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Biomedical subjects

Stefan Laufer

Publications and source records attributed to Stefan Laufer.

35 records · Page 2Linked to original sources
PubMed2004

Regulation of the expression of 5-lipoxygenase-activating protein/5-lipoxygenase and the synthesis of leukotriene B(4) in osteoarthritic chondrocytes: role of transforming growth factor beta and eicosanoids.

OBJECTIVE: To explore the modulation of 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase (5-LOX) expression in human osteoarthritic (OA) chondrocytes, their relative implications in leukotriene B(4) (LTB(4)) production, the effect of different factors on this system, and the influence of increased LTB(4) production on the synthesis of catabolic factors of cartilage. METHODS: FLAP and 5-LOX expression and LTB(4) production were monitored following treatment with transforming growth factor beta1 (TGFbeta1; 5 ng/ml) and 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3); 50 nM) alone or in combination with selective or nonselective cyclooxygenase (COX) inhibitors, naproxen (90 mug/ml), NS-398 (10 muM), or FR122047 (5 muM), or a dual inhibitor of COX/5-LOX activity, licofelone (2.6 muM). LTB(4), prostaglandin E(2) (PGE(2)), and matrix metalloprotease 1 (MMP-1) production were measured by specific enzyme-linked immunosorbent assays, nitric oxide by the Griess reaction, and FLAP and 5-LOX expression by quantitative polymerase chain reaction. RESULTS: Human OA chondrocytes expressed both FLAP and 5-LOX. TGFbeta1 and/or 1,25(OH)(2)D(3) induced a rapid and marked enhancement ( approximately 4-13-fold) in FLAP messenger RNA (mRNA) levels, which was associated with a subsequent and late increase in LTB(4) production and PGE(2) synthesis. Treatment with COX inhibitors in the absence or presence of TGFbeta1 and 1,25(OH)(2)D(3) induced a rapid increase in LTB(4) production; this response was mediated by the sustained and significant (P < 0.01) up-regulation ( approximately 1.5-fold) of 5-LOX mRNA levels. Conversely, treatment with licofelone showed no effect on 5-LOX but significantly reduced FLAP expression levels. Coincubation of licofelone with TGFbeta1 plus 1,25(OH)(2)D(3) did not affect FLAP or 5-LOX levels. In the presence of TGFbeta1 plus 1,25(OH)(2)D(3), naproxen, but not licofelone, induced MMP-1 production and both drugs decreased nitric oxide levels. CONCLUSION: Both the eicosanoids PGE(2) and LTB(4) are important cofactors in regulating FLAP/5-LOX expression; the inhibition of PGE(2) up-regulates 5-LOX while down-regulating FLAP gene expression, and LTB(4) appears to be an up-regulating factor on the 5-LOX gene. Importantly, nonsteroidal antiinflammatory drugs up-regulate the synthesis of LTB(4), supporting the shunt hypothesis from COX to 5-LOX. We also demonstrated that LTB(4) likely contributes to the up-regulation of important catabolic factors involved in the pathophysiology of OA, such as MMP.

5-Lipoxygenase-Activating Proteins↗
PubMed2004

Determination of (R)- and (S)-phenprocoumon in human plasma by enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry.

Phenprocoumon is a commonly used oral anticoagulant of the coumarin type, and has found extensive clinical use in the treatment of thrombophlebitis, pulmonary embolism and atrial fibrillation. In the course of a clinical study to investigate the influence of genetic polymorphisms of the CYP2C9 enzyme on phenprocoumon metabolism, we developed a new enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/MS/MS) method to quantify (R)- and (S)-phenprocoumon in human plasma. HPLC separation of the enantiomers was achieved on a Chira-Grom-2 column under isocratic conditions using a water/acetonitrile/formic acid eluent. For detection and quantification a triple-quadrupole MS system was used in the selected reaction monitoring (SRM) mode. As an internal standard the structurally homologous compound warfarin was chosen. The detector response was linear with a correlation coefficient of 0.988-0.999 for (R)-phenprocoumon and 0.989-0.999 for (S)-phenprocoumon in the investigated concentration range between 62.5 and 1000 ng/mL (per enantiomer). The limit of detection (LOD) was 12.5 ng/mL.

Anticoagulants↗
PubMed2004

The inhibition of subchondral bone resorption in the early phase of experimental dog osteoarthritis by licofelone is associated with a reduction in the synthesis of MMP-13 and cathepsin K.

