Standardisation of allergen products: 4. Validation of a candidate European Pharmacopoeia standard method for quantification of major grass pollen allergen Phl p 5.

BACKGROUND: The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5-specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. METHODS: A Phl p 5-specific ELISA system was assessed with respect to accuracy, precision, inter-assay (within laboratory) and inter-laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. RESULTS: The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter-assay variation (max. GCV 24%) and especially inter-laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. CONCLUSIONS: Based on the collaborative study results, the assessed Phl p 5-specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method.

Detection of hepatitis C virus and parvovirus B19 in human plasma pools by nucleic-acid amplification techniques - Trends in results of EDQM proficiency testing studies from 2004 to 2018.

The European Directorate for the Quality of Medicines & HealthCare (EDQM) has run proficiency testing schemes on the detection of viral contaminants in human plasma pools by nucleic-acid amplification techniques since 1999 for hepatitis C virus and since 2004 for parvovirus B19. A retrospective analysis was performed to assess their impact and identify trends and progress in the results obtained by participating laboratories over a 15-year span, from 2004 to 2018. The results demonstrate that overall performance improved over that time, especially among the regular participants. Participation in these proficiency testing schemes is therefore recommended for all interested control laboratories. This analysis also shows that hepatitis C virus detection now seems well established compared to that of parvovirus B19, which still appears more challenging.

Kreuth V initiative: European consensus proposals for treatment of hemophilia using standard products, extended half-life coagulation factor concentrates and non-replacement therapies.

This report contains the updated consensus recommendations for optimal hemophilia care produced in 2019 by three Working Groups (WG) on behalf of the European Directorate for Quality of Medicines and Healthcare in the frame of the Kreuth V Initiative. WG1 recommended access to prophylaxis for all patients, the achievement of plasma factor trough levels of at least 3-5% when extended half-life factor VIII (FVIII) and FIX products are used, a personalized treatment regimen, and a choice of chromogenic assays for treatment monitoring. It was also emphasized that innovative therapies should be supervised by hemophilia comprehensive care centers. WG2 recommended mandatory collection of postmarketing data to assure the long-term safety and efficacy of new hemophilia therapies, the establishment of national patient registries including the core data recommended by the European Medicines Agency and the International Society on Thrombosis and Haemostasis, with adequate support under public control, and greater collaboration to facilitate a comprehensive data evaluation throughout Europe. WG3 discussed methodological aspects of hemophilia care in the context of access decisions, particularly for innovative therapies, and recommended that clinical studies should be designed to provide the quality of evidence needed by regulatory authorities, HTA bodies and healthcare providers. The dialogue between all stakeholders in hemophilia care and patient organizations should be fostered to implement these recommendations.

Workshop on Animal free Detection of Pertussis Toxin in Vaccines--Alternatives to the Histamine Sensitisation Test.

The Paul-Ehrlich-Institut (PEI), the Nederlands Vaccin Instituut (NVI) and the European Directorate for the Quality of Medicines & HealthCare (EDQM) organised the international scientific workshop "Animal free Detection of Pertussis Toxin in Vaccines--Alternatives to the Histamine Sensitisation Test" at the PEI in Langen (Germany) on 09-10 June 2011. Twenty-seven experts (regulators, representatives from national control laboratories, vaccine manufacturers and academia) from 7 countries participated in this workshop. The meeting was triggered by the lack of satisfaction with the current safety testing for acellular pertussis vaccines, the "Histamine Sensitisation Test" (HIST) in mice, and the growing attention for the alternatives under development. The workshop objectives were: a) to review the current status of available alternative methods, b) to discuss the sensitivity that an alternative test needs, c) to plan experiments that allow for comparison of the alternative tests. The results of the workshop are summarised in this meeting report.

A locally active antiinflammatory macrolide (MLD987) for inhalation therapy of asthma.

