of AA is, in part, responsible for the antiinflammatory effects of EFAD and dietary supplementation with n-3 PUFA (Kinsella et al., 1990; Lefkowith et al., 1986a, b). EFAD has been used as an extreme dietary means to reduce AA in lipid pools and in some models of inflammation is disease modifying (Hurd et al., 1981; Mascolo et al., 1995; Wright et al., 1995). Macrophage and neutrophil function is compromised in EFAD and the correlative factor appears to be partial depletion and accompanying unavailability of AA in key lipid pools for eicosanoid synthesis (Lefkowith et al., 1986, 1987; Lefkowith, 1988).

AA is synthesized from LA (18:2 n-6) by a sequential series of enzymatic conversions occurring principally in the liver (fig. 1). Specifically, LA is converted to GLA (18:3 n-6) by the 6 desaturase, after which GLA is elongated to DGLA (20:3 n-6) by four discrete enzymes, collectively referred to as elongase, and, finally, DGLA is converted to AA by the 5 desaturase (Cinti et al., 1992; Holloway, 1983; Sprecher, 1983). Alleviation of inflammation might be attained in humans by decreasing the synthesis of AA via selective inhibition of the 6 or 5 desaturase. It was hypothesized that decreased synthesis and, thus, availability of AA would attenuate the inflammatory response by decreasing eicosanoid levels and/or by altering AA-mediated cell signaling (Di Marzo, 1995; Khan et al., 1995; Kinsella et al., 1990; Kinsella, 1990). Inhibition of the synthesis of AA could potentially go beyond amelioration of symptoms currently provided by NSAIDs and, similar to EFAD, be disease modifying. Unlike EFAD, desaturase inhibition might prevent some of the untoward side-effects associated with EFAD (e.g., hair loss, impaired fertility and psoriatic-like skin; Holman, 1968; Lefkowith et al., 1986a) because LA, an essential fatty acid, would be present in the diet.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   n-6 polyunsaturated fatty acid biosynthesis.

As a first step in concept evaluation of desaturase inhibition, CP-24879, a mixed 6/5 desaturase inhibitor, was shown to chronically inhibit combined 6 and 5 desaturase activities and cause partial depletion of AA and decreases in LTC₄ production in vitro (Obukowicz et al., 1998). When administered to mice for 6 days, CP-24879 inhibited combined 6 and 5 desaturase activities and caused partial depletion of AA in liver. The antiinflammatory properties of CP-24879 could not be evaluated, however, because of its overt side-effects after prolonged chronic dosing (i.e., lethargy accompanied by hypothermia and late-stage tremoring). Nonetheless, CP-24879 represented a novel, mixed 6/5 desaturase inhibitor. An emphasis was placed on identifying selective 5 desaturase inhibitors because DGLA, the substrate of the 5 desaturase, could serve as a surrogate of AA, but without being metabolized to proinflammatory eicosanoids. DGLA acid increases markedly and is accompanied by enhanced synthesis of PGE₁ when it is fed to animals at high levels (Knapp et al., 1978). Similarly, pharmacological inhibition of the synthesis of AA would allow for LA to be consumed in the diet and, if inhibited at the level of the 5 desaturase, DGLA to accumulate and serve as the source for PGE₁, thus potentially maintaining gastric and renal function (Narumiya, 1995). Furthermore, antiinflammatory properties have been ascribed to PGE₁, including its ability to reduce platelet aggregation, decrease blood pressure, and suppress the immune response (Kirtland, 1988; Zurier, 1990). Leukotrienes are not synthesized from DGLA because the 5 double bond is absent, thus eliminating another source of proinflammatory mediators.

This study describes the identification of novel, selective and potent 6 and 5 desaturase inhibitors for evaluation as antiinflammatory agents. By using quantitative and relatively high throughput in vitro and in vivo radioassays (Obukowicz et al., 1998), prototypic compounds representing five classes of 5 desaturase inhibitors and one class of 6 desaturase inhibitor were identified by screening available compound libraries. In the carrageenan paw edema model in the mouse, chronic dosing with the 6 desaturase inhibitor, SC-26196, decreased edema to the same extent as obtained with EFAD or acute dosing with indomethacin. The antiinflammatory properties of SC-26196 were consistent with its mechanism of action as a 6 desaturase inhibitor, providing the first evidence that this novel target has possible therapeutic value.

	Methods

Top Abstract Introduction Methods Results Discussion References

Materials and Reagents

Authentic fatty acids used as standards in GC or authentic fatty acid ethyl esters used in metabolic studies were purchased from Nu-Chek Prep, Inc. (Elysian, MN). Unless stipulated otherwise, the vehicle for i.g. dosing consisted of 0.5% methylcellulose + 0.025% Tween-20 (polyoxyethylene-sorbital monolaurate), both purchased from Sigma Chemical Co. (St. Louis, MO). For i.p. dosing, DMSO (5% final concentration) was added to the methylcellulose/Tween-20. Organic solvents were Optima grade from Fisher Scientific (Pittsburgh, PA). Carrageenan was kindly provided by FMC (Rockland, ME). Routine laboratory chemicals were purchased from Sigma or Fisher Scientific. [1-¹⁴C]-fatty acids (sp. act. approximately 55 mCi/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO). Precoated silica gel TLC plates (20 × 20 cm LK5D plates, 150-Å pore diameter, 250-µm thick, 19 channels, 500-µm thick preabsorbent strip) were purchased from Whatman (Clifton, NJ). The plates were immersed for 15 to 20 sec in a 10% AgNO₃ solution in water, after which they were drained and then air-dried for a minimum of 2 days. During storage, the plates were kept in the dark. Before use, the plates were activated for 1 hr at 110°C. Sample preparation of lipids/fatty acids was done in glass test tubes having Teflon-lined caps.

