Karina Kuczka

Analgesic efficacy of tramadol, pregabalin and ibuprofen in menthol-evoked cold hyperalgesia

ABSTRACT We investigated the analgesic efficacy of single doses of ibuprofen, tramadol and pregabalin in menthol‐evoked cold pain in a randomized, placebo‐controlled four‐way cross‐over study in 20 healthy volunteers. Tramadol 100 mg significantly reduced menthol‐evoked cold hyperalgesia. Effects of ibuprofen 600 mg and pregabalin 100 mg were not significant. Analgesic effects of tramadol were associated with minor side effects, particularly fatigue and nausea. Minor side effects also accompanied analgesic effects of pregabalin and ibuprofen in subjects responding to these drugs, mostly fatigue, dizziness and difficulties to concentrate for pregabalin and gastric upset for ibuprofen. Five out of 18 subjects had a ⩾50% reduction of cold hyperalgesia with tramadol, three of these additionally responded to pregabalin, and two with all three drugs. The numbers needed to treat (NNT ⩾ 50% for tramadol 4.5, for pregabalin 9) largely agree with the reported efficacy of tramadol and of moderate dosages of pregabalin in patients with peripheral or central neuropathic pain suggesting that menthol‐evoked cold pain hypersensitivity may represent a valid model for neuropathic pain, particularly cold allodynia.

The CX3C chemokine fractalkine mediates platelet adhesion via the von Willebrand receptor glycoprotein Ib

The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and is associated with the development of atherosclerosis. The potential of FKN in mediating platelet adhesion beyond platelet activation remains unexplored to date. A flow-based adhesion assay was used to study the adhesion of platelets to immobilized FKN under physiologic flow conditions. Platelet adhesion to von Willebrand factor (VWF) was increased in the presence of FKN at 600 inverse seconds. Additional platelet adhesion to FKN coimmobilized with VWF was dependent on the FKN receptor CX3CR1 and activation of glycoprotein (GP) IIb/IIIa. The number of platelets rolling on VWF was likewise enhanced in the presence of FKN. The enhancement of rolling on FKN and VWF was insensitive to anti-CX3CR1 antibody but was fully inhibited by neutralizing GPIbα function. The extracellular domain of GPIbα was covalently coupled to fluorescent microspheres, and microsphere binding was significantly higher in the presence of FKN. Platelet adhesion to activated endothelium in vitro and to intact human arteries was substantially increased in an FKN-dependent manner. These data demonstrate that endothelial expressed FKN activates platelets via its cognate receptor CX3CR1, whereas platelet adhesion is predominantly mediated by GPIbα and independent of CX3CR1.

Increased Bioavailability of 11-Keto-β-Boswellic Acid Following Single Oral Dose Frankincense Extract Administration After a Standardized Meal in Healthy Male Volunteers: Modeling and Simulation Considerations for Evaluating Drug Exposures

T use of gum resin extracts from the Boswellia serrata tree (frankincense) for the treatment of inflammatory conditions originates from traditional Ayurvedic medicine in India. In vitro studies suggests that 11-keto-β-boswellic acid (KBA) and 3-acetyl-11-keto-β-boswellic acid (AKBA) are the 2 lead boswellic acids with inhibition most pronounced in vitro for 5-lipoxygenase, thus reducing leukotrienes as inflammatory lipid mediators. Therapeutic effects of frankincense extracts were shown in laboratory animal disease models as well as in clinical trials studying inflammatory bowel diseases, arthritis, asthma, and cancer. However, valid recommendations regarding clinical use are scarce—perhaps not surprising, as natural products such as frankincense vary in their pharmaceutical quality and are available as different formulations with varying ratios of boswellic acids, and wellcontrolled clinical trials with unequivocal evidence of efficacy are missing. This suggests a cautious use of frankincense; however, clinical practice suggests the opposite. Frankincense extracts have been used within cancer support groups, supported by German import statistics for Gufic H 15, a Boswellia serrata gum resin extract. In addition, 36% of surveyed German patients have treated their inflammatory bowel disease with B serrata extracts.

