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1.
Support Care Cancer ; 26(1): 7-20, 2018 01.
Article in English | MEDLINE | ID: mdl-28939926

ABSTRACT

PURPOSE: Filgrastim (NEUPOGEN®) is the originator recombinant human granulocyte colony-stimulating factor widely used for preventing neutropenia-related infections and mobilizing hematopoietic stem cells. This report presents findings of a systematic literature review and meta-analysis of efficacy and safety of originator filgrastim to update previous reports. METHODS: A literature search of electronic databases, congress abstracts, and bibliographies of recent reviews was conducted to identify English-language reports of clinical trials and observational studies evaluating filgrastim in its US-approved indications up to February 2015. Two independent reviewers assessed titles/abstracts and full texts of publications, and extracted data from studies that compared originator filgrastim vs placebo or no treatment. For outcomes with sufficient homogeneous data reported across studies, meta-analysis was performed and relative risk (RR) determined. Data were summarized descriptively for all other evaluated outcomes. RESULTS: A total of 1194 unique articles evaluating originator filgrastim were identified, with 25 meeting eligibility criteria for data extraction: 18 randomized controlled trials, 2 nonrandomized clinical trials, and 5 observational studies. In chemotherapy-induced neutropenia (CIN), filgrastim vs placebo or no treatment significantly reduced febrile neutropenia incidence (RR 0.63, 95% CI 0.53-0.75) and grade 3 or 4 neutropenia incidence (RR 0.50, 95% CI 0.37-0.68). The most commonly reported adverse event (AE) with filgrastim was bone pain (RR 2.61, 95% CI 1.29-5.27 in CIN). Additional efficacy and safety outcomes are described within indications. CONCLUSIONS: This systematic literature review and meta-analysis confirms and updates previous reports on the efficacy and safety of originator filgrastim. Bone pain was the commonly reported AE associated with filgrastim use.


Subject(s)
Filgrastim/therapeutic use , Granulocyte Colony-Stimulating Factor/therapeutic use , Hematologic Agents/therapeutic use , Filgrastim/administration & dosage , Filgrastim/pharmacology , Granulocyte Colony-Stimulating Factor/administration & dosage , Granulocyte Colony-Stimulating Factor/pharmacology , Hematologic Agents/administration & dosage , Hematologic Agents/pharmacology , Humans
2.
Biotechnol Prog ; 27(4): 1054-67, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21656920

ABSTRACT

Crystallization holds the potential to be used for protein purification and low-viscosity drug substance and drug product formulations. Twenty-two different proteins (20 monoclonal antibodies and two Fc-fusions) were examined to determine the breadth of applicability of crystallization to these therapeutic proteins. Vapor diffusion technique and an evaporative screening method were used to identify crystallization conditions using around a 100 initial conditions based on reagents that are generally regarded as safe (GRAS). Of 16 IgG2 s examined, at least four formed diffraction-quality crystals and four others formed crystal-like particles. At least three of the IgG2 s that crystallized well were also crystallized under the same set of operating conditions using inexpensive GRAS reagents. The crystals were formed to high-yields in a few hours and were dissolved quickly without impacting product quality. Although only a fraction of the proteins examined crystallized, all exhibited liquid-liquid phase separation (LLPS), which could be used for their concentration or possibly purification. One of the Fc-fusions, for example, was concentrated by LLPS to a self-buffering solution at 150 g/L. Crystallization and LLPS in the salting-in region were shown to be feasible.


Subject(s)
Antibodies, Monoclonal/chemistry , Chemical Fractionation/methods , Immunoglobulin Fc Fragments/chemistry , Recombinant Fusion Proteins/chemistry , Chromatography, High Pressure Liquid , Crystallization , Immunoglobulin G/chemistry
3.
J Biol Chem ; 286(22): 19917-31, 2011 Jun 03.
Article in English | MEDLINE | ID: mdl-21464137

ABSTRACT

Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity.


