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1.
Genetics ; 197(1): 175-92, 2014 May.
Article in English | MEDLINE | ID: mdl-24558258

ABSTRACT

Insulin-producing cells (IPCs) in the Drosophila brain produce and release insulin-like peptides (ILPs) to the hemolymph. ILPs are crucial for growth and regulation of metabolic activity in flies, functions analogous to those of mammalian insulin and insulin-like growth factors (IGFs). To identify components functioning in IPCs to control ILP production, we employed genomic and candidate gene approaches. We used laser microdissection and messenger RNA sequencing to characterize the transcriptome of larval IPCs. IPCs highly express many genes homologous to genes active in insulin-producing ß-cells of the mammalian pancreas. The genes in common encode ILPs and proteins that control insulin metabolism, storage, secretion, ß-cell proliferation, and some not previously linked to insulin production or ß-cell function. Among these novelties is unc-104, a kinesin 3 family gene, which is more highly expressed in IPCs compared to most other neurons. Knockdown of unc-104 in IPCs impaired ILP secretion and reduced peripheral insulin signaling. Unc-104 appears to transport ILPs along axons. As a complementary approach, we tested dominant-negative Rab genes to find Rab proteins required in IPCs for ILP production or secretion. Rab1 was identified as crucial for ILP trafficking in IPCs. Inhibition of Rab1 in IPCs increased circulating sugar levels, delayed development, and lowered weight and body size. Immunofluorescence labeling of Rab1 showed its tight association with ILP2 in the Golgi of IPCs. Unc-104 and Rab1 join other proteins required for ILP transport in IPCs.


Subject(s)
Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Gene Expression Profiling , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Animals , Axons/metabolism , Carbohydrate Metabolism , Conserved Sequence , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Hemolymph/metabolism , Insulin Secretion , Insulin-Secreting Cells/cytology , Larva/cytology , Larva/genetics , Larva/growth & development , Larva/metabolism , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism
2.
Retina ; 27(9): 1260-6, 2007.
Article in English | MEDLINE | ID: mdl-18046235

ABSTRACT

PURPOSE: Ranibizumab (Lucentis) is a humanized antigen-binding fragment designed to inhibit all isoforms and active degradation products of vascular endothelial growth factor A (VEGF-A); it is in clinical development for the treatment of neovascular age-related macular degeneration (AMD). This study evaluated its pharmacokinetics (PK) and retinal distribution in rabbits when administered intravitreally (ITV). METHODS: A total of 27 New Zealand white rabbits received a single bilateral ITV injection of ranibizumab 625 muicrog/eye (Group 1, n = 24) or I-labeled ranibizumab 625 microg/eye, 22.5 microCi/eye (Group 2, n = 3). Ranibizumab concentration was determined in the vitreous, aqueous humor, and serum up to 60 days postdose by enzyme-linked immunosorbent assay in Group 1. Group 2 eyes were microautoradiographed on days 1-4. RESULTS: Ranibizumab has a terminal half-life of 2.9 days in the ocular compartments. Systemic exposure was low, measuring less than 0.01% of vitreous exposure when comparing AUC0-t values. Microautoradiography analysis demonstrated that ranibizumab penetrated all retinal layers, reaching the choriocapillaris on days 1, 2, and 4. CONCLUSIONS: This study demonstrates that following ITV injection, ranibizumab has a vitreous half-life of 2.9 days with minimal systemic exposure. Ranibizumab rapidly penetrates through the retina to reach the choroid, supporting its clinical development for neovascular AMD.


Subject(s)
Angiogenesis Inhibitors/pharmacokinetics , Antibodies, Monoclonal/pharmacokinetics , Aqueous Humor/metabolism , Retina/metabolism , Vitreous Body/metabolism , Animals , Antibodies, Monoclonal, Humanized , Autoradiography , Enzyme-Linked Immunosorbent Assay , Injections , Male , Rabbits , Ranibizumab , Tissue Distribution , Vascular Endothelial Growth Factor A/antagonists & inhibitors
3.
Exp Eye Res ; 85(4): 425-30, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17714704

ABSTRACT

Neovascular age-related macular degeneration (AMD) is the leading cause of blindness in older adults in the Western world. Ranibizumab (Lucentis), a humanized antibody fragment directed against vascular endothelial growth factor (VEGF-A), was recently approved by the US Food and Drug Administration (FDA) for the treatment of neovascular AMD. The objective of this study was to characterize the binding affinity and pharmacological activity of ranibizumab for 3 biologically active forms of VEGF-A: VEGF165, VEGF121, and VEGF110. The apparent equilibrium binding affinity of ranibizumab for VEGF-A molecules was determined by Biacore analysis; the biological activity of VEGF-A was demonstrated in a human umbilical vein endothelial cell (HUVEC) proliferation-inhibition assay. Inhibition of VEGF-A-induced vascular permeability by ranibizumab was assessed in vivo using hairless guinea pigs and a modified Miles assay. Ranibizumab was capable of binding to recombinant human VEGF165, VEGF121, and VEGF110 (KD < or = 192 pM), inhibiting VEGF-A-induced HUVEC proliferation in a concentration-dependent manner. Ranibizumab also exerted potent dose-dependent inhibition (IC(50) of 0.4-1.2 nM) of the vascular permeability-enhancing activity of VEGF165, VEGF121, and VEGF110 in the Miles assay. In conclusion, these results show that ranibizumab is capable of binding to and specifically inhibiting the activities of 3 biologically active forms of VEGF-A. As VEGF-A plays a pivotal role in the pathogenesis of neovascular AMD, ranibizumab activity, as demonstrated in this study, supports its clinical utility in the treatment of this disease.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Antibodies, Monoclonal/pharmacology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Angiogenesis Inhibitors/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal, Humanized , Capillary Permeability/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Guinea Pigs , Humans , Ranibizumab , Recombinant Proteins/metabolism , Skin/blood supply , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/pharmacology
4.
Int J Toxicol ; 24(5): 357-63, 2005.
Article in English | MEDLINE | ID: mdl-16257855