OBJECTIVE: To evaluate the morphological changes that take place in the subchondral bone and calcified cartilage zone in the experimental anterior cruciate ligament (ACL) dog model of osteoarthritis (OA) and analyze concomitant changes in the level of MMP-13 and cathepsin K, as well as examine the therapeutic effects of licofelone, a lipoxygenase (LO)/cyclooxygenase (COX) inhibitor, on these morphological and biochemical changes. METHODS: Experimental group 1 underwent sectioning of the ACL of the right knee with no active treatment (placebo group). Experimental groups 2 and 3 underwent sectioning of the ACL of the right knee and were administered therapeutic concentrations of licofelone (2.5 or 5.0 mg/kg/day p.o., respectively) for 8 weeks, beginning the day following surgery. Group 4 consisted of untreated dogs used as normal control. Specimens of subchondral bone including the calcified cartilage were selected from lesional and non-lesional areas of OA tibial plateaus. Specimens were processed for static morphometric analysis and immunohistochemical analysis for MMP-13 and cathepsin K. RESULTS: As indicated by a reduction in bone surface and trabecular thickness, a significant loss of subchondral bone occurred in OA dogs. These changes were associated with an increased level of MMP-13 synthesis by bone cells and an increase in the osteoclast population that stained strongly positive for cathepsin K and MMP-13. Changes were much more pronounced in the specimens taken from the lesional areas. Treatment with licofelone decreased, in a dose-dependent manner, the OA bone morphological changes at the same time it reduced the level of MMP-13 in bone cells and the number of cathepsin K and MMP-13 positive osteoclasts. CONCLUSIONS: Increased bone loss and bone resorption is associated with the development of OA cartilage lesions. Licofelone treatment was found to prevent the morphological and biochemical changes seen in early experimental OA effectively. These findings may help explain the mechanisms by which this drug could exert its possible effect on the development of OA.

Acetates↗
PubMed2004

The gastrointestinal tolerability of the LOX/COX inhibitor, licofelone, is similar to placebo and superior to naproxen therapy in healthy volunteers: results from a randomized, controlled trial.

OBJECTIVES: Concerns exist over the safety of conventional nonsteroidal antiinflammatory drugs (NSAIDs) and selective cyclooxygenase (COX)-2 inhibitors. In experimental models, licofelone, a competitive inhibitor of 5-lipoxygenase (5-LOX) and COX-1/-2, has been shown to have good gastrointestinal and general tolerability and analgesic and antiinflammatory properties. The aim of the present endoscopy trial was to investigate the gastroduodenal tolerability of licofelone compared to placebo and naproxen in healthy volunteers. METHODS: In this randomized, parallel-group trial, healthy volunteers received licofelone 200 mg b.i.d. (n = 30), licofelone 400 mg b.i.d. (n = 30), naproxen 500 mg b.i.d. (n = 30), or placebo (n = 31). Tolerability was assessed by gastro/duodenoscopy following 4 wk of treatment. Laboratory parameters and the incidence of ulcers and adverse events were recorded. RESULTS: Ulcers were observed in 20% of the naproxen-treated volunteers, compared with 0% of those receiving licofelone 200 mg, licofelone 400 mg, and placebo (p= 0.024). Posttreatment mean gastric Lanza scores were similar for volunteers who received placebo or either dose of licofelone, while Lanza scores were significantly worse following naproxen therapy (p < 0.00001). The gastric mucosa was normal in 93%, 89%, and 90% of volunteers who received licofelone 200 mg, licofelone 400 mg, or placebo, respectively, compared with 37% of volunteers receiving naproxen. The incidence of adverse events did not differ significantly between licofelone 200 mg or naproxen therapy. No clinically relevant changes in laboratory parameters were observed with licofelone or naproxen therapy. CONCLUSIONS: The results from this trial indicate that licofelone has a potential gastrointestinal safety advantage over conventional NSAID therapy, as licofelone was associated with significantly superior gastric tolerability and a lower incidence of ulcers compared with naproxen in healthy volunteers. Further trials will be required to investigate the safety and efficacy of licofelone in the treatment of diseases such as osteoarthritis.

Acetates↗
PubMed2003

Role of eicosanoids in structural degradation in osteoarthritis.

