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BACKGROUND: Prasugrel is an oral antiplatelet agent approved for the reduction of atherothrombotic cardiovascular events in patients presenting with acute coronary syndrome and undergoing percutaneous coronary intervention. Although the approved loading dose is 60 mg, earlier studies of prasugrel suggested that active-metabolite exposure and pharmacodynamic response may be higher in Asian subjects than in white subjects. OBJECTIVES: This study compared the pharmacodynamic response to a single 30-mg dose of prasugrel in healthy Chinese and white subjects and the response to a single 30-mg dose of prasugrel and a single 300-mg dose of clopidogrel in healthy Chinese subjects. The pharmacokinetics and tolerability of both drugs were also assessed. METHODS: This was an open-label, single-dose study conducted in Singapore. Chinese subjects were randomly allocated to receive prasugrel 30 mg or clopidogrel 300 mg; after a 14-day washout period, they received the alternative drug. White subjects received only prasugrel 30 mg. Blood samples for pharmaco-dynamic assessments were collected before dosing and at 0.5, 1, 2, 4, and 24 hours after dosing. Three methods were used to measure inhibition of platelet aggregation (IPA)-traditional light transmission aggregometry (LTA), the Verify Now P2Y12 (VN-P2Y12) assay, and a vasodilator-stimulated phosphoprotein (VASP) phosphorylation flow cytometry assay-and their results were compared. Blood samples for pharmacokinetic assessments were collected at 0.25, 0.5, 1, 1.5, 2, 4, 8, 12, and 24 hours after dosing. Concentrations of the active metabolite of prasugrel were measured using a validated LC-MS/MS method. RESULTS: The study enrolled 18 Chinese subjects and 14 white subjects. Chinese subjects had a mean (SD) age of 31 (10) years and a mean body weight of 65.2 (8.9) kg; 83% were male. The corresponding values for white subjects were 30 (10) years, 77.2 (12.4) kg, and 86%. Thirty of the 32 enrolled subjects completed the study. Two Chinese men were withdrawn from the study, one due to a low platelet-rich plasma count after receipt of prasugrel 30 mg and the other due to mild, intermittent rectal bleeding after bowel movements that began approximately 2 days after receipt of clopidogrel 300 mg. The mean IPA with prasugrel was significantly higher in Chinese than in white subjects at 0.5, 1, and 2 hours after dosing (P < 0.05), but not at 4 or 24 hours. In Chinese subjects, mean maximal IPA (87%) occurred 1 hour after prasugrel dosing; in white subjects, mean maximal IPA (78%) occurred 2 hours after prasugrel dosing. In Chinese subjects, the mean IPA was significantly higher at all time points after administration of prasugrel 30 mg than after administration of clopidogrel 300 mg (P <0.001). After administration of Clopidogrel 300 mg in Chinese subjects, mean maximal IPA (58%) occurred at 4 hours. The VN-P2Y12 and VASP phosphorylation assays yielded results comparable to those obtained by LTA. Mean exposure to prasugrel's active metabolite was higher in Chinese than in white subjects (geometric least squares mean ratio for AUC(0-t) = 1.47 (90% CI, 1.24-1.73). Both drugs were well tolerated. CONCLUSIONS: In this study, platelet inhibition was significantly higher in Chinese than in white subjects up to 2 hours after a single 30-mg dose of prasugrel. Platelet inhibition was significantly higher in Chinese subjects at all time points after a 30-mg dose of prasugrel than after a 300-mg dose of clopidogrel. Both treatments were generally well tolerated. [ABSTRACT FROM AUTHOR]

BACKGROUND: A substantial portion of patients at risk for acute coronary syndrome (ACS) are >65 years old. Prasugrel is a novel antiplatelet agent approved for the treatment of ACS patients undergoing percutaneous coronary intervention, and will be used in this population. OBJECTIVE: This study assessed the effect of age >or=65 years on the pharmacokinetics (PK) and pharmacodynamics (PD) of the active metabolite (R-138727) of prasugrel in healthy subjects taking aspirin (acetylsalicylic acid). METHODS: This was an open-label, single-sequence trial conducted in a single clinical research centre in the UK. A total of 17 subjects aged 65-80 years and 15 subjects aged 20-39 years received a prasugrel 5-mg once-daily maintenance dose for 10 days followed by 10-mg once daily maintenance doses for 10 days. All subjects also received aspirin 75 mg daily. Serial blood samples were collected pre-dose and at various times post-dose for measurement of the active metabolite of prasugrel in plasma on days 10 and 20, following the last 5- and 10-mg prasugrel dose, respectively. PK parameters of the active metabolite of prasugrel included area under the plasma concentration-time curve (AUC) from time zero to the time of the last quantifiable concentration (AUC(last)), maximum plasma concentration (C(max)) and time to C(max) (t(max)). Maximal platelet aggregation (MPA), assessed by light transmission aggregometry using adenosine diphosphate (ADP) 20 micromol/L, was assessed at baseline and on day 10 (5-mg maintenance dose) and day 20 (10-mg maintenance dose). Bleeding times (BTs) were determined on days -5, 1, 10, 11, 20 and 21 using a modified Ivy technique. RESULTS: AUC(last) did not differ significantly between age groups. The steady-state trough MPA to ADP 20 micromol/L during 10-mg maintenance dosing was 30.6% and 26.6% in elderly and young subjects, respectively. Mean MPA was consistently higher in elderly subjects compared with young subjects; however, differences were generally less than ten percentage points. BTs did not differ between the two populations during 5-mg maintenance dosing; however, during 10-mg maintenance dosing, BTs were up to 67% longer in young compared with elderly subjects. A higher frequency of minor bleeding during 10-mg maintenance dosing was observed in elderly subjects compared with young subjects. CONCLUSIONS: These data indicate that prasugrel PK and MPA were similar in healthy subjects regardless of age. Compared with younger subjects, elderly subjects had shorter BTs but a greater frequency of mild bleeding-related adverse events. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Clopidogrel is an oral thienopyridine antiplatelet agent indicated for the treatment of atherothrombotic events in patients with acute coronary syndrome (ACS). Prasugrel, a novel oral thienopyridine, is under investigation for the reduction of atherothrombotic events in patients with ACS undergoing percutaneous coronary intervention. Prasugrel's solubility decreases with increasing pH, suggesting that concomitantly-administered medications that increase gastric pH may lower the rate and/or extent of prasugrel absorption. This study evaluated the influence of ranitidine coadministration on the pharmacokinetics and pharmacodynamics of the respective active metabolite of prasugrel and clopidogrel. RESEARCH DESIGN AND METHODS: In this open-label, two-period, two-treatment, crossover study, 47 healthy male subjects were randomized to one of two study arms, receiving either prasugrel (60-mg loading dose [LD], 10-mg maintenance dose [MD] for 7 days; n = 23) or clopidogrel (600-mg LD, 75-mg MD for 7 days; n = 24). In one treatment period, subjects received prasugrel or clopidogrel alone, and in the alternate period received the same thienopyridine with ranitidine (150 mg twice daily, starting 1 day before the LD). Pharmacokinetic parameter estimates (AUC(0-t last), C(max), and t(max)) and inhibition of platelet aggregation (IPA) by light transmission aggregometry were assessed at multiple time points after the LD and final MD. RESULTS: Ranitidine had no clinically significant effect on the area under the plasma-concentration-time curve (AUC) and did not affect the time to C(max) (t(max)) for active metabolites of either prasugrel or clopidogrel. It reduced the geometric mean maximum concentrations of active metabolite (C(max)) after a prasugrel and clopidogrel LD by 14% and 10%, respectively, but these differences were not statistically significant. When coadministered with a 60-mg prasugrel LD, ranitidine did not affect the time to, or magnitude of, peak IPA, but did result in a modest reduction at 0.5 h from 67.4 to 55.1% (p < 0.001). Ranitidine did not affect prasugrel IPA during MD. For clopidogrel, IPA was not affected by ranitidine. Prasugrel and clopidogrel were both well-tolerated, with/without ranitidine. CONCLUSIONS: Results from this study suggest that there is no significant drug-drug interaction between oral ranitidine therapy and concomitantly-administered prasugrel or clopidogrel. [ABSTRACT FROM AUTHOR]