Preparation of Rat Liver Microsomes

Male Sprague-Dawley rats (150-175 g) were fasted for 3 days and then refed an EFAD diet for 2 days to induce 9 desaturase activity (Lefkowith, 1990). The rats were killed and their livers were subsequently removed and placed on ice. The livers were diced with scissors and then homogenized with a Polytron (PTA 10TS probe) in a 2× volume of homogenization buffer (150 mM KCl, 250 mM sucrose, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1.5 mM reduced glutathione) at 4°C. The homogenate was centrifuged at 1500 × g for 20 min at 4°C. The supernatant was filtered through gauze and centrifuged twice more at 10,000 × g for 20 min each at 4°C. The supernatant was saved and centrifuged one final time at 100,000 × g for 60 min at 4°C. The supernatant was discarded and the microsomal pellet was resuspended in homogenization buffer to a protein concentration of 10 mg/ml (Bradford, 1976). The microsomal preparation was aliquoted and stored at 80°C.

Desaturase Activity Assays

In vitro rat liver microsomal assay. All three desaturase activities, 5, 6 and 9, were assayed simultaneously. Assay of 9 desaturase activity was included as a control to evaluate inhibitor selectivity. The assay conditions were optimized in a 48-well microtiter plate format to achieve a relatively high throughput rate for screening. Into each well, the following, in order, were added: 1) 150 µl buffer/cofactors (250 mM sucrose, 150 mM KCl, 40 mM NaF, 100 mM sodium phosphate, pH 7.4, 1.3 mM ATP, 1.5 mM reduced glutathione, 0.06 mM reduced coenzyme A, 0.33 mM nicotinamide, 1 mg/ml MgCl₂ · 5H₂O and 0.67 mg/ml NADH), 2) 50 µl rat liver microsomes (approximately 0.5 mg total protein), 3) 2.2 µl test compound (dimethylsulfoxide stock; 1% final concentration), and 4) 20 µl of ¹⁴C-fatty- acid substrates (mixture of ¹⁴C-18:0, ¹⁴C-18:3 n-3 and ¹⁴C-20:3 n-6 in the buffer/cofactor solution). Listed below are the three ¹⁴C-fatty acid substrates and the respective desaturase and ¹⁴C-fatty acid product (scheme 1).

View larger version (6K): [in this window] [in a new window]   Scheme 1.

In Vivo Assay of 6 and 5 Desaturase Activities

An appropriate amount of ¹⁴C-LA (ethanolic solution) or ¹⁴C-DGLA was evaporated to dryness under nitrogen and immediately dissolved in 18.2 mM Na₂CO₃ (10-fold molar excess) to a specific activity of 100 µCi/ml. Mice (n = 3/group) received injections i.p. with 0.1 ml (10 µCi) of ¹⁴C-fatty acid and after 6 hr (¹⁴C-LA injection) or 2 hr (¹⁴C-DGLA injection) they were killed by CO₂ inhalation. Livers were removed quickly, frozen on dry ice, and then stored at 70°C. Total liver lipids were extracted from 100 to 200 mg liver tissue in chloroform:methanol:water and then saponified in 2.5 N KOH in methanol:water as described above. After evaporation of the chloroform layer under nitrogen gas, the fatty acids were transmethylated by the addition of 2 ml of methanolic-HCl (3-5% HCl, made by mixing 0.1 volume of acetyl chloride with 1 volume of cold methanol). After transmethylation, (65-70°C, 2 hr), 2 ml of water and 6 ml of hexane were added to each sample and the contents were mixed vigorously. The phases were separated by centrifugation at 1000 × g for 5 min at room temperature. The hexane layer was removed, transferred to a new tube and dried under nitrogen gas. The fatty acid-methyl esters (approximately 10⁵ dpm) were solubilized in 0.3 ml of acetonitrile and then transferred to a high-performance liquid chromatography autosampler vial. ¹⁴C-fatty acid substrate and ¹⁴C-fatty acid products were resolved and quantified using a Waters (Milford, MA) high-performance liquid chromatography system (Machery-Nagel C₁₈ reverse phase column) connected in-line with a FLO-ONE Beta (Radiomatic, Tampa, FL) radioactivity detector. Chromatography conditions were as follows: 70 to 90% acetonitrile gradient over 65 min followed by 90% acetonitrile for 10 min; flow rate = 1 ml/min. Combined 6 desaturase/elongase/5 desaturase activities were calculated as the % conversion of substrate (¹⁴C-LA) to products (¹⁴C-GLA + ¹⁴C-DGLA + ¹⁴C-AA). 5 desaturase activity was calculated as the % conversion of substrate (¹⁴C-DGLA) to product (¹⁴C-AA).

Fatty Acid Composition Analysis

GC analysis using electron capture detection was used to quantify the fatty acid composition of mouse liver, plasma or peritoneal cell samples. Mice were anesthetized with CO₂/O₂ (80/20), blood samples were obtained by retroorbital bleeding and plasma was prepared. Mice were killed by CO₂ inhalation and resident cells were isolated from the peritoneal cavity by injecting 4 ml of cold phosphate-buffered saline i.p. After gentle massaging, a small incision was made in the peritoneal cavity and the contents removed by aspiration with a Pasteur pipet. The cavity was rinsed and aspirated with an additional 2 ml of PBS and then pooled with the first lavage. The cells were pelleted by centrifugation. Cells from all mice in a group were pooled and prepared as a single sample to provide an adequate signal (>10⁶ cells/sample).

