Effect of arylamine acetyltransferase Nat3 gene knockout on N-acetylation in the mouse.

Arylamine N-acetyltransferases (NAT) catalyze the biotransformation of many important arylamine drugs and procarcinogens. NAT can either detoxify or activate procarcinogens, complicating the manner in which these enzymes may participate in enhancing or preventing toxic responses to particular agents. Mice possess three NAT isoenzymes: Nat1, Nat2, and Nat3. Whereas Nat1 and Nat2 can efficiently acetylate many arylamines, few substrates appear to be appreciably metabolized by Nat3. We generated a Nat3 knockout mouse strain and used it along with our double Nat1/2(-/-) knockout strain to further investigate the functional role of Nat3. Nat3(-/-) mice showed normal viability and reproductive capacity. Nat3 expression was very low in wild-type animals and completely undetectable in Nat3(-/-) mice. In contrast, greatly elevated expression of Nat3 transcript was observed in Nat1/2(-/-) mice. We used a transcribed marker polymorphism approach to establish that the increased expression of Nat3 in Nat1/2(-/-) mice is a positional artifact of insertion of the phosphoglycerate kinase-neomycin resistance cassette in place of the Nat1/Nat2 gene region and upstream of the intact Nat3 gene, rather than a biological compensatory mechanism. Despite the increase in Nat3 transcript, the N-acetylation of p-aminosalicylate, sulfamethazine, 2-aminofluorene, and 4-aminobiphenyl was undetectable either in vivo or in vitro in Nat1/2(-/-) animals. In parallel, no difference was observed in the in vivo clearance or in vitro metabolism of any of these substrates between wild-type and Nat3(-/-) mice. Thus, Nat3 is unlikely to play a significant role in the N-acetylation of arylamines either in wild-type mice or in mice lacking Nat1 and Nat2 activities.

In vivo and in vitro metabolism of arylamine procarcinogens in acetyltransferase-deficient mice.

Arylamine N-acetyltransferases (NATs) catalyze the biotransformation of a number of aromatic and heterocyclic amines, many of which are procarcinogenic agents. Interestingly, these enzymes are binary in nature, participating in both detoxification and activation reactions, and thus it is unclear what role NATs actually play in either preventing or enhancing toxic responses. The ultimate direction may be substrate-specific and dependent on its tissue-specific metabolism by competing, but genetically variable, drug-metabolizing enzymes. To investigate the effect of N-acetylation on the metabolism of some classical procarcinogenic arylamines, we have used our double knockout Nat1/2(-/-) mouse model to test both in vitro activity and the in vivo clearance of some of these agents. As expected, N-acetylation activity was undetectable in tissue cytosol preparations from Nat1/2(-/-) mice for 4-aminobiphenyl (ABP) and 2-aminofluorene (AF), whereas significant levels were measured in all wild-type tissue cytosols tested, indicating the widespread metabolism of these agents. Nat1/2(-/-) mice displayed a variable response with respect to in vivo pharmacokinetics. AF appeared to be most severely compromised, with a 3- to 4-fold increased area under the curve (AUC), whereas the clearance of ABP was found to be less dependent on N-acetylation, with no difference in ABP-AUC between wild-type and knockout animals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine was neither N-acetylated nor was its clearance affected by NAT genotype, signifying a dependence on other drug-metabolizing enzymes. The elucidation of the role that N-acetylation plays in the clearance of procarcinogenic agents is the first step in attempting to correlate metabolism by NATs to toxic outcome prevention or augmentation.

Different levels of gamma-synuclein mRNA in the cerebral cortex of dominant, neutral and submissive rats selected in the competition test.