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated channel involved in synaptic transmission, pain signalling, and several ischemia-associated pathological conditions. The spider venom-derived peptides PcTx1 and Hi1a are two of the most potent ASIC1a inhibitors known and have been instrumental in furthering our understanding of the structure, function, and biological roles of ASICs. To date, homologous spider peptides with different pharmacological profiles at ASIC1a have yet to be discovered. Here we report the characterisation of Hc3a, a single inhibitor cystine knot peptide from the Australian funnel-web spider Hadronyche cerberea with sequence similarity to PcTx1. We show that Hc3a has complex pharmacology and binds different ASIC1a conformational states (closed, open, and desensitised) with different affinities, with the most prominent effect on desensitisation. Hc3a slows the desensitisation kinetics of proton-activated ASIC1a currents across multiple application pHs, and when bound directly to ASIC1a in the desensitised conformation promotes current inhibition. The solution structure of Hc3a was solved, and the peptide-channel interaction examined via mutagenesis studies to highlight how small differences in sequence between Hc3a and PcTx1 can lead to peptides with distinct pharmacology. The discovery of Hc3a expands the pharmacological diversity of spider venom peptides targeting ASIC1a and adds to the toolbox of compounds to study the intricacies of ASIC1 gating., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The peptide recifin A is the inaugural member of the structurally intriguing new fold referred to as a tyrosine-lock. Its central four stranded β-sheet is stabilized by a unique arrangement in which three disulfide bonds and their interconnecting backbone form a ring that wraps around one of the strands, resulting in a Tyr side chain being buried in the molecular core. Here we aimed to establish a synthetic route to this complex class of natural products. Full length recifin A was successfully generated through native chemical ligation chemistry joining two 21 amino acid residue fragments. Surprisingly, reduced linear recifin A readily adopts the correct, topologically-complex fold via random oxidation of the cysteines, suggesting it is highly energetically favored. Utilizing our synthetic strategy, we generated five recifin A analogues to investigate the structural role of the central Tyr residue and provide the first insights into the structure activity relationship of recifin A towards its cancer target tyrosyl-DNA phosphodiesterase I., Competing Interests: LRHK, BRO, CIS and KJR are co-inventors on a patent relating to tyrosyl-lock peptides. CIS is a Genentech Inc employee and a shareholder of Roche., (This journal is © The Royal Society of Chemistry.)

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type Ca V 3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type Ca V s but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH 2 ) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH 2 to be a concentration-dependent partial inhibitor of Ca V 3.2 (IC 50 = 1.18 μM) and Ca V 3.3 (IC 50 = 0.49 μM) depolarized currents but was ineffective at Ca V 3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type Ca V 2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH 2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at Ca V 3.2 and Ca V 3.3. As both Ca V 3.2 and Ca V 3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH 2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH 2 produced antiallodynia in both mechanical and thermal pain.

Introduction: Subcutaneous (SC) injection is a common route of drug administration; however, injection site pain (ISP) might create a negative patient experience. We evaluated ISP, bioequivalence, and overall safety of the citrate-free (CF) formulation of ixekizumab, a high-affinity monoclonal antibody that selectively targets interleukin-17A., Methods: Two phase 1, single-blind studies were conducted in healthy participants. The crossover study A (NCT03848403) evaluated pain intensity on injection as measured by visual analog scale of pain (VAS) scores. Subjects (N = 70) were randomized 1:1:1 at the beginning to three possible treatment sequences and received a 1 mL SC injection of the three formulations sequentially in the abdomen on days 1, 8, and 15, respectively. A mixed-effects repeated measures analysis model was used to analyze VAS score by time post-injection. Study B (NCT04259346) evaluated the bioequivalence of a single 80 mg dose of CF formulation compared to the original commercial formulation. Subjects (N = 245) were randomized 1:1 to either commercial or CF formulation and received a single SC injection into the abdomen, arm, or thigh., Results: Primary endpoint was achieved in both studies. In study A, least-squares mean (LSM) difference of VAS scores immediately post injection between commercial (n = 61) and CF formulation (n = 63) was - 21.7 (p < 0.0001), indicating a lower degree of pain associated with CF formulation. In study B, bioequivalence of the CF formulation was established as 90% CIs for the ratio of geometric LSM AUC 0-tlast , AUC 0-∞ , and C max between treatments were contained within the prespecified limits of 0.8 and 1.25. Except for less ISP in the CF formulation, overall safety profile was comparable., Conclusion: Ixekizumab CF formulation proved to be bioequivalent, was associated with less ISP, and had no other notable differences in the safety profile compared to the original commercial formulation., Trail Registration: ClinicalTrials.gov identifier NCT03848403, NCT04259346., (© 2022. The Author(s).)

Natural product discovery by isolation and structure elucidation is a laborious task often requiring ample quantities of biological starting material and frequently resulting in the rediscovery of previously known compounds. However, peptides are a compound class amenable to an alternative genomic, transcriptomic, and in silico discovery route by similarity searches of known peptide sequences against sequencing data. Based on the sequences of barrettides A and B, we identified five new barrettide sequences (barrettides C-G) predicted from the North Atlantic deep-sea demosponge Geodia barretti (Geodiidae). We synthesized, folded, and investigated one of the newly described barrettides, barrettide C (NVVPCFCVEDETSGAKTCIPDNCDASRGTNP, disulfide connectivity I-IV, II-III). Co-elution experiments of synthetic and sponge-derived barrettide C confirmed its native conformation. NMR spectroscopy and the anti-biofouling activity on larval settlement of the bay barnacle Amphibalanus improvisus (IC 50 0.64 μM) show that barrettide C is highly similar to barrettides A and B in both structure and function. Several lines of evidence suggest that barrettides are produced by the sponge itself and not one of its microbial symbionts.

Plants are an excellent source of bioactive peptides, often with disulfide bonds and/or a cyclic backbone. While focus has predominantly been directed at disulfide-rich peptides, a large family of small, cyclic plant peptides lacking disulfide bonds, known as orbitides, has been relatively ignored. A recently discovered subfamily of orbitides is the PawL-derived peptides (PLPs), produced during the maturation of precursors for seed storage albumins. Although their evolutionary origins have been dated, in-depth exploration of the family's structural characteristics and potential bioactivities remains to be conducted. Here we present an extensive and systematic characterization of the PLP family. Nine PLPs were chosen and prepared by solid phase peptide synthesis. Their structural features were studied using solution NMR spectroscopy, and seven were found to possess regions of backbone order. Ordered regions consist of β-turns, with some PLPs adopting two well-defined β-turns within sequences as short as seven residues, which are largely the result of side chain interactions. Our data highlight that the sequence diversity within this family results in equally diverse structures. None of these nine PLPs showed antibacterial or antifungal activity.

The complement activation peptide C5a is a key mediator of inflammation that is associated with numerous immune disorders. C5a binds and activates two seven-transmembrane receptors, C5aR1 and C5aR2. Experimentally, C5a is utilized to investigate C5a receptor biology and to screen for potential C5aR1/C5aR2 therapeutics. Currently, laboratory sources of C5a stem from either isolation of endogenous C5a from human serum or most predominantly via recombinant expression. An alternative approach to C5a production is chemical synthesis, which has several advantages, including the ability to introduce non-natural amino acids and site-specific modifications whilst also maintaining a lower probability of C5a being contaminated with microbial molecules or other endogenous proteins. Here, we describe the efficient synthesis of both human (hC5a) and mouse C5a (mC5a) without the need for ligation chemistry. We validate the synthetic peptides by comparing pERK1/2 signaling in CHO-hC5aR1 cells and primary human macrophages (for hC5a) and in RAW264.7 cells (for mC5a). C5aR2 activation was confirmed by measuring β-arrestin recruitment in C5aR2-transfected HEK293 cells. We also demonstrate the functionalization of synthetic C5a through the introduction of a lanthanide chelating cage to facilitate a screen for the binding of ligands to C5aR1. Finally, we verify that the synthetic ligands are functionally similar to recombinant or native C5a by assessing hC5a-induced neutrophil chemotaxis in vitro and mC5a-mediated neutrophil mobilization in vivo . We propose that the synthetic hC5a and mC5a described herein are valuable alternatives to recombinant or purified C5a for in vitro and in vivo applications and add to the growing complement reagent toolbox., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Head-to-tail cyclic and disulfide-rich peptides are natural products with applications in drug design. Among these are the PawS-Derived Peptides (PDPs) produced in seeds of the daisy plant family. PDP-23 is a unique member of this class in that it is twice the typical size and adopts two β-hairpins separated by a hinge region. The β-hairpins, both stabilised by a single disulfide bond, fold together into a V-shaped tertiary structure creating a hydrophobic core. In water two PDP-23 molecules merge their hydrophobic cores to form a square prism quaternary structure. Here, we synthesised PDP-23 and its enantiomer comprising d-amino acids and achiral glycine, which allowed us to confirm these solution NMR structural data by racemic crystallography. Furthermore, we discovered the related PDP-24. NMR analysis showed that PDP-24 does not form a dimeric structure and it has poor water solubility, but in less polar solvents adopts near identical secondary and tertiary structure to PDP-23. The natural role of these peptides in plants remains enigmatic, as we did not observe any antimicrobial or insecticidal activity. However, the plasticity of these larger PDPs and their ability to change structure under different conditions make them appealing peptide drug scaffolds., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)

Purpose: The objective of this work was to systematically evaluate the effects of formulation composition on subcutaneous injection site pain (ISP) using matrices comprising of common pharmaceutical excipients., Methods: Two randomized, blinded, crossover studies in healthy subjects were conducted at a single site, where subjects received 1 mL SC injections of the buffer matrices. ISP intensity was measured using a 100 mm visual analogue scale (VAS), which was then analyzed via heatmap, categorical grouping, subgroup analysis, and paired delta analysis., Results: Buffer type, buffer concentration and tonicity agent showed a substantial impact on ISP. Citrate buffer demonstrated a higher ISP than acetate buffer or saline). The 20 mM citrate buffer was more painful than 10 or 5 mM citrate buffers. NaCl and propylene glycol were significantly more painful than sugar alcohols (mannitol, sucrose, trehalose or glycerol). Histidine buffers exhibited ISP in the descending order of 150 mM > 75 mM > 25 mM > 0 mM NaCl, while histidine buffers containing Arginine-HCl at 0, 50, or 150 mM all showed very low ISP. Histidine buffer at pH 6.5 showed a lower ISP than pH 5.7., Conclusions: This systematic study via orthogonal analyses demonstrated that subcutaneous ISP is significantly influenced by solution composition.

