WO2010033762A1 - Colivelin as a neuroprotective factor - Google Patents

Abstract

A method of treatment or prevention of alcohol induced neuronal toxicity is disclosed. The method comprises the step administering the synthetic peptide, Colivelin, in an amount effective to mitigate the toxic effects of prenatal alcohol exposure to neurons in a developing brain.

Description

COLIVELIN AS A NEUROPROTECTIVE FACTOR

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U. S. C. § 119(e) of U.S. provisional patent application serial no. 61/098,013, filed September 18, 2008, the entirety of the disclosure of which is incorporated herein by reference.

The present disclosure pertains to the fields of neurobiology and toxicology. More particularly, the present disclosure pertains to methods for use of Colivelin as a neuroprotective factor. Fetal alcohol exposure induces cell death. Alcohol exposure impedes cerebral and cerebellum growth (Bauer-Moffett and Altman, Exp. Neurol, 48:378-382, 1975; Brain Res., 119:249-268, 1977; Kornguth et al, Brain Res., 177:347-360, 1979; Samson and Diaz, Alcohol Clin. Exp. Res., 5:563-569, 1981; Sulik et al, Science, 214:936-938, 1981; Barren et al, Neurotoxical Teratol, 10:333-339, 1988; Bonthius and West, Alcohol Clin. Exp. Res., 14:107- 118, 1990) and causes morphological defects in fetal brain at different developmental stages (Sari and Gozes, Brain Res. Brain Res. Rev., 52:107-118, 2006). These toxic effects of alcohol may be mediated through an apoptotic mechanism (Ikonomidou et al., Science, 287:1056-1060, 2000).

Although studies have demonstrated that prenatal alcohol exposure (PAE) induces apoptosis, little is known about the signaling pathways involved in this mechanism.

Alcohol exposure may accelerate apoptosis in the developing brain through direct activation of intrinsic mitochondrial apoptotic signaling pathways or indirectly through extrinsic pathways such receptors systems (Cheema et al., Alcohol Clin. Exp. Res., 24:535-543, 2000; de Ia Monte et al., Alcohol Clin. Exp. Res., 24:716-726, 2000). The extrinsic signaling pathways may involve c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase ERK1/2 (Ashkenazi and Dixit, Science, 281:1305-1308, 1998). The intrinsic signaling pathways involve Bax protein that may be translocated from the cytosol to the outer mitochondrial membrane. This process induces an increase in the permeability of mitochondrial membrane and release of cytochrome c which binds to apoptotic protease activating factor- 1 (Apafl) and leads to activation of caspase-9 that cleaves caspase-3 (Green and Reed, Reprod. Fertil. Dev., 18:517-524, 2006). At the mitochondrial outer membrane, survival/apoptosis-related proteins Bcl-2, Bcl-xl, Bax, Bcl-xs, and Bad may regulate apoptosis during fetal brain development. Studies have focused on the identification of antagonists to block the toxic effects of alcohol. In the experiments described herein, a peptide, Colivelin, was tested for use as a neuroprotectant against the insult of fetal alcohol exposure in a mouse model of PAE. It was discovered that Colivelin protected neurons against the insult of alcohol exposure and prevented alcohol-induced apoptosis in several fetal brain regions.

Prenatal alcohol exposure induces mitochondrial dysfunction including decreased mitochondrial glutathione concentration, decreased activities of respiratory chain complex IV and ATP synthase, and increased mitochondrial permeability transition (Ramachandran et al, Alcohol Clin. Exp. Res., 2001; Spong et al, J. Pharmacol. Exp. Ther., 297:774-779, 2001; Xu et al, Birth Defects Res. A Clin. MoI. Teratol, 73:83-91, 2005; Green et al., Reprod. Fertil. Dev., 18:517-524, 2006). Investigation of the actions of PAE on signaling pathways that involve extrinsic and intrinsic mitochondrial factors may provide important information for the identification of possible mechanisms of neuroprotection and allow for the development of intervention procedures. The peptide, SALLRSIPA (SEQ ID NO: 1), known as SAL or ADNF-9, is derived from activity-dependent neurotrophic factor (ADNF) (Brenneman and Gozes, J. Clin. Invest., 97:2299-2307, 1996; Brenneman et al, J. Pharmacol. Exp. Ther., 285:619-627, 1998). Another peptide, NAPVSIPQ (SEQ ID NO: 2), known as NAP, is derived from activity- dependent neuroprotective protein (ADNP) (Bassan et al, J. Neurochem., 72:1283-1293, 1999; Zamostiano et al, J. Biol. Chem., 276:708-714, 2001). These peptides may protect against oxidative stress associated with alcohol exposure (Brain Res., 854:257-262, 2000; Steingart et al; J. MoI Neuroscl, 15:137-145, 2000).

A synthetic hybrid peptide named Colivelin composed of ADNF-9 (SALLRSIPA) and humanin (AGA-(C8R)HNG17 (PAGASRLLLTGEIDLP)) (SEQ ID NO: 3), a potent humanin derivative, has been found to prevent cell death by various familial Alzheimer's disease-causative genes and β-amyloid peptide (Chiba et al, J. Neuroscl, 25:10252-10261, 2005). In the experiments described herein, the ability of Colivelin to protect neurons against the toxic effects of prenatal alcohol exposure is demonstrated.

Figure 1 shows the protective effect of Colivelin (CLN) against alcohol (ALC) exposure in primary cortical neurons (PCNs). PCNs were treated with or without CLN together with or without alcohol. Relative viability measured with WST-8 is shown as means ± SD (n=3). * p<0.05. Figure 2 shows the effect of protein kinase inhibitors on ALC-mediated toxicity (A and B). Fl 1 neurohybrid cells were pre-treated with various inhibitors (vehicle, 0.5% dmso; PD98059 for MEK, Wortmannin for PI3-kinase, AG490 for JAK2, SP600125 for JNK, Nimesulide for COX-2, and Ro-20-1724 for PDE4) and then with ALC (1000 or 1500 mg/dl as indicated). Relative viability measured by WST- 8 is shown as means ± SD (n=6). (C) PCNs were also pre-treated with protein kinase inhibitors to examine the effect on ALC toxicity. n=3. * p<0.05, ** p< 0.01.

