Andrew Preece

cDNA sequence and chromosome localization of pig {alpha}1,3 galactosyltransferase

Human serum contains natural antibodies (NAb), which can bind to endothelial cell surface antigens of other mammals. This is believed to be the major initiating event in the process of hyperacute rejection of pig to primate xenografts. Recent work has implicated galoctosyl α1,3 galactosyl β1,4 N-acetyl-glucosaminyl carbohydrate epitopes, on the surface of pig endothelial cells as a major target of human natural antibodies. This epitope is made by a specific galactosyltransferase (α1,3 GT) present in pigs but not in higher primates. We have now cloned and sequenced a full-length pig α1,3 GT cDNA. The predicted 371 amino acid protein sequence shares 85% and 76% identity with previously characterized cattle and mouse α1,3 GT protein sequences, respectively. By using fluorescence and isotopic in situ hybridization, the GGTA1 gene was mapped to the region q2.10–q2.11 of pig chromosome 1, providing further evidence of homology between the subterminal region of pig chromosome 1q and human chromosome 9q, which harbors the locus encoding the AB0 blood group system, as well as a human pseudogene homologous to the pig GGTA1 gene.

Safety, Tolerability, Pharmacodynamics and Pharmacokinetics of Umeclidinium and Vilanterol Alone and in Combination: A Randomized Crossover Trial

Umeclidinium bromide (GSK573719; UMEC), a new long-acting muscarinic receptor antagonist (LAMA), is in development with vilanterol (GW642444; VI), a selective long-acting β2 agonist (LABA), as a once-daily inhaled combination therapy for the treatment of chronic obstructive pulmonary disease (COPD). A single dose healthy volunteer study was conducted to assess the safety and tolerability, pharmacodynamics (PD) and pharmacokinetics (PK) of inhaled umeclidinium (500 µg) and vilanterol (50 µg) when administered separately and in combination using a novel dry powder inhaler (NDPI). Co-administration of single inhaled doses of umeclidinium and vilanterol to healthy Japanese subjects was well tolerated and not associated with meaningful changes in systemic exposure or PD effects compared with administration of either compound individually. Pharmacokinetic assessments showed rapid absorption for both drugs (Tmax = 5 min for both umeclidinium and vilanterol) followed by rapid elimination with median tlast of 4–5 h for umeclidinium and median tlast of 1.5–2.0 h for vilanterol. Assessments of pharmacokinetic interaction were inconclusive since for umeclidinium, Cmax following combination was higher than umeclidinium alone but not AUC whereas for vilanterol, AUC following combination was higher than vilanterol alone but not Cmax. There were no obvious trends observed between individual maximum supine heart rate and umeclidinium Cmax or vilanterol Cmax when delivered as umeclidinium 500 µg and vilanterol 50 µg combination or when delivered as umeclidinium or vilanterol alone. Trial Registration Clinicaltrials.gov NCT00976144

Effect of verapamil on systemic exposure and safety of umeclidinium and vilanterol: a randomized and open-label study.

Background The combination of umeclidinium (UMEC), a long-acting muscarinic receptor antagonist, and vilanterol (VI), a selective long-acting β2 agonist, is in development for the treatment of chronic obstructive pulmonary disease (COPD). This study evaluated the pharmacokinetics, safety and tolerability, and pharmacodynamics of once-daily, inhaled UMEC and UMEC/VI when co-administered with oral verapamil, a moderate P-glycoprotein transporter and moderate cytochrome P450 3A4 (CYP3A4) inhibitor frequently used by patients with COPD and cardiovascular comorbidities. Methods Subjects were randomized to one of two 13-day treatment regimens: UMEC 500 μg or UMEC 500 μg/VI 25 μg. All subjects received a single tablet containing 240 mg verapamil on each of days 9–13. Results Repeat doses of UMEC and UMEC/VI in combination with and without verapamil were safe and well tolerated. There was no increase in systemic exposure of UMEC when administered in combination with VI compared to UMEC alone. UMEC maximum concentration was similar with or without verapamil; a moderate increase in UMEC area under the curve (approximately 1.4-fold) was observed with verapamil. Verapamil did not increase systemic exposure to VI following administration of the UMEC/VI combination. Conclusion Administration of UMEC and UMEC/VI combination was well tolerated and did not show clinically relevant increases in systemic exposure for either drug. The UMEC/VI combination is unlikely to have a clinically meaningful drug–drug interaction with moderate P-glycoprotein transporter and CYP3A4 inhibitor drugs.