Biomarkers and Coagulation Tests for Assessing the Biosimilarity of a Generic Low‐Molecular‐Weight Heparin: Results of a Study in Healthy Subjects With Enoxaparin

Low‐molecular‐weight heparins (LMWHs) differ considerably in their influence on clotting tests and release of tissue factor pathway inhibitor (TFPI). Biosimilarity therefore becomes an issue when generic forms of LMWHs are developed. So far, no bioequivalence study with a generic LMWH has been reported. A generic enoxaparin (test) was compared with the originator (reference) in 20 volunteers after single‐dose subcutaneous administration (40 mg enoxaparin sodium, 4000 IU/mL anti—factor Xa (anti‐FXa; activity). Target variables were anti‐FXa and anti‐FIIa activity, activated partial thromboplastin time (aPTT), prothrombinase‐induced clotting time (PiCT), and TFPI over 24 hours. The statistical evaluation of the anti‐FXa activity profile demonstrated bioequivalence of test and reference with confidence intervals of area under the plasma concentration‐time curve (AUC0‐tlast) (93%‐99%) and Amax (88%‐95%). Confidence intervals of AUC0‐tlast (89%‐102%) and Amax (90%‐103%) of anti‐FIIa activity also fulfill bioequivalence criteria. The 90% confidence interval for the maximum concentration of TFPI ranged from 90% to 113%. The claim of similarity was also supported by aPTT and PiCT profiles. Bioequivalence with the originator enoxaparin could be demonstrated by ex vivo inhibition of FXa and FIIa activity, by coagulation tests (aPTT and PiCT), and by in vivo release of TFPI. Whether such data also prove biosimilarity of the generic enoxaparin needs to be determined.

Platelet–leucocyte adhesion markers before and after the initiation of antiretroviral therapy with HIV protease inhibitors

INTRODUCTION Thromboembolic complications under antiretroviral therapy (ART) have been described in the past. In particular, the influence of protease inhibitors (PIs) on platelet activation and coagulation is currently under discussion. METHODS HIV-1-infected, PI-naive adults (n = 18) were investigated before and 4 weeks after the start of the ART, consisting either of boosted PI regimens (n = 13) plus reverse transcriptase inhibitors (RTIs) or a double PI regimen (n = 5) without RTI co-medication. Administered PIs were saquinavir (n = 15), lopinavir (n = 4), fosamprenavir (n = 2) and atazanavir (n = 2). Platelet CD62P, CD40L (%+ cells) and PAC-1 binding [mean fluorescence intensity (MFI)] as well as monocyte CD11b (MFI) and monocyte-associated CD41 (%+ cells and MFI) expression were assessed by flow cytometry with or without platelet stimulation. To investigate the influence of platelets on coagulation, the endogenous thrombin potential (ETP) [render fluorescence units (RFI)] was determined. RESULTS CD62P, PAC-1 binding and CD11b expression remained unchanged. In contrast, the mean+/-SD MFI of CD40L (from 18.2+/-9.0 to 25.5+/-10.4, P = 0.038) and CD41 (from 446.1+/-213.8 to 605.0+/-183.8, P = 0.010) as markers for increased platelet-leucocyte interaction increased significantly. The collagen-induced ETP time-to-peak was altered significantly from 23.8+/-11.4 to 17.0+/-4.2 min (P = 0.028), although the ETP RFI peak showed no evidence for increased procoagulatory capacity (47.1+/-18.6 to 57.3+/-19.9, P = 0.085). CONCLUSIONS Effects of the evaluated PI HIV therapy on platelet function assessed under field conditions seem to be minor, not affecting all investigated parameters. We found no evidence for increased platelet activation under PI-containing ART. However, CD41 as a marker for increased platelet-leucocyte interaction and CD40L, which can contribute to atherosclerosis, increased significantly.

Fluorescent Human EP3 Receptor Antagonists.

Exchange of the lipophilc part of ortho-substituted cinnamic acid lead structures with different small molecule fluorophoric moieties via a dimethylene spacer resulted in hEP3R ligands with affinities in the nanomolar concentration range. Synthesized compounds emit fluorescence in the blue, green, and red range of light and have been tested concerning their potential as a pharmacological tool. hEP3Rs were visualized by confocal laser scanning microscopy on HT-29 cells, on murine kidney tissues, and on human brain tissues and functionally were characterized as antagonists on human platelets. Inhibition of PGE2 and collagen-induced platelet aggregation was measured after preincubation with novel hEP3R ligands. The pyryllium-labeled ligand 8 has been shown as one of the most promising structures, displaying a useful fluorescence and highly affine hEP3R antagonists.

