Apo2L/TRAIL [Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand], a member of the TNF cytokine superfamily, induces cell death by apoptosis in a number of human cancer cells and is a potential agent for cancer therapy. We have characterized the in vitro stability of Apo2L/TRAIL in human serum and the tissue distribution and metabolism of Apo2L/TRAIL in a xenograft model of human colon carcinoma (COLO205). Apo2L/TRAIL was stable after incubation in human serum, with no significant high molecular weight complexes or degradation products observed. After i.v. administration of I-Apo2L/TRAIL to mice, a small percentage of the radiolabeled drug was seen as high molecular weight complex or as low molecular weight degradation products in plasma. However, the most abundant radioactive species corresponded to the intact Apo2L/TRAIL monomer, indicative of the relative stability of this recombinant protein in blood. Distribution of I-Apo2L/TRAIL to organs and solid xenograft tumors was limited. Intact I-Apo2L/TRAIL was detectable in the solid tumor at all time points and was the only tissue in which radioactivity transiently increased over time. Kidney contained the highest levels of radioactivity. Radioactive signal reached a tissue-toblood ratio of 18 in the kidney cortex region when I-Apo2L/ TRAIL was given in the presence of excess unlabeled ligand. In contrast to blood, extensive I-Apo2L/TRAIL degradation was observed in the kidney and, to a lesser degree, in the solid tumor and other organs, including liver, spleen, and lung. Our studies demonstrated that Apo2L/TRAIL is stable in the circulation, localizes to human solid xenograft tumors, and is primarily eliminated

Apo2L/TRAIL [Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand], a member of the TNF cytokine superfamily, induces cell death by apoptosis in a number of human cancer cells and is a potential agent for cancer therapy. We have characterized the in vitro stability of Apo2L/TRAIL in human serum and the tissue distribution and metabolism of Apo2L/TRAIL in a xenograft model of human colon carcinoma (COLO205). Apo2L/TRAIL was stable after incubation in human serum, with no significant high molecular weight complexes or degradation products observed. After i.v. administration of 125I-Apo2L/TRAIL to mice, a small percentage of the radiolabeled drug was seen as high molecular weight complex or as low molecular weight degradation products in plasma. However, the most abundant radioactive species corresponded to the intact Apo2L/TRAIL monomer, indicative of the relative stability of this recombinant protein in blood. Distribution of 125I-Apo2L/TRAIL to organs and solid xenograft tumors was limited. Intact 125I-Apo2L/TRAIL was detectable in the solid tumor at all time points and was the only tissue in which radioactivity transiently increased over time. Kidney contained the highest levels of radioactivity. Radioactive signal reached a tissue-to-blood ratio of 18 in the kidney cortex region when 125I-Apo2L/TRAIL was given in the presence of excess unlabeled ligand. In contrast to blood, extensive 125I-Apo2L/TRAIL degradation was observed in the kidney and, to a lesser degree, in the solid tumor and other organs, including liver, spleen, and lung. Our studies demonstrated that Apo2L/TRAIL is stable in the circulation, localizes to human solid xenograft tumors, and is primarily eliminated through the kidney.

Peripheral neuropathy is a significant complication of diabetes resulting in increased patient morbidity and mortality. Deficiencies of neurotrophic substances (e.g. NGF, NT-3, and IGF-I) have been proposed as pathogenetic mechanisms in the development of distal symmetrical sensory diabetic polyneuropathy, and salutary effects of exogenous NGF administration have been reported in animal models. In comparison, relatively little is known concerning the effect of NGF on experimental diabetic sympathetic autonomic neuropathy. We have developed an experimental animal model of diabetic autonomic neuropathy characterized by the regular occurrence of pathologically distinctive dystrophic axons in prevertebral sympathetic ganglia and ileal mesenteric nerves of rats with chronic streptozotocin (STZ)-induced diabetes. Treatment of STZ-diabetic rats for 2–3 months with pharmacologic doses of NGF or NT-3, neurotrophic substances with known effects on the adult sympathetic nervous system, did not normalize established neuroaxonal dystrophy (NAD) in diabetic rats in the prevertebral superior mesenteric ganglia (SMG) and ileal mesenteric nerves as had pancreatic islet transplantation and IGF-I in earlier experiments. NGF treatment of control animals actually increased the frequency of NAD in the SMG. New data suggests that, in adult sympathetic ganglia, NGF may contribute to the pathogenesis of NAD rather than its amelioration, perhaps as the result of inducing intraganglionic axonal sprouts in which dystrophic changes are superimposed. NT-3 administration did not alter the frequency of NAD in diabetic animals, although it resulted in a significant decrease in NAD in control SMG. Although deficiencies of neurotrophic substances may represent the underlying pathogenesis of a variety of experimental neuropathies, delivery of excessive levels of selected substances may produce untoward effects.