PURPOSE OF REVIEW: Osteoarthritis is characterized mainly by degenerative changes in joint cartilage, ultimately resulting in loss of cartilage, and alterations in the subchondral bone. Osteoarthritis osteoblasts show a number of metabolic alterations that may interfere with normal cell metabolism and signaling, possibly leading to altered extracellular matrix composition. This review examines the role of eicosanoids in this structural degradation. RECENT FINDINGS: Prostaglandins exert diverse modulatory roles in osteoarthritis, with prostaglandin E2 known to play an important role in inflammation. Prostaglandins and leukotriene B4 have been shown to regulate proinflammatory cytokine and interstitial collagenase synthesis in human osteoarthritis synovial membrane explants. Human osteoarthritis osteoblasts produce variable levels of prostaglandin E2 and leukotriene B4 compared with normal osteoblasts. Prostaglandin E2 levels can distinguish two types of patients with osteoarthritis: osteoblasts from one group produce low levels of prostaglandin E2 and interleukin-6, and the other shows an increase in production. In contrast, osteoarthritis osteoblasts that produce high levels of prostaglandin E2 produce low levels of leukotriene B4 and vice versa. This observation could be explained by the selective metabolism of arachidonic acid via the 5-lipoxygenase or cyclooxygenase pathways in osteoarthritis osteoblasts. SUMMARY: Prostaglandins play a significant role not only in joint physiology, but also in the pathogenesis of joint disorders. In addition, it has been identified that osteoarthritis subchondral osteoblasts can synthesize leukotriene B4, indicating a role of leukotrienes in bone remodeling associated with osteoarthritis. A therapeutic intervention that blocks lipoxygenase/cyclooxygenase pathways, thereby inhibiting production of prostaglandins and leukotrienes, may therefore be very attractive for the treatment of osteoarthritis patients.

Bone Remodeling↗
PubMed2003

International Conference on Inflammopharmacology -- VIII Side-Effects of Anti-inflammatory Drugs Symposium.

The International Conference on Inflammopharmacology with VIII Side-Effects of Anti-Inflammatory Drugs Symposium was held in Edinburgh, UK on 22 - 24 April 2003. The meeting was well attended, involving approximately 80 rheumatologists, pharmacologists and research scientists from both academia and industry with a special interest in osteoarthritis. Although few new drugs emerged at the meeting as treatment options for osteoarthritis, exciting data on the latest advances in novel inhibitors of the pathways involved in the pathogenesis of osteoarthritis were presented. This report describes recent clinical experiences with the unique lipoxygenase/cyclooxygenase inhibitor licofelone (ML3000; EuroAlliance, a partnership of Merckle [Germany], Alfa Wassermann [Italy] and Lácer [Spain]) and the latest developments in nitric oxide-donating non-steroidal anti-inflammatory drugs NMI-1093 and NMI1089 (Nitromed, Inc., USA) and AZD3582 (AstraZeneca, Sweden).

Acetates↗
PubMed2002

NO-donors (VII [1]): synthesis and cyclooxygenase inhibitory properties of N-and S-nitrooxypivaloyl-cysteine derivatives of naproxen -- a novel type of NO-NSAID.

Nitric oxide (NO)has been reported to subserve many of the same mucosal protection mechanisms as prostaglandins and is sufficient for acute gastroprotection and ulcer healing. In fact, NO-donating NSAID hybrid compounds such as the nitrooxybutyl ester of naproxen show reduced ulcerogenic activity while maintaining anti-inflammatory activity. We introduce two prototypes of novel triple-hybrid compounds consisting of cysteine which is known to enhance the activity of organic nitrates and to reduce nitrate tolerance, an NSAID (naproxen), and an organic nitrate (nitrooxypivaloic acid). L-Cysteine ethyl ester first was N-acylated in a CH(2)Cl(2)/H(2)O twophase system using the acid chlorides of naproxen or nitrooxypivaloic acid, respectively, and sodium acetate, or alternatively using the DCC-activated nitrooxy acid in absolute CH(2)Cl(2). The N-acylated intermediates were subsequently S-acylated using the acid chlorides or alternatively the carbonyldiimidazole (CDI)-activated acids again. The two naproxen-cysteine-nitrate hybrid prodrugs were screened in vitro for their cyclooxygenase inhibitory properties relative to naproxen. In this screening the N-nitrooxyacylcysteine derivative was found to be inactive in the concentration range of 0.1-10 micromol/L against both COX-1 and COX-2, while the S-nitrooxyacylcysteine derivative had only weak activity against COX-1.

Animals↗
PubMed2002

Antitubercular isoniazid and drug resistance of Mycobacterium tuberculosis--a review.

Isoniazid is one of the most potent drugs available for tuberculosis treatment. As a pro-drug it requires activation, which is performed by catalase/peroxidase. The active principle, whose identity has not yet been determined unambiguously, then acts on at least one target molecule, the enoyl-acyl carrier protein, required for the synthesis of the vital mycolic acids present in the cell wall of the bacterium. Some other targets have been proposed in order to explain the unusual potency of isoniazid; however, the supporting data are still controversial. We thoroughly discuss the action of isoniazid, resistance mechanisms, and the possible active product, which includes an isonicotinic acid-NADH adduct as well as a meta-isomer of NADH. Both structures have been probed positively in a 3D modeling analysis.