Head-to-tail cyclized peptides are intriguing natural products with unusual properties. The PawS-Derived Peptides (PDPs) are ribosomally synthesized as part of precursors for seed storage albumins in species of the daisy family, and are post-translationally excised and cyclized during proteolytic processing. Here we report a PDP twice the typical size and with two disulfide bonds, identified from seeds of Zinnia elegans . In water, synthetic PDP-23 forms a unique dimeric structure in which two monomers containing two β-hairpins cross-clasp and enclose a hydrophobic core, creating a square prism. This dimer can be split by addition of micelles or organic solvent and in monomeric form PDP-23 adopts open or closed V-shapes, exposing different levels of hydrophobicity dependent on conditions. This chameleonic character is unusual for disulfide-rich peptides and engenders PDP-23 with potential for cell delivery and accessing novel targets. We demonstrate this by conjugating a rhodamine dye to PDP-23, creating a stable, cell-penetrating inhibitor of the P-glycoprotein drug efflux pump., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Objective: To measure the nasal gas flow in infants treated with bubble continuous positive airway pressure (CPAP) and compare it with commonly used flows during nasal high flow (nHF) treatment., Design: This is a prospective, single-centre study. Bubble CPAP pressure was measured at the nasal prongs. Set gas flow was reduced until bubbling in the water chamber just ceased. Set gas flow without bubbling then approximated flow entering the infant's nose ('delivered flow')., Setting: Neonatal intensive care at The Royal Women's Hospital, Melbourne, Australia., Patients: Clinically stable preterm infants receiving bubble CPAP therapy., Main Outcome Measure: Delivered flow (L/min) when bubbling stopped at a range of clinically set CPAP pressures (cm H 2 O)., Results: Forty-four infants were studied, with a mean (SD) gestational age at birth of 28.4 (2.2) weeks and birth weight of 1154 (419) g. At the time of the study, infants had a median (IQR) age of 4.5 (2-12) days and a mean (SD) weight of 1205 (407) g. Delivered flow ranged from 0.5 to 9.0 L/min, and increased with higher set CPAP pressures (median 3.5 L/min at CPAP 5 cm H 2 O vs 6.3 L/min at CPAP 8 cm H 2 O) and heavier weights (median 3.5 L/min in infants <1000 g vs 6.5 L/min for infants >1500 g)., Conclusions: Nasal gas flows during bubble CPAP in preterm infants are similar to flows used during nHF and increase with higher set bubble CPAP pressures and in larger infants. Trial registration number ACTRN12619000197134., Competing Interests: Competing interests: CJM is a consultant for Fisher & Paykel Healthcare., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

The complement component C5 inhibitory peptide zilucoplan is currently in phase III clinical trials for myasthenia gravis (MG). Despite being at an advanced stage of clinical development, there have been no published reports in the literature detailing its chemical synthesis. In this work, we describe an approach for the chemical synthesis of zilucoplan and validate that the synthesised compound blocks LPS-induced C5a production from human blood.

The objective of this phase 1 study was to evaluate the pharmacokinetics, safety, and tolerability of baricitinib after single and multiple doses in healthy Chinese adults. Eligible subjects received a once-daily dose of baricitinib 2, 4, or 10 mg or placebo on day 1 (single dose) and days 4 through 10 for 7 consecutive days (multiple doses). Plasma pharmacokinetic samples were collected up to 48 hours after dosing on days 1 and 10, with predose samples collected before dosing on day 1 and days 4 through 10. Safety and tolerability were also assessed. Baricitinib was rapidly absorbed, reaching peak plasma concentrations within 0.5 to 1 hour (median). Plasma concentrations declined rapidly following the attainment of peak concentrations, with a mean terminal half-life of 5.7 to 7.3 hours. Steady-state plasma concentrations of baricitinib were achieved after the second day of once-daily dosing, with minimal accumulation of baricitinib in plasma (up to 10% increase in area under the plasma concentration-time curve). Single- and multiple-dose mean values for area under the plasma concentration-time curve from time zero to infinity and maximum plasma concentration appeared to increase in an approximately dose-proportional manner across the dose range. Single and multiple oral doses of once-daily baricitinib up to 10 mg were well tolerated by healthy Chinese subjects., (© 2020 The Authors. Clinical Pharmacology in Drug Development published by Wiley Periodicals LLC on behalf of American College of Clinical Pharmacology.)

Plants and their seeds have been shown to be a rich source of cystine-stabilized peptides. Recently a new family of plant seed peptides whose sequences are buried within precursors for seed storage vicilins was identified. Members of this Vicilin-Buried Peptide (VBP) family are found in distantly related plant species including the monocot date palm, as well as dicotyledonous species like pumpkin and sesame. Genetic evidence for their widespread occurrence indicates that they are of ancient origin. Limited structural studies have been conducted on VBP family members, but two members have been shown to adopt a helical hairpin fold. We present an extensive characterization of VBPs using solution NMR spectroscopy, to better understand their structural features. Four peptides were produced by solid phase peptide synthesis and shown to favor a helix-loop-helix hairpin fold, as a result of the I-IV/II-III ladderlike connectivity of their disulfide bonds. Interhelical interactions, including hydrophobic contacts and salt bridges, are critical for the fold stability and control the angle at which the antiparallel α-helices interface. Activities reported for VBPs include trypsin inhibitory activity and inhibition of ribosomal function; however, their diverse structural features despite a common fold suggest that additional bioactivities yet to be revealed are likely.

Cyclic peptides are reported to have antibacterial, antifungal, and other bioactivities. Orbitides are a class of cyclic peptides that are small, head-to-tail cyclized, composed of proteinogenic amino acids and lack disulfide bonds; they are also known in several genera of the plant family Rutaceae. Melicope xanthoxyloides is the Australian rain forest tree of the Rutaceae family in which evolidine, the first plant cyclic peptide, was discovered. Evolidine (cyclo-SFLPVNL) has subsequently been all but forgotten in the academic literature, so to redress this we used tandem MS and de novo transcriptomics to rediscover evolidine and decipher its biosynthetic origin from a short precursor just 48 residues in length. We also identified another six M. xanthoxyloides orbitides using the same techniques. These peptides have atypically diverse C termini consisting of residues not recognized by either of the known proteases plants use to macrocyclize peptides, suggesting new cyclizing enzymes await discovery. We examined the structure of two of the novel orbitides by NMR, finding one had a definable structure, whereas the other did not. Mining RNA-seq and whole genome sequencing data from other species of the Rutaceae family revealed that a large and diverse family of peptides is encoded by similar sequences across the family and demonstrates how powerful de novo transcriptomics can be at accelerating the discovery of new peptide families., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Fisher et al.)

The disposition and metabolism of prexasertib, a CHK-1 inhibitor was characterised over a 120 h period following a single 170-mg intravenous dose of [ 14 C]prexasertib (50 µCi) to 6 patients with advanced/metastatic solid tumours.The prexasertib safety profile was consistent with prior studies. Plasma, urine, and faeces were analysed for radioactivity, prexasertib, and metabolites. Geometric mean t 1/2 in plasma was 34.2 h for prexasertib and 73.8 h for total radioactivity. Unchanged prexasertib accounted for approximately 9% of plasma total radioactivity, indicating extensive metabolism by the presence of circulating metabolites. Both renal and faecal excretion were identified as important routes of elimination since 41.8% (±12.9%) of the total administered radioactivity was recovered in the renal excretions and 32.2% (±7.28%) in the faecal excretions. Mean renal clearance was approximately 15% of the total systemic clearance, while biliary clearance was also low. Prexasertib was cleared predominantly by metabolism with only 23% of the dose recovered in excreta as intact drug. Radioactivity was eliminated predominantly within 72 h in urine, but faecal elimination was protracted.The metabolism of prexasertib was complex while primary metabolic clearance pathways involved were oxidative deamination, O-dealkylation, mono-oxidation, and possibly direct glucuronide conjugation. Although prexasertib was the major component in plasma, up to 11 metabolites were observed. The most abundant metabolites identified in plasma were glucuronides and none of these are expected to contribute to the pharmacological activity or pose a safety concern.

