Particular fatty acids may be effective anti-inflammatory and immunomodulating agents. For example, certain plant seed oils, notably those extracted from the seeds of Oenothera biennis (evening primrose) and Borago officinalis (borage), contain relatively large amounts of gammalinolenic acid. Gammalinolenic acid is converted rapidly to dihomogammalinolenic acid Figure 1, the immediate precursor of prostaglandin E₁, an eicosanoid with known anti-inflammatory and immunoregulating properties [1-4].

View larger version (21K): [in this window] [in a new window]   Figure 1. Metabolic pathway of omega-6 (n-6) fatty acids. The pathway is one of progressive desaturation alternating with elongation (addition of two carbons). 18:2 = 18 carbons, 2 double bonds; (150H) DGLA = 15 hydroxy dihomogamma linolenic acid; LTC3 = leukotriene C3; LTB4 = leukotriene B4; PGE1 = prostaglandin E1; TXA1 = thromboxane A1.

In addition to their role as precursors of prostaglandin and leukotriene (eicosanoids), essential fatty acids are important for the maintenance of cell-membrane structure and function. Our previous studies [5, 6] suggest that dihomogammalinolenic acid can modulate immune responses, in a prostaglandin-independent manner, by acting directly on T cells.

Gammalinolenic acid also suppresses acute and chronic inflammation, including arthritis, in several animal models [7, 8]. Moreover, in a study designed to determine biochemical responses to gammalinolenic acid [9], administration of the fatty acid for 12 weeks (1.1 g/d in borage seed oil) in an open uncontrolled manner appeared to reduce synovitis in six of seven patients with rheumatoid arthritis. Taken together, these observations suggest that enrichment of cells with dihomogammalinolenic acid by administration of gammalinolenic acid may be a worthwhile strategy for the control of inflammation. We therefore did a trial of gammalinolenic acid treatment in patients with rheumatoid arthritis and active synovitis to assess the clinical efficacy and tolerability of gammalinolenic acid.

Methods

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Our study was a 24-week, randomized, double-blind comparison of gammalinolenic acid to placebo in patients with rheumatoid arthritis and with active synovitis. The protocol was reviewed and approved by the Committee on Human Experimentation at the University of Pennsylvania and at the University of Massachusetts and by the Food and Drug Administration. Written, informed consent was obtained from each patient.

Patients were eligible to participate in the study if they had rheumatoid arthritis according to the revised criteria of the American Rheumatism Association [10], were between the ages of 18 and 80 years, had functional class I, II, or III disease [11], and were treated with only aspirin or other nonsteroidal anti-inflammatory drugs or corticosteroids (not to exceed 10 mg/d of prednisone or an equivalent) or both. This represented approximately 10% of patients with rheumatoid arthritis followed at a university hospital rheumatology clinic. Most patients (about 90%) seen had been referred to the clinic, mainly by primary care physicians, orthopedic surgeons, and rheumatologists. Nonsteroidal anti-inflammatory drugs and corticosteroids were maintained at a stable dose for at least 1 month before entry into the study. Patients who had previously received therapy with either gold salts (oral or parenteral), penicillamine, hydroxychloroquine, methotrexate, sulfasalazine, or other second-line agents were eligible only if they had discontinued the medications at least 3 months before entering the study. All patients had disease activity as defined by the presence of at least three of the following four criteria: six or more painful or tender joints; three or more swollen joints; morning stiffness of at least 45 minutes' duration, and a Westergren erythrocyte sedimentation rate of at least 28 mm/h. Patients were instructed to maintain their pre-entry doses of nonsteroidal anti-inflammatory drugs or corticosteroids or both during the study. Medications required for coexisting medical conditions were permitted; these conditions were mild and well controlled. Patients were instructed to maintain their typical diet.

Patients were ineligible for entry if they had been treated with any investigational drug within 1 month of entry, had treatment with commercially available fish or plant seed oil products within 3 months of entry, or had intra-articular injection of corticosteroids within 1 month of entry.