Synucleins are small proteins regulating the filamentous network that in turn influences the release of dopamine and glutamate neurotransmitters involved in mood and motivation processes. We have studied the pattern of synuclein expression in animal models for mania and depression. Dominant behavior, as defined in a food competition test with dyads of rats, can serve as a model of mania and submissive behavior as a model of depression. The expression of alpha-, beta- and gamma-synuclein was analyzed in four regions of cortex from dominant, neutral and submissive rats using TaqMan reverse transcription-polymerase chain reaction technology. The expression levels of gamma-synuclein were elevated consistently in all regions of cerebral cortex of dominant rats (P <0.05; 23.5 +/- 1.1, normalized units) in contrast to the submissive rat group (10.3 +/- 1.2). Neutral rats had intermediate cerebral cortex levels of gamma-synuclein expression (15.7 +/- 1.4) that were significantly lower than that in dominant rats (P <0.05). No changes in alpha- or beta-synuclein expression were observed among the groups. These studies indicate that gamma-synuclein levels in the cerebral cortex were differentially associated with dominant and submissive behavior.

Neuropeptides: brain messengers of many faces.

Neuropeptides 2001, 2nd Joint Meeting of the European Neuropeptide Club and the American Summer Neuropeptide Conference (11th Annual Meeting). 6-11 May 2001 with Satellite Symposium, Israeli-French Symposium, Israel Ministry of Science, Culture and Sport, 6 May 2001, held at Maale Hachmicha and Tel Aviv University, Israel.

VIP receptor antagonists and chemotherapeutic drugs inhibit the growth of breast cancer cells.

The effects of vasoactive intestinal peptide (VIP) antagonists on breast cancer cells were investigated. (N-stearyl, norleucine17)VIP hybrid ((SN)VIPhyb) inhibited specific 125I-VIP binding to MCF7, SKBR3, T47D ZR75-1 and MDA-MB231 cells with high affinity (IC50 values of 0.03-0.06 microM). (SN)VIPhyb, 1 microM, inhibited the ability of 10 nM VIP to cause elevation of cAMP and to increase c-fos mRNA. Micromolar concentrations of (SN)VIPhyb inhibited the proliferation of MDA-MB231 or MCF7 cells using a MTT and clonogenic assay. Using a MTT assay, (SN)VIPhyb enhanced the ability of taxol and doxorubicin to inhibit breast cancer growth. Using nude mice bearing MDA-MB231 xenografts, VIPhyb potentiated the ability of taxol to inhibit proliferation. The results indicate that VIP receptor antagonists increase the ability of chemotherapeutic drugs to kill breast cancer cells.

A lipophilic vasoactive intestinal peptide analog enhances the antiproliferative effect of chemotherapeutic agents on cancer cell lines.

BACKGROUND: Vasoactive intestinal peptide (VIP) is one of several small neuropeptides that affect cancer growth. A lipophilic VIP analog, stearyl-Nle(17)-neuroten-sin(6-11)VIP(7-28) (SNH) that inhibited lung carcinoma growth has been described previously. The experiments performed were clonogenic assays in vitro and tumor xenografts in nude mice in vivo. These studies were now extended to colon carcinoma and to combination therapy with chemotherapeutic agents. METHODS: Assays were performed with cell lines, and tumor proliferation was assessed using the (3-[4,5-dimethylthiazol-2-yl-5]-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium) (MTS) colorimetric assay for mitochondrial function of living cells. RESULTS: The lipophilic analog (SNH) enhanced the antiproliferative activity of diverse chemotherapeutic agents: doxorubicine (antibiotic); vinorelbine (vinca alkaloid, antimicrotubule formation); paclitaxel (antimicrotubule agent); gemcitabine (antimetabolite); irinotecan (topoisomerase I inhibitor); and cisplatin (platinum compound acting as an alkylating agent). In all cases, the antiproliferative effect of SNH and the chemotheraputic agent was at least additive and for some combinations and concentrations even synergistic. For example, 2 microM of the antagonist that produced a 15-20% growth inhibition in the nonsmall cell lung carcinoma cell line reduced the IC(50) by 2-4-fold for most of the chemotherapeutic agents tested. Higher analog concentrations were even more efficacious. Similar results were obtained with colon carcinoma cell lines. CONCLUSIONS: Chemotherapeutic treatment of advanced solid tumors, such as nonsmall cell lung carcinoma, colon carcinoma, or prostate carcinoma, achieves a response rate of between 10% and 30% with significant toxicity. Combination therapy with the lipophilic VIP analog SNH and the preferred chemotherapeutic agent may greatly enhance the response rate, and by permitting a dose reduction, should significantly reduce side effects.