Figure 3 shows immunoblot analysis of p-ERKl/2 (A) and p-JNK (B). PCNs were treated with or without ALC at the dose of 1000 mg/dl for the indicated duration (h). Lower panel; densitometric analysis of signal for p-JNK levels.

Figure 4 shows the protective effect of Colivelin involving the MAP kinase pathways in PCNs treated with or without ALC at the dose of 1500 mg/dl for 24 or 48 h using the Meso Scale Discovery^® assay. Phospho-p38 (a), p-JNK (b), and p-ERKl/2 (c) are shown as means ± sem. Phospho-protein levels were standardized with the levels in the control PCNs with water treatment. * p<0.05, ** p< 0.01

Figure 5 shows the protective effect of Colivelin on fetal brain weight in Chow, Pair-fed, ALC, and ALC plus Colivelin prenatally treated groups of mice, including a Pair-fed plus Colivelin group run subsequently for comparison. One-way ANOVA demonstrated a significant difference between groups (p=0.0001). Prenatal alcohol exposure induced significant fetal brain reduction as compared to the Chow, PF, and PF/CLN control groups

(p<0.01, p<0.001, and p<0.001 respectively). CLN treatment prevented the reduction in fetal brain weight found in the ALC group (p<0.05). No significant differences were found between the Chow, PF, PF/CLN, and ALC/CLN groups. Values are shown as mean ± SEM. (Chow, n=5; PF, n=9; PF/CLN, n=5; ALC, n=8; ALC/CLN, n=6). * p<0.01, ** p<0.001 (Newman- Keul's post hoc test).

Figure 6 depicts the protective effect of Colivelin against the alcohol-induced increase in the number of TUNEL-positive cells in primordium frontal cortex (a, b, c) and quantification of TUNEL positive cells (d) in E13 fetal brains. Prenatal alcohol exposure increased the number of TUNEL-positive cells in primordium frontal cortex (a, b, c) of E13 fetal brains. Administration of Colivelin (CLN) along with alcohol exposure prevented alcohol-induced increases in TUNEL-positive cells. Arrowheads indicate cell undergoing apoptosis as indicated by cell processes, and arrows indicate the final stage of cell death. Scale bars: 100 μm. One-way ANOVA demonstrated a significant difference between groups (p=0.0032). An increase in TUNEL-positive cells was found in the ALC group as compared to the PF group (p<0.01) (D). CLN administration along with the alcohol exposure prevented a significant alcohol-induced increase in TUNEL-positive cells (p<0.01) (D). Values are shown as means + SEM. (PF, n=5; ALC, n=6; ALC/CLN, n=5). * p<0.01 (Newman-Keul's /rø^ hoc test).

Figure 7 shows caspase-3 activation in E13 fetal brains in Chow, Pair-fed, ALC, ALC plus Colivelin prenatally treated groups of mice, including a Pair-fed plus Colivelin group run subsequently for comparison. The neuroprotective effect of Colivelin (CLN) against the insult of prenatal alcohol exposure is likely mediated through caspase-3 activation as tested by caspase-3 colorimetric assay in E13 fetal brains. One-way ANOVA demonstrated a significant difference between groups (p=0.0039). Prenatal alcohol exposure induced a significant increase in the concentrations of active caspase-3 as compared to the Chow and PF control groups (p<0.05 and p<0.01, respectively). CLN administration prevented significantly the effect of alcohol-induced increases in the concentrations of active caspase-3 (p<0.05). There were no significant differences between the control and CLN treatment groups. Values are shown as mean + SEM. (Chow, n=5; PF, n=9; ALC, n=8; ALC/CLN, n=6). * p<0.05, ** p<0.01 (Newman-Keul's post hoc test).

Figure 8 shows cytosolic (a) and mitochondrial (b) cytochrome c measured by ELISA in El 3 fetal brains in Chow, Pair-fed, ALC, and ALC plus Colivelin prenatally treated groups of mice. The neuroprotective effect of Colivelin (CLN) against the insult of prenatal alcohol exposure is mediated through cytosolic cytochrome c as tested by ELISA in E13 fetal brains. The data generated with ELISA demonstrated a significant difference in the concentration of cytosolic and mitochondrial cytochrome c between groups (p<0.05) as shown by one-way ANOVA. (A) Prenatal alcohol exposure induced significant increase in the concentrations of cytosolic cytochrome c as compared to the Chow (p<0.01) and PF (p<0.01) control groups. CLN administration prevented significantly the increases in the concentrations of cytosolic cytochrome c as compared to the ALC group (p<0.01). (B) Prenatal alcohol exposure induced significant decrease in the concentration of mitochondrial cytochrome c as compared to the Chow and PF control groups (p<0.01 and p<0.001, respectively). CLN administration prevented the reduction in mitochondrial cytochrome c as compared to ALC

(p<0.01). There were no significant differences between the control and CLN treatment groups in the level of mitochondrial and cytosolic cytochrome c. Values are shown as mean ± SEM. (Chow, n=5; PF, n=5; ALC, n=6; ALC/CLN, n=6). * p<0.01, ** p<0.001 (Newman- Keul's post hoc test).

Figure 9 shows the protective effect of Colivelin involving MAP kinase pathways in the FAS/FAE model using the Meso Scale Discovery^® assay. Phospho-p38, p- JNK, and p-ERKl/2 levels (a) and p-/t-BAD levels (b) are shown as means ± SEM. Phospho- p38, p-JNK, and p-ERKl/2 levels (A) and p-/t-BAD levels (B) are shown as means ± sem. Phospho-protein levels were standardized with the levels in fetal brains from PF mice. (A) Prenatal alcohol exposure induced significant reduction in the phosphorylated form of BAD protein as compared to PF control group (p=0.0277) as indicated by the ratio of phosphorylated-BAD/total BAD protein (p-/t-BAD ratio). Colivelin prevented alcohol-induced down-regulation of p-/t-BAD ratio as compared to ALC group (p=0.0161). (B) Significant reduction in the phosphorylated form JNK protein was found in the ALC/Colivelin group as compared to PF group (p=0.0042 and 0.042). Moreover, significant reduction in phosphorylated form ERK1/2 was found in the ALC/Colivelin group as compared to the PF group (p=0.042).