Patients were randomized to receive either gammalinolenic acid or placebo capsules. Each capsule contained 0.6 mL of oil, 13.6 International Units of vitamin E, and provided 5.4 Kcal daily. Gammalinolenic acid was administered as borage seed oil in capsules (Boracelle Capsules with oil of borage, Bio Oil Research Ltd., Nantwich, Cheshire, England). The approximate composition of the borage seed oil was 23% gammalinolenic acid, 62% cis-linoleic acid, 8% oleic acid, and 7% other fatty acids. The total daily dose (four capsules three times daily after meals) provided 1.4 g of gammalinolenic acid. Placebo capsules, identical in appearance and taken according to the same schedule, contained cottonseed oil (Bio Oil Research Ltd.), which was 54% linoleic acid, 18% oleic acid, 24% palmitic acid, and 4% other fatty acids. Capsules were counted at each visit to monitor compliance. Fatty acid analysis of plasma or circulating cells was not used as a measure of compliance because the amount of gammalinolenic acid present would not necessarily reflect the number of capsules ingested. Gammalinolenic acid was administered under an Investigational New Drug approval (#28 867) issued to one of the investigators.

Disease activity, clinically assessed at baseline and at 6-week intervals, included 1) the physician's global assessment of disease activity rated as 0 (none), 1 (mild), 2 (moderate), 3 (severe), or 4 [very severe]; 2) the patient's global assessment of disease activity and pain assessment using the same scale plus a 100-mm visual analog scale for each; 3) the number of joints with tenderness or pain on pressure or passive motion or both [of a total of 68 diarthrodial joints]; 4) the number of joints with swelling [of a total of 66 diarthrodial joints]; 5) joint pain or tenderness score in which each joint was graded on a scale of 0 (none), 1 (mild), 2 (moderate), or 3 [severe]; 6) the joint swelling score, using the same scale; 7) the duration of morning stiffness [min]; 8) the ability to do vocational activity and household chores rated as 0 (without difficulty), 1 (with minimal difficulty), 2 (with difficulty), 3 [with great difficulty]; 9) the grip strength (mm Hg) in each hand using a sphygmomanometer inflated initially to 20 mm Hg.

A complete blood cell count (including differential cell count and platelet count), blood chemistry profile, urinalysis, and rheumatoid factor were measured at baseline and at week 24. The erythrocyte sedimentation rate was determined at baseline and at weeks 12 and 24. Radiographs were not used to assess disease progression because it is unlikely that changes would be detected during the relatively short (24 weeks) period of the study.

Patients were asked about adverse experiences at each visit. Criteria for patient withdrawal from the study included 1) adverse drug reaction that required missed doses for more than 7 days; 2) violation of study protocol; 3) the patient's desire to withdraw from the study; or 4) onset of a serious medical condition.

Categories of Response

The following definitions were used to characterize changes for several clinical measures. Important improvement in any measure was defined as at least a 25% improvement in continuous variables or an improvement of two levels or from a baseline value of 1 to a value of 0 in the scaled variables. Important worsening in any measure was defined as at least a 30% worsening in continuous variables or a worsening of two levels or from a baseline of 3 to 4 in the scaled variables.

Patients completing the study were classified into one of four overall response categories as described by several investigators [12-15]: remission, meaningful improvement, no meaningful change, or deterioration. A patient was considered in remission if global assessments by physician and patient were 0, no joints were tender or swollen, and the erythrocyte sedimentation rate was less than 20 mm/h for at least 2 consecutive months. Overall meaningful improvement (or deterioration) occurred if the patient had important improvement (or important worsening) in at least four of the following measures: joint tenderness count or score, joint swelling count or score, global assessment by physician, global assessment by patient, morning stiffness, and erythrocyte sedimentation rate.