Lipids from plasma (10 µl/sample) or peritoneal cells (>10⁶ cells/sample) were saponified directly in 200 µl of 2.5 N KOH in methanol:water (4:1) spiked with 2.5 µg of heneicosanoic acid (21:0; 0.1 mg/ml stock in hexane) as the internal standard.

Lipids from frozen liver tissue were prepared according to a modified Bligh and Dyer (1959) procedure. Briefly, liver tissue (100 mg) was added to 4 ml of chloroform:methanol:water (1:2:0.3) and homogenized with a hand-held homogenizer (Tissue Tearor, Biospec Products, Inc., Bartlesville, OK) at room temperature. The homogenate was centrifuged at 1000 × g for 5 min at room temperature. The supernatant was decanted and saved. To the residual tissue, 2.3 ml of chloroform:methanol:water (1:2:0.8) were added. The tissue was vortexed vigorously and then centrifuged at 1000 × g for 5 min at room temperature. The supernatant was decanted and pooled with the first supernatant. The pooled supernatants were diluted with 1.8 ml of chloroform and then 1.8 ml of water, followed by gentle, but thorough mixing. The chloroform and methanol/water phases were separated by centrifugation at 1000 × g for 5 min at room temperature. The chloroform layer was transferred to a new tube and spiked with 0.5 mg of 21:0. The lipids were saponified (60°C, 1 hr) by the addition of 1 ml of 2.5 N KOH in methanol:water (4:1). After saponification, fatty acids from liver, peritoneal cells or plasma were protonated by the addition of 2 ml of formic acid and then extracted into hexane by the addition of 2 ml of water + 6 ml of hexane followed by thorough mixing. The hexane layer was removed and transferred to a new tube containing a small amount of Na₂SO₄ to remove any residual water. A portion of the hexane layer (1/10 volume) was transferred to another new tube and evaporated to dryness under nitrogen gas. For electron capture detection, the fatty acids were derivatized to pentafluorobenzyl esters (Min et al., 1980) by the addition of 10 µl of diisopropyl ethylamine (Sigma) + 20 µl of 35% pentafluorobenzylbromide in acetonitrile (Pierce Chemical Co., Rockford, IL). The samples were incubated for 15 min in a 50°C water bath, after which the contents were evaporated under nitrogen gas. One ml hexane was added and the samples were washed twice with 1 ml water. The hexane layer was removed and evaporated under nitrogen gas. The sample residue was resolubilized in 1 ml of hexane and then transferred to a GC autosampler vial. Derivatized fatty acids were separated and identified using a Hewlett-Packard (Palo Alto, CA) model 5880 gas chromatograph equipped with an SP-2380 fused silica capillary column (30 m, 0.32 mm ID, 0.20-µ film thickness; Supelco, Bellefonte, PA), electron capture detector and an HP-5880A terminal integrator. Fatty acid pentafluorobenzyl esters were identified by comigration with authentic pentafluorobenzyl ester standards.

Dietary and Dosing Paradigms

Female Balb/C or Swiss/Webster mice were purchased at 8 to 10 wk of age and fed a standard rodent food diet (Teklad 2215 [W] rodent diet 8640, Harlan, Madison, WI) or a corn oil diet (AIN-76-based corn oil diet, DYETS, Inc., Bethlehem, PA). In studies evaluating the effects of EFAD, female Balb/C mice were purchased at 3 wk of age and fed the standard rodent chow diet or an EFAD diet (5803C, low essential fatty acid P.D., Purina Test Diets, Richmond, IN) for a minimum of 8 wk. The EFAD state was confirmed by fatty acid composition analysis of liver tissue; the ratio of Mead acid (20:3 n-9)/AA (20:4 n-6) was approximately four to five at the end of the study, much higher than the defined minimum value of 0.4 for EFAD (Holman, 1968). Water was provided ad libitum throughout.

Approximately 0.5 mg of AA is consumed per day in the standard Chow diet by a 20 g mouse (Lab Diet, The Richmond Standard, Animal Diet Reference guide, PMI Feeds, Inc., Richmond, IN). This level of AA may be high enough to circumvent reduction of AA during chronic inhibition of desaturase activity. To eliminate this possibility, mice initially fed a Chow diet were switched to a corn oil diet for at least 1 wk before initiating dosing with the desaturase inhibitors. The corn oil diet contains LA and oleic acid (OA; 18:1 n-9), but not AA (table 1). Furthermore, the high level of LA in the corn oil diet provides substrate for the in vivo synthesis of AA that, in the presence of a 5 and/or 6 desaturase inhibitor, would be blocked.

An appropriate amount of the ethyl ester of AA or OA was emulsified in vehicle by sonication and administered i.g. (0.1 ml/dosing), q.d. (pm). Control groups not receiving the fatty acid ethyl esters were gavaged with vehicle only.

The desaturase inhibitors or indomethacin were made as suspensions in vehicle by wet milling (Retsch, model MM2 wet miller). Compounds were administered as a 0.1 ml bolus dose, b.i.d., i.g. or i.p. Control groups were administered vehicle only.