Bacterial Proteins↗
PubMed2002

Study of the role of leukotriene B()4 in abnormal function of human subchondral osteoarthritis osteoblasts: effects of cyclooxygenase and/or 5-lipoxygenase inhibition.

OBJECTIVE: To compare the effect of licofelone, NS-398 (an inhibitor of cyclooxygenase 2 [COX-2]), and BayX-1005 (an inhibitor of 5-lipoxygenase activating protein) on the production of leukotriene B(4) (LTB(4)) and prostaglandin E(2) (PGE(2)), and on cell biomarkers by human osteoarthritis (OA) subchondral osteoblasts. METHODS: Primary in vitro osteoblasts were prepared from subchondral bone specimens obtained from OA patients and autopsy subjects. LTB(4) and PGE(2) levels were measured by enzyme-linked immunosorbent assay in conditioned media of osteoblasts incubated in the presence or absence of licofelone, NS-398, or BayX-1005. The effect of these drugs or of the addition of LTB(4) on alkaline phosphatase (AP) activity and osteocalcin release by OA and normal osteoblasts was determined. The presence of LTB(4) receptors in normal and OA osteoblasts was evaluated by Western blot analysis. RESULTS: OA osteoblasts produced variable levels of PGE(2) and LTB(4) compared with normal osteoblasts. Licofelone, at the maximal dose used, inhibited production of PGE(2) and LTB(4) by OA osteoblasts by a mean +/- SEM of 61.2 +/- 6.4% and 67.0 +/- 7.6%, respectively. NS-398 reduced PGE(2) production by 75.8 +/- 5.3%. BayX-1005 inhibited LTB(4) production in OA osteoblasts by 38.7 +/- 14.5% and marginally affected PGE(2) levels (reduction of 14.8 +/- 5.3%). Licofelone dose-dependently stimulated 1,25-dihydroxyvitamin D-induced AP activity while inhibiting osteocalcin release. BayX-1005 partly reproduced these effects, but NS-398 failed to affect them. LTB(4) dose-dependently inhibited AP activity in OA osteoblasts, while its effect on osteocalcin depended on endogenous LTB(4) levels in these cells. In normal osteoblasts, LTB(4) dose-dependently stimulated osteocalcin, whereas it failed to influence AP. LTB(4) receptors BLT1 and BLT2 were present in normal and OA osteoblasts. CONCLUSION: Licofelone inhibits the production of PGE(2) and LTB(4). Selective effects of licofelone on AP and osteocalcin occur via its role on LTB(4) production. Because LTB(4) can modify cell biomarkers in OA and normal osteoblasts, our results suggest licofelone could modify abnormal bone remodeling in OA.

Acetates↗
PubMed2002

Studies on the anti-inflammatory activity of phytopharmaceuticals prepared from Arnica flowers.

Phytopharmaceuticals prepared from flowerheads of Arnica montana of Spanish origin and of the new type "Arbo", which can be easily and economically cultivated, were studied for their capability to impair activation of the transcription factors NF-kappa B and NF-AT. Both proteins are responsible for the transcription of genes encoding various inflammatory mediators. Additionally, their influence on the release of the cytokines IL-1 and TNF-alpha were examined. The inhibitory activities correlate with their quantitative and qualitative content of sesquiterpene lactones (Sls). Moreover, it was shown that the inhibitory potency of 11 alpha,13-dihydrohelenalin derivatives being the main Sls in the Spanish flowers depend on their esterfication. Compounds with unsaturated acyl moieties, such as methacrylate and tiglinate, exhibited a stronger activity in the NF-kappa B EMSA as well as in the croton oil ear test in mice than the acetate derivative.

Animals↗
PubMed2002

Synthesis of interleukin 1beta, tumor necrosis factor-alpha, and interstitial collagenase (MMP-1) is eicosanoid dependent in human osteoarthritis synovial membrane explants: interactions with antiinflammatory cytokines.