Statistical Analysis

Statistical analysis was done using chi-square testing (with Yates correction for continuity when appropriate), Mann-Whitney U-test for comparisons between groups, and the Wilcoxon signed-rank test for within-group comparisons of final visit to baseline data. Nonparametric tests were used because most variables were not normally distributed. Data were analyzed for both the entire group of patients enrolled in the study and for patients who completed the study. Results are expressed as means ± standard deviation. Statistical significance was defined as P < 0.05, measured in a two-tailed manner. We used SPS + software (SPS, Inc., Chicago, Illinois) for the analysis. Overall responses were compared using the Fisher exact test.

Results

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Of 37 patients enrolled in the study, 19 received gammalinolenic acid and 18 received placebo. Demographic and clinical characteristics, laboratory data, and treatment at entry were similar for the two groups (Table 1). Although more patients in the placebo group had previously taken second-line agents, only hydroxychloroquine reached statistical significance. Five patients in each group withdrew from the study because of lack of efficacy (two patients in the placebo group and three in the gammalinolenic-acid group), rash (one placebo-treated patient), and violation of the study protocol (two patients from each group).

Clinical responses are presented as absolute changes and percentage changes (Table 2). All patients entered were analyzed (data not shown), and the group treated with gammalinolenic acid showed significant improvement from baseline in the joint tenderness score, joint swelling score, and platelet count. The placebo group did not have a significant improvement in any measure. When compared with patients treated with placebo, those treated with gammalinolenic acid treatment had significantly better joint tenderness counts and scores, joint swelling scores, global assessments by physician, pain by scaled assessment, and reduction of platelet count.

The group of patients who completed the study (27 patients) included 14 patients in the gammalinolenic-acid group and 13 in the placebo group. Demographic characteristics did not differ between the two groups but baseline measures of morning stiffness and joint tenderness score were significantly higher in the gammalinolenic-acid group (data not shown). Whereas the placebo group did not show significant improvement in any measure, the gammalinolenic acid treatment group showed significant improvement at week 24, compared with baseline, in joint tenderness count and score, joint swelling count and score, global assessment by physician, pain assessment by visual analog, morning stiffness, and platelet count (Table 2). The degree of improvement was moderate; the tender joint count was reduced by a mean of 36%; the tenderness score by 45%; the swollen joint count by 28%; the swollen joint score by 41%; and morning stiffness by 33%. When compared with placebo, gammalinolenic acid treatment was significantly superior in joint tenderness count and score Table 2, Figure 2, joint swelling count and score Table 2, Figure 3, global assessment by physician, and pain by visual analog and by scaled assessment (Table 2).

View larger version (21K): [in this window] [in a new window]   Figure 2. Joint tenderness scores in patients completing the study. Mean percentage change from baseline at each evaluation for placebo (; n = 13) and gammalinolenic acid (GLA) treatment (\#9632;;n = 14) groups. Error bars are ± 1 standard error of the mean.

View larger version (20K): [in this window] [in a new window]   Figure 3. Joint swelling scores in patients completing the study. Mean percentage change from baseline at each evaluation for placebo (; n = 13) and gammalinolenic acid (GLA) treatment (\#9632;;n = 14) groups. Error bars are ± 1 standard error of the mean.

Adverse reactions included soft stools (two patients in the gammalinolenic acid group, one patient in the placebo group), constipation (one patient in the placebo group), flatulence (one gammalinolenic-acid-treated patient), and belching (one patient treated with gammalinolenic acid). No patients in the gammalinolenic acid treatment group withdrew from the study because of adverse reactions.

No patients went into remission during the study. In the gammalinolenic acid-treatment-group, seven patients had meaningful improvement (important improvement in at least four measures as described in the Methods section), and seven had no meaningful change. No patient in the gammalinolenic-acid-treatment group showed deterioration (Table 3). In the placebo group, one patient had meaningful improvement, eight patients had no meaningful change, and four patients met criteria for deterioration. When the overall response data were collapsed into meaningful improvement and no improvement (including no meaningful change and deterioration), seven patients treated with gammalinolenic acid improved and seven did not. In contrast, one patient in the placebo group improved and 12 did not (P = 0.02; gammalinolenic acid compared with placebo).