Subject(s)
Endoplasmic Reticulum/metabolism , Gene Expression , Immunoglobulin G/biosynthesis , Protein Folding , Recombinant Proteins/biosynthesis , Animals , CHO Cells , Cricetinae , Cricetulus , Endoplasmic Reticulum/genetics , Humans , Hydrogen-Ion Concentration , Immunoglobulin G/genetics , Recombinant Proteins/genetics
4.
Bioorg Med Chem ; 15(20): 6574-95, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-17697781

ABSTRACT

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase that plays a critical role in the early development of the nervous system. Deregulation of CDK5 is believed to contribute to the abnormal phosphorylation of various cellular substrates associated with neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and ischemic stroke. Acyclic urea 3 was identified as a potent CDK5 inhibitor and co-crystallographic data of urea 3/CDK2 enzyme were used to design a novel series of 3,4-dihydroquinazolin-2(1H)-ones as CDK5 inhibitors. In this investigation we present our synthetic studies toward this series of compounds and discuss their biological relevance as CDK5 inhibitors.


Subject(s)
Cyclin-Dependent Kinase 5/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Quinazolinones/chemistry , Quinazolinones/pharmacology , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Benzyl Compounds/chemical synthesis , Benzyl Compounds/chemistry , Benzylamines/chemical synthesis , Benzylamines/chemistry , Binding Sites , Crystallography, X-Ray , Cyclin-Dependent Kinase 5/metabolism , Hydrogen/chemistry , Inhibitory Concentration 50 , Isomerism , Models, Molecular , Molecular Structure , Oxadiazoles/chemical synthesis , Oxadiazoles/chemistry , Protein Kinase Inhibitors/chemistry , Quinazolinones/chemical synthesis , Structure-Activity Relationship , Thiadiazoles/chemical synthesis , Thiadiazoles/chemistry , Thiazoles/chemical synthesis , Thiazoles/chemistry
5.
Bioorg Med Chem Lett ; 17(19): 5384-9, 2007 Oct 01.
Article in English | MEDLINE | ID: mdl-17709247

ABSTRACT

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine protein kinase and its deregulation is implicated in a number of neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and ischemic stroke. Using active site homology modeling between CDK5 and CDK2, we explored several different chemical series of potent CDK5 inhibitors. In this report, we describe the design, synthesis, and CDK5 inhibitory activities of quinolin-2(1H)-one derivatives.


Subject(s)
Cyclin-Dependent Kinase 5/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Quinolines/chemical synthesis , Quinolines/pharmacology , Binding Sites , Cyclin-Dependent Kinase 2/chemistry , Cyclin-Dependent Kinase 5/chemistry , Drug Design , Indicators and Reagents , Models, Molecular , Protein Binding , Structure-Activity Relationship
6.
Protein Eng Des Sel ; 19(4): 147-53, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16478751

ABSTRACT

Native sequence keratinocyte growth factor (KGF) is fairly unstable, as manifested by the loss of the monomeric native protein accompanied by the accumulation of aggregated species during storage at moderate temperatures. Several different types of analogs were generated and the storage stability of the protein assessed. In the first type of analog one or more of the five cysteinyl residues in KGF were replaced; in the second class the N-terminal residues that included the first disulfide bond were deleted. Both of these types of analogs involved removal of the disulfide bond between cysteines 1 and 15. The third group involved mutating one of the basic amino acids located in a cluster of positive charges (involved in heparin binding) around Arg144 to a neutral or acidic amino acyl residue. Among the cysteine replacement analogs, the double mutation of Cys1 and 15 to Ser resulted in significantly increased stability without compromising the mitogenic activity, while Cys to Ser mutations at other positions were either destabilizing or had no effect. Deletion of the 15, 23 or 27 N-terminal amino acyl residues also increased the stability of the protein. The activity of the analogs was not affected by the deletion of 15 or 23 amino acids, but it was significantly decreased upon removal of the 27 N-terminal amino acyl residues. Much greater stability was achieved by mutation of the basic amino acids, especially Arg144, to Glu or Gln, but this increase in stability was accompanied by large decrease in activity. The analog with the 23 N-terminal amino acyl residues deleted represents one of the best compromises between increased stability and retention of activity.