Pharmacokinetics of 5 and 6 Desaturase Inhibitors

Desaturase inhibitors were prepared as solutions in DMSO (i.v. dosing) or as suspensions in methylcellulose/Tween-20 (i.g. dosing). Female Balb/C mice (8-10 wk old) that were fed a standard Chow diet were used for i.v. or i.g. dosing. For i.v. dosing, mice were injected in the tail vein with 0.1 ml of a 10 mpk bolus dose. For i.g. dosing, mice were gavaged with a 0.1 ml of a 100 mpk bolus dose. Bioavailability was calculated from the area under the curve (AUC) ratio of i.g. vs. i.v. dosing, corrected for dosage. For i.v. dosing, time points were taken at 0 (DMSO control), 1, 2, 5, 15, 30, 60, 120, 240 and 360 min after injection of the compound. For i.g. dosing, time points were taken at 0 min (methylcellulose/Tween-20 control), 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr and 24 hr after gavaging of the compound. Five mice were included per time point. At the end of each time point, the mice in each group were anesthetized by CO₂/O₂ (80/20) inhalation and blood was collected by retroorbital eye bleeds. Approximately 0.5 ml blood/mouse was collected into a heparinized tube. The heparinized blood samples were kept on ice until the end of the experiment, after which plasma was prepared. A standard curve was generated by spiking in known amounts of a given desaturase inhibitor into mouse plasma. A portion of plasma (50 µl) from each sample was fractionated on a C₁₈ SepPak column. After washing to remove nonspecific binding components, bound desaturase inhibitor was eluted with 100% acetonitrile. The samples were dried and resolubilized in chloroform:methanol (1:1). Intact desaturase inhibitor was detected by electrospray mass spectrometry using the multiple reaction monitoring mode. The amount of desaturase inhibitor in each plasma sample was quantified by interpolation from the standard curve. Terminal phase elimination half-lives were calculated from plasma concentration vs. time data using the CSTRIP computer program (Sedman and Wagner, 1976). Whole body clearance and volume of distribution were calculated using an AUC computer program developed in-house in which initial parameter estimates were obtained from the CSTRIP computer program.

Pharmacodynamics of SC-26196

Mice were dosed with SC-26196 (100 mpk, i.g.) at t = 0 hr, after which they were injected with ¹⁴C-LA (10 µCi, i.p.) at t = 2 hr, 6 hr or 18 hr. After a further 6-hr incubation time, the mice were killed by CO₂ inhalation and liver samples were prepared for quantification of the conversion of ¹⁴C-LA to ¹⁴C-GLA + ¹⁴C-DGLA + ¹⁴C-AA by radiometric reverse phase-high performance liquid chromatography (see above). Taking into account the 6 hr in vivo incubation time, the conversion of ¹⁴C-LA to ¹⁴C-GLA + ¹⁴C-DGLA + ¹⁴C-AA was quantified at 8, 12 or 24 hr after a single oral dose with SC-26196.

Carrageenan Paw Edema Model of Inflammation

The carrageenan paw edema model of inflammation in the mouse (Levy, 1969) was used to evaluate the antiinflammatory properties of desaturase inhibitors compared to essential fatty acid deficiency or non-steroidal anti-inflammatory drug (i.e., indomethacin) treatment. Carrageenan (25 µl of a 1% solution in saline) was injected sub-plantar into the right and left hind footpads of 8- to 10-wk-old, female Balb/C mice (n = 5/group). Hind footpad thickness was measured with a pocket thickness gauge (no. 7309, Mitutoyo Corp., Sakato, Japan). The edema component of inflammation was quantified by measuring the difference in hind footpad thickness before carrageenan injection and at the time point (3-4 hr) when swelling is maximal (Levy, 1969). Food and water were provided ad libitum.

MPO Assay

Neutrophil infiltration was measured by quantifying MPO activity, an enzyme marker of neutrophils (Bradley et al., 1982). Amputated hind footpads were frozen in liquid nitrogen and the paws were then pulverized into small pieces. The pieces were homogenized in phosphate buffer and subjected to centrifugation at 35,000 × g for 20 min. The supernatant was discarded and the homogenization was repeated in the presence of 0.5% hexadecyltrimethlammonium bromide (Sigma), followed by sonication using a W385 ultrasonic processor (Heat Systems-Ultrasonics, Inc., Farmingdale, NY). The samples were subjected to freeze-thaw three times, followed by sonication and centrifugation. The supernatant was used for assay. The final assay buffer was a mixture of 16.7 mg o-dianisidine HCl (Sigma) per 10 ml distilled water, 1.6 µl of 30% hydrogen peroxide (Sigma) and 90 ml of 50 mM phosphate buffer (pH 6.0). A portion (7 µl) of sample was added to 200 µl of buffer and then analyzed using an EL 340 Biokinetics Reader (450 nM wavelength). Standard curves were made using MPO purified from human neutrophils (Calbiochem, La Jolla, CA).

Statistics

Statistical analyses were performed on the effects of SC-26196 on liver fatty acid composition (table 3) and the AA content of peritoneal cells (fig. 5). The data are expressed as log₁₀ of total fatty acid content or as square root of arc sin of fatty acid relative percent. The log and arc sin square root transformations were chosen because they successfully resolved nonconstant variance problems in the data. (The transformations are reasonable choices based on theoretical arguments.) A one-way analysis of variance was fit to estimate the pooled, within-treatment group variation.

In the evaluation of the effect of SC-26196 on liver fatty acid composition (table 3), parametric trend tests were performed for each fatty acid and the total fatty acid content. Slope contrasts were constructed and tested in a particular order. The testing started with the largest group of samples and then proceeded to the next smaller group, only if the larger group had a significant slope. The reported values are the slope and corresponding S.E. for the smallest group that produced a significant slope (P < .05). The slope should be interpreted as the expected change in the log₁₀ of the total fatty acid content or arc sin square root of the relative % of an individual fatty acid with a 3-fold increase in dose of SC-26196.

Comparisons of the effects of SC-26196 ± dietary AA or OA on the level of AA in plasma (fig. 5) were evaluated using the pooled, within-treatment variation estimated in the analysis of variance. Significance was determined by calculating the difference in square root of arc sin of relative % AA.