Initial assessment of single and repeat doses of inhaled umeclidinium in patients with chronic obstructive pulmonary disease: Two randomised studies

To characterise the safety, tolerability, pharmacodynamics (bronchodilatory effect) and pharmacokinetics of inhaled umeclidinium in patients with chronic obstructive pulmonary disease (COPD). The first investigation was a single dose, randomised, double-blind, placebo-controlled study (clinicaltrials.gov: NCT00515502) in which ipratropium bromide-sensitive patients received umeclidinium (250μg, 500μg, and 1000μg), tiotropium bromide 18μg or placebo. Patients were randomised to receive four out of five possible treatments as an incomplete block four-way cross-over. A subsequent study (clinicaltrials.gov: NCT700732472) was focused on assessment of safety, tolerability and pharmacokinetics of umeclidinium (250μg and 1000μg) administered once-daily for 7 days in a randomised, double-blind, placebo-controlled, parallel-group design. Of the 24 patients randomised for the single dose study, 20 completed; 31 out of 38 patients completed the repeat dose study. Most adverse events were mild-to-moderate and transient. Examination of heart rate, QTc interval, blood pressure and clinical laboratory assessments raised no concern over the safety of umeclidinium. Evidence of pharmacology was demonstrated in first study by statistically significant increases in specific airway conductance (sGaw) for up to 24h for all active treatments compared with placebo. Increases in forced expiratory volume in 1s were also observed. Pharmacokinetic analysis demonstrated that maximum observed plasma umeclidinium concentration (Cmax) was reached rapidly (time to Cmax: ∼5-15min) after single and repeat doses; 1.5-1.9-fold accumulation was observed after repeat-dosing. Single and repeat doses of umeclidinium were well tolerated and produced clinically relevant lung function improvements over 24h in patients with COPD.

Effect of Disease Severity in Asthma and Chronic Obstructive Pulmonary Disease on Inhaler-Specific Inhalation Profiles Through the ELLIPTA® Dry Powder Inhaler.

Abstract Background: Two studies were undertaken to characterize the maximal effort inhalation profiles of healthy subjects and patients with asthma or chronic obstructive pulmonary disease (COPD) through a moderate-resistance dry powder inhaler (DPI). Correlations between inhaler-specific inhalation characteristics and inhaler-independent lung function parameters were investigated. Methods: Healthy subjects (n = 15), patients with mild, moderate, or severe asthma (n = 45), and patients with mild, moderate, severe, or very-severe COPD (n = 60) were included in the studies. Inhalation pressure drop versus time profiles were recorded using an instrumented ELLIPTA® DPI or bespoke resistor component with equivalent resistivity. Inhaler-independent lung function assessments included pharyngometry, spirometry, plethysmography, and diffusion. Results: For the inhaler-specific inhalation profiles, the mean maximal effort peak inspiratory flow rates (PIFRs) varied across the subgroups from 65.8–110.6 L/min (range: 41.6–142.9). Peak pressure drop, PIFR, inhaled volume, and average inhalation flow rate (primary endpoints) did not differ markedly between healthy subjects and patients with asthma or mild COPD. Moderate, severe, and very-severe COPD patients demonstrated lower mean peak pressure drops, PIFRs and inhaled volumes, which tended to decrease with increasing COPD severity. Severe and very-severe COPD patients demonstrated shorter mean inhalation times compared with all other participants. Inhaler-independent lung function parameters were consistent with disease severity, and statistically significant (p < 0.05) strong correlations (R > 0.7) with components of the inhaler-specific inhalation profiles were observed in the COPD cohort; correlations in the asthma cohort tended to be weaker. Conclusions: All participants achieved a maximal effort PIFR ≥ 41.6 L/min through the moderate resistance of the ELLIPTA inhaler. Patients with asthma achieved similar inhalation profiles to healthy subjects, but increasing COPD severity tended to reduce a patients inhalation capability. Correlation analyses suggest that some lung function parameters may be a useful indicator of ability to inhale efficiently through a moderate-resistance DPI, such as the ELLIPTA inhaler.