OBJECTIVE: To determine the level of leukotriene B4 (LTB4) synthesized and released by synovium of patients with osteoarthritis (OA), and to study the role of lipoxygenase (LO)/cyclooxygenase (COX) products on proinflammatory cytokine and interstitial collagenase (MMP-1) synthesis. METHODS: Human OA synovial explants were cultured in the presence of lipopolysaccharide (L) and the ionophores ionomycin (I) and thapsigargin (T) (LIT) for 72 h at 37 degrees C, and LTB4 released into the culture medium was measured in the absence or presence of a COX-2-specific inhibitor, NS-398, or the 5-LO activating protein inhibitor Bay-x-1005. Increasing concentrations of LTB4 (10(-9) to 10(-6) M) were incubated with explants for 24 h at 37 degrees C, and interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in the conditioned medium were quantitated by ELISA. The effect of endogenous eicosanoids on basal and induced levels of IL-1beta, TNF-alpha, and MMP-1 synthesis was examined by incubating explants in the presence of NS-398 and Bay-x-1005. The effect of antiinflammatory cytokines rhIL-4, IL-10, and IL-13 on basal and LTB4 dependent stimulation of IL-1beta/TNF-alpha synthesis was studied under titration conditions. RESULTS: Physiologically relevant concentrations (10(-10) to 10(-9) mol/l) of LTB4 were produced in the presence of LIT. Bay-x-1005 abrogated LTB4 release, while NS-398 was without effect. LTB4 stimulated IL-1beta and TNF-alpha synthesis with an EC50 of 190 +/- 35 and 45 +/- 9 nmol/l, respectively. Significant concentrations of IL-1beta and TNF-alpha were released (100-200 and 500-600 pg/ml, respectively). Basal and LIT induced IL-1beta and TNF-alpha production were inhibited by Bay-x-1005 in a dose dependent manner, while the addition of NS-398 caused a potent stimulatory effect. The preferential COX-2 inhibitor also induced MMP-1 synthesis in a manner essentially identical to the proinflammatory cytokines. The antiinflammatory cytokine IL-4 blocked LTB4 dependent stimulation of IL-1beta and TNF-alpha synthesis. In contrast, IL-10 markedly stimulated both cytokines when incubated alone or in the presence of LTB4 where the effect was additive. CONCLUSION: Endogenous and locally produced eicosanoids regulate proinflammatory cytokine and MMP-1 synthesis under basal and stimulated conditions in vitro, with leukotrienes and prostaglandins having opposite effects in general. The clinical use of antiinflammatory drugs that inhibit eicosanoid synthesis requires an appreciation of their relative capacity to inhibit LO/COX in order to predict their effect on the synthesis of proinflammatory cytokines and matrix metalloproteases. IL-10 stimulated proinflammatory cytokine synthesis in our ex vivo culture system.

Aged↗
PubMed2002

Licofelone (ML-3000), a dual inhibitor of 5-lipoxygenase and cyclooxygenase, reduces the level of cartilage chondrocyte death in vivo in experimental dog osteoarthritis: inhibition of pro-apoptotic factors.

OBJECTIVE: To evaluate in vivo therapeutic efficacy of licofelone, a novel competitive dual inhibitor of 5-lipoxygenase (5-LOX) and cyclooxygenase (COX) in chondrocyte death in the canine ligament transection model of osteoarthritis (OA), and to explore its effect on factors involved in the apoptotic phenomenon, i.e., caspase-3, COX-2, and inducible nitric oxide synthase (iNOS). METHODS: Cartilage specimens were obtained from 3 experimental groups of dogs: Group 1, dogs subjected to sectioning of the anterior cruciate ligament of the right knee and given placebo treatment; Groups 2 and 3, operated dogs that received oral treatment with licofelone (2.5 or 5.0 mg/kg/day, respectively) for 8 weeks starting immediately after surgery. All dogs were killed 8 weeks postsurgery. The cartilage level of chondrocyte death was detected by TUNEL reaction. Cartilage distribution of caspase-3, COX-2, and iNOS was documented by immunohistochemistry using specific antibodies, and other levels were quantified by morphometric analysis. RESULTS: In cartilage specimens from placebo treated dogs a large number of chondrocytes in the superficial layers stained positive for TUNEL reaction. Treatment with therapeutic concentrations of licofelone (2.5 and 5.0 mg/kg/day) markedly reduced the level of chondrocyte apoptosis to the same extent in both therapeutic groups (p < 0.0001, p < 0.002, respectively). In these groups, the levels of caspase-3, COX-2, and iNOS in cartilage from both condyles and plateaus were also significantly decreased (p < 0.0001, p < 0.0001, p < 0.0002, respectively) compared to the control (placebo) group. CONCLUSION: Licofelone is an effective treatment in vivo, capable of reducing the level of OA chondrocyte death. This effect is likely mediated by a decrease in the level of caspase-3 activity, which may be related to the reduced production of 2 major factors involved in chondrocyte apoptosis, NO and prostaglandin E2. These findings may explain some of the mechanisms by which licofelone reduces the progression of experimental OA.

Acetates↗