Discussion

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Treatment with 1.4 g/d of gammalinolenic acid (in borage seed oil) for 6 months resulted in clinically relevant and statistically significant reduction in signs and symptoms of disease activity in patients with rheumatoid arthritis. In contrast, patients given a placebo (cottonseed oil) showed worsening or no significant improvement of disease activity. Most patients were treated with nonsteroidal anti-inflammatory drugs, and some patients were taking prednisone, as much as 10 mg/d; the trial did not include any patients taking second-line drugs such as gold or methotrexate.

Patients who completed 24 weeks of gammalinolenic acid treatment showed statistically significant improvement in global assessment by physician, patient assessment of pain using both a 5-point scale and visual analog scale, ability to perform vocational tasks, number of tender and swollen joints, and associated tender and swollen joint scores. Data analyzed from all patients entered into the study, regardless of whether they completed the protocol, confirmed that the results seen in the patients treated with gammalinolenic acid were not caused by bias introduced by patients leaving the study prematurely. Overall clinical responses were also significantly better in the gammalinolenic acid treatment group than in the placebo group. The three patients in the gammalinolenic acid group who withdrew because of lack of efficacy all did so within the first 2 months of the study. Because the therapeutic effects of gammalinolenic acid usually are not apparent for at least 6 to 12 weeks (see Figures 2 and 3) [18], a favorable clinical response might not be expected in these patients.

Although adverse effects of gammalinolenic acid administration were negligible, potential problems cannot be dismissed. Administration of long-chain polyunsaturated fatty acids increases the likelihood of lipid peroxidation with its associated toxic effects on cells. It is not known whether an increased requirement for an antioxidant (such as vitamins E and C) accompanies increased intake of long-chain unsaturated fatty acids. Our own experiments (Baker D, Tate G, Zurier RB. Unpublished data) indicate that no increase in lipid peroxidation occurs after addition of dihomogammalinolenic acid to human cells in tissue culture. Vitamin E, which alone does not benefit rheumatoid patients [16], was added to our gammalinolenic-acid and placebo preparations.

Lower doses of gammalinolenic acid (480 to 540 mg/d), used in previous studies, were either not effective [17] or reduced pain only, without an effect on objective physical findings [18]. Clearly, long-term, multicenter, placebo-controlled studies of large numbers of patients are needed to determine whether any form of treatment is useful for patients with rheumatoid arthritis. The results of our study suggest, however, that a higher dose of gammalinolenic acid (1.4 g/d) is necessary to reduce active synovitis in patients with rheumatoid arthritis. Indeed, the relatively rapid response to gammalinolenic acid seen in some patients at 6 weeks of treatment (see Figures 2 and 3) may have been caused by the more substantial dose of gammalinolenic acid than was used in previous studies in which patients did not appear to respond until 12 to 16 weeks of therapy [18]. Although a positive response to placebo might be expected during the first few weeks of the study, continued improvement in the placebo group after 1 month would not be anticipated [12]. The beneficial effects of gammalinolenic acid were sustained during 24 weeks of treatment, whereas patients in the placebo group, as expected, had greater variability in their clinical findings and had progressive worsening of disease activity (Figures 2 and 3). Some patients in the placebo group did respond favorably, but these individual responses are not reflected in the mean values for the entire group. It is not possible to discount entirely the notion that the placebo (cottonseed oil) increases signs and symptoms of synovitis in patients with rheumatoid arthritis. However, analysis of the overall responses indicates that most patients in the placebo group did not deteriorate (see Table 3).

Our results indicate that gammalinolenic acid can reduce joint swelling and tenderness in patients with active rheumatoid arthritis and suggest that administration of gammalinolenic acid is a worthwhile strategy for treatment of chronic synovitis. Gammalinolenic acid is generally available in health food stores in preparations of evening primrose and borage seed oils. Capsules usually contain approximately 45 mg of gammalinolenic acid so that the amount of the fatty acid taken is usually far less than used in this study. Although gammalinolenic acid treatment of atopic eczema is approved by the National Health Service in the United Kingdom, gammalinolenic acid is not approved therapy for any condition in the United States. When given in doses used in this study, it should be administered by a physician to patients in an approved study protocol. Further controlled studies of gammalinolenic acid treatment of patients with rheumatoid arthritis are warranted.