One of the characteristic features of asthma is a persistent pulmonary inflammation, with increased numbers of eosinophils and activated T-lymphocytes in the airways. T-helper cells of the Th2 phenotype play a pivotal role in the pathogenesis of asthma, and they are believed to orchestrate the asthmatic response by releasing a wide repertoire of cytokines. Herein, we describe the design, synthesis, and evaluation in models of allergic asthma of a locally active T-cell modulator, MLD987 (1). Compound 1 is a potent immunosuppressant that inhibits the activation, proliferation, and release of cytokines from T-cells with IC(50) values in the low nanomolar range. In a Brown-Norway rat model of allergic asthma, 1, when given into the airways by intratracheal administration (ED(50) = 1 mg/kg) or by inhalation (ED(50) = 0.4 mg/kg), potently reduced the influx of leukocytes into bronchoalveolar lavage fluid samples obtained from antigen-challenged animals. In contrast, 1 had an appreciably weaker activity in this model when given orally or intravenously. Pharmacokinetic evaluation in rat and rhesus monkey showed that 1 had both a low oral (2-4%) and a low pulmonary (7%, monkey) bioavailability. These findings are consistent with a local site of action of the compound and rule out that its antiinflammatory activity in the lung was caused by systemically absorbed material, which had been swallowed during inhalation or which had entered the circulation via the airways. Local administration and the metabolically soft structure of 1, which favors rapid systemic metabolism to less immunosuppressive metabolite 2, are the main reasons for the low exposure and weak systemic activity of the compound. Administration of a locally active compound such as 1, by inhalation, should reduce systemic side effects. Our results indicate that 1 has the potential to serve as an alternative to inhaled glucocorticosteroids for the long-term therapy of asthma of all grades of severity.

Effect of the K+(ATP) channel opener, KCO912, on baseline and allergen induced airway hyperresponsiveness in allergic rabbits.

The effect of the adenosine triphosphate sensitive K+ (K(ATP)) channel opener (3S,4R)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-piperidinyl)-N-phenyl-1-benzopyran-6-sulphonamide (KCO912) on airway hyperresponsiveness induced using either a combination of allergen immunization (i.p.) followed by aerosol allergen challenge or immunization alone was investigated. Rabbits were immunized with Alternaria tenuis for the first 3 months of life. Airway responsiveness to histamine was measured 24 h before and after A. tenuis aerosol challenge. Fifteen minutes before the second challenge, rabbits were pre-treated with 10 microg of KCO912 or vehicle by inhalation. Allergen challenge induced airway hyperresponsiveness in vehicle pre-treated rabbits and pre-treatment with KCO912 abolished the airway hyperresponsiveness. The effect of KCO912 (10 microg) or vehicle on baseline airway hyperresponsiveness to the adenosine A(1) receptor agonist, cyclopentyl adenosine (CPA), induced by immunization with A. tenuis alone, was also assessed. Rabbits, immunized with A. tenuis alone, exhibited baseline airway hyperresponsiveness as demonstrated by an increase in airway resistance to CPA. Treatment with KCO912 did not alter the allergen-induced airway responsiveness to CPA. This study demonstrates that KCO912 can inhibit allergen-induced exacerbations of airway hyperresponsiveness.

KCO912: a potent and selective opener of ATP-dependent potassium (K(ATP)) channels which suppresses airways hyperreactivity at doses devoid of cardiovascular effects.