Figure 10 shows the comparative effects of Colivelin (IpM, 10OpM, and 1OnM), C8A-humnanin, and ADNF-9 against alcohol exposure on cell viability in primary cortical neurons (PCNs). PCNs were treated with or without the indicated concentrations of peptides along with ALC. Cell index, which reflects cell viability, measured by xCELLigence system, is shown as mean ± SD (n=6). Cell index of PCNs without alcohol exposure is equal to 1.0 [the baseline]. Control (water), bottom trace, 6^th from top; 1 pM CLN, 4^th trace from top; 100 pM CLN, top trace, 1^st; 10 nM CLN, 2^nd trace from top; 10 nM C8A-humanin (HNA), 3^rd trace from top; and 100 pM ADNF-9, 5^th trace from top. The one-way ANOVA for the 8 hours revealed a significant difference between groups (F_(5j35)=7.940 [p<0.0001]). * p<0.05, ** p<0.01, *** p<0.001, n.s. means no significant difference (Dunnett's post hoc test).

Figure 11 shows in vivo optical imaging of Colivelin (CLN) labeled with Alexa680 (CLN- Alexa680). Pregnant female mice (E14) were i.p. administered with saline or saline containing 7 nmol of CLN-Alexa680. Thirty minutes after the injection, mice were optically imaged with Optix MX2 (A). The uterus was then dissected from the mice and imaged (B). The embryos were further dissected from the uterus and imaged (C). H, head; T, tail.

While the invention is susceptible to various modifications and alternative forms, specific embodiments will herein be described in detail. It should be understood, however, that there is no intent to limit the invention to the particular forms described, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

In accordance with one illustrative embodiment of the invention, a method of using Colivelin, or a pharmaceutical salt thereof, to inhibit cellular toxicity is disclosed.

In accordance with another illustrative embodiment of the invention, a method of using Colivelin, or a pharmaceutical salt thereof, to treat or prevent the teratogenic effects of alcohol on neurons is disclosed.

In accordance with another illustrative embodiment of the invention, a method of using Colivelin, or a pharmaceutical salt thereof, to treat or prevent a neurodevelopmental disorder is disclosed.

In accordance with another illustrative embodiment of the invention, a method of using Colivelin, or a pharmaceutical salt thereof, to treat or prevent fetal alcohol syndrome (FAS) or fetal alcohol effects (FAE) is disclosed. Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. See, e.g., P. Stahl, et al, HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE, (VCHA/Wiley-VCH, 2002); S.M. Berge et al, "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977.

Compounds employed in the invention are preferably formulated as pharmaceutical compositions administered by a variety of routes. Such pharmaceutical compositions and processes for preparing them are well known in the art. See, e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (A. Gennaro et al, eds., 19^th ed., Mack Publishing Co., 1995).

Any suitable route of administration may be employed for providing a human with an effective dosage. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms may include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, suppositories and the like.

The dose administered will depend upon perhaps many factors, such as the age and weight of the patient, and the route of administration. The effective amount of an individual compound is determined, in the final analysis, by the physician in charge of the case, but depends on factors such as the severity of the disease and other diseases or conditions from which the patient suffers, the chosen route of administration other drugs and treatments which the patient may concomitantly require, and other factors in the physician's judgment. In general, the daily dose lies within the range of from about 0.001 mg to about 1000 mg per kg body weight of a human, preferably 0.01 mg to about 500 mg per kg, and more preferably 0.1 to 200 mg per kg, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases. Further, the dosing period may be acute or chronic, and may be one day, a week, two weeks, a month, several months, or ongoing.

As used herein, the phrase "therapeutically effective amount of Colivelin" is understood to encompass an amount of Colivelin sufficient to obtain the goals of the invention, including inhibiting cellular toxicity or ameliorating at least one symptom associated with neurodevelopmental disorders such as fetal alcohol syndrome or fetal alcohol effects. Symptoms of fetal alcohol syndrome, or fetal alcohol effects, are understood to include cell loss, poor body growth in the womb and after birth, decreased muscle tone and poor coordination, deficient, impaired, or delayed brain development, impaired vision, impaired cognition, heart defects such as ventricular septal defect (VSD) or atrial septal defect (ASD), as well as physical deformities associated with abnormal development such as structural problems with the head and face, including narrow, small eyes with large epicanthal folds, small head, small upper jaw, smooth groove in upper lip, smooth and thin upper lip.

As used herein, the term "developmental cellular toxicity" is understood to encompass a decrease in cell number, viability, or function.

As used herein, the term "inhibiting" is understood to encompass preventing, blocking, stopping, or slowing the progression in any manner, including partially or completely reversing.

EXAMPLE 1 In vitro studies Peptides, antibodies, and materials

Colivelin (SALLRSIPAPAGASRLLLLTGEIDLP) (SEQ ID NO: 4) was synthesized as described previously (Chiba et al, J. Neurosci., 25:10252-10261, 2005). Rabbit antibodies against phospho-STAT3 (Tyr⁷⁰⁵), phospho-ERKl/2 (Thr²⁰²/Tyr²⁰⁴), and total STAT3 (79D7) were from Cell Signaling Technology. Rabbit polyclonal antibodies against total- ERKl were from Santa Cruz Biotechnology. PD98059, Wortmannin, SP600125, AG490, Ro- 20-1724 were from Calbiochem. Nimesulide was from Sigma- Aldrich. Other reagents described herein are commercially available. CeIl culture and phosphorylation assays

FI l neurohybrid cells are hybrids of mouse neuroblastoma N18TG2 and primary rat dorsal root ganglion cells. Fl 1 cells were grown in Ham's F12 medium containing 18% FBS. For one set of cell viability assays, Fl 1 cells (cultured at 3.0 x 10³ per well in a 96- well plate) were incubated with the indicated concentrations of chemical inhibitors (5 μM

PD98059 for MEK, 10 nM Wortmannin for PI3-kinase, 10 nM SP600125 for c-Jun N-terminal kinase (JNK), 1 μM AG490 for JAK2, Nimesulide for cyclooxygenase 2 (COX-2), Ro-20-1724 for phosphodiesterase 4 (PDE4) containing 0.5% DMSO) for 30 min. Cells were treated with alcohol (ALC, 1000 or 1500 mg/dl) for 24 h and cell viability was measured by the WST-8 assay. The WST-8 assay was performed with 2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5- (2,4- disulfophenyl)-2H- tetrazolium, monosodium salt, using Cell Counting kit-8 (Wako Pure Chemicals Industries), as previously described (Chiba et al., J. Neurosci. Res., 78:542-552, 2004).