In this study, we have characterized the metabolism, tissue disposition, excretion routes, and plasma pharmacokinetics of recombinant human nerve growth factor after single and multiple s.c. administration in male cynomolgus monkeys. Unlabeled nerve growth factor (NGF; 2 mg/kg) was administered three times a week for 4 weeks and a full pharmacokinetic profile was obtained for doses 1 and 12. For the tissue distribution studies, 0.8 microg/kg of trace (125)I-labeled recombinant human nerve growth factor was dosed. Histological analysis of emulsion-microautoradiography indicated that specific (125)I-NGF labeling was confined to sections of nerves most frequently localized adjacent to large vessels in sections of kidney, spleen, liver, and salivary gland. A small percentage of large neurons within the sympathetic ganglia were intensely labeled, as well as large neurons within the dorsal root ganglia. We found an increased disposition of (125)I-NGF in parts of the peripheral nervous system (including sympathetic ganglia) from 8 to 24 h postdose. In contrast, radioactivity in most non-neuronal tissues declined. This suggests specific uptake in these target tissues known to express specific receptors for NGF. We also identified changes in pharmacokinetic parameters after single versus chronic s. c. administration. These studies demonstrated that s.c. administration of NGF at 0.8 microg/kg doses in monkeys is capable of accessing and localizing in the target tissues.

The purpose of this investigation was to characterize the stability, activity, and interactions of recombinant human nerve growth factor (rhNGF) in various biological matrices in vitro and in vivo. rhNGF (10 microg/ml) remained stable in human plasma for up to 4 days at 37 degrees C. There was a decrease in the recovery of rhNGF after incubation at lower concentrations (20 ng/ml) and for longer time periods (3 and 5 days at 37 degrees C). Size exclusion HPLC analysis indicated that rhNGF forms high molecular weight (HMW) complexes after long incubation periods. We confirmed that alpha(2)-macroglobulin (alpha(2)M) is the major plasma component that binds to rhNGF. Furthermore, this interaction was considerably increased by treatment of plasma with primary amines such as CH(3)NH(2). Changes in the pH environment did not affect the interaction of rhNGF with alpha(2)M. We also determined that the binding of rhNGF to CH(3)NH(2)-treated pure alpha(2)M or alpha(2)M present in human plasma substantially diminished its immunoreactivity and bioactivity detection. The interaction of rhNGF with activated alpha(2)M was reversed and inhibited by coincubation with dimethyl sulfoxide. Released rhNGF under these conditions was fully bioactive. (125)I-rhNGF also binds to alpha(2)M by forming similar (125)I-rhNGF/HMW complexes in plasma after i.v. administration in rats and mice. Sixty minutes after dosing in rats, most of the labeled material was in the form of a (125)I-rhNGF/HMW complex. These studies have provided a better understanding of the nature of the interactions of rhNGF with plasma components as well as methods to enhance, reverse, and inhibit these interactions.