Virus recognition by specific natural antibodies and complement results in MHC I cross-presentation.

Natural antibodies (NAb) and complement (C’) are important regulators of immune system activation. We have shown previously that the galactosyl‐α1,3‐galactosyl (Galα1,3Gal) xenoantigen and the similar ABO histo‐blood group antigens are transferred onto virus from the producer cell, resulting in sensitisation of the virus to the respective NAb in a C’‐dependent manner. Here we show that measles virus (Mv) that expresses Galα1,3Gal termini can drive the proliferation of human T cells in the presence of serum and autologous DC, whereas without such targets, measles, as expected, suppress T cell reactivity. The use of affinity‐purified NAb to Galα1,3Gal and rabbit C’ demonstrated the components in human serum responsible for this effect. Proteasome inhibition and blocking of antigen presentation showed that the increased T cell proliferation was mediated by MHC class I cross‐presentation of immune complexes. These results lend further support to the idea that polymorphic carbohydrates of the Galα1,3Gal/ABO type serve as important targets for NAb and C’ and that their expression on virus has influenced their evolution by contributing to protection against viral transmission within as well as between species. The adjuvance effect of this recognition, acting as a bridge between the natural innate and adaptive immune systems, also has important implications for vaccine development.

Effects of Moderate Hepatic Impairment on the Pharmacokinetic Properties and Tolerability of Umeclidinium and Vilanterol in Inhalational Umeclidinium Monotherapy and Umeclidinium/Vilanterol Combination Therapy: An Open-Label, Nonrandomized Study

BACKGROUND The long-acting muscarinic antagonist umeclidinium (UMEC) is approved as a monotherapy, and in combination with the long-acting β2-agonist vilanterol (VI), as a once-daily inhaled maintenance bronchodilator therapy for chronic obstructive pulmonary disease in the US and EU; they are not indicated for the treatment of asthma. Preclinical and clinical data suggest that UMEC and VI are predominantly eliminated by the liver. OBJECTIVES The objectives of the study were to evaluate the effects of moderate hepatic impairment on the plasma and urinary pharmacokinetic properties of each drug, and on the tolerability of inhalational UMEC/VI 125/25 µg and UMEC 125 µg. METHODS This open-label, nonrandomized study was conducted in patients with moderate hepatic impairment (Child-Pugh score, 7-9) and in healthy volunteers (control). Patients and volunteers were administered a single dose of UMEC/VI 125/25 µg, and, after a 7- to 14-day washout period, repeat-dose UMEC 125 µg once daily for 7 days. Primary end points were the plasma pharmacokinetic properties of single- and repeat-dose UMEC and VI. Secondary end points were the urinary pharmacokinetic properties of UMEC, and the tolerability of each treatment. RESULTS All 18 enrolled patients and volunteers (12 men, 6 women; mean age, 53.6 years) completed the study. Mean systemic exposures of UMEC and VI were similar or numerically lower in patients with moderate hepatic impairment compared with those in healthy volunteers, but the differences were not clinically significant. UMEC accumulations with 7-day dosing of UMEC were similar between patients with moderate hepatic impairment and healthy volunteers. UMEC/VI 125/25 µg and UMEC 125 µg were well-tolerated, with no safety concerns identified. CONCLUSIONS The administration of UMEC/VI 125/25 µg or UMEC 125 µg in patients with moderate hepatic impairment did not result in clinically relevant increases in UMEC or VI exposures compared with those in healthy volunteers. Based on these findings, no dose adjustment for UMEC/VI or UMEC is warranted in patients with moderate hepatic impairment.