	Results

Top Abstract Introduction Methods Results Discussion References

Identification of 6 and 5 Desaturase Inhibitors

The rat liver microsomal assay was utilized to identify selective 5 or 6 desaturase inhibitors from the Monsanto/Searle library of compounds. Five classes of compounds were identified as potent and selective 5 desaturase inhibitors, while one class of compounds was identified as a potent and selective 6 desaturase inhibitor (table 2). The prototypic compounds from each chemical class had IC₅₀ values 1 µM and had selectivity ratios >700× relative to the two nontargeted desaturases (i.e., 9 and 6 desaturases for 5 desaturase inhibition or 9 and 5 desaturases for 6 desaturase inhibition).

View this table: [in this window] [in a new window]   TABLE 2 Desaturase inhibitors. The potency and selectivity of the 6 and 5 desaturases were determined in the rat liver microsomal assay. Desaturase activity was determined directly in mouse liver by quantifying the conversion of 14C-DGLA  14C-AA (5 desaturase activity) or 14C-LA  14C-AA (combined 6 desaturase + elongase + 5 desaturase activities). Mice were first dosed i.p. with vehicle or 100 mpk of inhibitor and after 2 hr received injections i.p. with 10 µCi of 14C-DGLA or 14C-LA (see "Methods" for details). Desaturase activity in vivo is expressed as % conversion of substrate to product(s) (mean ± S.E., n = 3) relative to vehicle-injected control mice.

Inhibition of the conversion of ¹⁴C-DGLA to ¹⁴C-AA (5 desaturase activity) or ¹⁴C-LA to ¹⁴C-AA (combined 6 desaturase/elongase/5 desaturase activities) in vivo varied after a single dosing (100 mpk, i.p.), depending on the inhibitor (table 2). The 6 desaturase inhibitor, SC-26196, inhibited conversion of ¹⁴C-LA to ¹⁴C-GLA + ¹⁴C-DGLA + ¹⁴C-AA by 95%, whether the compound was administered i.g. or i.p. Also, SC-26196 did not inhibit the conversion of ¹⁴C-DGLA to ¹⁴C-AA (table 2), demonstrating selective inhibition of 6 desaturase activity in vivo. Complete inhibition of 5 desaturase activity in vivo was not obtained with any of the five prototypic 5 desaturase inhibitors (range of 66 to 26% inhibition), even though three of them, CP-186682, CP-74006 and CP-214339, were more potent than SC-26196 in vitro (table 2).

Pharmacokinetic Profiles of the Desaturase Inhibitors

The lack of correlation between potency in vitro and acute inhibition of desaturase activity in vivo suggested that the desaturase inhibitors were cleared differentially in mice. As a prelude to evaluating the antiinflammatory properties of the 5 and 6 desaturase inhibitors in chronic studies, the pharmacokinetic and bioavailability profiles of the prototypic compounds from each chemical class were determined. The 6 desaturase inhibitor, SC-26196, had the best pharmacokinetic profile; based on AUC calculations, the half-life was 1.2 hr and bioavailability was approximately 60%. After a 100-mpk i.g. dose of SC-26196, the plasma level (0.9 µg/ml = 2 µM) was approximately 10-fold more than the in vitro IC₅₀ value (0.2 µM) over a 12-hr period, but was undetectable by 24 hr (fig. 2). None of the 5 desaturase inhibitors had acceptable pharmacokinetic profiles for chronic, oral dosing in mice (data not shown). CP-186682, CP-214339 and CP-74006 were cleared rapidly, having half-lives shorter than 10 min. SC-60714 and TDLFT were cleared less rapidly, having half-lives of approximately 1 hr, similar to that for SC-26196. However, after a 100-mpk oral dose, the bioavailability of SC-60714 and TDLFT was very low, 15 and 2%, respectively, and, more importantly, plasma levels higher than the in vitro IC₅₀ value were not achieved over a 12-hr period. None of the 5 desaturase inhibitors was evaluated further in vivo because they were either cleared too quickly, lacked oral availability, never achieved a plasma level near the in vitro IC₅₀ value over a 12-hr period, or only minimally inhibited 5 desaturase activity in vivo (table 2).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Pharmacokinetic and pharmacodynamic profiles of the 6 desaturase inhibitor, SC-26196. Plasma levels of SC-26196 were quantified by mass spectrometry following a 10 mpk i.v. or 100 mpk i.g. administration. Half-life and bioavailability values were calculated using CSTRIP and AUC programs (see "Methods"). The pharmacodynamic profile of SC-26196 was determined by measuring the degree of inhibition of desaturase activity with increasing time increments (8, 12 and 24 hr) after a 100 mpk i.g. dose. Plasma levels correlated with the degree of desaturase inhibition. Results are expressed as mean ± S.E. (five mice/time point for the pharmacokinetic analysis; three mice/time point for the pharmacodynamic analysis).

Pharmacodynamic Profile of SC-26196

To determine whether a correlation existed between plasma level and inhibition of liver 6 desaturase activity, the pharmacodynamic profile of SC-26196 was determined. Inhibition of conversion of ¹⁴C-LA to ¹⁴C-GLA + ¹⁴C-DGLA + ¹⁴C-AA in mouse liver was quantified at 8, 12 or 24 hr after a single 100-mpk i.g. dose. In conjunction with the pharmacokinetic results, the degree of inhibition of conversion of ¹⁴C-LA to ¹⁴C-GLA + ¹⁴C-DGLA + ¹⁴C-AA at 8, 12 or 24 hr could then be used to determine whether a t.i.d., b.i.d. or q.d. dosing regimen was required.

