9-Carboxymethyl-5H,10H-imidazo[1,2-a]indeno[1,2-e]pyrazin-4-one-2-carbocylic acid (RPR117824): selective anticonvulsive and neuroprotective AMPA antagonist

Excessive release of glutamate, a potent excitatory neurotransmitter, is thought to play an important role in a variety of acute and chronic neurological disorders, suggesting that excitatory amino acid antagonists may have broad therapeutic potential in neurology. Here, we describe the synthesis, pharmacological properties and neuroprotective activity of 9-carboxymethyl-imidazo-[1-2a]indeno[1-2e]pyrazin-4-one-2-carboxylic acid (RPR117824), an original selective AMPA antagonist. RPR117824 can be obtained through a six-step synthesis starting from (1-oxo-indan-4-yl) acetic acid, which has been validated on a gram-scale with an overall yield of 25%. Monosodium or disodium salts of the compound exhibit excellent solubility in saline (> or = 10 g/L), enabling intravenous administration. RPR117824 displays nanomolar affinity (IC(50)=18 nM) for AMPA receptors and competitive inhibition of electrophysiological responses mediated by AMPA receptors heterologously expressed in Xenopus oocytes (K(B)=5 nM) and native receptors in rat brain slices (IC(50)=0.36 microM). In in vivo testing, RPR117824 behaves as a powerful blocker of convulsions induced in mice or rats by supramaximal electroshock or chemoconvulsive agents such as pentylenetetrazole, bicuculline, isoniazide, strychnine, 4-aminopyridine and harmaline with half maximal effective doses ranging from 1.5 to 10 mg/kg following subcutaneous or intraperitoneal administration. In disease models in rats and gerbils, RPR117824 possesses significant neuroprotective activity in global and focal cerebral ischemia, and brain and spinal cord trauma.     

Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints

The synovium from patients with rheumatoid arthritis (RA) and LEW/N rats with streptococcal cell wall (SCW) arthritis, an experimental model resembling RA, is characterized by massive proliferation of synovial connective tissues and invasive destruction of periarticular bone and cartilage. Since heparin binding growth factor (HBGF)-1, the precursor of acidic fibroblast growth factor (FGF), is a potent angiogenic polypeptide and mitogen for mesenchymal cells, we sought evidence that it was involved in the synovial pathology of RA and SCW arthritis. HBGF-1 mRNA was detected in RA synovium using the polymerase chain reaction technique, and its product was immunolocalized intracellularly in both RA and osteoarthritis (OA) synovium. HBGF-1 staining was more extensive and intense in synovium of RA patients than OA and correlated with the extent and intensity of synovial mononuclear cell infiltration. HBGF-1 staining also correlated with c-Fos protein staining. In SCW arthritis, HBGF-1 immunostaining was noted in bone marrow, bone, cartilage, synovium, ligamentous and tendinous structures, as well as various dermal structures and developed early in both T-cell competent and incompetent rats. Persistent high level immunostaining of HBGF-1 was only noted in T-cell competent rats like the disease process in general. These observations implicate HBGF-1 in a multitude of biological functions in inflammatory joint diseases.     

Production of interleukin (IL)-1beta, IL-1 receptor antagonist and IL-10 by blood mononuclear cells in chronic arthritis

Joint erosion is a prevalent feature of rheumatoid arthritis (RA) but not of many other chronic inflammatory arthritides (non-RA). Joint destruction is mediated by cytokines, primarily interleukin (IL)-1 and tumour necrosis factor. Less erosive activity in patients with non-RA compared to RA might be related to factors that inhibit production and/or function of IL-1. Release of IL-1beta, and the two antagonists, IL-1 receptor antagonist (IL-1ra) and IL-10 from blood mononuclear cells were therefore quantitated by ELISA in 22 patients with RA, 11 with non-RA and 15 healthy age-matched controls. Release of IL-1beta was comparable between the three groups but only detectable in cultures stimulated with lipopolysaccharide; it decreased in patients treated with prednisolone: 3.8 ng/10(6)monocytes (median) vs 11.7 (P=0.045). Release of IL-1ra was in all but IgG-stimulated cultures comparable between groups. The ratio of IL-1ra/IL-1beta was elevated in LPS-stimulated cells from RA patients only: 2.0 versus 1.3 (P=0.02). In contrast, IgG-induced IL-1ra release was significantly elevated only in non-RA patients: 95 ng/10(6)monocytes vs 40 (P=0.014), and the levels correlated positively to those of blood CRP (P=0.02). Though stimulated release of IL-10 was similar between the three groups, the levels were lower in non-erosive than erosive arthritis patients, and controls (P=0. 05). In conclusion, increased IgG-stimulated IL-1ra release and elevated IL-1ra/IL-1beta ratio may protect against actions of IL-1 in vivo, and decreased release of IL-10 might be related to features of non-erosive arthritis.     