Cyclic peptides are abundant in plants and have attracted interest due to their bioactivity and potential as drug scaffolds. Orbitides are head-to-tail cyclic peptides that are ribosomally synthesized, post-translationally modified, and lack disulfide bonds. All known orbitides contain 5-12 amino acid residues. Here we describe PLP-53, a novel orbitide from the seed of Ratibida columnifera . PLP-53 consists of 16 amino acids, four residues larger than any known orbitide. NMR structural studies showed that, compared to previously characterized orbitides, PLP-53 is more flexible and, under the studied conditions, did not adopt a single ordered conformation based on analysis of NOEs and chemical shifts.

New proteins can evolve by duplication and divergence or de novo, from previously noncoding DNA. A recently observed mechanism is for peptides to evolve within a "host" protein and emerge by proteolytic processing. The first examples of such interstitial peptides were ones hosted by precursors for seed storage albumin. Interstitial peptides have also been observed in precursors for seed vicilins, but current evidence for vicilin-buried peptides (VBPs) is limited to seeds of the broadleaf plants pumpkin and macadamia. Here, an extensive sequence analysis of vicilin precursors suggested that peptides buried within the N-terminal region of preprovicilins are widespread and truly ancient. Gene sequences indicative of interstitial peptides were found in species from Amborellales to eudicots and include important grass and legume crop species. We show the first protein evidence for a monocot VBP in date palm seeds as well as protein evidence from other crops including the common tomato, sesame and pumpkin relatives, cucumber, and the sponge loofah ( Luffa aegyptiaca). Their excision was consistent with asparaginyl endopeptidase-mediated maturation, and sequences were confirmed by tandem mass spectrometry. Our findings suggest that the family is large and ancient and that based on the NMR solution structures for loofah Luffin P1 and tomato VBP-8, VBPs adopt a helical hairpin fold stapled by two internal disulfide bonds. The first VBPs characterized were a protease inhibitor, antimicrobials, and a ribosome inactivator. The age and evolutionary retention of this peptide family suggest its members play important roles in plant biology.

Baricitinib, an oral selective Janus kinase 1 and 2 inhibitor, undergoes active renal tubular secretion. Baricitinib was not predicted to inhibit hepatic and renal uptake and efflux drug transporters, based on the ratio of the unbound maximum eliminating-organ inlet concentration and the in vitro half-maximal inhibitory concentrations (IC 50 ). In vitro, baricitinib was a substrate for organic anion transporter (OAT)3, multidrug and toxin extrusion protein (MATE)2-K, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP). Probenecid, a strong OAT3 inhibitor, increased the area under the concentration-time curve from time zero to infinity (AUC [0-∞] ) of baricitinib by twofold and decreased renal clearance to 69% of control in healthy subjects. Physiologically based pharmacokinetic (PBPK) modeling reproduced the renal clearance of baricitinib and the inhibitory effect of probenecid using the in vitro IC 50 value of 4.4 μM. Using ibuprofen and diclofenac in vitro IC 50 values of 4.4 and 3.8 μM toward OAT3, 1.2 and 1.0 AUC (0-∞) ratios of baricitinib were predicted. These predictions suggest clinically relevant drug-drug interactions (DDIs) with ibuprofen and diclofenac are unlikely., (© 2017 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Platelets of patients with sickle cell disease (SCD) show evidence of mild activation in the non-crisis steady state and greater activation during vaso-occlusive crises (VOC). Prasugrel, a potent inhibitor of ADP-mediated platelet activation and aggregation, may be useful in attenuating VOC. We compared platelet responses to ADP stimulation in patients with SCD and healthy subjects before and after treatment with prasugrel. In a phase 1 study, platelet biomarker levels were assessed in 12 adult patients with SCD and 13 healthy subjects before and after 12 ± 2 days of 5.0 or 7.5 mg/day prasugrel. The following were determined in whole blood samples stimulated with 20 µM ADP: (i) percentages of monocytes and neutrophils with adherent platelets (cell-platelet aggregates); (ii) the relative number (mass) of platelets associated with each monocyte and neutrophil as reported by CD61 mean fluorescence intensity (MFI) of the monocyte-platelet and neutrophil-platelet aggregates; (iii) the percentages of platelets positive for surface expression of CD40 ligand (CD40L), P-selectin (CD62p) and activated glycoprotein IIb-IIIa (GPIIb-IIIa); and (iv) the percentages of platelets and monocyte-platelet aggregates positive for surface tissue factor (TF) expression. At baseline, there were no significant differences between cohorts in the percentages of platelets expressing activation biomarkers. Following 12 days of prasugrel administration, the percentages of platelets expressing activation biomarkers following ADP stimulation were reduced in both cohorts, and there were no significant differences between groups. Both patients with SCD and healthy subjects had significant reductions in the monocyte-platelet and neutrophil-platelet aggregate MFI and the percentage of platelets expressing P-selectin and activated GPIIb-IIIa (all p < 0.05). Healthy subjects also had significant reductions in monocyte-platelet aggregate percentages (p = 0.004), neutrophil-platelet aggregate percentages (p = 0.011) and the percentage of CD40L-positive platelets (p = 0.044) that were not observed in patients with SCD. Prasugrel administration to SCD patients attenuates ex vivo ADP-stimulated platelet activation as measured by the percentage of platelets positive for P-selectin and GPIIb-IIIa, thus reducing the proportion of platelets that may participate in aggregates. Furthermore, prasugrel decreases ex vivo ADP-stimulated platelet aggregation with monocytes and neutrophils as measured by the monocyte-platelet and neutrophil-platelet aggregate MFI. This implies that in the presence of prasugrel, fewer platelets adhere to monocytes and neutrophils, which may result in reducing cell-platelet aggregate size. Therefore, reduced platelet reactivity and decreased size of leukocyte-platelet aggregates suggest additional mechanisms by which prasugrel may provide benefit to patients with SCD and support further investigation of possible therapeutic benefits of prasugrel in this population.

Aim: The aim of this study was to assess the safety and tolerability, pharmacokinetics and pharmacodynamics of LY3000328 when administered as single escalating doses to healthy volunteers., Methods: This was a phase 1, placebo-controlled, dose escalation study with LY3000328 in 21 healthy male volunteers. Subjects were administered escalating LY3000328 doses up to 300 mg with food in this single dose study. Blood samples were collected at set times post-dose for the assessment of LY3000328 pharmacokinetics and the measurement of cathepsin S (CatS) activity, CatS mass and calculated CatS specific activity., Results: All doses of LY3000328 were well tolerated, with linear pharmacokinetics up to the 300 mg dose. The pharmacodynamic activity of LY3000328 was measured ex vivo showing a biphasic response to LY3000328, where CatS activity declines, then returns to baseline, and then increases to a level above baseline. CatS mass was also assessed post-dose which increased in a dose-dependent manner, and continued to increase after LY3000328 had been cleared from the body. CatS specific activity was additionally calculated to normalize CatS activity for changes in CatS mass. This demonstrated the increase in CatS activity was attributable to the increase in CatS mass detected in plasma., Conclusion: A specific inhibitor of CatS which is cleared quickly from plasma may produce a transient decrease in plasma CatS activity which is followed by a more prolonged increase in plasma CatS mass which may have implications for the future clinical development of inhibitors of CatS., (© 2014 The British Pharmacological Society.)

Introduction: Prasugrel, a P2Y₁₂ adenosine diphosphate (ADP) receptor antagonist effectively inhibits ADP-mediated platelet activation and aggregation, and may be useful in reducing vaso-occlusive crises in sickle cell disease (SCD). In this study, we assess the effect of prasugrel on biomarkers of platelet activation and coagulation in patients with SCD., Materials and Methods: Twelve adult patients with SCD and 13 healthy subjects were examined before and after 12 ± 2 days of 5.0 or 7.5 mg/day oral prasugrel. Assessed cellular biomarkers included monocyte- and neutrophil-platelet aggregates, activated glycoprotein IIb-IIIa (GPIIbIIIa), P-selectin, CD40 ligand (CD40L), tissue factor (TF) expression on circulating platelets and on monocyte-platelet aggregates, and platelet-erythrocyte aggregates. Soluble biomarkers included CD40L, prothrombin fragment 1.2 (F1.2), thromboxane B₂ (TXB₂), P-selectin, and TF., Results: Patients with SCD had increased platelet baseline activation compared to healthy subjects, as measured by percentages of monocyte-platelet aggregates, neutrophil-platelet aggregates, and platelets expressing CD40L. Likewise, baseline levels of soluble F1.2 and TXB₂ were elevated in patients with SCD compared to healthy subjects. After 12 days of prasugrel, patients with SCD had a significant reduction in platelet-monocyte aggregates that was not observed in healthy subjects. Following prasugrel administration, those with SCD maintained higher levels of monocyte-platelet aggregates and soluble F1.2, but had lower levels of platelet-erythrocyte aggregates and soluble TF compared to healthy subjects., Conclusions: These results provide evidence for chronic platelet activation in the SCD steady state, activation that was in part attenuated by prasugrel, thereby suggesting that ADP may mediate platelet activation in SCD., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The potential interactive effects of iron (Fe) limitation and Ocean Acidification in the Southern Ocean (SO) are largely unknown. Here we present results of a long-term incubation experiment investigating the combined effects of CO2 and Fe availability on natural phytoplankton assemblages from the Weddell Sea, Antarctica. Active Chl a fluorescence measurements revealed that we successfully cultured phytoplankton under both Fe-depleted and Fe-enriched conditions. Fe treatments had significant effects on photosynthetic efficiency (Fv/Fm; 0.3 for Fe-depleted and 0.5 for Fe-enriched conditions), non-photochemical quenching (NPQ), and relative electron transport rates (rETR). pCO2 treatments significantly affected NPQ and rETR, but had no effect on Fv/Fm. Under Fe limitation, increased pCO2 had no influence on C fixation whereas under Fe enrichment, primary production increased with increasing pCO2 levels. These CO2-dependent changes in productivity under Fe-enriched conditions were accompanied by a pronounced taxonomic shift from weakly to heavily silicified diatoms (i.e. from Pseudo-nitzschia sp. to Fragilariopsis sp.). Under Fe-depleted conditions, this functional shift was absent and thinly silicified species dominated all pCO2 treatments (Pseudo-nitzschia sp. and Synedropsis sp. for low and high pCO2, respectively). Our results suggest that Ocean Acidification could increase primary productivity and the abundance of heavily silicified, fast sinking diatoms in Fe-enriched areas, both potentially leading to a stimulation of the biological pump. Over much of the SO, however, Fe limitation could restrict this possible CO2 fertilization effect.