Inhibition of 6 desaturase activity by SC-26196 was time-dependent (fig. 2). The plasma level of SC-26196 generally corresponded to the degree of 6 desaturase inhibition and was also time-dependent (fig. 2). Alternatively, some degree of complexity may be present because it could be argued that the plasma levels of SC-26196 at 8 and 12 hr did not correspond exactly to the degree of 6 desaturase inhibition. A reasonable explanation was that SC-26196 accumulated in liver tissue to levels that did not reflect liver perfusion. This interpretation is further supported by results showing that SC-26196 accumulated dose dependently in liver tissue after chronic i.p. dosing (data not shown). Overall, these data demonstrated that with SC-26196 there was a correlation between plasma level, the degree of inhibition of desaturase activity in vivo, and the in vitro IC₅₀ value. SC-26196 was thus chosen to evaluate the antiinflammatory effects of desaturase inhibition. It was the only desaturase inhibitor that had combined bioavailability, a sufficiently long half-life, sustained plasma levels that were higher than the in vitro IC₅₀ value, and 90% inhibition of liver desaturase activity following a single bolus dose. Based on the pharmacokinetic/pharmacodynamic profile of SC-26196, a chronic dosing regimen of 100 mpk, b.i.d., i.g. or i.p., provided 65% inhibition of 6 desaturase activity over a 24-hr period.

Antiinflammatory Properties of SC-26196

The carrageenan paw edema model in the mouse (Levy, 1969) was used to evaluate whether SC-26196 was antiinflammatory. The model was appropriate for testing the effects of desaturase inhibition because, after subplantar injection of carrageenan, an acute and localized inflammatory response occurs in the footpad, characterized by increased edema and neutrophil infiltration. Inhibition of edema was obtained with aspirin and indomethacin and was comparable to that obtained in the rat model.

A. SC-26196 inhibited 6 desaturase activity and decreased edema. SC-26196 inhibited desaturase activity in vivo (ED₅₀ = 20 mpk) and decreased edema dose-dependently (fig. 3). Nearly complete inhibition of the conversion of ¹⁴C-LA to ¹⁴C-GLA + ¹⁴C-DGLA + ¹⁴C-AA in mouse liver was obtained with a single 100 mpk i.p. or i.g. dose (90-100% inhibition). At the highest dose of SC-26196 administered (100 mpk, i.p., b.i.d., 9 days), the decrease in edema (50%) was comparable to that obtained with EFAD mice (58%). Indomethacin administered as a single 10 mpk i.g. bolus dose 2 hr before the injection of carrageenan into the hind paws was more efficacious (71% inhibition) than SC-26196 or EFAD. However, there was no further decrease in edema when indomethacin treatment was combined with SC-26196 (70% inhibition) or EFAD (68% inhibition).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Dose-dependent inhibition of mouse liver desaturase activity and mitigation of edema by SC-26196. Desaturase inhibition. SC-26196 was administered to mice in doses ranging from 1 to 100 mpk, i.p. Two hours later, 10 µCi of 14C-LA was injected, i.p. After a 6-hr in vivo incubation period, the mice were killed and desaturase activity was measured directly in liver by measuring the conversion of 14C-LA to 14C-GLA + 14C-DGLA + 14C-AA by radiometric RP-HPLC (see "Methods"). Carrageenan paw edema. SC-26196 was administered to mice at doses of 10, 30 or 100 mpk, i.p. After 2 hr, carrageenan was injected into both hind paws and edema was measured 3 hr later (see "Methods"). Results are expressed as mean ± S.E. (three mice/group for desaturase activity assay; five mice/group for carrageenan paw edema).

B. The onset of mitigation of edema by SC-26196 was time dependent. Based on results with EFAD, it was hypothesized that chronic inhibition of de novo synthesis of AA by desaturase inhibition would be required to deplete AA in lipid pools before an antiinflammatory response would be manifested. As such, the onset of a decrease in edema should not occur acutely and, instead, should first become manifest after a certain period of time. In agreement with the proposed mechanism, the onset of the decrease in edema by SC-26196 was time dependent (fig. 4). A marked decrease (27% inhibition) was first observed after 36 hr (three b.i.d. dosings) that decreased further (34% inhibition) after 9 days (18 b.i.d. dosings). In contrast, no decrease in edema was obtained with a single bolus dose 2 or 10 hr before injecting the hind paws with carrageenan. These results differed from the acute action of indomethacin, a standard NSAID, in which edema was inhibited by 71% after a single 10 mpk i.g. dosing 2 hr before injecting the hind paws with carrageenan.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Time-dependent inhibition of edema by SC-26196. Mice were dosed with SC-26196 (30 mpk, i.p.) at 2, 10 and thereafter at 12-hr intervals, to 3 days and, finally, to 9 days before injecting carrageenan into the hind paws. Edema was measured 3 hr later. Results are expressed as mean ± S.E. (five mice/group). Note that the ordinate scale is compressed (60-100% of control) and that there was only 35% maximal inhibition of edema when SC-26196 was dosed at 30 mpk, i.p., b.i.d., for 9 days. Reference values for mitigation of edema by indomethacin, EFAD, combined indomethacin + SC-26196 and combined EFAD + SC-26196 are provided in "Results." Antiinflammatory Properties of SC-26196, subsection A.