RPR203494 a pyrimidine analogue of the p38 inhibitor RPR200765A with an improved in vitro potency

Following the discovery of RPR200765, a series of pyrimidine analogues have been prepared as backups. Amongst them, RPR203494 was identified with a better in vitro profile than RPR200765A.     

Urokinase,a constitutive component of the inflamed synovial fluid,induces arthritis

Urokinase plasminogen activator (uPA) is an important regulator of fibrinolysis in synovial fluid. An increase of uPA activity and expression of its receptor have been reported in joints of patients with rheumatoid arthritis (RA). The aim of the present study was to assess the arthritogenic capacity of uPA and the mechanisms by which this effect is mediated. uPA was injected into the knee joints of healthy mice, and morphological signs of arthritis were assessed 4 days after the injection. The prerequisite of different leukocyte populations for the development of uPA-triggered arthritis was assessed by selective cell depletion. The inflammatory capacity of uPA was assessed in vitro. Finally, levels of uPA were measured in 67 paired blood and synovial fluid samples from RA patients. The synovial fluid from RA patients displayed higher levels of uPA compared with blood samples. Morphological signs of arthritis were found in 72% of uPA-injected joints compared with in only 18% of joints injected with PBS (P < 0.05). Synovitis was characterised by infiltration of CD4-Mac-1+ mononuclear cells, by the formation of pannus and by occasional cartilage destruction. The absence of monocytes and lymphocytes diminished the frequency of synovitis (P < 0.01), indicating an arthritogenic role of both these leukocyte populations. Synthetic uPA inhibitor downregulated the incidence of uPA-triggered arthritis by 50%. uPA induced arthritis, stimulating the release of proinflammatory cytokines IL-6, IL-1beta and tumour necrosis factor alpha. Accumulation of uPA locally in the joint cavity is a typical finding in erosive RA. uPA exerts potent arthritogenic properties and thus may be viewed as one of the essential mediators of joint inflammation.     

A potent and selective nonpeptide antagonist of CXCR2 inhibits acute and chronic models of arthritis in the rabbit

Much evidence implicates IL-8 as a major mediator of inflammation and joint destruction in rheumatoid arthritis. The effects of IL-8 and its related ligands are mediated via two receptors, CXCR1 and CXCR2. In the present study, we demonstrate that a potent and selective nonpeptide antagonist of human CXCR2 potently inhibits (125)I-labeled human IL-8 binding to, and human IL-8-induced calcium mobilization mediated by, rabbit CXCR2 (IC(50) = 40.5 and 7.7 nM, respectively), but not rabbit CXCR1 (IC(50) = >1000 and 2200 nM, respectively). These data suggest that the rabbit is an appropriate species in which to examine the anti-inflammatory effects of a human CXCR2-selective antagonist. In two acute models of arthritis in the rabbit induced by knee joint injection of human IL-8 or LPS, and a chronic Ag (OVA)-induced arthritis model, administration of the antagonist at 25 mg/kg by mouth twice a day significantly reduced synovial fluid neutrophils, monocytes, and lymphocytes. In addition, in the more robust LPS- and OVA-induced arthritis models, which were characterized by increased levels of proinflammatory mediators in the synovial fluid, TNF-alpha, IL-8, PGE(2), leukotriene B(4), and leukotriene C(4) levels were significantly reduced, as was erythrocyte sedimentation rate, possibly as a result of the observed decreases in serum TNF-alpha and IL-8 levels. In vitro, the antagonist potently inhibited human IL-8-induced chemotaxis of rabbit neutrophils (IC(50) = 0.75 nM), suggesting that inhibition of leukocyte migration into the knee joint is a likely mechanism by which the CXCR2 antagonist modulates disease.     

The discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1 ,5-a]-pyrimidine: a corticotropin-releasing factor (hCRF1) antagonist

Structure activity relationship studies led to the discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazo lo-[1,5-a]-pyrimidine 11-31 (DMP904), whose pharmacological profile strongly supports the hypothesis that hCRF1 antagonists may be potent anxiolytic drugs. Compound 11-31 (hCRF1 Ki = 1.0+/-0.2 nM (n = 8)) was a potent antagonist of hCRF1-coupled adenylate cyclase activity in HEK293 cells (IC50= 10.0+/-0.01 nM versus 10 nM r/hCRF, n = 8); alpha-helical CRF(9-41) had weaker potency (IC50 = 286+/-63 nM, n = 3). Analogue 11-31 had good oral activity in the rat situational anxiety test; the minimum effective dose for 11-31 was 0.3 mg/kg (po). Maximal efficacy (approximately 57% reduction in latency time in the dark compartment) was observed at this dose. Chlordiazepoxide caused a 72% reduction in latency at 20 mg/kg (po). The literature compound 1 (CP154526-1, 30 mg/kg (po)) was inactive in this test. Compound 11-31 did not inhibit open-field locomotor activity at 10, 30, and 100 mg/kg (po) in rats. In beagle dogs, this compound (5 mg/kg, iv, po) afforded good plasma levels. The key iv pharmacokinetic parameters were t1/2, CL and Vd,ss values equal to 46.4+/-7.6 h. 0.49+/-0.08 L/kg/h and 23.0+/-4.2 L/kg, respectively. After oral dosing, the mean Cmax, Tmax t1/2 and bioavailability values were equal to 1260+/-290 nM, 0.75+/-0.25 h. 45.1+/-10.2 h and 33.1%, respectively. The overall rat behavioral profile of this compound suggests that it may be an anxiolytic drug with a low motor side effect liability.     

Therapeutic effects of lactosyl derivative Gu-4 in a collagen-induced arthritis rat model

Rheumatoid arthritis (RA) is an inflammatory disorder that is characterized by persistent recurrence of joint inflammation leading to cartilage and bone destruction. The present anti-arthritis therapies failed to achieve satisfactory remission in all patients; therefore, it is still necessary to develop novel approaches to fulfill the demand in clinic. Here, we reported the therapeutic effects of lactosyl derivative Gu-4, a synthetic compound that was previously identified as a selective inhibitor against leukocyte integrin CD11b, in a bovine type II collagen induced arthritis (CIA) rat model. First, prophylactic administration of Gu-4 (1.2728?mg/kg) to rats by intraperitoneal injection every 2?days from the first day of collagen immunization significantly decreased the incidence of CIA, diminished the mean paw volume increase, and reduced the number of swollen paws. Second, administration of Gu-4 (1.2728?mg/kg) to rats at early-onset stage of CIA prevented the progression of the pathological process of RA, accelerated the remission of paw edema, and declined the arthritis score; after 5?weeks treatment, X-ray and histological examinations were carried out, the ankle joint of hind limb of Gu-4 treated CIA rats exhibited slighter bone erosion and much less inflammatory cell infiltration compared to those of saline treated animals; furthermore, Gu-4 remarkably attenuated the production of rheumatoid factor (RF) in the serum of CIA rats as determined by ELISA. Moreover, we performed in vitro lymphocyte proliferation assay and found that Gu-4 significantly inhibited the proliferation of splenic lymphocytes isolated from CIA rats in a dose-dependent manner. Our results suggest that Gu-4 can effectively ameliorate CIA and might be an alternative option for the treatment of RA.     

The human renin infused rat: use as an in vivo model for the biological evaluation of human renin inhibitors

The human renin infused rat model (HRIRM) was used as an in vivo small-animal model for evaluating the efficacy of a collection of inhibitors of human renin. The intravenous infusion of recombinant human renin (2.4 microg x kg(-1) x min(-1)) in the ganglion-blocked, nephrectomized rat produced a mean blood pressor response of 47+/-3 mm Hg (1 mm Hg = 133.3 Pa), which was reduced by captopril, enalkiren, and losartan in a dose-dependent manner following oral administration, with ED50 values of 0.3+/-0.1, 2.5+/-0.9, and 5.2+/-1.6 mg/kg, respectively. A series of peptidomimetic P2-P3 butanediamide renin inhibitors inhibited purified recombinant human renin in vitro in a concentration-dependent manner, with IC50 values ranging from 0.4 to 20 nM at pH 6.0, with a higher range of IC50 values (0.8-80 nM) observed at pH 7.4. Following i.v. administration of renin inhibitors, the pressor response to infused human renin in the HRIRM was inhibited in a dose-dependent manner, with ED50 values ranging from 4 to 600 microg/kg. The in vivo inhibition of human renin following i.v. administration in the rat correlated significantly better with the in vitro inhibition of human renin at pH 7.4 (r = 0.8) compared with pH 6.0 (r = 0.5). Oral administration of renin inhibitors also resulted in a dose-dependent inhibition of the pressor response to infused human renin, with ED50 values ranging from 0.4 to 6.0 mg/kg and the identification of six renin inhibitors with an oral potency of <1 mg/kg. The ED50 of renin inhibitors for inhibition of angiotensin I formation in vivo was highly correlated (r = 0.9) with the ED50 for inhibition of the pressor response. These results demonstrate the high potency, dose dependence, and availability following oral administration of the butanediamide series of renin inhibitors.     