We did not address mechanisms responsible for the anti-inflammatory effects of gammalinolenic acid in this clinical trial, but several may be possible. The time-honored putative mechanism for the anti-inflammatory effects of both marine and botanical lipids is through a dual action to reduce inflammatory products of arachidonic acid oxygenation and to form less biologically active eicosanoids [3]. Gammalinolenic acid is converted rapidly to dihomogammalinolenic acid, the immediate precursor of the monoenoic prostaglandin E₁, which has potent anti-inflammatory activity [1]. In addition, dihomogammalinolenic acid cannot be converted to inflammatory leukotrienes by 5-lipoxygenase; instead, it is converted to 15-hydroxy-dihomogammalinolenic acid (see Figure 1), which in turn suppresses 5- and 12-lipoxygenase activity [19].

Prostaglandin E₁ has a range of desirable effects distinct from other prostaglandins that might be used therapeutically [20]. An approach to prostaglandin E₁ therapy, first suggested by Willis [21], is provision of the prostaglandin E₁ precursors gammalinolenic acid and dihomogammalinolenic acid. The extremely short half-lives of natural prostaglandins have a purpose: they allow moment-to-moment regulation of cell function in response to external stimuli and internal messengers. Exogenous administration of prostaglandin E₁, which may be useful in the short term [22], cannot mimic the physiologic variations of endogenous prostaglandin E₁. Enrichment of cells with dihomogammalinolenic acid should enable prostaglandin E₁ concentrations to be raised as needed without overriding the physiologic controls that modulate rapid changes in its synthesis and degradation. Dihomogammalinolenic acid suppresses interleukin-1 induced proliferation of human synovial cells in culture by virtue of increasing synthesis of prostaglandin E₁ and subsequent concentrations of cellular cyclic AMP [4]. Administration of gammalinolenic acid to human volunteers and to patients with rheumatoid arthritis does result in increased dihomogammalinolenic acid in phospholipids of plasma, mononuclear cells, and platelets [9], increased prostaglandin E₁ production by stimulated monocytes [3], and marked reduction in prostaglandin E₂ and leukotriene B₄ synthesis [9]. Moreover, cellular arachidonic acid is not increased appreciably after gammalinolenic acid administration [9].

We have shown previously [9] that leukocyte prostaglandin E production is not suppressed completely by nonsteroidal anti-inflammatory drug therapy. Nonetheless, it may be asked why nonsteroidal anti-inflammatory drug treatment does not block the therapeutic effect of gammalinolenic acid. In addition to their role as eicosanoid precursors, fatty acids are of major importance in maintaining cell membrane structure and are key determinants of the behavior of membrane-bound enzymes and receptors [23]. The fatty acid precursors can exert these functions directly and therefore may themselves be important regulators of immune responses. For example, dihomogammalinolenic acid suppresses interleukin-2 production by human peripheral blood mononuclear cells in vitro, suppresses proliferation of interleukin-2-dependent T lymphocytes, and reduces expression of activation markers on T lymphocytes directly, in a manner independent of its conversion to prostaglandins [5, 6]. These observations indicate that fatty acids can modulate immune responses by acting directly on T cells and suggest that alteration of cellular fatty acids may be a worthwhile approach to control inflammation. Other relevant direct effects of unsaturated fatty acids include regulation of ion channels [24] and suppression of smooth muscle cell proliferation [25].

The potential regulation of cell activation, immune responses, and inflammation by particular fatty acids is exciting to consider at the clinical, cellular, and molecular levels. A better understanding of how fatty acids modulate function of cells involved in host defense might lead to development of new, benign treatment for diseases such as rheumatoid arthritis that are characterized by acute and chronic inflammation.