GP2015, a proposed etanercept biosimilar: Pharmacokinetic similarity to its reference product and comparison of its autoinjector device with prefilled syringes.

AIMS: To assess pharmacokinetics (PK) and safety of GP2015, a proposed etanercept biosimilar, in two studies: comparison with etanercept originator (ETN, bioequivalence study) and comparison of GP2015 administered via an autoinjector (AI) or prefilled syringes (PFS, delivery study). METHODS: Both studies were randomized, two-sequence, two-period, crossover studies conducted in healthy male subjects. In the bioequivalence study, subjects were randomized to receive a single 50 mg subcutaneous (s.c.) injection of GP2015 or ETN. In the delivery study, subjects were randomized to receive a single 50 mg s.c. injection of GP2015 via AI or PFS. Following a wash-out period of 35 days, subjects in the bioequivalence study received single 50 mg s.c. injection of GP2015 or ETN, and subjects in the delivery study received single 50 mg s.c. injection of GP2015 via AI or PFS. RESULTS: The geometric mean ratios (90% confidence interval) of GP2015/ETN for Cmax (1.11 [1.05-1.17]), AUC0-tlast (0.98 [0.94-1.02]) and AUC0-inf (0.96 [0.93-1.00]) were within the predefined bioequivalence range of 0.80-1.25. The geometric mean ratios (90% confidence interval) of AI/PFS for Cmax (1.01 [0.94-1.08]), AUC0-tlast (1.01 [0.95-1.07]) and AUC0-inf (1.01 [0.96-1.07]) were also within the range 0.80-1.25. No new safety issues were reported. Three subjects had low titres of non-neutralising anti-drug antibodies during a follow-up visit in the bioequivalence study. CONCLUSIONS: The PK of GP2015 was similar to ETN, demonstrating bioequivalence. The safety profile of GP2015 was consistent with previous reports for ETN. The GP2015 AI provided equivalent dosing and tolerability to the GP2015 PFS.

Protein chaperones mediating copper insertion into the CuA site of the aa3-type cytochrome c oxidase of Paracoccus denitrificans.

The biogenesis of the mitochondrial cytochrome c oxidase is a complex process involving the stepwise assembly of its multiple subunits encoded by two genetic systems. Moreover, several chaperones are required to recruit and insert the redox-active metal centers into subunits I and II, two a-type hemes and a total of three copper ions, two of which form the CuA center located in a hydrophilic domain of subunit II. The copper-binding Sco protein(s) have been implicated with the metallation of this site in various model organisms. Here we analyze the role of the two Sco homologues termed ScoA and ScoB, along with two other copper chaperones, on the biogenesis of the cytochrome c oxidase in the bacterium Paracoccus denitrificans by deleting each of the four genes individually or pairwise, followed by assessing the functionality of the assembled oxidase both in intact membranes and in the purified enzyme complex. Copper starvation leads to a drastic decrease of oxidase activity in membranes from strains involving the scoB deletion. This loss is shown to be of dual origin, (i) a severe drop in steady-state oxidase levels in membranes, and (ii) a diminished enzymatic activity of the remaining oxidase complex, traced back to a lower copper content, specifically in the CuA site of the enzyme. Neither of the other proteins addressed here, ScoA or the two PCu proteins, exhibit a direct effect on the metallation of the CuA site in P. denitrificans, but are discussed as potential interaction partners of ScoB.

Pimasertib, a selective oral MEK1/2 inhibitor: absolute bioavailability, mass balance, elimination route, and metabolite profile in cancer patients.

AIM: This trial (NCT: 01713036) investigated the absolute bioavailability, mass balance and metabolite profile of pimasertib in a new design combining these investigations in a single group of patients. METHODS: Six male patients with pathologically confirmed, locally advanced or metastatic solid tumours were enrolled. Exclusion criteria included Eastern Cooperative Oncology Group performance status >1. In Part A of the trial, patients received a 60 mg oral dose of unlabelled pimasertib followed by an intravenous (i.v.) tracer dose of [14 C]pimasertib 2 µg (equalling 9 kBq) as a bolus injection, one hour after the oral dose, on Day 1. On Day 8, all patients received 60 mg pimasertib capsules spiked with 2.6 MBq of [14 C]pimasertib. Patients received 60 mg oral unlabelled pimasertib twice daily from Day 3 to Day 21 of Part A and in subsequent 21-day cycles in Part B. RESULTS: Following i.v. administration, [14 C]pimasertib exhibited a geometric mean total body clearance of 45.7 l h-1 (geometric coefficient of variation [geometric CV]: 47.2%) and a volume of distribution of 229 l (geometric CV: 42.0%). Absolute bioavailability was 73%. The majority of the oral [14 C] dose (85.1%) was recovered in excreta. Total radioactivity was mainly excreted into urine (52.8%) and faeces (30.7%) with 78.9% of the [14 C] dose recovered as metabolites. Two major circulating metabolites were identified in plasma: a carboxylic acid (M445) and a phosphoethanolamine conjugate (M554). The safety profile was in line with the published pimasertib trials. CONCLUSION: Pimasertib showed a favourable pharmacokinetic profile with high absolute bioavailability and a unique metabolic pathway (conjugation with phosphoethanolamine).