In another set of cell viability assays, primary cortical neurons (PCNs) (5.0 x 10⁴ cells per well in poly-L-lysine-coated 96-well plates) were prepared from E14 mouse embryos as described (Chiba et al., J. Neurosci., 25:10252-10261, 2005). At 3 days in vitro (DIV3), PCNs were similarly pre-treated with CLN peptide (100 pM) or kinase inhibitors (5 μM PD98059 for MEK, 10 nM Wortmannin for PI3-kinase, 1 μM AG490 for JAK2, 10 nM SP600125 for JNK; all inhibitors contain 0.5% DMSO) for 30 minutes and then further cultured for 72 h with or without alcohol (ALC, 1000 mg/dl). Cultured media containing inhibitors and ethanol were refreshed on DIV6 and DIV7. Cell viability was measured with the WST-8 assay.

In addition, PCN cell viability was monitored using the xCELLigence system (Roche Applied Science and ACEA Biosciences, Inc.). Use of the xCELLigence system allowed for monitoring of cellular events in real time without the need to incorporate labels. The system measures the electrical impedance across interdigitated microelectrodes integrated on the bottom of tissue culture E- Plates (96 well). The impedance measurement provides quantitative information about the biological status of the cells, including cell number, viability, and morphology. Using the xCELLLigence system, the "Cell Index", derived from the measured impedances and reflective of cell viability, PCN cell viability was continuously monitored under ethanol exposure together with peptide treatment (CLN at 1 pM, 100 pM, or 10 nM; HNA at 10 nM; ADNF-9 at 100 pM, n= 6). Electrochemiluminescence assay

To detect the intracellular phosphorylation signaling pathways, the Meso Scale Discovery^® (MSD) electrochemiluminescence assay (MAPK Panel pERKl/2 Vl, pJNK, pp38 multiplex assay (MULTI-SPOT 4, 96-well plate; Meso Scale Discovery^® and MSD Phospho (Serl 12)/Total BAD assay (Duplex assay)) was performed using a MSD Sector Imager 2400 according to the manufacturer's protocol. The MSD assay is based on the sandwich immunoassay utilizing electrochemiluminescence (ECL) to measure protein levels. Mouse fetal brain samples from ALC treated, pair-fed (PF), or ALC plus CLN treated pregnant mice were first homogenized in the Tris lysis buffer containing protease inhibitors and phosphatase inhibitors (included in the kit) and then were centrifuged at 15,000 rpm at 4⁰C for 15 min to obtain the soluble fraction. BCA assays were performed to determine the protein concentrations in the lysates. After blocking the wells for I h, 100 μg-protein lysates in 25 μl- lysis buffer were added to the multi- spotted ELISA plates. After incubation at 4 ⁰C overnight, the plates were washed four times with 150 μl of the provided wash buffer. Then, 25 μl of the appropriate SULFO-TAG-labeled detection antibody solution was added to the wells and the plates were incubated at room temperature for 2 h. The SULFO-TAG (Ruthenium (II) trisbiphyridine iV-hydroxysuccinimide ester) emits light following electrical stimulation of the plates when in close proximity to the bottom of the well. The plates were again washed four times with the wash buffer and then 150 μl of the provided read buffer T with surfactant was added to the wells, avoiding the introduction of any bubbles. The plates were analyzed with the SECTOR Imager 2400, in which a voltage was applied to the plate electrodes to cause the SULFO-TAG bound to the electrode surface to emit light. BAD phosphorylation levels (% Phosphoprotein) were calculated using the following equation: (2*(Phospho signal))/(Phospho signal + Total signal) *100. Immunoblot analysis

For immunoblot (IB) analysis, PCNs (at 2 x 10⁶ per well in a poly-L-lysine- coated 6- well plate on DIV4) were incubated with or without 1000 mg/dl ALC for the indicated time. Cells were harvested in a lysis buffer (50 mM Tris HCl (pH7.4), 150 mM NaCl, 1% Triton-X 100, protease inhibitors, 1 mM EDTA, phosphatase inhibitor cocktails 1 and 2 (Sigma)). Samples (35 μg/lane) were subjected to normal SDS-PAGE and were then blotted onto PVDF membranes. The membranes were soaked with appropriate primary antibodies (p- ERK1/2, p-JNK, at 1:1000) and then with HRP-labeled secondary antibodies (BioRad Laboratories). Immunoreactive bands were detected with ECL Western Blotting Detection Reagents (Amersham Bioscience). Total protein levels were detected on the identical membranes using anti-total protein antibodies (t-ERKl/2, t-JNK, at 1:3000) after stripping the anti-phospho-protein antibodies. Statistical Analyses All values in the figures are shown as means ± SEM (or means ± SD as indicated for Figs 1, 2 & 10). Statistical analysis was performed with either one-way analysis of variance (ANOVA) or two-way ANOVA in which the/? value was set at 0.05 as indicated in the figure legends. Newman-Keul's or Dunnett' s post hoc tests were used for multiple comparisons between individual groups. In vitro data analyses

Colivelin protected primary cortical neurons from alcohol-induced neurotoxicity

Colivelin (CLN) is a hybrid neuroprotective peptide consisting of a humanin derivative and ADNF-9, and has been reported to suppress neuronal death related to Alzheimer's disease (AD) by activating STAT3 (signal transducer and activator of transcription 3) and Ca²⁺/calmodulin-dependent protein kinase 4 (CaMKIV) (Chiba et ah, J. Neurosci.,

25:10252-10261, 2005). In the experiments described herein, primary cortical neurons (PCNs) were treated with/without 1500 mg/dl ALC together with/without 100 pM CLN. ALC treatment for 72 h significantly decreased the viability of PCNs as shown in Figure 1. Simultaneous treatment with CLN recovered the viability of ALC-treated PCNs to normal levels. In this case, CLN treatment itself did not increase the viability of PCNs in the absence of ALC treatment.