Oral buprenorphine is anti-inflammatory and modulates the pathogenesis of streptococcal cell wall polymer-induced arthritis in the Lew/SSN rat

This study was carried out to determine an effective regimen for pain management in streptococcal cell wall (SCW)-induced arthritis in female Lew/SSN rats. Forty weanling rats lin 2 groups) were trained to accept disks of jelly as part of their dietary regimen. At 8 weeks of age weighing 150 g, SCW arthritis was induced and sublingual buprenorphine tablets were incorporated into the jelly disks to alleviate the pain of acute arthritis, which developed 24 h post-induction. Group A rats received buprenorphine at a rate of 1 mg/kg 12 hourly. Group B rats received buprenorphine at a rate of 2 mg/kg 12 hourly. Both groups of rats were monitored for symptoms of distress using an adaptation of the Morton and Griffin scale of adverse reactions. Group A rats with severe arthritis required additional subcutaneous (s.c.) injections of buprenorphine to alleviate the adverse effects of arthritis. Group B rats, with twice the dose of buprenorphine did not require additional s.c. injections of buprenorphine. Histological sections of rat hocks indicated that the inflammation was suppressed in Group B rats. We concluded that oral administration of buprenorphine is an effective method of pain management in the pathogenesis of SCW-induced arthritis in Lew/SSN rats. In this model of arthritis, oral buprenorphine has a significant anti-inflammatory effect and appears to modulate the destructive arthritic phase in joints in this animal model of arthritis.     

New non-steroidal aromatase inhibitors: focus on R76713

R76713 is a novel triazole derivative which selectively blocks the cytochrome P450-dependent aromatase. In human placental microsomes, in FSH-stimulated rat and human granulosa cells and in human adipose stromal cells, 50% inhibition of estradiol biosynthesis was obtained at drug concentrations of 2-10 nM. In PMSG-injected female rats, R76713 lowered plasma estradiol levels by 50 and 90% 2 h after single oral doses of 0.005 and 0.05 mg/kg respectively. After 1 mg/kg, estradiol levels were suppressed by 90% for 16 h. In male cynomolgus monkeys, R76713 dose-dependently (0.03-10 micrograms/kg) inhibited peripheral aromatization with an ED50 of 0.13 microgram/kg without altering metabolic clearance rates and conversion ratios. In vitro R76713 had no effect on other P450-dependent steroidogenic enzymes up to 1000 nM at least. In rats, LHRH-, ACTH- and sodium-deprived diet stimulated plasma testosterone, corticosterone and aldosterone levels were not modified 2 h after single oral administrations of R76713 (up to 20 mg/kg). Furthermore, R76713 did not show any in vitro or in vivo estrogenic or antiestrogenic property. R76713 also induced regression of DMBA-induced mammary tumors after daily oral administration of 1 mg/kg b.i.d. In male volunteers (n = 4), a single oral dose of 5 and 10 mg lowered median plasma estradiol levels from 70 pM to the detection limit of the assay (40 pM) 4, 8 and 24 h after intake whereas no changes were detected after placebo administration. In premenopausal women (n = 15), receiving a single oral dose of 20 mg, median plasma estradiol levels decreased from 389 pM (before) to 168, 133 and 147 pM, 4, 8 and 24 h after intake whereas they remained above 420 pM after placebo (n = 7).     