Silica nanoparticles for the oriented encapsulation of membrane proteins into artificial bilayer lipid membranes.

An artificial bilayer lipid membrane system is presented, featuring the oriented encapsulation of membrane proteins in a functionally active form. Nickel nitrilo-triacetic acid-functionalized silica nanoparticles, of a diameter of around 25 nm, are used to attach the proteins via a genetically engineered histidine tag in a uniform orientation. Subsequently, the proteins are reconstituted within a phospholipid bilayer, formed around the particles by in situ dialysis to form so-called proteo-lipobeads (PLBs). With a final size of about 50 nm, the PLBs can be employed for UV/vis spectroscopy studies, particularly of multiredox center proteins, because the effects of light scattering are negligible. As a proof of concept, we use cytochrome c oxidase (CcO) from P. denitrificans with the his tag genetically engineered to subunit I. In this orientation, the P side of CcO is directed to the outside and hence electron transfer can be initiated by reduced cytochrome c (cc). UV/vis measurements are used in order to determine the occupancy by CcO molecules encapsulated in the lipid bilayer as well as the kinetics of electron transfer between CcO and cc. The kinetic data are analyzed in terms of the Michaelis-Menten kinetics showing that the turnover rate of CcO is significantly decreased compared to that of solubilized protein, whereas the binding characteristics are improved. The data demonstrate the suitability of PLBs for functional cell-free bioassays of membrane proteins.

Proteo-lipobeads for the oriented encapsulation of membrane proteins.

As a surrogate of live cells, proteo-lipobeads are presented, encapsulating functional membrane proteins in a strict orientation into a lipid bilayer. Assays can be performed just as on live cells, for example using fluorescence measurements. As a proof of concept, we have demonstrated proton transport through cytochrome c oxidase.

The protein effect in the structure of two ferryl-oxo intermediates at the same oxidation level in the heme copper binuclear center of cytochrome c oxidase.

Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O2 activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O2 with the mixed valence form (CuA(2+), heme a(3+), heme a3(2+)-CuB(1+)) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm(-1) at the peroxy oxidation level (PM). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the PM intermediate is also formed in the reaction of Y167F with O2. These results suggest that in the fully reduced enzyme, the proton pumping ν(Fe(IV)=O) = 804 cm(-1) to ν(Fe(IV)=O) = 790 cm(-1) transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a3 but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a3. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a3-Asp-399-H2O site and, thus, to the exit/output proton channel (H2O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a3 controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.

'Totality of Evidence' Approach in the Development of GP2017, an Approved Adalimumab Biosimilar.

INTRODUCTION: Hyrimoz®, (GP2017 [SDZ-ADL]), is a biosimilar to Humira® (REF-ADL). SDZ-ADL was approved in 2018 by both the United States Food and Drug Administration (US FDA) and European Medicines Agency (EMA) for the indications of REF-ADL not protected by orphan exclusivity. In 2023, the US FDA and EMA also approved a citrate-free high-concentration formulation (HCF) of SDZ-ADL. TOTALITY OF EVIDENCE-THE APPROACH: Approval of SDZ-ADL was based on data gathered using the US FDA, EMA and World Health Organization (WHO)-recommended step-wise Totality of Evidence approach. This approach is a robust dataset confirming high confidence in analytical, functional, pharmacokinetic (PK) and clinical biosimilarity between the biosimilar and reference medicine determined through analytical and clinical investigation. EVIDENCE OF BIOSIMILARITY: Evidence supporting the biosimilarity of SDZ-ADL and REF-ADL was reported at each stage of investigation. Comprehensive comparative analytical and functional assessments demonstrated that SDZ-ADL was analytically indistinguishable from REF-ADL in required critical quality attributes, including receptor binding. Phase I clinical data showed PK similarity of SDZ-ADL and REF-ADL in healthy volunteers, with similar safety, tolerability and immunogenicity profiles. Phase III confirmatory efficacy and safety studies, ADACCESS (included in US/EU dossiers) and ADMYRA (separate to US/EU dossiers), both confirmed that SDZ-ADL's efficacy, safety, and immunogenicity matched REF-ADL in all patient groups with no clinically meaningful differences. More recently, this data package was the basis for a citrate-free HCF of SDZ-ADL to be developed, and its PK, safety and immunogenicity were confirmed against the initially approved formulation of SDZ-ADL. CONCLUSION: Overall, the Totality of Evidence provided for biosimilar adalimumab, SDZ-ADL, confirmed the analytical, functional and clinical similarity of SDZ-ADL to REF-ADL, supporting its regulatory approval and providing a data bridge with which to evaluate and support the approval of citrate-free HCF SDZ-ADL for clinical use.