Real time measurement of PCN cell viability under alcohol exposure was also performed using the xCELLigence system, which detects changes in electric impedance caused by the attachment of cells to culture plates integrated with interdigitated micro-electrodes. Alcohol exposure caused a rapid decrease in Cell Index, which reflects impedance and cell viability (Fig. 10). CLN dose-dependently suppressed a decrease in Cell Index induced by alcohol exposure, indicating that CLN inhibited alcohol-induced neural cell toxicity. In contrast, both C8A-HN at 1OnM and ADNF-9 at lOOpM (considered active concentrations) only showed marginal effects on alcohol-induced toxicity in PCNs (Fig. 10). Pharmacological characterization of ALC neurotoxicity

The effects of various protein kinase inhibitors on the ALC-induced decrease in cell viability were examined to characterize the cellular toxicity induced by ALC. ALC treatment for 24 h at the concentration of 1000 mg/dl did not decrease the viability of Fl 1 neurohybrid cells (Fig. 2a). Pretreatment with a MEK inhibitor, PD98059, which antagonizes the MEK1/2-ERK1/2 signaling pathway, for 30 min together with ALC treatment at 1000 mg/dl, decreased the viability of both FI l cells. These data indicate that the MEK-ERK pathway may be a neuroprotective signaling pathway against neurotoxicity induced by ALC. Pretreatment with inhibitors for the PI3-kinase/Akt pathway (Wortmannin) and the

JAK2/STAT3 pathway (AG490) moderately increased the neurotoxicity induced by ALC treatment on Fl 1 cells.

ALC treatment for 24 h at the concentration of 1500 mg/dl significantly decreased the viability of Fl 1 cells as shown in Figure 2b. Pretreatment with a c-Jun N- terminal kinase (JNK) inhibitor, SP600125, antagonized the ALC neurotoxicity and recovered the viability of Fl 1 cells to the control levels. Pretreatment with either a COX-2 inhibitor, Nimesulide, or a phosphodiesterase 4 (PDE4) inhibitor, Ro-20-1724, however, did not result in recovery of cell viability. This indicates that the activation of JNK may be involved in the neurotoxicity induced by ALC. The effects of the inhibitors were also examined on PCNs. Alcohol treatment for 72 h at a concentration of 1000 mg/dl significantly decreased the viability of PCNs (Fig. 2c). Treatment with a MEK inhibitor, PD98059, significantly augmented the neurotoxicity induced by ALC in PCNs (Fig. 2c). Wortmannin and AG490 did not increase the loss in viability of PCNs treated with ALC. Pretreatment with SP600125 recovered the viability of PCNs treated with ALC to the control levels as it did in Fl 1 cells. These data indicate that the MEK-ERK pathway may be a neuroprotective signaling pathway against ALC neurotoxicity and that the JNK pathway may transduce the neurotoxic signals induced by ALC. Time-course analysis of phosphorylation levels of the MAP kinase pathways in PCNs treated with alcohol and role of Colivelin in neuroprotection A time-course analysis was performed of phosphorylation levels of the MAP kinase pathways in PCNs treated with ALC at the concentration of 1000 mg/dl. Phospho- (p-) ERK1/2 levels in PCNs were acutely decreased by ALC treatment at 1-2 h as shown in Figure 3a. On the contrary, ALC treatment for more than 24 h substantially increased p-ERKl/2 levels as compared to control PCNs treated with water. Total- (t-) ERK1/2 levels did not change substantially by the treatments. ALC treatment did not affect p-JNK levels acutely (1-2 h) (Fig. 3b). After chronic treatment (24-48 h or more), a significant increase in p-JNK levels was observed (Fig. 3, lower panel, densitometric analysis). It was next examined if the MAP kinase pathways are involved in the CLN- mediated neuroprotection against ALC treatment. Phosphorylation levels of the MAP kinase pathways were quantified in PCNs treated with/without ALC for 24 or 48 h together with/without 100 pM CLN using the MSD multi-spot electrochemiluminescence assay. There were no significant changes in phospho-p38 MAP kinase levels (Fig. 4a). At 24 h, p-JNK levels were significantly increased by CLN in PCNs treated with water, while CLN moderately decreased p-JNK levels in PCNs treated with ALC at 48 h (Fig. 4b). CLN also induced significant upregulation of p-ERKl/2 levels in PCNs treated with water at 24 h (Fig. 4c). ALC treatment significantly increased p-ERKl/2 levels at 48 h in accordance with the immunoblot analysis (Fig. 3a). CLN suppressed the increase in p-ERKl/2 levels at 48 h caused by ALC treatment to the control levels. CLN treatment may protect PCNs from ALC neurotoxicity by modulating the MAP kinase pathways.

EXAMPLE 2 In vivo studies Animals

Mice (C57BL/6) were used in these studies. Both male and female mice were obtained at 6-7 weeks of age from Harlan Laboratories at Indianapolis, Indiana. All mice were housed in the departmental animal colony in a vivarium with a controlled climate (temperature 22⁰C, 30% humidity) with a 12 h light/dark cycle with lights on at 07:00. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Indiana University Bloomington and are in accordance with the guidelines of the Institutional Animal Care and Use Committee of the NIH, and the Guide for the Care and Use of Laboratory Animals. Breeding and treatment procedure

Female mice were placed into male home cages for 2 h, and the females were then checked for a sperm plug by vaginal smear immediately afterwards. If the plugs were positive, this time point was designated as embryonic day 0 (EO). Weight-matched pregnant females were assigned to the following groups: (1) Ethanol liquid diet group (ALC, n = 8) fed with chocolate SUSTACAL (supplemented with vitamins and minerals) liquid diet 25%

(4.49%, v/v) ethanol derived calories (EDC), (2) Pair-fed control group (PF, pair-fed to ethanol- fed group, n = 9) fed with a maltose-dextrin solution which was made isocaloric to the dose of ethanol used, (3) Chow group (Chow, 5) fed with mouse chow pellets and water, (4) treatment group with alcohol along with injections of Colivelin (ALC/CLN, 20 μg/ 20 g, n = 6). The PF and Chow served as control groups. Subsequently, a fifth treatment group, Pair-fed along with Colivelin (PF/CLN, 20 μg/20 g, n=5) was added for comparison with the previously run treatment groups. The fortified liquid diet contained 237 ml of chocolate-flavored SUSTACAL