Optimization of cytokine stability in stored amniotic fluid.

OBJECTIVE: Many studies use stored amniotic fluid samples to assay cytokines and other proteins for outcome-based research; however, there is little information on the optimal methods of storage. The objective of our study was to evaluate cytokine stability in amniotic fluid stored at different temperatures both with and without a proteolytic enzyme inhibitor. STUDY DESIGN: Patients undergoing midtrimester genetic amniocentesis for routine indications gave consent for the study. After the sample was centrifuged, the acellular portion of the sample was mixed to homogeneity and aliquoted in 0.5-mL increments and stored for 1 year at 4 degrees C, -20 degrees C, and -80 degrees C with and without the protease inhibitor aprotinin. Enzyme-linked immunoassays for angiogenin, interleukin-6, and vascular endothelial growth factor were performed simultaneously on each aliquot. RESULTS: Thirty samples were assayed for each storage condition. Results were calculated as the percentage of its own sister aliquot stored at -80 degrees C without aprotinin. In all samples, there was a significant relation between storage temperature and cytokine levels, with the lowest levels found at 4 degrees C and the highest at -80 degrees C (angiogenin, P =.004; interleukin-6, P <.001; vascular endothelial growth factor, P =.02). The addition of aprotinin improved stability only for angiogenin at all temperatures (all P <.05). CONCLUSIONS: Degradation of cytokines occurs when amniotic fluid samples are stored for prolonged periods at temperatures greater than -80 degrees C. The addition of a protease inhibitor helps stem the degradation of some cytokines.

IGF-I as a mediator of VIP/activity-dependent neurotrophic factor-stimulated embryonic growth.

IGF-I and the IGF-I receptor are necessary for normal embryonic growth. VIP is an important regulator of early postimplantation growth and acts indirectly through the release of other factors, including activity-dependent neurotrophic factor. The relationship of IGF-I growth regulation to VIP/activity-dependent neurotrophic factor-stimulated growth was examined with whole cultured embryonic d 9.5 mouse embryos. Somite numbers and DNA and protein contents were measured in embryos treated with IGF-I, anti-IGF-I, VIP, activity-dependent neurotrophic factor, and anti-activity-dependent neurotrophic factor-14 (antiserum to an activity-dependent neurotrophic factor agonist). IGF-I mRNA content was measured after incubation with and without VIP for 30 and 60 min using competitive RT-PCR. IGF-I induced a significant, dose-dependent increase in growth as measured by somite number, DNA levels, and protein content. Furthermore, anti-IGF-I inhibited embryonic growth and also prevented exogenous IGF-mediated growth. Both VIP- and activity-dependent neurotrophic factor-stimulated growth were blocked by anti-IGF-I, whereas anti-activity-dependent neurotrophic factor-14 had no detectable effect on IGF-I-induced growth. Treatment with VIP resulted in a 2-fold increase in embryonic IGF-I mRNA. These data suggest that IGF-I is a downstream mediator of VIP and activity-dependent neurotrophic factor in a regulatory pathway coordinating embryonic growth and that VIP may function as a regulator of IGF-I gene expression in the embryo.

Vasoactive intestinal peptide and related molecules induce nitrite accumulation in the extracellular milieu of rat cerebral cortical cultures.