Aims: Prasugrel is a novel thienopyridine P2Y12 adenosine diphosphate (ADP) receptor antagonist that inhibits ADP-mediated platelet activation and aggregation. Accordingly, it may be useful in reducing platelet-related ischaemia in sickle cell disease (SCD). Exposure to prasugrel's active metabolite (Pras-AM) and its antiplatelet activity in SCD have not been investigated., Methods: Thirteen adult patients with SCD and an equal number of matched healthy control subjects were studied before and after 12 days of 5.0 or 7.5 mg day(-1) prasugrel treatment. Platelet reactivity was assessed by light transmission aggregometry (LTA), impedance aggregometry (MEA), VerifyNow® P2Y12, vasodilator-stimulated phosphoprotein (VASP) phosphorylation and Plateletworks. Exposure to Pras-AM was also assessed., Results: At baseline, patients with SCD showed increased platelet reactivity vs. healthy control subjects with VerifyNow (408 vs. 323 P2Y12 reaction units (PRU), respectively, P = 0.003) and MEA (106 vs. 77 area under the aggregation curve (AU.min), P = 0.002); lower platelet reactivity index with VASP flow cytometry (59 vs. 79% platelet reactivity index (PRI), P = 0.018); and no significant differences with LTA, VASP enzyme-linked immunosorbent assay or Plateletworks. Relative to baseline, prasugrel significantly reduced platelet reactivity by all assays in both populations (all P < 0.05). Prasugrel was well tolerated, with no bleeding-related events in patients with SCD. The mean concentration-time profiles of Pras-AM were comparable between healthy subjects and patients with SCD following a single 10 mg prasugrel dose and following the 12th dose of 7.5 or 5 mg prasugrel., Conclusions: Results demonstrate that in response to prasugrel, patients with SCD and healthy subjects have similar degrees of platelet inhibition and exposure to Pras-AM, and provide a basis for further study of prasugrel in patients with SCD., (© 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.)

An integrated analysis of pharmacokinetic (PK) parameter estimates for prasugrel, from 16 phase I clinical pharmacology studies, consolidated exposure estimates from 506 healthy male and female participants and evaluated the effect of specific intrinsic and extrinsic factors on exposure to prasugrel's active metabolite (AM, R-138727). A linear mixed effect model was fitted with study and subject nested within study as random effects and subject factors as fixed effects. Prasugrel AM exposure was similar in males and females, in participants <65 years and ≥65 years, in smokers and nonsmokers, in drinkers and nondrinkers of alcohol, and in Asians, Caucasians, and participants of African and Hispanic descent. Prasugrel AM exposure increased as body weight decreased and was 40% higher in participants <60 kg than in participants ≥60 kg. Exposure in Asians was 40% higher than that in Caucasians, but this difference could be explained by the lower overall weight of the Asian participants and a disproportionately large difference between ethnic groups in lighter participants, especially those <60 kg. These results characterize prasugrel's PK across a range of studies and highlight body weight as the most influential covariate on prasugrel AM exposure, with implications for prasugrel maintenance dosing in clinical practice.

Thienopyridines are inactive prodrugs that are converted in vivo to active metabolites, which irreversibly bind to and inactivate platelet P2Y(12) receptors, and inhibit platelet activation and aggregation. Prasugrel is a third-generation thienopyridine, recently approved for prevention of thrombotic cardiovascular complications in patients with an acute coronary syndrome undergoing percutaneous coronary intervention. Prasugrel is converted to its active metabolite (Pras-AM; compound R-138727) in two sequential steps: (i) rapid and complete hydrolysis by intestinal human carboxylesterase-2 to form a thiolactone intermediate; and (ii) oxidation of the thiolactone by cytochrome P450 (CYP) enzymes in the gut and/or the liver. CYP3A and CYP2B6 are the primary CYPs contributing to Pras-AM formation, with smaller contributions from CYP2C9 and CYP2C19. Prasugrel is rapidly absorbed and metabolized, with Pras-AM plasma concentrations peaking at about 0.5 hours after oral administration; this helps to account for the rapid onset of inhibition of platelet aggregation (IPA) achieved by prasugrel. In the clinical pharmacology programme for prasugrel, bodyweight had the greatest effect of all covariates that were tested. In the phase III TRITON-TIMI 38 trial, the mean exposure to Pras-AM was 42% greater in patients weighing < 60 kg than in patients with the study population median bodyweight of 85 kg. In a pharmacodynamic meta-analysis of data from healthy subjects a decrease of 1 kg in bodyweight was associated with an increase in IPA of approximately 0.26 percentage points (p < 0.0001). Pras-AM exposure was greater in subjects aged ≥ 75 years, but exposure differences were not as large as those for bodyweight. Pras-AM exposure was greater in Asians than in Caucasians, but this appeared to result from a disproportionately greater exposure difference in Asian subjects with low bodyweight. Sex and allelic variation in CYPs 1A2, 2B6, 2C19, 2C9, 3A4 and 3A5 appeared to have no clinically relevant effect on Pras-AM exposure or IPA. Consistent with the lack of association between genetic status and these pharmacokinetic and pharmacodynamic results in healthy subjects, no significant association was detected between these allelic variants and the composite primary endpoint (cardiovascular death, non-fatal myocardial infarction or non-fatal stroke) in the TRITON-TIMI 38 trial. Studies in renally impaired subjects and subjects with mild or moderate hepatic impairment have indicated that dose adjustment is not required in these patient populations. Prasugrel has few clinically significant drug-drug interactions. Potent CYP3A inhibitors, gastric acid suppressants and food have been shown to reduce the rate of formation of Pras-AM but not its overall exposure. This pharmacokinetic effect reduced the rate of onset of IPA after a loading dose but did not affect the peak IPA after a loading dose or the IPA during maintenance dosing. Potent induction of CYP3A, as well as smoking--which induces CYP1A2--did not affect Pras-AM exposure or IPA. Prior treatment with clopidogrel did not influence tolerability to prasugrel and did not appear to alter IPA during prasugrel treatment. Prasugrel did not affect the activities of CYP2C9, CYP2C19 or P-glycoprotein, but it weakly inhibited CYP2B6. The inhibition of CYP2B6 is potentially clinically significant only for drugs that have a narrow therapeutic window and have CYP2B6 as the primary elimination pathway. No interaction was detected between prasugrel and heparin. Although prasugrel did not alter warfarin pharmacokinetics, prasugrel and warfarin should not be used together, because of an increased bleeding risk associated with their concomitant use.

We compared platelet function results obtained with the VerifyNow P2Y12 (VN-P2Y12) point-of-care device and the light transmission aggregometry (5 and 20 microM adenosine diphosphate) method using an integrated database of eight clinical trials with a total of 591 subjects. The study was performed in healthy subjects, patients with coronary artery disease, patients with end-stage renal disease, and patients with acute coronary syndrome after treatment with prasugrel or clopidogrel. Analyses focused on loading doses of 60 mg prasugrel or 600 mg clopidogrel and daily maintenance doses of 10 mg prasugrel or 75 or 150 mg clopidogrel. Similar patterns of platelet inhibition were observed for light transmission aggregometry versus VN-P2Y12 and assay results were well correlated (r approximately 0.7), although a sigmoidal model may more accurately represent the relationship between light transmission aggregometry and VN-P2Y12, because VN-P2Y12 was relatively less sensitive to low and high levels of inhibition. The percentage of poor responders was less with prasugrel compared with clopidogrel by both assays, but the percentages tended to differ between the assays. The VN-P2Y12 "BASE" channel appeared to be susceptible to high levels of P2Y12 blockade, which would underestimate the VN-P2Y12-reported percent inhibition in individuals who respond well to loading doses of thienopyridines. This integrated analysis supports the findings of earlier individual studies comparing these methodologies that assess platelet function.