Urokinase,a constitutive component of the inflamed synovial fluid,induces arthritis

Urokinase plasminogen activator (uPA) is an important regulator of fibrinolysis in synovial fluid. An increase of uPA activity and expression of its receptor have been reported in joints of patients with rheumatoid arthritis (RA). The aim of the present study was to assess the arthritogenic capacity of uPA and the mechanisms by which this effect is mediated. uPA was injected into the knee joints of healthy mice, and morphological signs of arthritis were assessed 4 days after the injection. The prerequisite of different leukocyte populations for the development of uPA-triggered arthritis was assessed by selective cell depletion. The inflammatory capacity of uPA was assessed in vitro. Finally, levels of uPA were measured in 67 paired blood and synovial fluid samples from RA patients. The synovial fluid from RA patients displayed higher levels of uPA compared with blood samples. Morphological signs of arthritis were found in 72% of uPA-injected joints compared with in only 18% of joints injected with PBS (P < 0.05). Synovitis was characterised by infiltration of CD4^-Mac-1⁺ mononuclear cells, by the formation of pannus and by occasional cartilage destruction. The absence of monocytes and lymphocytes diminished the frequency of synovitis (P < 0.01), indicating an arthritogenic role of both these leukocyte populations. Synthetic uPA inhibitor downregulated the incidence of uPA-triggered arthritis by 50%. uPA induced arthritis, stimulating the release of proinflammatory cytokines IL-6, IL-1β and tumour necrosis factor alpha. Accumulation of uPA locally in the joint cavity is a typical finding in erosive RA. uPA exerts potent arthritogenic properties and thus may be viewed as one of the essential mediators of joint inflammation.     

NT-702 (parogrelil hydrochloride, NM-702), a novel and potent phosphodiesterase inhibitor, improves reduced walking distance and lowered hindlimb plantar surface temperature in a rat experimental intermittent claudication model

NT-702 (parogrelil hydrochloride, NM-702), 4-bromo-6-[3-(4-chlorophenyl)propoxy]-5-[(pyridin-3-ylmethyl)amino]pyridazin-3(2H)-one hydrochloride, a novel phosphodiesterase (PDE) inhibitor synthesized as a potent vasodilatory and antiplatelet agent, is being developed for the treatment of intermittent claudication (IC) in patients with peripheral arterial disease. We assessed the efficacy of NT-702 in an experimental IC model as compared with cilostazol and additionally investigated the pharmacological property in vitro and ex vivo. NT-702 selectively inhibited PDE3 (IC(50)=0.179 and 0.260 nM for PDE3A and 3B) more potently than cilostazol (IC(50)=231 and 237 nM for PDE3A and 3B) among recombinant human PDE1 to PDE6. NT-702 inhibited in vitro human platelet aggregation induced by various agonists (IC(50)=11 to 67 nM) and phenylephrine-induced rat aortic contraction (IC(50)=24 nM). Corresponding results for cilostazol were 4.1 to 17 microM and 1.0 microM, respectively. NT-702 (3 mg/kg or more) significantly inhibited ex vivo rat platelet aggregation after a single oral dose. For cilostazol, 300 mg/kg was effective. In a rat femoral artery ligation model, NT-702 at 5 and 10 mg/kg repeated oral doses twice a day (BID) for 13 days significantly improved the reduced walking distance while the lowered plantar surface temperature was improved at 2.5 mg/kg and more. Cilostazol also improved the walking distance and surface temperature at 300 mg/kg BID but significant difference was only observed for surface temperature on day 8. These results suggest that NT-702 can be expected to have therapeutic advantage for IC.     

Prolactin increases tumor necrosis factor alpha expression in peripheral CD14 monocytes of patients with rheumatoid arthritis

Tumor necrosis factor (TNF)-α is one of the major proinflammatory mediators of rheumatic arthritis (RA); the regulatory factors for TNF-α release is not fully understood. This study aims to investigate the role of prolactin receptor (PRLR) activation in regulating the expression and release of TNF-α from CD14⁺ monocytes. The results showed that the expression of PRLR was detectable in CD14⁺ monocytes of healthy subjects, which was markedly increased in RA patients. Exposure to PRL in the culture increased the expression and release of TNF-α from CD14⁺ monocytes, which was abolished by the PRLR gene silencing or blocking the mitogen activated protein (MAPK) pathway. We conclude that exposure to PRL increases TNF-α release from CD14⁺ monocytes of RA patients, which can be abolished by PRLR gene silencing or treating with MAPK inhibitor.     