Nanomolar concentrations of vasoactive intestinal peptide (VIP), picomolar concentrations of stearyl-norleucine17-VIP (SNV) and femtomolar concentrations of NAPVSIPQ (NAP), an 8-amino-acid peptide derived from the VIP-responsive activity-dependent neuroprotective protein, provide broad neuroprotection. In rat cerebral cortical cultures, 10(-16)-10(-7) M NAP increased intracellular cyclic guanosine monophosphate (cGMP) (2.5-4-fold) and 10(-10) M NAP increased extracellular nitric oxide (NO) by 60%. In the same culture system, VIP and SNV (at micromolar concentrations) increased extracellular NO by 45-55%. The NAP dose required for cGMP increases correlated with the dose providing neuroprotection. However, the concentrations of NAP, SNV and VIP affecting NO production did not match the neuro-protective doses. Thus, NO may mediate part of the cell-cell interaction and natural maintenance activity of VIP/SNV/NAP, while cGMP may mediate neuroprotection.

Prevention of fetal demise and growth restriction in a mouse model of fetal alcohol syndrome.

Two peptides [NAPVSIPQ (NAP) and SALLRSIPA (ADNF-9)], that are associated with novel glial proteins regulated by vasoactive intestinal peptide, are shown now to provide protective intervention in a model of fetal alcohol syndrome. Fetal demise and growth restrictions were produced after intraperitoneal injection of ethanol to pregnant mice during midgestation (E8). Death and growth abnormalities elicited by alcohol treatment during development are believed to be associated, in part, with severe oxidative damage. NAP and ADNF-9 have been shown to exhibit antioxidative and antiapoptotic actions in vitro. Pretreatment with an equimolar combination of the peptides prevented the alcohol-induced fetal death and growth abnormalities. Pretreatment with NAP alone resulted in a significant decrease in alcohol-associated fetal death; whereas ADNF-9 alone had no detectable effect on fetal survival after alcohol exposure, indicating a pharmacological distinction between the peptides. Biochemical assessment of the fetuses indicated that the combination peptide treatment prevented the alcohol-induced decreases in reduced glutathione. Peptide efficacy was evident with either 30-min pretreatment or with 1-h post-alcohol administration. Bioavailability studies with [(3)H]NAPVSIPQ indicated that 39% of the total radioactivity comigrated with intact peptide in the fetus 60 min after administration. These studies demonstrate that fetal death and growth restriction associated with prenatal alcohol exposure were prevented by combinatorial peptide treatment and suggest that this therapeutic strategy be explored in other models/diseases associated with oxidative stress.

Effectiveness of antidepressant pharmacotherapy: the impact of medication compliance and patient education.

This study was designed to investigate the impact of a time-phased patient education program (RHYTHMS) on medication compliance and treatment outcomes of primary care patients diagnosed with major depression and started on antidepressant pharmacotherapy. Two hundred forty-six depressed patients, diagnosed and treated at one of three outpatient clinics affiliated with the Kaiser-Permanente Northwest Region (KPNW) healthcare system, were randomly assigned to either receive or not receive (usual care) the educational materials by mail. Depression severity and functional impairment affecting patients' quality of life were assessed at baseline and 4, 12, and 30 weeks later. Self-reported impressions of improvement and patient satisfaction with treatment were also assessed at follow-up. Clinical assessment data were obtained using an interactive voice response (IVR) system. Study subjects were compensated $5, $10, $15, and $25 for completing each assessment (Baseline to Week 30, respectively). Upon study completion, prescription fill data of the subjects were extracted from the KPNW Pharmacy System for analysis of medication compliance. Most of the study subjects (63.5%) responded to the pharmacotherapy treatment by study end-point. Few statistically significant differences in either treatment outcomes or duration of medication compliance were found between the treatment groups, and significant differences found were of fairly small magnitude. Patients not receiving the educational materials initially exhibited a more positive response to treatment (Week 4), but this difference did not persist at later follow-ups and was associated with significantly higher relapse rates. A strong time-dose relationship was evident between the duration of the initial treatment episode and treatment outcomes at follow-up, but randomized treatment assignment did not influence the duration of initial medication compliance. Educational programs designed to encourage depressed patients to obtain adequate pharmacotherapy likely provide medical benefits. Such benefits appear to be relatively subtle and methodological differences between studies contribute to inconsistent conclusions concerning observed benefits. The intent of providing time-phased educational materials to patients is to maximize the relevance of such information by synchronizing it with typical recovery processes and issues. This study suggests that additional efforts at engaging patients earlier after the initiation of treatment might be of most benefit.

