The market of biopharmaceutical medicines: A snapshot of a diverse industrial landscape

Background: Biopharmaceutical medicines represent a growing share of the global pharmaceutical market, and with many of these biopharmaceutical products facing loss of exclusivity rights, also biosimilars may now enter the biopharmaceutical market. Objectives: This study aims to identify and document which investment and development strategies are adopted by industrial players in the global biopharmaceutical market. Methods: A descriptive analysis was undertaken of the investment and development strategies of the top 25 pharmaceutical companies according to 2015 worldwide prescription drug sales. Strategies were documented by collecting data on manufacturing plans, development programs, acquisition and collaboration agreements, the portfolio and pipeline of biosimilar, originator and next-generation biopharmaceutical products. Data were extracted from publicly available sources. Results: Various investment and development strategies can be identified in the global biopharmaceutical market: (a) development of originator biopharmaceuticals, (b) investment in biotechnology, (c) development of next-generation biopharmaceuticals, (d) development of biosimilars, (e) investment in emerging countries, and (f) collaboration between companies. In the top 25 pharmaceutical companies almost every company invests in originator biopharmaceuticals and in biotechnology in general, but only half of them develops next-generation biopharmaceuticals. Furthermore, only half of them invest in development of biosimilars. The companies' biosimilar pipeline is mainly focused on development of biosimilar monoclonal antibodies and to some extent on biosimilar insulins. A common strategy is collaboration between companies and investment in emerging countries. Conclusions: A snapshot of investment and development strategies used by industrial players in the global biopharmaceutical market shows that all top 25 pharmaceutical companies are engaged in the biopharmaceutical market and that this industrial landscape is diverse. Companies do not focus on a single strategy, but are involved in multiple investment and development strategies. A common strategy to market biopharmaceuticals is collaboration between companies. These collaborations can as well be used to gain access in regions the company has less experience with. With patents expiring for some of the highest selling monoclonal antibodies, this snapshot highlights the interest of companies to invest in the development of these molecules and/or enter into collaborations to create access to these molecules

The arrival of biosimilar monoclonal antibodies in oncology: clinical studies for trastuzumab biosimilars

The monoclonal antibody trastuzumab (Herceptin®), which targets the human epidermal growth factor receptor 2 (HER2), is approved for the treatment of early breast and advanced breast and gastric cancer in which HER2 is overexpressed. Several biosimilar versions of trastuzumab are expected to enter the European market over the course of 2018 and 2019. The biosimilar development pathway consists of a comprehensive comparability exercise between the biosimilar candidate and the reference product, primarily focussing on data from analytical studies. Clinical studies for biosimilar candidates follow a different design to those for a new biological, as the aim is not to independently establish clinical benefit, but to confirm biosimilarity between the two agents. The different trastuzumab biosimilar candidates have followed diverse pathways in their clinical development, with differences in clinical trial design (equivalence or non-inferiority design), patient population (those with metastatic or early breast cancer) and endpoint (overall response rate or pathological complete response). These differences in approach in phase 3 testing must be viewed in the totality of evidence demonstrating biosimilarity. Adequate information on the biosimilar approval pathway, the nature of the biosimilarity exercise and how the clinical development of a biosimilar is tailored to meet the licensing requirements can help informed decision making in clinical practice

Quantification of Myocardial Blood Flow in Absolute Terms Using (82)Rb PET Imaging: The RUBY-10 Study.

OBJECTIVES: The purpose of this study was to compare myocardial blood flow (MBF) and myocardial flow reserve (MFR) estimates from rubidium-82 positron emission tomography ((82)Rb PET) data using 10 software packages (SPs) based on 8 tracer kinetic models. BACKGROUND: It is unknown how MBF and MFR values from existing SPs agree for (82)Rb PET. METHODS: Rest and stress (82)Rb PET scans of 48 patients with suspected or known coronary artery disease were analyzed in 10 centers. Each center used 1 of 10 SPs to analyze global and regional MBF using the different kinetic models implemented. Values were considered to agree if they simultaneously had an intraclass correlation coefficient >0.75 and a difference <20% of the median across all programs. RESULTS: The most common model evaluated was the Ottawa Heart Institute 1-tissue compartment model (OHI-1-TCM). MBF values from 7 of 8 SPs implementing this model agreed best. Values from 2 other models (alternative 1-TCM and Axially distributed) also agreed well, with occasional differences. The MBF results from other models (e.g., 2-TCM and retention) were less in agreement with values from OHI-1-TCM. CONCLUSIONS: SPs using the most common kinetic model-OHI-1-TCM-provided consistent results in measuring global and regional MBF values, suggesting that they may be used interchangeably to process data acquired with a common imaging protocol