Antagonism of peptidoleukotrienes and inhibition of systemic anaphylaxis by RG 12525 in guinea pigs

RG 12525 was determined to be a specific, competitive and orally effective antagonist of the peptidoleukotrienes, LTC4, LTD4 and LTE4, in several assays utilizing guinea pigs. In vitro, RG 12525 competitively inhibited 3H-LTD4 binding to lung membranes (Ki = 3.0 +/- 0.3 nM) and competitively antagonized the spasmogenic activity of LTC4, LTD4 and LTE4 on lung strips (KB values = 3 nM) with greater than 8000 fold selectivity. In vivo, RG 12525 orally inhibited LTD4 induced wheal formation (ED50 = 5 mg/kg with a t1/2 = 10 hrs at 9 mg/kg), LTD4 induced bronchoconstriction (ED50 = 0.6 mg/kg), and anaphylactic death (ED50 = 2.2 mg/kg with a t1/2 = 7 hrs at 10 mg/kg) and antigen induced bronchoconstriction (ED50 = 0.6 mg/kg). RG 12525 represents a significant improvement in receptor affinity and oral efficacy and thus, is a valuable pharmacological tool to evaluate peptidoleukotrienes in allergic diseases.     

CP-481,715, a potent and selective CCR1 antagonist with potential therapeutic implications for inflammatory diseases

The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5'-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases.     

More than just B-cell inhibition

Despite tremendous advances in the therapy of rheumatoid arthritis (RA), there remains interest in oral agents that may offer benefits that are similar to, or better than, those of biologic therapies. In their paper, Chang and colleagues demonstrate the effectiveness of a Bruton tyrosine kinase (Btk) inhibitor in two models of RA. Btk inhibition impacts several pathways affecting both B-cell and macrophage activation, making it a promising target in RA. However, other kinase inhibitors have failed to transition from animal models to human therapy, so it remains to be seen whether a Btk inhibitor will have a role in the RA treatment armamentarium.     

Interleukin-1 production by mononuclear cells from rheumatoid synovial effusions

The polypeptide interleukin-1 (IL-1) is a cytokine that may mediate inflammation and connective tissue damage in rheumatoid arthritis (RA). We examined cytokine production by normal blood and by rheumatoid synovial mononuclear cells with sensitive (picomolar) assays. The assays were immunolabeling and immunoblotting with rabbit anti-IL-1 beta sera, and proliferation of the murine D10 cell line to IL-1. Little or no cytokine was detected in rheumatoid joint fluid or in exudate mononuclear cells from patients with acute rheumatoid flares. The mononuclear cells could be induced to make IL-1 upon stimulation with lipopolysaccharide (LPS). The responsive cells were monocytes, since all could be double-labeled with anti-IL-1 and the monocyte-specific CD14 antibody. More than 80% of the synovial fluid monocytes made IL-1 beta after 24 hr in 2 ng/ml LPS. Other agents failed to induce IL-1 from enriched populations of monocytes including interferon gamma (IFN-gamma), poly (I/C), phorbol myristate acetate (PMA), concanavalin A (Con A), phytohemagglutinin (PHA), and anti-CD3 antibodies. Relatively high levels of dendritic cells (DC) were present in RA effusions, but these did not produce IL-1 in response to any of the above stimuli. Blood dendritic cells also did not make IL-1, whereas blood monocytes responded comparably to synovial exudate cells. The data indicate that rheumatoid exudate monocytes make very little IL-1 during acute flares of arthritis and that this cytokine is primarily a macrophage rather than a dendritic cell product.     

Transforming growth factor-beta production by synovial tissues from rheumatoid patients and streptococcal cell wall arthritic rats. Studies on secretion by synovial fibroblast-like cells and immunohistologic localization

The growth of synovial fibroblast-like cells from patients with rheumatoid arthritis and rats with streptococcal cell wall (SCW)-induced arthritis in vitro under anchorage-independent conditions is inhibited by transforming growth factor-beta (TGF-beta). Because this growth factor is present in rheumatoid synovial fluids, we studied whether this cytokine might be secreted by cells in rheumatoid synovial tissue. We show that synovial tissues from patients with rheumatoid arthritis and osteoarthritis, and rats with SCW-induced arthritis, contain TGF-beta-1 mRNA. TGF-beta, predominantly type 1, was spontaneously secreted in vitro by synovial tissue explants and synovial fibroblast-like cells. In addition, TGF-beta could be detected immunohistochemically in cells throughout rheumatoid and SCW-induced arthritic rat synovial tissues. Finally, exogenous TGF-beta induced collagen and inhibited collagenase mRNA levels by cultured synoviocytes. These data support an autocrine role for TGF-beta in the regulation of synoviocytes in rheumatoid arthritis and, in light of its demonstrated effects on the immune system, suggest that TGF-beta might also have important paracrine effects on infiltrating inflammatory cells.