C. Mitigation of edema by SC-26196 was correlated with a decrease of AA. In the same mice in which SC-26196 decreased edema dose dependently (fig. 3), there was a corresponding decrease of AA in liver tissue (table 3). As predicted by chronic inhibition of 6 desaturase activity, there was a dose-dependent decrease of AA and an accompanying dose-dependent increase of LA in liver tissue (table 3). Total fatty acid content increased dose dependently (table 3), in agreement with dose-dependent lipidosis observed histologically (data not shown). In a separate study, SC-26196 caused a dose-dependent decrease of AA in peritoneal cells and plasma (fig. 5), showing that inhibition of de novo synthesis of AA could affect the AA content of peripheral inflammatory cells, the ultimate target, and blood, the fatty acid transport medium. Coadministration of AA (30 mg/day), but not oleic acid (30 mg/day), along with the highest dose of SC-26196 (100 mpk) for 9 days caused complete repletion of AA in peritoneal cells and plasma (fig. 5). These results showed that dietary AA could selectively circumvent a decrease of AA mediated by 6 desaturase inhibition, supporting the proposed mechanism.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Depletion of arachidonic acid by SC-26196 in mouse peritoneal cells and plasma and reversal by dietary AA. Female Swiss/Webster mice were dosed with vehicle (400 mM glycine-HCl, 40% ethanol) or 10, 30 or 100 mpk SC-26196 (i.g., b.i.d.) for 8 days. In addition, ethyl-arachidonate (30 mg, i.g., q.d.) or ethyl-oleate (30 mg, i.g., q.d.) was coadministered along with vehicle or 100 mpk SC-26196, starting at day 0. After 8 days of dosing, plasma was prepared from blood obtained by retroorbital bleeding. The mice were then killed and peritoneal cells were isolated and pooled within a given group of mice to provide an adequate signal (>106 cells/sample). The fatty acid content of individual plasma samples and pooled peritoneal cell samples was determined by gas chromatography (see "Methods"). Results are expressed as mean ± S.E. for plasma (five mice/group) and mean for peritoneal cells (pooled from five mice/group). The following statistical comparisons were made: 1) vehicle vs. vehicle + AA, 2) vehicle vs. vehicle + OA, 3) 100 mpk SC-26196 + AA vs. vehicle + AA, 4) 100 mpk SC-26196 + OA vs. 100 mpk SC-26196 and 5) slope of response to four doses of SC-26196 (0, 10, 30 or 100 mpk). For comparison 5, the difference in slope should be interpreted as the expected change in the level of AA for a 3-fold increase in the dose of SC-26196. Only comparisons 1 and 5 showed a statistically significant effect (P < .05); 30 mg AA/day increased the level of plasma AA and SC-26196 caused a dose-dependent decrease in the level of plasma AA.

D. Dietary AA restored edema and MPO activity. The antiinflammatory properties conferred by chronic administration of SC-26196 were reversed by coadministration of dietary AA. Mitigation of both edema and MPO activity by SC-26196 (100 mpk) in carrageenan-injected hind paws were reversed dose-dependently by AA (0.03-1 mg/day; fig. 6). There was selectivity with AA because OA, also co-administered with SC-26196 at the highest dose of AA tested (1 mg/day), did not reverse the decreases in edema and MPO activity (fig. 6). These results suggest that SC-26196 blocks de novo synthesis of AA, resulting in accompanying reduction of AA in key lipid pools that are utilized during the inflammatory response in this model.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Dietary arachidonic reverses the antiinflammatory effects of SC-26196. Female Balb/C mice were dosed concurrently with SC-26196 (100 mpk, i.p., b.i.d.) and ethyl-arachidonate (0.01, 0.03, 0.1, 0.3 or 1.0 mg, i.g., q.d.) or ethyl-oleate (1.0 mg/day, i.g., q.d.) for 9 days. As controls, mice were dosed with vehicle (i.p., b.i.d.) alone or concurrently with ethyl-arachidonate or ethyl-oleate (1.0 mg, i.g., q.d.) for 9 days. After 9 days, carrageenan was injected into the hind paws, after which edema and myeloperoxidase (MPO) activity were measured as parameters of inflammation. Results are expressed as mean ± S.E. (five mice/group).

	Discussion

Top Abstract Introduction Methods Results Discussion References

The central hypothesis that chronic desaturase inhibition would lead to an antiinflammatory response by blocking de novo synthesis of AA was validated with the selective 6 desaturase inhibitor, SC-26196. Data in support of a mechanism-based effect with SC-26196 were obtained in the mouse by correlating inhibition of 6 desaturase activity with significant reduction of AA and mitigation of edema in the carrageenan paw edema model. Four key results with SC-26196 support a mechanism-based effect: 1) A dose-dependent relationship existed in vivo between inhibition of 6 desaturase activity and decreases in edema. 2) The onset of the decrease in edema by 6 desaturase inhibition was time dependent, being consistent with a mechanism dependent first on reduction of AA in tissues. 3) A selective, dose-dependent decrease of AA by SC-26196 occurred in liver, plasma and peritoneal cells. 4) Controlled refeeding of AA resulted in repletion of AA in peritoneal cells and plasma and, concomitantly, restored edema and MPO activity in carrageenan-injected hind paws. In contrast, refeeding of oleic acid had no effect on AA repletion, edema, and MPO activity.