Antimetastatic Potential of PAI-1 Specific RNA Aptamers

The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is increased in several cancers, including breast, where it is associated with a poor outcome. Metastatic breast cancer has a dismal prognosis, as evidenced by treatment goals that are no longer curative but are largely palliative in nature. PAI-1 competes with integrins and the urokinase plasminogen activator receptor on the surface of breast cancer cells for binding to vitronectin. This results in the detachment of tumor cells from the extracellular matrix, which is critical to the metastatic process. For this reason, we sought to isolate RNA aptamers that disrupt the interaction between PAI-1 and vitronectin. Through utilization of combinatorial chemistry techniques, aptamers have been selected that bind to PAI-1 with high affinity and specificity. We identified two aptamers, WT-15 and SM-20, that disrupt the interactions between PAI-1 and heparin, as well as PAI-1 and vitronectin, without affecting the antiprotease activity of PAI-1. Furthermore, SM-20 prevented the detachment of breast cancer cells (MDA-MB-231) from vitronectin in the presence of PAI-1, resulting in an increase in cellular adhesion. Therefore, the PAI-1 aptamer SM-20 demonstrates therapeutic potential as an antimetastatic agent and could possibly be used as an adjuvant to traditional chemotherapy for breast cancer.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78126/1/oli.2008.0177.pd

Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin

Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples

Molecular cloning and characterization of an antibody fragment (SCFV) that converts plasminogen activator inhibitor-1 from inhibitor to substrate

Plasminogen activator inhibitor-1 (PAI-1) can occur in three different interconvertible conformations: an active, a latent and a substrate form. We have previously characterized monoclonal antibodies (MAs) with neutralizing properties towards PAI-1. However, these antibodies (Mr=150 kDa) are unlikely to be succcsful for use as therapeutic agents with PAI-1 modulating properties. In this study we report the cloning of the variable domains of the heavy and light chain of one of these antibodies (MA-8H9D4), the expression (in E.coli) as a single-chain variable fragment (scFv) and the characterization of its functional properties. The percentage inhibition of the PAI-1 activity by scFv-8H9D4 (5-fold molar excess) was similar to that observed with the intact antibody (16-fold molar excess) (97 \ub116 % and 88 \ub110 %, respectively, mean SD, n=3). Furthermore, their influence on the reaction products formed during interaction of PAI-1 with tissue-type plasminogen activator (t-PA), as evaluated by SDS-PAGE followed by densitometric scanning, was also comparable. control MA-8H9D4 scFv-8H9D4 active PAI-1 51\ub111* 14\ub15 15\ub15 latent PAI-1 40\ub110 45\ub13 37\ub112 substrate PAI-1 9\ub12 42\ub13 45\ub14 * expressed as % of total PAI-1 antigen; mean \ub1 SD (n=3); MA-8H9D4 and scFv8H9D4 in 3-fold molar and equimolar excess respectively. In conclusion, we have demonstrated that scFv-8H9D4 fully retains the functional properties of the original MA-8H9D4. This smaller antibody fragment (Mr - 24 kDa) may serve as a starting point for the design of pharmacological compounds for the treatment of patients with increased PAI-1 levels

Cloning of a single-chain variable fragment (scFv) switching active plasminogen activator inhibitor-1 to substrate