Peripheral sensory neuropathies result from many known etiologies, including diabetes and other metabolic disorders; toxicity from chemotherapeutic agents, such as taxol, cisplatin and vincristine; mechanical compression; ischemia; HIV and other viral infections; hereditary causes; and inflammatory and immune diseases. The etiology of some peripheral neuropathies remains obscure, despite thorough evaluations of the known causes; these are classified as idiopathic neuropathies. Diabetic polyneuropathy comprises the largest group of peripheral sensory neuropathies world-wide and, thus, is the targeted population in many ongoing clinical trials, including NGF. However, as will be illustrated below, neurotrophins, such as NGF, have the potential to be effective therapies for various types of peripheral sensory neuropathies, regardless of etiology.

Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis and in pathological processes such as tumor growth, rheumatoid arthritis, and ocular neovascularization. A recombinant humanized monoclonal antibody (rhuMAb), rhuMAb VEGF, has been developed to inhibit the effects of VEGF in the treatment of solid tumors. Intravenous and s.c. pharmacokinetic studies were conducted in mice, rats, and cynomolgus monkeys. In addition, the tissue distribution of i.v. 125I-rhuMAb VEGF was investigated in rabbits. At a dose of approximately 10 mg/kg, the clearance of rhuMAb VEGF from the serum was 15.7 ml/day/kg in mice, 4.83 ml/day/kg in rats, and 5.59 ml/day/kg in cynomolgus monkeys, and the terminal half-life ranged from 6 to 12 days in all species. After s.c. administration, rhuMAb VEGF had a bioavailability of 69% in rats and 100% in mice and cynomolgus monkeys. Pharmacokinetic data in mice, rats, and cynomolgus monkeys were used to predict the pharmacokinetics of rhuMAb VEGF using allometric scaling in humans. The predicted serum clearance of rhuMAb VEGF in humans was 2.4 ml/day/kg and the terminal half-life was 12 days. Two hours after i.v. bolus administration of 125I-rhuMAb VEGF in rabbits, trichloroacetic acid-precipitable radioactivity was noted primarily in the plasma, with lesser amounts in highly perfused tissues such as kidneys, testes, spleen, heart, and lungs. At 48 h after dosing, trichloroacetic acid-precipitable radioactivity was noted in plasma with minimal distribution to testes, bladder, heart, lungs, and kidneys. Tissue distribution and pharmacokinetic data indicate that rhuMAb VEGF is cleared slowly and distributes to specific sites in the body.

The temporal patterns of BDNF andtrkBexpression in the developingXenopus laevistadpole, and the responsiveness of retinal ganglion cells to BDNF, both in culture andin vivo,suggest significant roles for this neurotrophin during visual system development (Cohen-Cory and Fraser,Neuron12, 747–761, 1994;Nature378, 192–196, 1995). To examine the potential roles of this neurotrophin within the developing retina and in its target tissue, the optic tectum, we studied the cellular sites of BDNF expression byin situhybridization. In the developing optic tectum, discrete groups of cells juxtaposed to the tectal neuropil where retinal axons arborize expressed BDNF, supporting the target-derived role commonly proposed for this neurotrophin. In the retina, retinal ganglion cells, ciliary margin cells, and a subset of cells in the inner nuclear layer expressed the BDNF gene. The expression of BDNF coincided with specifictrkBexpression by both retinal ganglion cells and amacrine cells, as well as with the localization of functional BDNF binding sites within the developing retina, as shown byin situhybridization and BDNF cross-linking studies. To test for a possible role of endogenous retinal BDNF during development, we studied the effects of neutralizing antibodies to BDNF on the survival of retinal ganglion cells in culture. Exogenously administered BDNF increased survival, whereas neutralizing antibodies to BDNF significantly reduced baseline retinal ganglion cell survival and differentiation. This suggests the presence of an endogenous retinal source of neurotrophic support and that this is most likely BDNF itself. The retinal cellular patterns of BDNF andtrkBexpression as well as the effects of neutralizing antibodies to this neurotrophin suggest that, in addition to a target-derived role, BDNF plays both autocrine and/or paracrine roles during visual system development.