ABSTRACT

Bevacizumab (Avastin) is a humanized monoclonal antibody against vascular endothelial growth factor approved for use in combination with 5-fluorouracil (5-FU)-based chemotherapy for first-line treatment of metastatic colorectal cancer. The Saltz regimen (irinotecan/5-FU/leucovorin [LV]) is a first-line treatment for this indication. The objective of this study was to evaluate the safety of bevacizumab when administered concomitantly with the Saltz regimen to cynomolgus monkeys, and to determine if the pharmacokinetics of bevacizumab, irinotecan, SN38 (the active metabolite of irinotecan), or 5-FU were affected by combined administration. Male cynomolgus monkeys were intravenously administered the Saltz regimen (125 mg/m2 irinotecan, 500 mg/m2 5-FU, 20 mg/m2 LV) alone (n = 4) or concomitantly with 10 mg/kg bevacizumab (n = 5) on days 1 and 8. All animals survived to euthanasia on day 15. Adverse effects associated with the Saltz regimen included diarrhea and neutropenia. Macroscopically, two animals from each group had small thymus glands that correlated microscopically with lymphoid depletion. Myeloid hypoplasia and/or erythroid hyperplasia was observed in the sternal bone marrow of most animals. These effects were considered to be associated with the Saltz regimen; concomitant bevacizumab administration did not alter the severity of these findings. Irinotecan and 5-FU were observed to be rapidly eliminated (t1/2 = 1 h and 0.5 h, respectively). Although the number of animals in each group was small and no statistical comparison between groups was performed, bevacizumab did not affect the disposition of either agent. These results indicate that bevacizumab can be safely administered in combination with the Saltz regimen without pharmacokinetic interaction.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacokinetics , Prodrugs , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Area Under Curve , Bevacizumab , Camptothecin/administration & dosage , Camptothecin/analogs & derivatives , Camptothecin/pharmacokinetics , Fluorouracil/administration & dosage , Fluorouracil/pharmacokinetics , Half-Life , Irinotecan , Leucovorin/administration & dosage , Macaca fascicularis , Male , Metabolic Clearance Rate , Random Allocation
5.
Invest Ophthalmol Vis Sci ; 46(2): 726-33, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15671306

ABSTRACT

PURPOSE: Ranibizumab (rhuFab V2; Lucentis, Genentech, South San Francisco, CA) is a humanized monoclonal antibody fragment designed to bind all forms of VEGF, thereby blocking vessel permeability and angiogenesis in neovascular age-related macular degeneration. This study evaluated the pharmacokinetic (PK) and serum bioavailability of ranibizumab after a single intravitreal (ITV) or intravenous (IV) dose in cynomolgus monkeys. METHODS: Monkeys received ranibizumab as either a bilateral ITV dose (500 or 2000 microg/eye; n = 6/group) or a single IV dose (1000 or 4000 microg/animal; n = 4/group). After ITV administration, ranibizumab concentrations were measured in several ocular compartments and in serum for 10 days and, after IV administration, for 48 hours. Pharmacokinetic parameters were estimated by compartmental and noncompartmental methods. RESULTS: Ranibizumab cleared in parallel from all ocular compartments, with a terminal half-life of approximately 3 days. It distributed rapidly to the retina (6-24 hours), and concentrations were approximately one third that in the vitreous. After ITV injection, bioavailability (F) was 50% to 60%. Serum concentrations were very low, reflecting wider distribution and faster clearance when ranibizumab reached the serum. After IV administration, the terminal half-life was approximately 0.5 day. CONCLUSIONS: This study demonstrates that ranibizumab has a PK profile that is favorable for its clinical use in treating neovascular AMD by monthly ITV injection.


Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Aqueous Humor/metabolism , Retina/metabolism , Vascular Endothelial Growth Factor A/immunology , Vitreous Body/metabolism , Animals , Antibodies, Monoclonal, Humanized , Area Under Curve , Biological Availability , Drug Evaluation, Preclinical , Female , Half-Life , Immunoglobulin Fab Fragments/immunology , Injections , Macaca fascicularis , Male , Ranibizumab , Recombinant Fusion Proteins , Vitreous Body/drug effects
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