(Mead Johnson), 1.44 g Vitamin Diet Fortification Mixture and 1.2 g Salt Mixture XIV. For the ethanol diet, 15.3 ml (4.49% v/v; 25% EDC) of 95% ethanol was added to the fortified SUSTACAL formula with water that was added to make 320 ml of diet with 1 cal/ml (ethanol). For the isocaloric control diet, 20.2 Maltose Dextrin was added to the fortified SUSTACAL formula with water that was added to bring it to 1 cal/ml (Middaugh et al, Neurotoxicol. Teratol, 10:175-180, 1988; Middaugh and Boggan, Alcohol Clin. Exp. Res., 19:1351-1358, 1995). One day before treatment, the ALC, PF, PF/CLN and ALC/CLN dams were adapted to the liquid diet. Between 9:00 and 10:00, the dams were weighed, the volume of liquid diet consumed during the previous 24 h was recorded from 30-ml graduated screw-cap tubes, and freshly prepared diet was provided. The PF subjects had unlimited access to the EDC liquid diet each day to match the drinking of ALC or ALC/CLN subjects. Maternal Blood Alcohol Levels

Maternal blood alcohol levels were tested in a separate group of C57B1/6 dams with the 25% (4.49%, v/v) ethanol derived calorie (EDC) diet as described previously (Sari and Zhou, Alcohol CHn. Exp. Res., 28:941-948, 2004). Briefly, pregnant mice were given the same feeding protocol as the other experimental dams (EDC diet provided on E7 at 700 h), and two 50-μl tail blood samples were obtained (at 900 and 1100 h in a reverse dark cycle) on E8 and El 1. The blood samples were collected in heparinized capillary tubes and centrifuged, and 5-μl plasma samples were analyzed for alcohol concentration using the ANALOX Alcohol Analyzer, calibrated with a 100 mg/dl ethanol standard. Blood alcohol concentrations (BACs) were evaluated at 2 and 4 h on E8 and El l. The BACs were consistently higher at 2 h exposure. The average peaks obtained in the 25% EDC group at 2 h were about 40 mg/dl on E8 and about 55 mg/dl on El 1. Fetal brains At E13, pregnant mice were euthanized by CO₂ followed by cervical dislocation and the fetuses were removed. The fetal brains were further dissected from the base of the primordium olfactory bulb to the base of the metencephalon. From the same litter, one group of fetal brains was frozen and stored at -70 ⁰C until used for chemical assays and another group of fetal brains was fixed in 4% paraformaldehyde for TUNEL-positive cell detection and fetal brain weight analysis. In vivo optical imaging

A pregnant female CD-I mouse (E14) administered i.p. with ALEXA FLUOR 680 (Invitrogen, Carlsbad, CA)-labeled CLN (CLN- Alexa680), was imaged with an Optix

MX2 system (ART, Inc., Montreal, Canada), allowing for real time-scanning of fluorophores in vivo. To avoid strong auto-fluorescence from the hair, the abdominal side was shaved before imaging. A 679-nm pulse laser was used for excitation and a 700-nm long-pass filter on the emission side. Regions of interest (ROI) were manually set for each mouse, and each ROI was scanned with the resolution of a 1.5-mm grid. The laser setting (power of the laser and integration time) for each ROI was automatically optimized by the Optix system and collected data were analyzed by OPTIVIEW software (Ver.2.0.1, ART, Inc.). The experiment was repeated and the same results were obtained. Measurement of Caspase-3 concentration For tissue extracts, frozen forebrain from E13 Chow, PF, ALC, ALC/CLN, and

PF/CLN were ground to a powder with a pestle. The powdered tissue was mixed with TNE buffer (10 mM Tris, pH 7.4; 0.15 M NaCl; 1 mM ethylenediaminetetraacetic acid) supplemented with protease inhibitor cocktail (Sigma) under continuous grinding until the suspension was homogeneous. The suspension was centrifuged at 14,000 rpm for 10 min at 4 ⁰C. The supernatant was collected and frozen at -80 ⁰C until a later date for detection of protein or active caspase-3 concentration. The total protein estimation in each sample was evaluated with the Bio-Rad protein assay.

The concentration of active caspase-3 was determined by using a caspase-3 colorimetric assay kit (Assay Designs, Inc.). The kit involves the conversion of a specific chromogenic substrate for caspase-3 followed by the colorimetric detection of the colored product of a reaction that absorbs visible light at 405 nm. The samples in variant dilutions, standards, p-nitroaniline calibrator (pNA), and blank controls were plated in duplicate in 96 microplates. The blank control was a mixture of active caspase-3 reaction buffer and caspase-3 substrate. The conversion of substrate into the colored product was measured after 3 h incubation at 37 ⁰C, and the reaction was stopped by a 1 N solution of hydrochloric acid. The multiple samples, standards, pNA, and blank controls were read rapidly by an absorbance reader (SUNRISE, Phoenix Research Products). The average net optical density (OD) for each standard and sample was calculated by subtracting the average blank OD from the average OD for each standard and sample. The activity measurements were quantitated by comparisons of the optical densities obtained with standards or with the pNA. Using graphing software (GRAPHPAD PRISM, HalloGram Publishing), the concentration of active caspase-3 in the samples was determined by interpolation of the average net OD for each standard versus the actual concentration of active caspase-3 substrate for the standards. The concentration of active caspase-3 in the samples for all groups is expressed as units per milligram. Analysis of cytosolic and mitochondrial fractions of cytochrome c