Subject(s)
Fibroblast Growth Factor 7/genetics , Recombinant Proteins/genetics , Amino Acid Sequence , Animals , Calorimetry, Differential Scanning , Cell Line , Circular Dichroism , Drug Stability , Fibroblast Growth Factor 7/metabolism , Fibroblast Growth Factor 7/pharmacology , Heparin/metabolism , Hot Temperature , Mice , Mitogens/analysis , Molecular Sequence Data , Mutagenesis , Protein Binding , Protein Conformation , Protein Denaturation
7.
Biochemistry ; 44(18): 6948-57, 2005 May 10.
Article in English | MEDLINE | ID: mdl-15865440

ABSTRACT

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of 11-dehydrocorticosterone to its active form corticosterone in rodents (or cortisone to cortisol in humans). The reductive reaction of the 11-keto to 11-hydroxyl is the pivotal switch in the activation of glucocorticoids. An excess of active glucocorticoids has been shown to play a key role in metabolic disorders such as diabetes and obesity. Therefore, 11beta-HSD1 represents an important therapeutic target for the treatment of these diseases. To facilitate the iterative design of inhibitors, we have crystallized and determined the three-dimensional structures of a binary complex of murine 11beta-HSD1 with NADP(H) to a resolution of 2.3 A and of a ternary complex with corticosterone and NADP(H) to a resolution of 3.0 A by X-ray crystallography. The enzyme forms a homodimer in the crystal and has a fold similar to those of other members of the family of short chain steroid dehydrogenases/reductases (SDRs). The structure shows a novel folding feature at the C-terminus of the enzyme. The C-terminal helix insertions provide additional dimer contacts, exert an influence on the conformations of the substrate binding loops, and present hydrophobic regions for potential membrane attachment. The structure also reveals how 11beta-HSD1 achieves its selectivity for its substrate.


Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 1/chemistry , 11-beta-Hydroxysteroid Dehydrogenase Type 1/metabolism , Catalytic Domain , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/enzymology , Drug Delivery Systems , 11-beta-Hydroxysteroid Dehydrogenase Type 1/antagonists & inhibitors , Amino Acid Sequence , Animals , Binding Sites , Corticosterone/chemistry , Corticosterone/metabolism , Crystallography, X-Ray , Dimerization , Drug Delivery Systems/methods , Humans , Mice , Molecular Sequence Data , NADP/chemistry , NADP/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , Substrate Specificity
8.
Bioorg Med Chem Lett ; 14(11): 2905-9, 2004 Jun 07.
Article in English | MEDLINE | ID: mdl-15125957

ABSTRACT

Novel alphavbeta3 antagonists based on the N-aryl-gamma-lactam scaffold were prepared. SAR studies led to the identification of potent antagonists for alphavbeta3 receptor with excellent selectivity against the structurally related alpha(IIb)beta3 receptor. Additional interactions of N-aryl-gamma-lactam derivatives with alphavbeta3 were found when compared to c(-RGDf[NMe]V-) peptide antagonist. The effects of the conformation and configuration of the gamma-lactam core on the binding were also assessed.


Subject(s)
Integrin alphaVbeta3/antagonists & inhibitors , Lactams/pharmacology , Animals , Binding Sites , Binding, Competitive , Humans , Lactams/chemical synthesis , Ligands , Molecular Conformation , Platelet Glycoprotein GPIIb-IIIa Complex/drug effects , Structure-Activity Relationship
9.
Nat Biotechnol ; 21(4): 414-21, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12612588

ABSTRACT

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).


Subject(s)
Drug Delivery Systems/methods , Glycoproteins/biosynthesis , Glycoproteins/genetics , Protein Engineering/methods , Proteins/therapeutic use , Anemia/drug therapy , Animals , CHO Cells/metabolism , COS Cells/metabolism , Cricetinae , Erythropoietin/genetics , Erythropoietin/metabolism , Erythropoietin/therapeutic use , Excipients/chemistry , Female , Genetic Enhancement/methods , Glycoproteins/metabolism , Humans , Leptin/biosynthesis , Leptin/deficiency , Leptin/genetics , Leptin/therapeutic use , Mice , Mice, Inbred BALB C , Proteins/administration & dosage , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/therapeutic use , Thrombopoietin/biosynthesis , Thrombopoietin/therapeutic use
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