Members of the NGF family of proteins act as neurotrophic agents for defined populations of peripheral and central neurons during embryonic and postnatal development. We have studied the presence of receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 and - 4/5 (NT-3, NT-4/5) by cross-linking radioiodinated neurotrophins to specific cell surface receptors. We have identified neurotrophin receptors representing full-length TrkB and TrkC and their truncated forms (lacking a functional cytoplasmic kinase domain) in neuronal as well as in non-neuronal tissues. During chicken embryonic and early postnatal brain development, expression of full-length TrkB and TrkC proteins preceded the onset of the truncated forms of these receptors. A similar pattern was also observed in mouse embryonic and early postnatal brain. The relative levels of neurotrophin receptors in the basal forebrain and in the hippocampus did not change significantly with age in mice. High levels of receptors for the three neurotrophins were detected in the nigrostriatal system. Full-length TrkB and TrkC receptors were found in chicken and rat embryonic ventral spinal cord, as well as on purified motoneurons. Again, truncated TrkB appeared significantly later than the full-length form on spinal motoneurons. In chicken embryonic retina and optic tectum we detected full-length TrkB and TrkC; however, the optic tectum also expressed large amounts of the truncated form of TrkB. TrkC but not TrkB was detected in chicken embryonic skeletal muscle, suggesting that NT-3 may have a novel function in this tissue. The presence of neurotrophin receptors in a wide variety of embryonic and postnatal tissues underlines the significant role of BDNF, NT-3, and NT-4/5 in embryonic and postnatal development. The regulation of the ratio of full-length versus truncated neurotrophin receptors may play an important role in the development, maturation, and maintenance of various neuronal networks.

Cell division, cell death, and remodeling of connections are major features of the construction of the mammalian CNS. We have begun to address the role of neurotrophins in these events through characterization of the expression of their receptors in the developing ferret visual system. By use of chemical cross-linking of iodinated neurotrophins, proteins corresponding to trkB, trkC, and p75 were identified as receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) throughout development. BDNF was also cross- linked to a truncated form of trkB that lacks the tyrosine kinase domain (trkB. T1) in retinal target tissues and in cortex. At the earliest developmental age examined (E24), the ratio of full-length to truncated trkB is > > 1 in the retinal target tissues, LGN and superior colliculus. During the ensuing period of retinal ganglion cell death and segregation into eye-specific layers, the amount of truncated trkB increases markedly relative to full-length trkB. By P27, truncated trkB is the predominant receptor for BDNF in the retinal target tissues and this pattern is maintained into adulthood. Within all subdivisions of visual cortex including the ventricular zone (VZ), intermediate zone (IZ), and cortical plate (CP), similar profiles of bands are observed. The developmental increase in abundance of truncated trkB relative to full-length occurs earliest in the VZ, with a major increase between E30 and P3. In the IZ, this shift to a predominance of truncated trkB occurs between P15 and P30, while in the CP the shift is even further delayed, not occurring until after P30. Within each subdivision of cortex, the shift to a predominance of truncated trkB occurs at times that correlate with the onset of cell death and maturation of axonal connections. This study demonstrates that members of the trk family, previously identified in the CNS on the basis of mRNA transcripts, are present as receptors with specific binding affinities for BDNF and NT- 3. Moreover, the correspondence between the developmental shift from full-length to truncated trkB and the critical periods for cell fate determination, cell death, and axonal remodeling suggests an important role for neurotrophic factors in the development of the visual system.

Survival and maintenance of vertebrate neurons are influenced by neurotrophic factors which mediate their signal by binding to specific cell surface receptors. We determined the binding sites of human neurotrophin‐3 (NT‐3) to its receptors trkC and gp75 by mutational analysis and compared them to the analogous interactions of nerve growth factor (NGF) with trkA and gp75. The trkC binding site extends around the central beta‐strand bundle and in contrast to NGF does not make use of non‐conserved loops and the six N‐terminal residues. The gp75 epitope is dominated by loop residues and the C‐terminus of NT‐3. A novel rapid biological screening procedure allowed the identification of NT‐3 mutants that are able to signal efficiently through the non‐preferred receptors trkA and trkB, which are specific for NGF and BDNF respectively. Mutation of only seven residues in NT‐3 resulted in a human neurotrophin variant which bound to all receptors of the trk family with high affinity and efficiently supported the survival of NGF‐, BDNF‐ and NT‐3‐dependent neurons. Our results suggest that the specificity among neurotrophic factors is not solely encoded in sequence diversity, but rather in the way each neurotrophin interacts with its preferred receptor.