Frozen brains were homogenized with digitonin (0.05%) in a lysis buffer (250 mM Sucrose, 20 mM HEPES, 10 mM KCl, 5 mM MgCl₂, 1 mM EGTA, ImM EDTA, 1:100 and protease cocktail inhibitor). The homogenates were then centrifuged for 12 min (12,000 rpm, 4 ⁰C). The supernatant (cytosolic fraction) was removed and stored at - 80 ⁰C. The pellet was resuspended in a second lysis buffer for 30 min (133 mM NaCl, 50 mM Tris pH 8.0, SDS 0.1% (w/v), sodium deoxycholate 0.5% (w/v), Igepal CA630 1.0% (v/v) and protease cocktail inhibitor 1:100). The mixture was then centrifuged and the supernatant (mitochondrial fraction) was collected and stored at - 80 ⁰C until testing for cytochrome c. Cytosolic and mitochondrial fractions were assayed by ELISA for determination of cytochrome c. In brief, an anti- cytochrome c monoclonal coating antibody was adsorbed onto a 96 microtiter plate. Samples or standards containing cytochrome c were incubated with the adsorbed antibody for 2 h at room temperature. After incubation, unbound anti-cytochrome c was removed with wash buffer and then the samples and standards were incubated with a biotin-conjugated monoclonal anti-cytochrome c antibody for 2 h at room temperature. Following that incubation, unbound biotin-conjugated anti-cytochrome c was removed by several steps of washes with wash buffer. The samples and standards were then incubated with Streptavidin-HRP for 1 h at room temperature. After washes, a substrate solution reactive with HRP was added to each well for 5 min at room temperature. The enzymatic reaction was stopped with stop solution and the absorbance was read immediately on a spectrophotometer at 450 nm. To standardize for differences in concentration due to differences in tissue mass, the total protein concentration in the cytosolic or mitochondrial fraction in each sample was evaluated with the Bio-Rad protein assay. This allowed for calculation of the average nanograms cytochrome c per milligram of tissue protein in cytosolic and/or mitochondrial fractions.

TUNEL immuno staining reaction for cell death detection and cell counts

Cell death was determined using TUNEL reaction (TdT-mediated dUTP Nick End Labeling). To keep consistent conditions of immunostaining for both the experimental and the control groups, the fetal brains from ALC, ALC/CLN, PF, and Chow groups were embedded in 10% gelatin. All fetal brains were aligned at the same level in gelatin, and serial 50-μm coronal sections were then cut using a vibrating microtome apparatus. Fetal brain sections were treated with Proteinase K (10-20 μg/ml) for 5 min at 37 ⁰C, rinsed with PBS 3 times for 5 min and then incubated with 3% H₂O₂ in methanol for 10 min at room temperature. The sections were again rinsed with PBS 3 times for 5 min and then incubated in a permeabilization solution (0.1% TX-100 in 0.1% sodium citrate) for 2 min at 4⁰C. After the sections were rinsed twice in PBS for 5 min, they were incubated with a TUNEL reaction mixture (50 μl from bottle 1 and 450 μl from bottle 2, Roche Pharmaceuticals, Inc.) for 1 h at 37 ⁰C. The control was prepared by incubation in solution from bottle 2 only. The sections were rinsed 3 times for 5 min with PBS and incubated in converter-POD for 30 min at 37 ⁰C. Next, the sections were rinsed with TBS, followed by incubation in 0.05% 3'-3'- diaminobenzidine tetrahydrochloride and 0.003% H₂O₂ in TBS to reveal the peroxidase activity. Afterwards, sections were Nissl-counterstained with 0.5% cresyl violet to further reveal the cellular profile.

TUNEL-positive cells were counted by a blind experimenter in the primordium frontal cortex of E13 fetal brains from PF, ALC, and ALC/CLN groups. The penetration of TUNEL- staining through a thickness of 50 μm was verified at IOOX magnification. The expected shrinkage of a 50 μm- thick section in the z plane was averaged to approximately 14 μm. The number of sections in the selected brain region for TUNEL-positive cell counting was also considered and controlled in this study to avoid the bias of any missing sections from PF, ALC, and ALC/CLN groups. The entire population of TUNEL-positive cells was counted manually in every other section of the primordium frontal cortex, and this measure overcomes the bias of over-counting the number of TUNEL-positive cells in adjacent sections. The number of TUNEL-positive cells was averaged per section and analyzed statistically. In vivo data analyses

Protective effects of Colivelin administration on fetal brain weight against the insult of alcohol exposure

Morphological observations of fetal brains showed reduction in the size of fetal brains in the ALC group as compared to the control Chow (p<0.01), PF (p<0.001), and PF/CLN (p<0.001) groups (Fig. 5). Statistical analyses showed significant differences in the fetal brain weights between ALC group and the Chow and PF control groups (Fig. 5). Administration of Colivelin along with prenatal alcohol exposure prevented such weight reduction and stabilized brain weights comparable to control groups (Chow, PF, and PF/CLN); however, there were significant differences between the Colivelin treatment group (ALC/CLN) and the ALC group. There were no significant differences between Chow, PF and ALC/CLN groups. These results demonstrate a role for Colivelin in protection against alcohol-induced restriction of brain growth.

Biodistribution of CLN into the fetal brain by crossing the placenta

Significant uterus-like fluorescence intensity was observed in the abdominal region of CLN-Alexa680-injected mice as compared to control pregnant mice injected with saline (Fig. 11). Upon further dissection, fluorescence intensity was also detected directly in the uterus of CLN-Alexa680-injected mice. Furthermore, it was found that CLN-Alexa680 was distributed in the fetal brains (Fig. 11). Neuroprotective effect of Colivelin administration against prenatal alcohol-induced apoptosis

To determine if Colivelin plays a role in prevention of alcohol-induced apoptosis, TUNEL- staining was used to evaluate cell death in the primordium frontal cortex, one of the fetal brain regions examined morphologically. In this region, alcohol exposure induced increases of TUNEL-positive cells (Fig. 6b) as compared to PF (Fig. 6a). Colivelin administration prevented the alcohol-induced increase in TUNEL positive cells (Fig. 6c). Quantification of TUNEL-positive cells per section revealed a significant increase in the ALC group compared to PF (p<0.01) and ALC/CLN (p<0.01) groups (Fig. 6d). Neuroprotective effect of Colivelin administration against prenatal alcohol-induced caspase-3 activation

A Caspase-3 colorimetric assay was used to detect the concentration of active caspase-3 in the E13 fetal brain to determine if the neuroprotective effect of Colivelin is mediated through a caspase-3 downstream apoptotic pathway. The activity of the caspase-3 enzyme indicated that cells entered an apoptotic pathway. Statistical analyses show significant differences in the concentrations of active caspase-3 in the fetal brain between ALC and control groups (Chow and PF) (Fig. 7). Administration of Colivelin prevented the effect of alcohol- induced increases in the concentration of active caspase-3. No significant difference was found in the concentration of active caspase-3 in fetal brains between the Colivelin treatment group (ALC/CLN) and control groups (Chow and PF) (Fig. 7).