A biosimilar is a type of medicine that is designed to match the structure and function of a 'reference' biologic medicine. Hyrimoz^® (SDZ-ADL) is a biosimilar of the adalimumab reference medicine, Humira^® ([REF-ADL]). SDZ-ADL was approved in the US and Europe in 2018. For SDZ-ADL to be approved, a collection of evidence needed to be created, called the 'Totality of Evidence.' The purpose of this collection of data is to show there is a high confidence that the new biosimilar medicine matches the reference medicine, from the structure of the medicine to the effect of the medicine on the human body. For SDZ-ADL, this investigation started with comparing the physical structure and other functional properties of SDZ-ADL versus REF-ADL and ended with clinical studies in both healthy volunteers and in patients with diseases treated with adalimumab. This Totality of Evidence gathered for biosimilar adalimumab, SDZ-ADL, confirmed the similarity of SDZ-ADL to REF-ADL and therefore supported the approval of SDZ-ADL. In 2018, a citrate-free high-concentration version (high concentration formulation [HCF]) of REF-ADL was launched that matched REF-ADL. HCF REF-ADL has since become the primary formulation of REF-ADL used in practice. In 2023, a HCF version of SDZ-ADL was also approved in the US and EU based on evidence confirming that HCF SDZ-ADL matched SDZ-ADL. As SDZ-ADL had been previously confirmed to match the reference medicine, this meant that HCF SDZ-ADL could be directly compared against SDZ-ADL to confirm biosimilarity and support its approval.

Signal-to-noise ratio to assess magnitude, kinetics and impact on pharmacokinetics of the immune response to an adalimumab biosimilar.

Background: The antidrug antibody (ADA) signal-to-noise (S/N) ratio was explored as a novel immunogenicity measure to evaluate the immune response of healthy subjects to a single dose of GP2017, an adalimumab biosimilar. Methodology/results: Bioanalytical methods used for the analysis of ADA S/N ratios and ADA titers were validated for sensitivity, precision and drug interference. ADA S/N ratios strongly correlated with ADA titers. Correlations between ADA area under the curve and ADAmax and pharmacokinetics (PK) were stronger for ADA S/N ratio than for ADA titers. Conclusion: ADA S/N ratio allowed for a more sensitive evaluation of the magnitude and kinetics of the immune response, was better correlated with adalimumab PK and was superior to ADA titers in assessing the impact of the immune response on PK.

Introducing the Biosimilar Paradigm to Neurology: The Totality of Evidence for the First Biosimilar Natalizumab.

A biosimilar medicine is a successor to a reference ('originator'/'original-brand') biologic medicine brought to market once the patent and exclusive marketing rights for the reference have expired. Biosimilar natalizumab (PB006 [biosim-NTZ]; developed by Polpharma Biologics S.A. and marketed globally as Tyruko®; Sandoz) has been developed as a successor to reference natalizumab (Tysabri® [ref-NTZ]; Biogen) and is the first US Food and Drug Administration (FDA)-approved and European Medicines Agency (EMA)-approved biosimilar in neurology. As per the FDA and EMA indications for ref-NTZ, biosim-NTZ is approved to treat relapsing forms of multiple sclerosis (USA, EU) and Crohn's disease (USA only). Approval of biosim-NTZ was based on the 'totality of evidence', a comprehensive body of data collected during the development process, demonstrating similarity to its reference medicine. The foundational step of demonstrating structural and functional similarity between biosim-NTZ and ref-NTZ confirmed identical primary and indistinguishable higher order structures, as well as matching binding affinity to α4ß1/α4ß7 integrins. Following the confirmation of matching structure and function, pharmacokinetic/pharmacodynamic similarity of biosim-NTZ to ref-NTZ in healthy subjects was demonstrated, with no clinically meaningful differences identified in safety and immunogenicity. A comparative, double-blind, randomized study (Antelope) was also conducted in patients with relapsing-remitting multiple sclerosis and demonstrated matching efficacy, safety, and immunogenicity with no clinically meaningful differences between biosim-NTZ and ref-NTZ. This review presents the totality of evidence that confirmed the biosimilarity of biosimilar natalizumab to its reference medicine, which supported its approval by the FDA and the EMA. [Graphical plain language summary available].

Allosteric interactions and proton conducting pathways in proton pumping aa(3) oxidases: heme a as a key coupling element.

In this paper allosteric interactions in protonmotive heme aa(3) terminal oxidases of the respiratory chain are dealt with. The different lines of evidence supporting the key role of H(+)/e(-) coupling (redox Bohr effect) at the low spin heme a in the proton pump of the bovine oxidase are summarized. Results are presented showing that the I-R54M mutation in P. denitrificans aa(3) oxidase, which decreases by more than 200mV the E(m) of heme a, inhibits proton pumping. Mutational amino acid replacement in proton channels, at the negative (N) side of membrane-inserted prokaryotic aa(3) oxidases, as well as Zn(2+) binding at this site in the bovine oxidase, uncouples proton pumping. This effect appears to result from alteration of the structural/functional device, closer to the positive, opposite (P) surface, which separates pumped protons from those consumed in the reduction of O(2) to 2 H(2)O.