Purpose: Prasugrel is a novel thienopyridine prodrug metabolised to an active metabolite that binds irreversibly to the platelet P2Y(12) receptor and inhibits adenosine diphosphate (ADP)-induced platelet aggregation. We compared prasugrel pharmacokinetics, pharmacodynamics, and tolerability in healthy Chinese, Japanese, Korean and Caucasian subjects., Methods: In an open-label, single-centre, parallel-design study, 89 healthy subjects (25 Chinese, 20 Japanese, 22 Korean and 22 Caucasian) aged 20-65 years were given a prasugrel 60-mg loading dose (LD) followed by daily 10-mg maintenance doses (MD) for 7 days and then 5-mg MD for 10 days. Plasma concentrations of prasugrel's active metabolite and inhibition of ADP-induced platelet aggregation (IPA) were determined., Results: Mean exposure to prasugrel's active metabolite in all treatment regimens was higher in each of the Asian groups than in the Caucasian group, although there was considerable overlap between individual exposure estimates in Asians and Caucasians. The mean IPA was also higher in Asians than in Caucasians following a prasugrel 60-mg LD, although the difference did not consistently achieve statistical significance. Prasugrel 10-mg or 5-mg MD produced statistically significantly higher IPA in each Asian group compared with that in the Caucasians. Prasugrel was well tolerated during the LD and MD regimens by all groups., Conclusions: Mean exposure to the prasugrel active metabolite following prasugrel 60-mg LD and during daily 10-mg or 5-mg MD was higher in each of the Asian groups than in the Caucasian group, which resulted in greater platelet inhibition.

Objective: The pharmacokinetic (PK) and pharmacodynamic (PD) responses to prasugrel were compared in three studies of healthy subjects vs. those with moderate or end-stage renal impairment., Methods: Two of the three protocols were parallel-design, open-label, single dose (60-mg prasugrel) studies in subjects with end-stage renal disease (ESRD; n = 12) or moderate renal impairment (n = 10) and matched healthy subjects with normal renal function (n = 10). The third protocol was an open-label, single-dose escalation (5, 10, 30 and 60 mg prasugrel) study in subjects with ESRD (n = 16) and matched healthy subjects with normal renal function (n = 16). Plasma concentrations of prasugrel's active metabolite were determined and pharmacokinetic parameter estimates were derived. Maximum platelet aggregation (MPA) was measured by light transmission aggregometry using 20 mum adenosine diphosphate as agonist., Results: Across all studies, prasugrel's C(max) and AUC(0-t) were 51% and 42% lower in subjects with ESRD than in healthy subjects. AUC(0-t) did not differ between healthy subjects and subjects with moderate renal impairment. The magnitude of change and time-course profiles of MPA was similar for healthy subjects compared with subjects with moderate renal impairment and those with ESRD. Prasugrel was well-tolerated in all subjects., Conclusion: There was no difference in pharmacokinetics or PD responses between subjects with moderate renal impairment and healthy subjects. Despite significantly lower exposure to prasugrel's active metabolite in subjects with ESRD, MPA did not differ between healthy subjects and those with ESRD.

Background and Objective: Prasugrel is a thienopyridine antiplatelet agent under investigation for the prevention of atherothrombotic events in patients with acute coronary syndrome who undergo percutaneous coronary intervention. Patients with chronic liver disease are among those in the target population for prasugrel. As hepatic enzymes play a key role in formation of prasugrel's active metabolite, hepatic impairment could affect the safety and/or efficacy of prasugrel in such patients., Methods: This was a parallel-design, open-label, multiple dose study of 30 subjects, 10 with moderate hepatic impairment (Child-Pugh Class B) and 20 with normal hepatic function. Prasugrel was administered orally as a 60-mg loading dose (LD) and daily 10-mg maintenance doses (MDs) for 5 days. Pharmacokinetic parameters (AUC(0-t), C(max) and t(max)) and maximal platelet aggregation (MPA) by light transmission aggregometry were assessed after the LD and final MD., Results and Discussion: Exposure to prasugrel's active metabolite was comparable between healthy subjects and those with moderate hepatic impairment. Point estimates for the ratios of geometric least square means for AUC(0-t) and C(max) after the LD and last MD ranged from 0.91 to 1.14. MPA to 20 microm ADP was similar between subjects with moderate hepatic impairment and healthy subjects for both the LD and MD. Prasugrel was well tolerated by all subjects, and adverse events were mild in severity., Conclusion: Moderate hepatic impairment appears to have no effect on exposure to prasugrel's active metabolite. Furthermore, MPA results suggest that moderate hepatic impairment has little or no effect on platelet aggregation relative to healthy controls. Overall, these results suggest that a dose adjustment would not be required in moderately hepatically impaired patients taking prasugrel.

This integrated analysis compared speed of onset, level of platelet inhibition, and response variability to prasugrel and clopidogrel in healthy subjects and in patients with stable coronary artery disease with data pooled from 24 clinical pharmacology studies. Data from subjects (N = 846) were categorized into the following treatment groups: prasugrel 60 mg loading dose (LD)/10 mg maintenance dose (MD), clopidogrel 300 mg LD/75 mg MD, or clopidogrel 600 mg LD/75 mg MDs. Maximum platelet aggregation (MPA) and inhibition of platelet aggregation (IPA) to 5 and 20 muM ADP were assessed by turbidimetric aggregometry. A linear mixed-effect model compared the MPA and IPA between treatments over time points evaluated in the integrated database, and covariates affecting platelet inhibition were identified. Prasugrel 60 mg LD resulted in faster onset, greater magnitude, and more consistent levels of inhibition of platelet function compared to either clopidogrel 300 mg or 600 mg LDs. Greater and more consistent levels of platelet inhibition were observed with the prasugrel 10 mg MD compared to the clopidogrel 75 mg MD. This integrated analysis confirms the findings of earlier individual studies, that prasugrel achieves faster onset of greater extent and more consistent platelet inhibition compared to the approved and higher loading doses of clopidogrel. Gender, race, body weight, and age were identified as statistically significant covariates impacting platelet inhibition.

Objective: Prasugrel is a thienopyridine antiplatelet agent for the prevention of atherothrombotic events in patients with acute coronary syndrome undergoing percutaneous coronary intervention. Since cytochrome P450 enzymes CYP3A4 and CYP2B6 play a major role in prasugrel's active metabolite formation, the effect of potent CYP induction by rifampin on the pharmacokinetics of prasugrel and on the pharmacodynamic response to prasugrel was evaluated in healthy male subjects., Research Design and Methods: This was an open-label, two-period, fixed-sequence study conducted at a single clinical research center. In the first treatment period, subjects received prasugrel as an oral 60-mg loading dose (LD) on the first day followed by ten oral, 10-mg daily maintenance doses. After a 2-week washout period, subjects received oral rifampin alone (600 mg once daily) for 8 days, followed by coadministration of oral rifampin with prasugrel, given as a 60-mg LD on the first day followed by five daily 10-mg MDs. Blood collection for pharmacokinetic and pharmacodynamic analyses occurred after the LD and fifth MD of prasugrel in both periods. CLINICAL TRIAL SYNOPSIS: clinicalstudyresults.org ID #8976, Results: Rifampin coadministration (600 mg daily) did not affect exposure to prasugrel's active metabolite (R-138727). However, at 2 and 4 h after the prasugrel loading dose (60 mg), rifampicin coadministration was associated with a 6-9 percentage point decrease (p < 0.01) in the magnitude of platelet inhibition; similarly, a 5-17 percentage point decrease (p < 0.05) was observed with rifampin coadministration during the prasugrel maintenance dose (10 mg) period. Post hoc in vitro experiments demonstrated a dose-dependent R-138727-rifampin interaction at the P2Y(12) level unrelated to enzyme induction. A limitation of this study is that while results of the in vitro post hoc study indicate a pharmacodynamic interaction with rifampin, the mechanism underlying this interaction has not been elucidated., Conclusions: Dose adjustment should not be necessary when prasugrel is administered with CYP inducers since formation of prasugrel's active metabolite is not affected by potent enzyme induction with rifampin.