Neurotrophic factors regulate the developmental survival and differentiation of specific neuronal populations. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of the nerve growth factor (NGF) protein family, also known as the neurotrophins. Insights into the different roles of neurotrophins can be gained by studying the expression of their functional receptors. Here we report the development of procedures for their radiolabeling and efficient crosslinking to specific cell-surface receptors. BDNF and NT-3 receptors in cell lines and tissue preparations expressing receptors for the 2 neurotrophins have been identified using this affinity crosslinking procedure. Like NGF, BDNF and NT-3 crosslinked to the low affinity NGF receptor (p75NGFR) on PC12 cells. BDNF and NT-3 also crosslinked to cells expressing p145trkB protein, producing an approximately 160 kD neurotrophin-receptor complex. Crosslinking of the 2 neurotrophins in vivo to specific trk family members in many areas of the central nervous system also produced a 160 kD receptor complex. However, in all brain regions a complex of approx. 100 kD could also be identified, all or most of which represents crosslinking to a truncated form of trkB. The broad distribution of BDNF and NT-3 receptors throughout the CNS suggests that neurotrophins may have yet unrecognized functions on specific neuronal populations. BDNF and NT-3 receptors were also found in brain areas in which the neurotrophins themselves are also synthesized, suggesting that beyond long-range trophic effects, these proteins may also act as autocrine or short-range paracrine regulators.

Members of the Trk tyrosine kinase family have recently been identified as functional receptors of the NGF family of neurotrophins. Here we show the rat trkC locus to be complex, encoding at least four distinct polypeptides. Three of the encoded polypeptides are full-length receptor tyrosine kinases that differ by novel amino acid insertions in the kinase domain. A fourth protein is a truncated receptor that lacks the catalytic domain. Tyrosine phosphorylation, cross-linking, and ligand binding assays indicate that TrkC receptors interact with NT-3 and not with the related neurotrophins NGF, BDNF, xNT-4, or hNT-5. Furthermore, high and low affinity NT-3-binding sites are associated with the TrkC receptors. Stable and transient expression of TrkC receptors in PC12 cells indicates that the neurite outgrowth response elicited by NT-3 is dramatic in receptors lacking the novel kinase insert (gp150trkC) but absent in receptors containing the 14 amino acid insert in the kinase domain (gp150trkC14). These data suggest that the trkC locus encodes receptors that may be capable of mediating different biological responses within the cell. This could have important implications in understanding the role of neurotrophins in the development of the vertebrate nervous system.

The expression of neurotrophin (NGF, BDNF, and NT-3) mRNAs in 24 cell lines derived from human malignant gliomas was studied by Northern analysis. Widespread expression of neurotrophin genes was found with BDNF being the most abundantly expressed. Nearly all cell lines expressed BDNF, and about two-thirds of the cell lines expressed NGF and NT-3. Half of the cell lines analyzed expressed all three neurotrophins. Secretion of NGF into the medium of several cell lines could be detected by ELISA and a PC12 neurite outgrowth assay. Immuno- and bioactive NGF was isolated from conditioned medium of one cell line. No evidence of expression of the neurotrophin receptors trk and trkB by Northern analysis was found. Receptor crosslinking with radiolabeled cognate ligands failed to detect functional receptors in all but one cell line. In this cell line a receptor complex for BDNF was found that corresponded to truncated trkB receptors that lack the signal transducing tyrosine kinase domain. Neurotrophins did not stimulate mitosis of the glioma cultures. The findings suggest that production of neurotrophins by glioma cells is a general phenomenon, although neurotrophins made by gliomas lacking their receptors may not play an autocrine but rather a paracrine role.