Neuroprotective effect of Colivelin administration against prenatal alcohol-induced increase in the level of cytosolic cytochrome c and mitochondrial cytochrome c It was also investigated whether cytosolic cytochrome c release is correlated with a change in the concentration of active caspase-3 and whether the neuroprotective effects of Colivelin are mediated through the downstream signaling pathways that involved cytosolic cytochrome c. Statistical analyses showed that prenatal alcohol exposure induced a significant increase in cytosolic cytochrome c level as compared to Chow and PF groups (p<0.01); Fig. 8a). Colivelin administration along with prenatal alcohol exposure prevented this increase in the levels of cytosolic cytochrome c (p<0.01; Fig. 8a). No significant difference was found in the concentration of cytosolic cytochrome c in fetal brains between the Colivelin treatment group and control groups (Chow and PF). Also examined was whether the changes in the cytosolic cytochrome c are correlated with the mitochondrial cytochrome c. Statistical analyses revealed that prenatal alcohol exposure induced a significant decrease in mitochondrial cytochrome c as compared to Chow (p<0.01) and PF (p<0.01) controls (Fig. 8b). Administration of Colivelin along with prenatal alcohol exposure prevented the reduction in mitochondrial cytochrome c in ALC/CLN as compared to ALC groups (Fig. 8b). No significant differences were found between ALC/CLN, Chow, and PF.

Neuroprotective effect of Colivelin administration against prenatal alcohol exposure is mediated through MAP kinase pathway

The MSD assay was also performed on the lysates from fetal brains prenatally exposed to alcohol. Phospho-p38 MAPK levels were not affected by ALC treatment or

ALC/CLN treatment (Fig. 9a). CLN along with ALC treatment significantly decreased both p- JNK and p-ERKl/2 levels as compared with the control lysates from the pair-fed (PF) group (Fig. 9a), suggesting that CLN protects against PAE by modulating the levels of p-ERKl/2 and p-JNK as observed in vitro. A significant increase in p-ERKl/2 and p-JNK by ALC exposure was not observed, possibly due to the existence of a number of non-neuronal cells around neurons in vivo, a difference between in vitro experimental systems such as PCNs and in vivo systems. Considering that there are about 10-times more glial cells in the brain, neuronal upregulation of the phospho-MAP kinases may not be easily detected in vivo. Further, sampling time may be another reason why ALC did not appear to increase p-MAPKs in vivo. To examine the role of the MAP kinase pathways in the ALC neurotoxicity, were quantified the p-Bad levels were quantified in the fetal brain lysates. The phosphorylation of Bad by p90 ribosomal S6 kinase, one of the targets of p-ERK, is reported to be involved in neuronal apoptosis (Jin et al, J. Neurochem., 80:119-125, 2002; Koh, Neurosci. Lett., 412:68- 72, 2007). PAE significantly decreased p-Bad levels in the fetal brains as shown in Figure 9b. CLN treatment recovered the p-Bad levels in lysates from fetal brains prenatally exposed to ALC to the level of controls. Since decreases in p-Bad levels results in neuronal apoptosis (Wang et al, Science, 284:339-343, 1999), MEK-ERK pathway-induced phosphorylation of Bad is a possible mechanism of CLN-mediated neuroprotection against ALC neurotoxicity. The increases of phosphorylated BAD protein were linked to down-regulation of the phosphorylated forms of JNK and ERK proteins which lead to neuroprotection. In vitro data in primary cortical neurons confirm the involvement of ERK and JNK proteins in Colivelin neuroprotection against the insult of alcohol exposure. Colivelin was found to be effective in protecting fetal brain neurons against the insult of alcohol exposure. Colivelin prevented cellular toxicity induced by alcohol as shown by examination of TUNEL-positive cells. Further, administration of Colivelin was found to prevent the loss in fetal brain weight induced by prenatal exposure to alcohol. The downstream signaling pathways involved caspase-3, and both cytosolic and mitochondrial cytochrome c, suggesting that Colivelin may affect mitochondria. Colivelin-mediated neuroprotection against the effects of ALC exposure involved up-regulation of the phosphorylated form of BAD protein, which in turn down-regulated the phosphorylated forms of JNK and ERK1/2 downstream signaling pathways. The experiments described herein demonstrate that Colivelin is effective in preventing the teratogenic effects of fetal alcohol exposure. While the invention has been illustrated and described in detail in the foregoing description, such an illustration and description is to be considered as exemplary and not restrictive in character, it being understood that only the illustrative embodiments have been described and that all changes and modifications that come within the spirit of the invention are desired to be protected. Those of ordinary skill in the art may readily devise their own implementations that incorporate one or more of the features described herein, and thus fall within the spirit and scope of the present invention.

Claims

CLAIMS:

1. A method of inhibiting developmental cellular toxicity in an individual in need thereof comprising the step of administering a therapeutically effective amount of Colivelin or pharmaceutically acceptable salt thereof.

2. The method of Claim 1 wherein the cells reside in the central nervous system.

3. The method of Claim 1 wherein the cellular toxicity is caused in part from exposure to a teratogen.

4. The method of Claim 3 where the teratogen is ethanol.

5. A method of treating a neurodevelopmental disorder in an individual in need thereof comprising administering a therapeutically effective amount of Colivelin or pharmaceutically acceptable salt thereof to said individual.

6. The method of Claim 5 wherein the disorder is caused in part by exposure to a teratogen.

7. The method of Claim 6 wherein the teratogen is ethanol.

8. The method of Claim 6 wherein the Colivelin is administered in utero.

9. A method of treating fetal alcohol syndrome or fetal alcohol effects in an individual in need thereof comprising administering a therapeutically effective amount of Colivelin or pharmaceutically acceptable salt thereof to said individual.

10. A method of treating a pregnant female to inhibit fetal alcohol syndrome or fetal alcohol effects in the fetus of said pregnant female comprising administering a therapeutically effective amount of Colivelin or pharmaceutically acceptable salt thereof to said pregnant female.

11. The method of claim 10 wherein the Colivelin is administered to the pregnant female during the first trimester of pregnancy.

12. The method of claim 10 wherein the Colivelin is administered to the pregnant female during the second trimester of pregnancy.

13. The method of claim 10 wherein the Colivelin is administered to the pregnant female during the third trimester of pregnancy.