Study Objective: To investigate the potential effect of atorvastatin 80 mg/day on the pharmacokinetics and pharmacodynamics of the thienopyridines prasugrel and clopidogrel., Design: Open-label, randomized, crossover, two-arm, parallel-group study., Setting: Single clinical research center in the United Kingdom., Participants: Sixty-nine healthy men aged 18-60 years. Intervention. Subjects received either a loading dose of prasugrel 60 mg followed by a maintenance dose of 10 mg/day or a loading dose of clopidogrel 300 mg followed by 75 mg/day. The drug was given as monotherapy for 10 days, and after a 6-day run-in period with atorvastatin 80 mg/day, the same dosage of atorvastatin was continued with the respective thienopyridine for 10 days. A 14-day washout period separated the treatment regimens., Measurements and Main Results: Blood samples were collected before and at various time points after dosing on days 1 and 11 for determination of plasma concentrations of metabolites and for measurement of platelet aggregation induced by adenosine 5'-diphosphate 20 microM and vasodilator-stimulated phosphoprotein (VASP). Coadministration of atorvastatin did not alter exposure to active metabolites of prasugrel or clopidogrel after the loading dose and thus did not alter inhibition of platelet aggregation (IPA). During maintenance dosing, atorvastatin administration resulted in 17% and 28% increases in the area under the plasma concentration-time curve (AUC) values of prasugrel's and clopidogrel's active metabolites, respectively. These small changes in AUC did not result in a significant change in IPA response to prasugrel but did result in a significant increase in IPA during clopidogrel maintenance dosing at some, but not all, of the time points on day 11. Coadministration of atorvastatin with either prasugrel or clopidogrel had no effect on VASP phosphorylation relative to the thienopyridine alone after the loading dose., Conclusion: Coadministration of atorvastatin 80 mg/day with prasugrel or clopidogrel did not negatively affect the antiplatelet response to either drug after a loading dose or during maintenance dosing. The lack of a clinically meaningful effect of high-dose atorvastatin on the pharmacodynamic response to prasugrel after the loading or maintenance dose indicates that no dosage adjustment should be necessary in patients receiving these drugs concomitantly.

Prasugrel, a novel P2Y(12) antagonist, achieves faster onset and greater inhibition of platelet aggregation than clopidogrel 300 and 600 mg loading doses (LD). We studied the safety, time course, and level of platelet inhibition when switching directly from clopidogrel 75 mg maintenance dose (MD) to a prasugrel 60 mg LD/10 mg MD or 10 mg MD regimen. Healthy subjects (n = 39) on aspirin (81 mg/d) received a clopidogrel 600 mg LD followed by 10 days of clopidogrel MD (75 mg/d). Subjects were then randomized without a washout period to prasugrel 60 mg LD (n = 16) followed by 10 days of prasugrel MD (10 mg/d) or to prasugrel MD (10 mg/d, n = 19) for 11 days. Maximal platelet aggregation (MPA) to 20 microM ADP was measured by turbidimetric aggregometry. In subjects on clopidogrel 75 mg MD, mean MPA decreased from 39 to 12% by 30 minutes, and to 5% by 1 hour after a prasugrel 60 mg LD (p < 0.001 for both) and from 37 to 28% (p < 0.001) by 1 hour after a prasugrel 10 mg MD. During prasugrel MD, a new pharmacodynamic steady state MPA of approximately 24% (p < 0.01 vs. clopidogrel MD) occurred within four to five days of switching from clopidogrel. Changing from clopidogrel to prasugrel did not increase bleeding episodes or other adverse events. Switching directly from clopidogrel MD to either prasugrel LD or MD was well tolerated and resulted in significantly greater levels of platelet inhibition than a clopidogrel 75 mg MD.

Prasugrel and clopidogrel, thienopyridine prodrugs, are each metabolized to an active metabolite that inhibits the platelet P2Y(12) ADP receptor. In this open-label, 4-period crossover study, the effects of the proton pump inhibitor lansoprazole on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel were assessed in healthy subjects given single doses of prasugrel 60 mg and clopidogrel 300 mg with and without concurrent lansoprazole 30 mg qd. C(max) and AUC(0-tlast) of prasugrel's active metabolite, R-138727, and clopidogrel's inactive carboxylic acid metabolite, SR26334, were assessed. Inhibition of platelet aggregation (IPA) was measured by turbidimetric aggregometry 4 to 24 hours after each treatment. Lansoprazole (1) decreased R-138727 AUC(0-tlast) and C(max) by 13% and 29%, respectively, but did not affect IPA after the prasugrel dose, and (2) did not affect SR62334 exposure but tended to lower IPA after a clopidogrel dose. A retrospective tertile analysis showed in subjects with high IPA after a clopidogrel dose alone that lansoprazole decreased IPA, whereas IPA was unaffected in these same subjects after a prasugrel dose. The overall data suggest that a prasugrel dose adjustment is not likely warranted in an individual taking prasugrel with a proton pump inhibitor such as lansoprazole.

Variability in response to antiplatelet agents has prompted the development of point-of-care (POC) technology. In this study, we compared the VerifyNow P2Y12 (VN-P2Y12) POC device with light transmission aggregometry (LTA) in subjects switched directly from clopidogrel to prasugrel. Healthy subjects on aspirin were administered a clopidogrel 600 mg loading dose (LD) followed by a 75 mg/d maintenance dose (MD) for 10 days. Subjects were then switched to a prasugrel 60 mg LD and then 10 mg/d MD for 10 days (n = 16), or to a prasugrel 10 mg/d MD for 11 days (n = 19). Platelet function was measured by LTA and VN-P2Y12 at baseline and after dosing. Clopidogrel 600 mg LD/75 mg MD treatment led to a reduction in P2Y(12) reaction units (PRU) from baseline. A switch from clopidogrel MD to prasugrel 60 mg LD/10 mg MD produced an immediate decrease in PRU, while a switch to prasugrel 10 mg MD resulted in a more gradual decline. Consistent with the reduction in PRU, device-reported percent inhibition increased during both clopidogrel and prasugrel regimens. Inhibition of platelet aggregation as measured by LTA showed a very similar pattern to that found with VN-P2Y12 measurement, irrespective of treatment regimens. The dynamic range of VN-P2Y12 appeared to be narrower than that of LTA. With two different thienopyridines, the VN-P2Y12 device, within a somewhat more limited range, reflected the overall magnitude of change in aggregation response determined by LTA. The determination of the clinical utility of such POC devices will require their use in clinical outcome studies.

Prasugrel, a thienopyridine prodrug, is hydrolyzed in vivo by esterases to a thiolactone followed by a single cytochrome P450 (CYP)-dependent step to an active metabolite that is a potent inhibitor of adenosine diphosphate-induced platelet aggregation. This open-label, multiple-dose, 2-period, fixed-sequence study assessed CYP2B6 inhibition by prasugrel using bupropion as a probe substrate, where its active metabolite, hydroxybupropion, is almost exclusively formed by CYP2B6. Thirty healthy subjects received a single 150-mg oral dose of sustained-release bupropion. After a 7-day washout, a 60-mg prasugrel loading dose, followed by a 10-mg daily maintenance dose for 10 days, was administered. Bupropion (150 mg) was given with prasugrel on day 7 of this phase. Prasugrel weakly inhibited CYP2B6 activity as it increased bupropion Cmax and AUC0-infinity by 14% and 18%, respectively, and decreased hydroxybupropion Cmax and AUC0-infinity by 32% and 23%. These results are consistent with patients receiving prasugrel not requiring dose adjustments when treated with drugs primarily metabolized by CYP2B6.

Platelet inhibition as measured by vasodilator-stimulated phosphoprotein (VASP) and light transmission aggregometry (LTA) have shown concordance following dosing of clopidogrel. No reports have directly compared the VASP assay and LTA at the levels of P2Y(12) blockade after loading doses (LDs) of prasugrel or high dose clopidogrel (600 and 900 mg). The aim was to compare the VASP assay and LTA during the loading dose phase of a comparative study of prasugrel and clopidogrel. Prasugrel 60 mg LD/10 mg maintenance dose (MD) and clopidogrel 300 mg/75 mg and 600 mg/75 mg LD/MD regimens were compared in a 3-way crossover study in 41 healthy, aspirin-free subjects. Each LD was followed by seven daily MDs and a 14-day washout period. P2Y(12) receptor blockade was estimated using the VASP assay, expressed as platelet reactivity index (VASP-PRI). Platelet aggregation was assessed by light transmission aggregometry (20 and 5 microM ADP). Twenty-four hours after prasgurel 60 mg or clopidogrel 300 mg and 600 mg, respectively, VASP-PRI decreased from approximately 80% to 8.9%, 54.7%, and 39.0%, and maximal platelet aggregation (MPA) decreased from approximately 79% to 10.8%, 42.7%, and 31.2%, with an overall VASP:MPA correlation of 0.88 (p < 0.01). VASP assay responses after the clopidogrel LDs showed a wider range of values (300 mg: 0-93%; 600 mg: 0-80%) than prasugrel (0-13%); MPA responses followed a similar trend. Pearson's correlation suggested a strong agreement between VASP and LTA (20 microM ADP) for MPA (r = 0.86, p < 0.0001). VASP and LTA demonstrated concordance across the response range of P2Y(12) receptor blockade following thienopyridine LDs.