A vasoactive intestinal peptide antagonist inhibits the growth of glioblastoma cells.

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist (VIPhyb) on human glioblastoma cells were characterized. Pituitary adenylate cyclase activating polypeptide (125I-PACAP-27) bound with high affinity to U87, U118, and U373 cells. Specific 125I-PACAP-27 binding to U87 cells was inhibited, with high affinity, by PACAP but not VIP or VIPhyb (IC50 = 10, 1500, and 500 nM, respectively). By reverse transcriptase-polymerase chain reaction (RT-PCR), a major 305 bp band was observed indicative of PAC1 receptors. PACAP-27 caused cAMP elevation and the increase in cAMP caused by PACAP-27, was inhibited by the VIPhyb. Also, PACAP-27 caused cytosolic Ca2+ elevation in Fura-2AM loaded U87 cells and the VIPhyb inhibited this increase. Using the MTT growth assay, the VIPhyb was shown to inhibit glioblastoma growth in a concentration-dependent manner. Using a clonogenic assay in vitro, 10 microM VIPhyb significantly inhibited proliferation of U87, U118, and U373 cells. In vivo, 0.4 microg/kg VIPhyb inhibited U87 xenograft proliferation in nude mice. These results suggest that the VIPhyb antagonizes PAC1 receptors on glioblastoma cells and inhibits their proliferation.

A peptide derived from activity-dependent neuroprotective protein (ADNP) ameliorates injury response in closed head injury in mice.

Brain injury induces disruption of the blood-brain barrier, edema, and release of autodestructive factors that produce delayed neuronal damage. NAPSVIPQ (NAP), a femtomolar-acting peptide, is shown to be neuroprotective in a mouse model of closed head injury. NAP injection after injury reduced mortality and facilitated neurobehavioral recovery (P < 0.005). Edema was reduced by 70% in the NAP-treated mice (P < 0.01). Furthermore, in vivo magnetic resonance imaging demonstrated significant brain-tissue recovery in the NAP-treated animals. NAP treatment decreased tumor necrosis factor-alpha levels in the injured brain and was shown to protect pheochromocytoma (PC12 cells) against tumor necrosis factor-alpha-induced toxicity. Thus, NAP provides significant amelioration from the complex array of injuries elicited by head trauma.

Cloning and characterization of the human activity-dependent neuroprotective protein.

We have recently cloned the mouse activity-dependent neuroprotective protein (ADNP). Here, we disclose the cloning of human ADNP (hADNP) from a fetal brain cDNA library. Comparative sequence analysis of these two ADNP orthologs indicated 90% identity at the mRNA level. Several single nucleotide polymorphic sites were noticed. The deduced protein structure contained nine zinc fingers, a proline-rich region, a nuclear bipartite localization signal, and a homeobox domain profile, suggesting a transcription factor function. Further comparative analysis identified an ADNP paralog (33% identity and 46% similarity), indicating that these genes belong to a novel protein family with a nine-zinc finger motif followed by a homeobox domain. The hADNP gene structure spans approximately 40 kilobases and includes five exons and four introns with alternative splicing of an untranslated second exon. The hADNP gene was mapped to chromosome 20q12-13.2, a region associated with aggressive tumor growth, frequently amplified in many neoplasias, including breast, bladder, ovarian, pancreatic, and colon cancers. hADNP mRNA is abundantly expressed in distinct normal tissues, and high expression levels were encountered in malignant cells. Down-regulation of ADNP by antisense oligodeoxynucleotides up-regulated the tumor suppressor p53 and reduced the viability of intestinal cancer cells by 90%. Thus, ADNP is implicated in maintaining cell survival, perhaps through modulation of p53.

