Therapeutic proteins have revolutionized the treatment of many diseases but low activity or rapid clearance limits their utility. New approaches have been taken to design drugs with enhanced in vivo ...activity and half-life to reduce injection frequency, increase convenience, and improve patient compliance. One recently used approach is glycoengineering, changing protein-associated carbohydrate to alter pharmacokinetic properties of proteins. This technology has been applied to erythropoietin and resulted in the discovery of darbepoetin alfa (DA), a hyperglycosylated analogue of erythropoietin that contains two additional N-linked carbohydrates, a threefold increase in serum half-life and increased in vivo activity compared to recombinant human erythropoietin (rHuEPO). The increased serum half-life allows for less frequent dosing to maintain target hemoglobin levels in anemic patients. Carbohydrates on DA and other molecules can also increase molecular stability, solubility, increase in vivo biological activity, and reduce immunogenicity. These properties are discussed.
Clinical development of erythropoiesis-stimulating agents (ESAs) revolutionized the management of anemia. The major clinical benefits of ESAs are effective treatment of anemia and avoidance of blood ...transfusion risks. Erythropoietin (EPO) interacts directly with the EPO receptor on the red blood cell (RBC) surface, triggering activation of several signal transduction pathways, resulting in the proliferation and terminal differentiation of erythroid precursor cells and providing protection from RBC precursor apoptosis. The magnitude of increase in RBC concentration in response to administration of recombinant human EPO products (rhEPO) is primarily controlled by the length of time EPO concentrations are maintained, not by the EPO concentration level. Subcutaneous (SC) EPO administration results in slower absorption than intravenous (IV) administration, leading to lower peak plasma levels and an apparent extended terminal half-life. However, SC administration requires additional needle-sticks and is associated with an increased risk of immunogenicity compared with IV administration. Multiple pathways may play a role in EPO clearance from the body. Epoetin alfa was the first rhEPO produced and approved for pharmaceutical use, followed by several related products and by newer ESAs with the same mechanism but more prolonged action. Darbepoetin alfa is a hyperglycosylated EPO analog with an extended terminal half-life and a greater relative potency compared with rhEPO at extended dosing intervals. PEGylation of EPO (addition of polyethylene glycol) has been used to further extend the terminal half-life. Also, new strategies are under investigation for stimulating erythropoiesis through activation of the EPO receptor.
Research Problems Elliott, Steve
British journal for the philosophy of science,
12/2021, Letnik:
72, Številka:
4
Journal Article
Recenzirano
Odprti dostop
To identify and conceptualize research problems in science, philosophers and often scientists rely on classical accounts of problems that focus on intellectual problems defined in relation to ...theories. Recently, philosophers have begun to study the structures and functions of research problems not defined in relation to theories. Furthermore, scientists have long pursued research problems often labeled as practical or applied. As yet, no account of problems specifies the description of both so-called intellectual problems and so-called applied problems. This article proposes a new conceptual framework of problems that accounts both for intellectual aspects of problems and for their practical or applied aspects. I illustrate the account with an example of a recent research project from evolutionary biology, and I indicate further routes by that to develop the account, especially in connection to empirical studies of science.
Proof of Concept Research Elliott, Steve
Philosophy of science,
04/2021, Letnik:
88, Številka:
2
Journal Article
Recenzirano
Researchers often pursue proof of concept research, but criteria for evaluating such research remain poorly specified. This article proposes a general framework for proof of concept research that ...knits together and augments earlier discussions. The framework includes prototypes, proof of concept demonstrations, and post facto demonstrations. With a case from theoretical evolutionary genetics, the article illustrates the general framework and articulates some of the reasoning strategies used within that field. This article provides both specific tools with which to understand how researchers evaluate models in theoretical evolutionary genetics and general tools that apply to proof of concept research more generally.
Erythropoietin (Epo) is a cytokine that binds and activates an Epo receptor (EpoR) expressed on the surface of erythroid progenitor cells to promote erythropoiesis. While early studies suggested EpoR ...transcripts were expressed exclusively in the erythroid compartment, low-level EpoR transcripts were detected in nonhematopoietic tissues and tumor cell lines using sensitive RT-PCR methods. However due to the widespread use of nonspecific anti-EpoR antibodies there are conflicting data on EpoR protein expression. In tumor cell lines and normal human tissues examined with a specific and sensitive monoclonal antibody to human EpoR (A82), little/no EpoR protein was detected and it was not functional. In contrast, EpoR protein was reportedly detectable in a breast tumor cell line (MCF-7) and breast cancer tissues with an anti-EpoR polyclonal antibody (M-20), and functional responses to rHuEpo were reported with MCF-7 cells. In another study, a functional response was reported with the lung tumor cell line (NCI-H838) at physiological levels of rHuEpo. However, the specificity of M-20 is in question and the absence of appropriate negative controls raise questions about possible false-positive effects. Here we show that with A82, no EpoR protein was detectable in normal human and matching cancer tissues from breast, lung, colon, ovary and skin with little/no EpoR in MCF-7 and most other breast and lung tumor cell lines. We show further that M-20 provides false positive staining with tissues and it binds to a non-EpoR protein that migrates at the same size as EpoR with MCF-7 lysates. EpoR protein was detectable with NCI-H838 cells, but no rHuEpo-induced phosphorylation of AKT, STAT3, pS6RP or STAT5 was observed suggesting the EpoR was not functional. Taken together these results raise questions about the hypothesis that most tumors express high levels of functional EpoR protein.
Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have ...increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).
Darbepoetin alfa, a novel erythropoiesis-stimulating protein, is a glycosylation analog of recombinant human erythropoietin (rHuEPO) with two additional N-linked carbohydrates. Used to treat anemia ...of cancer, chemotherapy, and kidney disease, it has a three-fold longer serum half-life and increased in vivo activity, but decreased receptor-binding activity. Glycosylation analogs with altered N-linked carbohydrate content were compared with rHuEPO to elucidate the relationship between carbohydrate content and activity.
EPO glycosylation analogs and rHuEPO were expressed and, in some cases, purified from Chinese hamster ovary cells and carbohydrate characterized by Western blotting. Assays were performed to compare in vitro receptor binding and in vivo activity of rHuEPO, darbepoetin alfa, and analogs.
Reduced receptor binding of darbepoetin alfa could be accounted for entirely by increased sialic acid content and not by carbohydrate-related stearic hindrance or by amino acid differences. Shapes of dose-response curves, maximal responses in proliferation and colony assays, and magnitude and duration of downstream signaling events were comparable in vitro for rHuEPO and darbepoetin alfa. The in vivo response correlated with the number of N-linked carbohydrates. The number of carbohydrates was a more significant determinant for in vivo activity than position. The differences in in vivo erythropoietic activity among glycosylation analogs were more evident with increased time following administration in exhypoxic polycythemic mice.
Carbohydrate increases persistence of EPO, resulting in a prolonged and increased biological response in vivo, and overcoming reduced receptor-binding activity.
Scientific progress is dependent on accumulation of quality data with appropriate data analysis. Unfortunately, there are a troubling number of accounts describing an inability to replicate published ...work. Some explanations are lack of access to proprietary reagents and equipment, or lack of expertise and know how. However, it is clear that there are many publications that are fatally flawed, and it is difficult to ascertain which ones they are, but there are clues. Many articles are improperly controlled, resulting in false-positive or -negative results. Reagents and procedures are used without verifying their specificity. There is also confirmation bias, a tendency to seek and find conclusions that we like, which is exacerbated by faithful acceptance by readers of the publication record without assessment of merit. These and other issues have slowed progress, resulted in waste of scarce funds, and even put patients at risk when clinical decisions are made according to flawed data. Solving these and related problems requires recognition of the problem and better training. We also need to take personal responsibility for not only our own work, but also for the accuracy of information in the scientific domain.
Several clinical trials in oncology have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents. One hypothesis proposes that erythropoiesis-stimulating ...agents directly stimulate tumor proliferation and/or survival through cell-surface receptors. To test this hypothesis and examine if human tumors utilize the erythropoietin receptor pathway, the response of tumor cells to human recombinant erythropoietin was investigated in disaggregated tumor cells obtained from 186 patients with colorectal, breast, lung, ovarian, head and neck, and other tumors. A cocktail of well characterized tumor growth factors (EGF, HGF, and IGF-1) were analyzed in parallel as a positive control to determine whether freshly-isolated tumor cells were able to respond to growth factor activation ex vivo. Exposing tumor cells to the growth factor cocktail resulted in stimulation of survival and proliferation pathways as measured by an increase in phosphorylation of the downstream signaling proteins AKT and ERK. In contrast, no activation by human recombinant erythropoietin was observed in isolated tumor cells. Though tumor samples exhibited a broad range of cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface erythropoietin receptor was detected in tumor cells from the 186 tumors examined (by flow cytometry or Western blot). Erythropoiesis-stimulating agents did not act directly upon isolated tumor cells to stimulate pathways known to promote proliferation or survival of human tumor cells isolated from primary and metastatic tumor tissues.
Increasingly, science diaspora networks are managed by formal organizations such as embassies or non-profit organizations. Researchers have studied these networks to understand how they influence ...international collaborations and science diplomacy, and to determine which network activities foster those outcomes and which do not. In this perspective, we suggest that many of these network organizations confront an underappreciated conundrum for managing resources: organizations with few resources must learn how to obtain more resources despite lacking means to do so. To substantiate our suggestion, we do the following. We review exploratory results from a study of network organizations that indicate that these organizations generally lack resources, learn too little from each other, and struggle to overcome the resource conundrum. We also show that this conundrum is expected from organizational theory based on bounded rationality. To help organizations confront the issue, we do the following. First we provide a new database of operating science diaspora networks. We encourage managers of network organizations to use it as a resource to identify peers with whom to regularly exchange knowledge about securing resources. We also suggest that other scientific organizations should infuse network organizations with fresh resources. Ultimately, we urge all relevant stakeholders to recognize that the conundrum results not from the shortcomings of individual managers, but rather is a legitimate organizational phenomena that must be addressed by organizational design.
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