ATP-sensitive potassium (K(ATP)) channel openers can obviate experimental airways hyperreactivity (AHR) and have shown therapeutic benefit in asthma. However, the clinical potential of such compounds has been compromised by cardiovascular side-effects. We report here the pharmacological properties of (3 S,4 R)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-piperidinyl)- N-phenyl-2 H-1-benzopyran-6-sulphonamide (KCO912), a K(ATP) channel opener which suppresses AHR at doses devoid of cardiovascular effects.Specific interaction of KCO912 with the native vascular channel and the sulphonylurea receptor subunit (SUR2B) of the vascular K(ATP) channel was shown in radioligand binding assays. In rat aortic strips, KCO912 inhibited specific binding of [3H]P1075 and [3H]glibenclamide with up to 100% efficacy and with p Ki values of 8.28 and 7.96, respectively. In HEK cells transfected with the recombinant vascular K(ATP) channel (Kir6.1 + SUR2B), the compound elicited a concentration-dependent outward current (pEC50 6.8) and in preloaded rat aortic rings it induced a concentration-dependent glibenclamide-sensitive 86Rb+ efflux (pEC50 7.51). Following intratracheal (i.t.) administration of KCO912 to guinea pigs, AHR induced by immune complexes or ozone was rapidly (<5 min) reversed (ED50 values 1 microg/kg and 0.03 microg/kg, respectively). Changes in blood pressure were seen only at doses =100 microg/kg yielding 'therapeutic ratios' of 100 and 3333, respectively. In addition, KCO912 reversed AHR induced by lipopolysaccharide (LPS; ED50 0.5 microg/kg i.t.) and a dose of 1 microg/kg i.t. fully reversed AHR induced by subchronic treatment with salbutamol. At doses which suppressed AHR, KCO912 had no anti-bronchoconstrictor effects in normoreactive guinea pigs. In spontaneously hyperreactive rhesus monkeys, KCO912, given by inhalation, inhibited methacholine-induced bronchoconstriction (ED50 1.2 microg/kg) but had no significant effects on blood pressure or heart rate at all doses tested (therapeutic ratio >100). In rats given 3 mg/kg of KCO912 by inhalation, the ratio of the area under the concentration-time curve (AUC) for lung to the AUC in blood was 190 and the compound was rapidly cleared (initial t1/2 approximately 30 min). Thus, the wide therapeutic window following administration of KCO912 to the lung seems likely to reflect slow or incomplete passage of KCO912 from the lung into the systemic circulation coupled with rapid removal from the systemic circulation.Thus, when given locally to the airways in both guinea pigs and monkeys, KCO912 suppresses AHR at doses devoid of cardiovascular effects and has a significantly better therapeutic window than representative earlier generation K(ATP) channel openers defined in the same models. Given the pivotal role of AHR in the pathophysiology of asthma and the preclinical profile of KCO912, this compound was selected for clinical evaluation.

In vivo pharmacology of SDZ 249-665, a novel, non-pungent capsaicin analogue.

Capsaicin and analogues are valuable analgesic agents when administered to mammals, including humans. However, their pungency and the effects on the cardiovascular and respiratory systems through their general activation of small calibre (nociceptive) primary afferents severely limit their use. Recently, structure activity analysis revealed that the initial pungent and general excitatory effects can be prevented by structural modifications in such a way that the analgesic activity is retained. In this paper we present SDZ 249-665, a capsaicin analogue which produced analgesia in the mouse and anti-hyperalgesic effects in the rat and guinea pig. SDZ 249-665 was administered p.o., s.c. and i.v. in models of nociceptive pain, such as tail flick latency in response to a noxious thermal stimulus and acetic acid-induced writhing in mice, and in models of inflammatory mechanical hyperalgesia induced by turpentine or carrageenan in the rat and guinea pig, respectively. SDZ 249-665 was effective in the tail flick and the writhing assays and produced significant anti-hyperalgesic effects in the inflammatory models. The efficacy of SDZ 245-665 was similar to that of capsaicin, however, it was significantly more potent. SDZ 249-665 did not produce any irritancy in a nose wipe assay in guinea pigs or an eye irritancy assay in rats, while capsaicin was clearly irritant in both cases. Furthermore, unlike capsaicin, SDZ 249-665 did not produce unwanted side effects such as bronchoconstriction and blood pressure changes in the analgesic/anti-hyperalgesic dose range. Thus, a clear analgesic therapeutic window exists for SDZ 249-665. In summary, SDZ 249-665 is a potent orally active, analgesic/anti-hyperalgesic agent in mouse, rat and guinea pig. It lacks the excitatory effects associated with capsaicin and other close analogues, and therefore provides a clear therapeutic window for use in painful conditions. In addition to this favourable profile, no sign of tolerance was detected after a 5 day repeated dose treatment.