A novel peptide prevents death in enriched neuronal cultures.

We have recently cloned a novel protein (activity-dependent neuroprotective protein, ADNP) containing an 8-amino-acid, femtomolar-acting peptide, NAPVSIPQ (NAP). Here we show, for the first time, that NAP exerted a protective effect on glia-depleted neurons in culture. The number of surviving neurons was assessed in cerebral cortical cultures derived from newborn rats. In these cultures, a 24-h treatment with the beta-amyloid peptide (the Alzheimer's disease associated toxin) induced a 30-40% reduction in neuronal survival that was prevented by NAP (10(-13)-10(-11) M). Maximal survival was achieved at NAP concentrations of 10(-12) M. In a second set of experiments, a 5-day incubation period, with NAP added once (at the beginning of the incubation period) exhibited maximal protection at 10(-10) M NAP. In a third set of experiments, a 10-min period of glucose deprivation resulted in a 30-40% neuronal death that was prevented by a 24-h incubation with NAP. Glucose deprivation coupled with beta-amyloid treatment did not increase neuronal death, suggesting a common pathway. We thus conclude, that NAP can prevent neurotoxicity associated with direct action of the beta-amyloid peptide on neurons, perhaps through protection against impaired glucose metabolism.

A glia-derived signal regulating neuronal differentiation.

Astrocytes are present in large numbers in the nervous system, are associated with synapses, and propagate ionic signals. Astrocytes influence neuronal physiology by responding to and releasing neurotransmitters, but the mechanisms that establish the close interaction between these cells are not defined. Here we use hippocampal neurons in culture to demonstrate that vasoactive intestinal polypeptide (VIP) promotes neuronal differentiation through activity-dependent neurotrophic factor (ADNF), a protein secreted by VIP-stimulated astroglia. ADNF is produced by glial cells and acts directly on neurons to promote glutamate responses and morphological development. ADNF causes secretion of neurotrophin 3 (NT-3), and both proteins regulate NMDA receptor subunit 2A (NR2A) and NR2B. These data suggest that the VIP-ADNF-NT-3 neuronal-glial pathway regulates glutamate responses from an early stage in the synaptic development of excitatory neurons and may also contribute to the known effects of VIP on learning and behavior in the adult nervous system.

Protective peptides derived from novel glial proteins.

In studying the mediators of VIP neurotrophism in the central nervous system, two glial proteins have been discovered. Both of these proteins contain short peptides that exhibit femtomolar potency in preventing neuronal cell death from a wide variety of neurotoxic substances. Extension of these peptides to models of oxidative stress or neurodegeneration in vivo have indicated significant efficacy in protection. These peptides, both as individual agents and in combination, have promise as possible protective agents in the treatment of human neurodegenerative disease and in pathologies involving oxidative stress.

CREB contributes to the increased neurite outgrowth of sensory neurons induced by vasoactive intestinal polypeptide and activity-dependent neurotrophic factor.

Our recent experiments suggest that vasoactive intestinal polypeptide (VIP) enhances neurite outgrowth of dissociated rat dorsal root ganglion cells, indirectly, via the release of a trophic factor from the spinal cord. In this study, we have examined the possible contribution of activity-dependent neurotrophic factor (ADNF) to the trophic actions of VIP. In addition, as we have shown that the factor mediating the trophic actions of VIP acts via protein kinase A we have also examined the contribution of CREB, which is a transcription factor activated by protein kinase A. As previously shown, supernatant taken from spinal cord incubated with VIP, significantly increased the percentage of sensory neurons with neurites. Antiserum against ADNF attenuated the trophic effect of the VIP-conditioned supernatant. Consistently, the ADNF agonist, ADNF(14) (0.001-0.1 fM), significantly enhanced the percentage of cells with neurite outgrowth. Furthermore, the trophic action of ADNF(14) was attenuated by a protein kinase A inhibitor, Rp-cAMPS, whereas the inactive isomer, Sp-cAMPS, had no effect. Preincubation of cells with 5 mcM CREB antisense oligonucleotides, attenuated the increase in neurite outgrowth induced by either the supernatant or ADNF(14). The sense oligonucleotide had no influence on the enhanced neurite outgrowth. We also found that both the supernatant and ADNF(14) induced an increase in the percentage of cells expressing phosphorylated CREB. The data suggests that VIP induces a release of neurotrophic factors, such as ADNF, which enhance neurite outgrowth. In addition, protein kinase A and CREB appear to contribute to the neurotrophic actions of VIP and ADNF. The mechanisms underlying the neurotrophic action of VIP, may have important implications for sprouting and/or synaptic reorganization of central terminals of sensory neurons, which may contribute to neuropathic pain that commonly occurs following peripheral nerve damage.