Long term administration of LMWH - pharmacodynamic parameters under therapeutic or prophylactic regimen of enoxaparin or tinzaparin in neurological rehabilitation patients

We investigated anti-FXa- and anti-FIIa-activity, thrombin generation (ETP), tissue factor pathway inhibitor (TFPI) - and D-dimer in patients exhibiting high bleeding risk in early neurological rehabilitation over 2 months in an observational study. Blood of 64 patients under LMWH administration due to therapeutic (cohort 1 [tinzaparin 90 IE/kg BID, N = 18] and 2 [enoxaparin 100 IE/kg BID; N = 15]) or prophylactic (cohort 3 [tinzaparin 4500 IE; N = 16] and 4 [enoxaparin 4000 IE; N = 15]) indication was drawn before and 4h after injection on day 7 (V1) and 2 months (follow up [V2]). Although the dose in cohort 1 and 2 was similar (median 7000 IE BID), a-FXa-activity was significantly larger under enoxaparin than under tinzaparin (e.g. median at V2: 0.70 IU/ml vs. 0.33 IU/ml). Also, prophylactic enoxaparin exhibited larger a-FXa-activity than tinzaparin (e.g. median at V2: 0.37 IU/ml vs. 0.22 IU/ml). The a-FXa/a-FIIa-ratio in plasma samples at 4h p.a. was about 4 (tinzaparin) and 8 (enoxaparin), respectively. No differences were seen for TFPI and ETP between cohort 1 and 2 or between cohort 3 and 4. D-dimer levels decreased significantly between V1 (e.g. cohort 4 median 1940 ng/ml) and V2 (median 652 ng/ml). Minimal bleeding events occurred in 6 patients (2 under tinzaparin, 4 under enoxaparin) and were associated with significantly higher anti-FXa-activity. In conclusion, although marked differences between tinzaparin and enoxaparin based on anti-FXa-activity were seen, markers of in vivo biological activity such as TFPI and D-dimer were not different. Furthermore, BID tinzaparin is a feasible option for therapeutic anticoagulation in patients with high bleeding risk.

The integrin antagonist, cilengitide, is a weak inhibitor of αIIbβ3 mediated platelet activation and inhibits platelet adhesion under flow

Abstract The RGD cyclic pentapetide, cilengitide, is a selective inhibitor of αvβ3 and αvβ5 integrins and was developed for antiangiogenic therapy. Since cilengitide interacts with platelet αIIbβ3 and platelets express αv integrins, the effect of cilengitide on platelet pro-coagulative response and adhesion is of interest. Flow-based adhesion assays were performed to evaluate platelet adhesion and rolling on von Willebrand factor (vWf), on fibrinogen and on human umbilical vein endothelial cells (HUVECs). Flow cytometry was used to detect platelet activation (PAC1) and secretion (CD62P) by cilengitide and light transmission aggregometry was used to detect cilengitide-dependent platelet aggregation. Cilengitide inhibited platelet adhesion to fibrinogen at concentrations above 250 µM [which is the Cmax in human studies] and adhesion to vWf and HUVECs at higher concentrations under physiologic flow conditions. Platelet aggregation was already impaired at cilengitide concentrations >10 µM. Activation of αIIbβ3 integrin was inhibited by 250 µM cilengitide, whereas platelet secretion was unaffected by cilengitide. No evidence of cilengitide-induced platelet activation was found at all tested concentrations (0.01–1500 µM). At higher concentrations, platelet activation was inhibited, predominantly due to αIIbβ3 inhibition.

Phenotypic differences of human neutrophils of carriers of the PSGL-1 A and B-allele in binding to immobilised P-selectin under flow conditions

P-selectin glycoprotein ligand-1 (PSGL-1) interacts with P-selectin expressed on endothelial cells and platelets. PSGL-1 extracellular mucin-like domain displays a variable number of tandem repeats (VNTRs) polymorphism. The wildtype consists of 16 decameric repeats (designated A isoforms) and variants with 15 (B allele) and 14 (C allele) repeats that are assumed to be associated with reduced risk of vascular disease. We investigated the adhesion of these natural variants to P-selectin in native human neutrophils. Healthy volunteers were genotyped and the adhesion of neutrophils expressing the PSGL-1 isoforms A/A, A/B and B/B were studied under static and physiologic flow conditions. Homozygous B/B neutrophils attached significantly weaker to P-selectin at elevated shear rates from 24 up to 64 dyn/cm(2) than A/A and A/B neutrophils. No difference in adhesion rate was found under static conditions and shear stress below 24 dyn/cm(2), but B/B neutrophils rolled significantly faster than A/A neutrophils at shear stress ≥ 12 dyn/cm(2). There was no difference in the adhesive capacity between A/A an A/B neutrophils. These data support the view that the role of the decamers is to extend the ligand binding domain far above the cell surface to support stable leukocyte adhesion and rolling.