Effect of severe renal impairment on umeclidinium and umeclidinium/vilanterol pharmacokinetics and safety: a single-blind, nonrandomized study

Background Umeclidinium and vilanterol, long-acting bronchodilators for the treatment of chronic obstructive pulmonary disease, are primarily eliminated via the hepatic route; however, severe renal impairment may adversely affect some elimination pathways other than the kidney. Objectives To evaluate the effect of severe renal impairment on the pharmacokinetics of umeclidinium and umeclidinium/vilanterol. Methods Nine patients with severe renal impairment (creatinine clearance <30 mL/min) and nine matched healthy volunteers received a single dose of umeclidinium 125 μg; and after a 7- to 14-day washout, a single dose of umeclidinium/vilanterol 125/25 μg. Results No clinically relevant increases in plasma umeclidinium or vilanterol systemic exposure (area under the curve or maximum observed plasma concentration) were observed following umeclidinium 125 μg or umeclidinium/vilanterol 125/25 μg administration. On average, the amount of umeclidinium excreted in 24 hours in urine (90% confidence interval) was 88% (81%–93%) and 89% (81%–93%) lower in patients with severe renal impairment compared with healthy volunteers following umeclidinium 125 μg and umeclidinium/vilanterol 125/25 μg administration, respectively. Treatments were well tolerated in both populations. Conclusion Umeclidinium 125 μg or umeclidinium/vilanterol 125/25 μg administration to patients with severe renal impairment did not demonstrate clinically relevant increases in systemic exposure compared with healthy volunteers. No dose adjustment for umeclidinium and umeclidinium/vilanterol is warranted in patients with severe renal impairment.

Pig alpha1, 3galactosyltransferase: a major target for genetic manipulation in xenotransplantation.

Terminal carbohydrate residues of glycolipids and glycoproteins display polymorphism among as well as within various species. With the exception of Old World monkeys, great apes and man, the Gala1,3Gal structure is widely expressed in all mammals examined so far. The lack of expression of the glycosyltransferase responsible for the synthesis of Gala1,3Gal leads to the production of high titers of natural antibodies (NAb) against the Gala1,3Gal of other species. The inactivation of this gene occurred during early evolution of primates. Neutralization of viruses (e.g. retroviruses) carrying the epitope, by the pre-formed human NAb, indicates one possible evolutionary reason for the polymorphism of terminal carbohydrates among as well as within species. It has been shown that this epitope constitutes the major target, on pig endothelial cells (EC), for the pre-formed human NAb resulting in a hyperacute rejection (HAR) response. This currently makes transplantation of e.g. pig organs to humans impossible. Efforts are currently underway to prevent or to eradicate the expression of this epitope in transgenic pigs. Such pigs are likely to display a greatly increased resistance to the HAR.

A Randomized Clinical Trial Comparing the ELLIPTA and HandiHaler Dry Powder Inhalers in Patients With COPD: Inhaler-Specific Attributes and Overall Patient Preference

ABSTRACT This randomised, open-label, cross-over, placebo-containing inhaler study assessed patient preference indicators for ELLIPTA and HandiHaler dry powder inhalers in patients with COPD (NCT02786927; GSK identifier: 204983). The primary objective of this study was to assess patient preference between ELLIPTA and HandiHaler based on the number of steps needed to use the inhaler. Eligible patients ≥40 years of age with COPD were randomised 1:1 to receive their current COPD medication plus a placebo-containing ELLIPTA or HandiHaler inhaler once daily for 7 ± 2 days (treatment period 1); this was followed by a 7 ± 2-day placebo treatment with the alternative inhaler. A 5-item questionnaire assessed inhaler-related patient preferences. A total of 212 patients (mean age, 65.1 years) were enrolled at 22 US sites; 73% had a COPD duration ≥5 years. Median (range) exposure was 8 (5, 13) days for ELLIPTA and 8 (1, 16) days for HandiHaler. Significantly more patients preferred ELLIPTA to HandiHaler in terms of the number of steps to use and all secondary attributes (size, comfort of the mouthpiece, remaining doses, and ease of use of the two inhalers; all p < 0.001). Similar results were observed irrespective of the order of inhaler use. Eighteen patients (8%) reported at least one AE and two (<1%) patients reported four non-fatal SAEs; none were related to the study treatment. Patient attitude toward a particular inhaler and their experiences in using it can affect adherence to therapy, which can in turn strongly influence effectiveness of inhaled medications. This study uses a robust methodology to assess patient preference.