A new concept in the pharmacology of neuroprotection.

Vasoactive intestinal peptide (VIP), originally discovered in the intestine as a peptide of 28 amino acids, was later found to be a major brain peptide having neuroprotective activities. To exert neuroprotective activity, VIP requires glial cells secreting neuroprotective proteins. Activity-dependent neurotrophic factor (ADNF) is a recently isolated factor secreted by glial cells under the action of VIP. This protein, isolated by sequential chromatographic methods, was named activity-dependent neurotrophic factor since it protected neurons from death associated with blockade of electrical activity. A fourteen-amino-acid fragment of ADNF (ADNF-14) and the more potent, nine-amino-acid derivative (ADNF-9), exhibit activity that surpasses that of the parent protein with regard to potency and a broader range of effective concentration. Furthermore, the peptides exhibit protective activity in Alzheimer's disease-related systems (e.g., beta-amyloid toxicity and apolipoprotein E deficiencies, genes that have been associated with Alzheimer's disease onset and progression). ADNP is another glial mediator of VIP-associated neuroprotection. NAP, an eight-amino-acid peptide derived from ADNP (sharing structural and functional similarities with ADNF-9), was identified as the most potent neuroprotectant described to-date in an animal model of apolipoprotein E-deficiency (knock-out mice). These femtomolar-acting peptides form a basis for a new concept in pharmacology: femtomolar neuroprotection.

Activity-dependent neurotrophic factor: intranasal administration of femtomolar-acting peptides improve performance in a water maze.

Activity-dependent neurotrophic factor (ADNF) is a glia-derived protein that is neuroprotective at femtomolar concentrations. A nine-amino acid peptide derived from ADNF (Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala; ADNF-9) captured the activity of the parent protein and has been reported to protect cultured neurons from multiple neurotoxins. Antibodies recognizing ADNF-9 produced neuronal apoptosis, and identified an additional, structurally related, glia-derived peptide, Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (NAP). Previous comparative studies have characterized s.c.-injected NAP as most efficacious in protecting against developmental retardation and learning impairments in apolipoprotein E-deficient mice. This study was designed to assess 1) neuroprotection after intranasal administration of ADNF-9 and NAP to rats treated with the cholinotoxin ethylcholine aziridium; and 2) bioavailability and pharmacokinetics after intranasal administration. Results showed significant improvements in short-term spatial memory, as assessed in a water maze, after daily intranasal administration of 1 microg of peptide (ADNF-9 or NAP) per animal. However, a 5-day pretreatment with ADNF-9 did not improve performance measured after cessation of treatment. Compared with rats treated with ADNF-9, NAP-pretreated animals exhibited a significantly better performance. Furthermore, NAP (and not ADNF-9) protected against loss of choline acetyl transferase activity. Significant amounts of (3)H-labeled NAP reached the brain, remained intact 30 min after administration, and dissipated 60 min after administration. This study revealed efficacy for ADNF-related peptides in rodent models for neurodegeneration. The small size of the molecules, the low dosage required, the noninvasive administration route, and the demonstrated activity in a relevant paradigm suggest NAP as a lead compound for future drug design.