Background: Thienopyridines are metabolized to active metabolites that irreversibly inhibit the platelet P2Y(12) adenosine diphosphate receptor. The pharmacodynamic response to clopidogrel is more variable than the response to prasugrel, but the reasons for variation in response to clopidogrel are not well characterized., Objective: To determine the relationship between genetic variation in cytochrome P450 (CYP) isoenzymes and the pharmacokinetic/pharmacodynamic response to prasugrel and clopidogrel., Methods: Genotyping was performed for CYP1A2, CYP2B6, CYP2C19, CYP2C9, CYP3A4 and CYP3A5 on samples from healthy subjects participating in studies evaluating pharmacokinetic and pharmacodynamic responses to prasugrel (60 mg, n = 71) or clopidogrel (300 mg, n = 74)., Results: In subjects receiving clopidogrel, the presence of the CYP2C19*2 loss of function variant was significantly associated with lower exposure to clopidogrel active metabolite, as measured by the area under the concentration curve (AUC(0-24); P = 0.004) and maximal plasma concentration (C(max); P = 0.020), lower inhibition of platelet aggregation at 4 h (P = 0.003) and poor-responder status (P = 0.030). Similarly, CYP2C9 loss of function variants were significantly associated with lower AUC(0-24) (P = 0.043), lower C(max) (P = 0.006), lower IPA (P = 0.046) and poor-responder status (P = 0.024). For prasugrel, there was no relationship observed between CYP2C19 or CYP2C9 loss of function genotypes and exposure to the active metabolite of prasugrel or pharmacodynamic response., Conclusions: The common loss of function polymorphisms of CYP2C19 and CYP2C9 are associated with decreased exposure to the active metabolite of clopidogrel but not prasugrel. Decreased exposure to its active metabolite is associated with a diminished pharmacodynamic response to clopidogrel.

Prasugrel pharmacodynamics and pharmacokinetics after a 60-mg loading dose (LD) and daily 10-mg maintenance doses (MD) were compared in a 3-way crossover study to clopidogrel 600-mg/75-mg and 300-mg/75-mg LD/MD in 41 healthy, aspirin-free subjects. Each LD was followed by 7 days of daily MD and a 14-day washout period. Inhibition of platelet aggregation (IPA) was assessed by turbidometric aggregometry (20 and 5 microM ADP). Prasugrel 60-mg achieved higher mean IPA (54%) 30 minutes post-LD than clopidogrel 300-mg (3%) or 600-mg (6%) (P < 0.001) and greater IPA by 1 hour (82%) and 2 hours (91%) than the 6-hour IPA for clopidogrel 300-mg (51%) or 600-mg (69%) (P < 0.01). During MD, IPA for prasugrel 10-mg (78%) exceeded that of clopidogrel (300-mg/75-mg, 56%; 600-mg/75-mg, 52%; P < 0.001). Active metabolite area under the concentration-time curve (AUC0-tlast) after prasugrel 60-mg (594 ng.hr/mL) was 2.2 times that after clopidogrel 600-mg. Prasugrel active metabolite AUC0-tlast was consistent with dose-proportionality from 10-mg to 60-mg, while clopidogrel active metabolite AUC0-tlast exhibited saturable absorption and/or metabolism. In conclusion, greater exposure to prasugrel's active metabolite results in faster onset, higher levels, and less variability of platelet inhibition compared with high-dose clopidogrel in healthy subjects.

Existing definitions of poor response to the antiplatelet effect of clopidogrel are empiric. Bayesian classification theory is widely used to classify subjects into non-overlapping groups based on observed responses. The purpose of this analysis is to objectively define pharmacodynamic poor responders to clopidogrel using Bayesian classification methodology. An integrated database of turbidometric platelet aggregometry data (5 and 20 microM ADP) was analyzed from 112 healthy subjects who participated in three Phase 1 clinical pharmacology studies. Change in maximum platelet aggregation (DeltaMPA) from baseline and percent inhibition of platelet aggregation (IPA) were evaluated at both 4-5 and 24 hours after a clopidogrel 300 mg loading dose (LD). Clopidogrel poor responders were predefined as having a response similar to that of drug-free baseline, derived from multiple MPA values prior to clopidogrel administration. The model identified a clopidogrel poor responder as a subject who failed to achieve and maintain an IPA >or= 25% (or DeltaMPA >or= 20%) with 5 microM ADP at 4-5 and 24 hours after dosing or who failed to achieve and maintain an IPA >or= 20% (or DeltaMPA >or= 15%) with 20 microM ADP at 4-5 and 24 hours after dosing. Application of these thresholds indicated that, depending on the concentration of ADP used, 25% to 45% of subjects were classified as clopidogrel poor responders. Using thresholds from the published literature resulted in 17% to 56% of subjects being classified as poor responders. Objective thresholds for pharmacodynamic poor responders to clopidogrel should consider the concentration of the agonist used and may help assess the consistency of pharmacodynamic response to novel ADP receptor antagonists.

Multiple studies report response variability to a 300-mg clopidogrel loading dose (LD). Pooled platelet aggregometry data compared responses (change in maximal platelet aggregation [DeltaMPA] or inhibition of platelet aggregation [IPA]) to clopidogrel 300-mg (n = 131) or prasugrel 60-mg (n = 109) LDs. Poor responder rates were determined using empiric criteria (IPA < 10% and DeltaMPA < 10% for 20 microM and 5 microM adenosine diphosphate [ADP]) and Bayesian model-based criteria (IPA < 20% and DeltaMPA < 15% for 20 microM ADP; IPA < 25% and DeltaMPA < 20% for 5 microM ADP). Prasugrel achieved greater DeltaMPA and IPA from 2 to 24 hours post-LD (P < .001). For 20 microM ADP, poor responder rates for clopidogrel ranged from 17% to 43%; no prasugrel poor responders were observed. Regardless of the criterion, prasugrel 60 mg achieved greater IPA and fewer poor responders than the clopidogrel 300-mg LD.

Failure to achieve an adequate level of platelet inhibition during percutaneous coronary intervention is associated with an increased risk for periprocedural myocardial injury. This study was conducted to compare the initial rate of platelet inhibition after a loading dose (LD) of prasugrel or clopidogrel and determine the association between the initial rate of inhibition and pharmacodynamic responder status. Data were pooled from 3 studies in which healthy subjects received LDs of prasugrel (60 mg; n = 76) or clopidogrel (300 mg; n = 87). Maximum platelet aggregation (MPA; 20 mumol/L adenosine diphosphate) was measured by turbidimetric aggregometry (0.25 to 24 hours after dosing). A mechanistic model was used to estimate the initial rate of decrease in MPA per hour (fast onset: MPA decrease >20%/hour). Subjects were defined as pharmacodynamic poor responders if the absolute decrease in MPA from baseline was <15% at either 4 to 5 or 24 hours after dosing. The median initial rate of decrease in MPA was greater after prasugrel (203%/hour) than with clopidogrel (23%/hour) (p <0.001). Overall, 76 subjects (100%) receiving prasugrel had fast onset of platelet inhibition compared with 47 subjects (54%) receiving clopidogrel. The initial rate of decrease in MPA was highly correlated with responder status (p <0.001). After prasugrel, subjects had a lower median MPA compared with clopidogrel (p <0.001; from >0.25 to 24 hours after dosing), and intersubject variability in MPA response was less after prasugrel compared with clopidogrel (p <0.001; from >1 to 24 hours after dosing). In conclusion, platelet inhibition after a 60-mg LD of prasugrel was more rapid in onset, less variable, and greater in magnitude than with a 300-mg LD of clopidogrel. After a thienopyridine LD, the initial rate of platelet inhibition was predictive of pharmacodynamic responder status.

Prasugrel and clopidogrel inhibit platelet aggregation through active metabolite formation. Prasugrel's active metabolite (R-138727) is formed primarily by cytochrome P450 (CYP) 3A and CYP2B6, with roles for CYP2C9 and CYP2C19. Clopidogrel's activation involves two sequential steps by CYP3A, CYP1A2, CYP2C9, CYP2C19, and/or CYP2B6. In a randomized crossover study, healthy subjects received a loading dose (LD) of prasugrel (60 mg) or clopidogrel (300 mg), followed by five daily maintenance doses (MDs) (15 and 75 mg, respectively) with or without the potent CYP3A inhibitor ketoconazole (400 mg/day). Subjects had a 2-week washout between periods. Ketoconazole decreased R-138727 and clopidogrel active metabolite Cmax (maximum plasma concentration) 34-61% after prasugrel and clopidogrel dosing. Ketoconazole did not affect R-138727 exposure or prasugrel's inhibition of platelet aggregation (IPA). Ketoconazole decreased clopidogrel's active metabolite AUC0-24 (area under the concentration-time curve to 24 h postdose) 22% (LD) to 29% (MD) and reduced IPA 28% (LD) to 33% (MD). We conclude that CYP3A4 and CYP3A5 inhibition by ketoconazole affects formation of clopidogrel's but not prasugrel's active metabolite. The decreased formation of clopidogrel's active metabolite is associated with reduced IPA.