A time-dependent decrease in the onset of edema by desaturase inhibition and circumvention by dietary AA suggest that newly synthesized or recently acquired dietary AA is involved in the inflammatory response. The combined results imply that a significant reduction of AA in key lipid pools had to be attained before mitigation of edema was observed and that AA was mobilized rapidly and selectively into or out of discrete lipid species. A selective decrease of AA and a corresponding increase of LA in mouse liver suggested that lipid remodeling was occurring in vivo. A precedent for rapid and preferential acylation/deacylation and accompanying mobilization of newly synthesized AA into or out of discrete lipid pools exists. Notable examples include results showing that recently synthesized AA is primarily released from phospholipids after stimulation (Capriotti et al., 1988) and that mobilization and accompanying rapid remodeling of AA in lipid species occurs during cell activation (Fonteh and Chilton, 1993; Furth et al., 1989). Because PUFAs are incorporated into lipoprotein species that are transported out of the liver in VLDL particles, a decreased plasma level of AA may be primarily responsible for the reduction of AA in peripheral tissues/cells, particularly in leukocytes that cannot synthesize their own AA (Chapkin et al., 1988). Remodeling and not necessarily reduction of AA in lipid species has been shown to affect eicosanoid production (Laposata et al., 1988). Unfortunately, a correlation between reduction of AA and a reduction in eicosanoid production was not established in this study because extraction and quantification of eicosanoids from inflamed or control hind paws were not reproducible.

Reversal of the antiinflammatory properties of SC-26196 selectively by dietary AA suggests the possibility that desaturase inhibition and accompanying attenuation of inflammation could be circumvented by dietary AA. Ingestion of varying amounts of LA or AA in humans and effects on plasma fatty acid composition, desaturase activity, urinary eicosanoid levels and platelet function have been reported (Adam, 1992; Ferretti et al., 1985; James et al., 1993; Kinsella et al., 1990; Lands et al., 1990; Mathias and DuPont, 1985). Three key points were elaborated in these studies: 1) Linoleic acid is the major PUFA consumed in the Western diet; approximately 20 g/day vs. the estimated minimum requirement of 2 to 4 g/day. In sharp contrast, the amount of AA consumed is only approximately 200 mg/day. 2) Few foods contain measurable amounts of AA. Organs and meat, notably poultry, are exceptions (Kinsella et al., 1990; Li et al., 1994; U.S.D.A. Handbook no. 8, Composition of Foods). 3) It is unknown whether the source of AA in tissues is from de novo synthesis (i.e., desaturation and elongation of LA) or directly from dietary AA. Seemingly, only a relatively small amount of dietary AA (1 mg/day) was required to reverse the decreases in edema and MPO activity in the paws of mice dosed with SC-26196 (fig. 6). When scaled allometrically to a human equivalent dose based on caloric intake (energy percentage), 1 mg AA/day in a 20-g mouse consuming 20 kcal/day is approximately equivalent to 100 mg AA/day in a 70-kg human consuming 2000 kcal/day. Based on the estimated dietary intake of 200 mg AA/day in humans, these results suggest that dietary AA could reverse the antiinflammatory effects of chronic desaturase inhibition. Allometric scaling, however, may not accurately predict these effects in mice and humans. It is thus purely conjectural that dietary restrictions to curtail the intake of AA would be required in conjunction with chronic desaturase inhibition in humans.

Reduction of AA by desaturase inhibition and the accompanying antiinflammatory response mimic EFAD. EFAD, however, has never been a viable option for treating chronic inflammatory diseases (Lefkowith et al., 1986a; Holman, 1968). It is too severe in that there is a dramatic remodeling of the fatty acid composition of lipid species. It is plagued with untoward side-effects (e.g., hair loss, impaired fertility, fatty liver and psoriatic-type skin). The necessary depletion of AA can only be attained if weanling animals are maintained on an EFAD diet for a minimum of 8 wk. In contrast, the antiinflammatory effects manifested by inhibition of 6 or 5 desaturase activity may obviate most of the limitations imposed by EFAD because LA, the precursor n-6 essential fatty acid, would be present in the standard Western diet (approximately 20 g consumed per day; Kinsella et al., 1990) and would be incorporated into lipid species. However, chronic inhibition of the 6 desaturase may not be the ideal therapeutic target because LA is not sufficient to ameliorate all of the symptoms of EFAD (Lefkowith et al., 1986a; Holman, 1968) and inadequate 6 desaturase activity has been correlated with chronic inflammatory disease, viz, atopic eczema and diabetic neuropathy (Horrobin, 1993). Also, inhibition of 6 desaturase activity by SC-26196 caused a dose-dependent increase in total fatty acid content and accompanying lipidosis in mouse liver (i.e., fatty liver). It is possible that lipidosis was mediated by decreased levels of n-6 polyunsaturated fatty acids, being analogous mechanistically to lipidosis observed in the livers of EFAD mice. Alternatively, liver lipidosis could have been caused indirectly by toxicity associated with SC-26196.

PG-dependent gastric and renal function could possibly be compromised with a 6 desaturase inhibitor because LA does not serve as a substrate for prostaglandin synthesis via cyclooxygenase (fig. 1). Instead, selective inhibition of the 5 desaturase may be preferred because LA would still be consumed in the diet and metabolized to DGLA, but not to AA. DGLA has been shown to increase markedly and to be accompanied by enhanced synthesis of PGE₁ when it is fed to animals at high levels (Knapp et al., 1978). Mouse peritoneal macrophages, lacking 5 desaturase activity, elongate ¹⁴C-GLA to ¹⁴C-DGLA and, after stimulation, synthesize ¹⁴C-PGE₁, but not ¹⁴C-PGE₂ (Chapkin and Coble, 1991). DGLA could thus serve as the substrate for PGE₁ synthesis in order to maintain gastric and renal function. In addition, beneficial properties have been ascribed to PGE₁, including its ability to reduce platelet aggregation, decrease blood pressure and suppress the immune response (Kirtland, 1988; Zurier, 1990). These issues should be addressed on the identification of 5 desaturase inhibitors that are more amenable for chronic evaluation in vivo.