Increased levels of plasminogen activator inhibitor-1 (PAI-1) are a well-known risk for cardiovascular diseases. A significant number of investigations are aimed at lowering plasma levels of PAI-1 to enhance endogenous fibrinolysis. We have recently generated monoclonal antibodies that neutralize PAI-1 activity by switching the inhibitory conformation to a substrate conformation. However, intact murine antibodies have quite some disadvantages for therapeutic use in man. In the current study, we describe the construction of a smaller antibody fragment derived from a monoclonal antibody (MA-8H9D4) with PAI-1 neutralizing properties. The cDNAs encoding the variable domains of the heavy and light chain were amplified, linked and cloned into a phagemid vector. Resulting clones were expressed as a single-chain variable fragment (scFv, VH-(Gly4Ser)3-VL) on the surface of a phage and selected for binding to PAI-1. Subsequently, a positive phage was used for the production of soluble scFv-8H9D4. Following purification, the characteristics of the scFv-8H9D4 were compared to those of the original MA-8H9D4. The scFv inhibited PAI-1 activity to a similar extent as MA-8H9D4 and by a similar mechanism, i.e., induction of a conformational switch. Thus, this smaller antibody fragment, exhibiting the same properties as the parent molecule may constitute a useful starting point for the design of PAI-1 neutralizing therapeutics

Elucidation of the molecular mechanisms of two nanobodies that inhibit thrombin‐activatable fibrinolysis inhibitor activation and activated thrombin‐activatable fibrinolysis inhibitor activity

Background Thrombin-activatable fibrinolysis inhibitor (TAFI) is converted to activated TAFI (TAFIa) by thrombin, plasmin, or the thrombin-thrombomodulin complex (T/TM). TAFIa is antifibrinolytic, and high levels of TAFIa are associated with an increased risk for cardiovascular disorders. TAFI-inhibitory nanobodies represent a promising approach for developing profibrinolytic therapeutics. Objective To elucidate the molecular mechanisms of inhibition of TAFI activation and TAFIa activity by nanobodies with the use of X-ray crystallography and biochemical characterization. Methods and results We selected two nanobodies for cocrystallization with TAFI. VHH-a204 interferes with all TAFI activation modes, whereas VHH-i83 interferes with T/TM-mediated activation and also inhibits TAFIa activity. The 3.05-angstrom-resolution crystal structure of TAFI-VHH-a204 reveals that the VHH-a204 epitope is localized to the catalytic moiety (CM) in close proximity to the TAFI activation site at Arg92, indicating that VHH-a204 inhibits TAFI activation by steric hindrance. The 2.85-angstrom-resolution crystal structure of TAFI-VHH-i83 reveals that the VHH-i83 epitope is located close to the presumptive thrombomodulin-binding site in the activation peptide (AP). The structure and supporting biochemical assays suggest that VHH-i83 inhibits TAFIa by bridging the AP to the CM following TAFI activation. In addition, the 3.00-angstrom-resolution crystal structure of the triple TAFI-VHH-a204-VHH-i83 complex demonstrates that the two nanobodies can simultaneously bind to TAFI. Conclusions This study provides detailed insights into the molecular mechanisms of TAFI inhibition, and reveals a novel mode of TAFIa inhibition. VHH-a204 and VHH-i83 merit further evaluation as potential profibrinolytic therapeutics

A naturally heterogeneous landscape can effectively slow down dispersal of aquatic microcrustaceans

dispersers in a variety of landscapes, whereas others have indicated dispersal limitation at large spatial scales or under specific circumstances. Based on a survey of a set of recently created ponds in an area of approximately 18×25 km, we found multiple indications of dispersal limitation affecting the community assembly of microcrustacean communities. Spatial patterns in the community composition were better explained by the geomorphological structure of the landscape than by mere geographic distances. This suggests that ridges separating the network of valleys act as dispersal barriers, and as such may channel the dispersal routes of the studied taxa and, likely, of their animal vectors as well. Dispersal limitation was further supported by a strong positive relationship between species richness and the abundance of neighbouring water bodies, suggesting that isolation affects colonization rates. Finally, the apparent dispersal limitation of microcrustaceans is further corroborated by the observation of low colonization rates in newly dug experimental ponds in the study area