CTAP glycopeptides were prepared using published methods . CTAP: dPhe-Cys-Tyr-dTrp-Arg-Thr-Pen-Thr (parent peptide); glycosylated congener gCTAP5: dPhe-Cys-Tyr-dTrp-Arg-Thr-Pen-Thr-Gly-Gly-Ser-b Glucopyranoside.
The peptides were assembled on Rink resin using Fmoc (fluorenylmethoxycarbonyl protecting group) methodology. Couplings were accomplished with 1-hydroxy-benzotriazole, N,N′-diisopropyl-carbodiimide and the desired amino acid. Coupling times ranged from 40 min up to 4 h for the more sterically encumbered sequences. For the serine glucoside, 1.3 equiv of the amino acid, and the couplings were monitored with the Kaiser ninhydrin test. For all other amino acids, 3.0 equiv were used. For tyrosine and cysteine, penicillamine O-tert-butyl and S-trityl protecting groups were employed. Then, the N-terminal FMOC-groups were removed, and the acetate groups of the glycoside were removed with hydrazine hydrate in MeOH while on the solid support. After ester cleavage, the resin was washed several times to remove excess hydrazine and vacuum-dried to obtain mass determination. The dried peptide resins were cleaved with a cocktail mixture (9.0 mL trifluoroacetic acid (TFA), 1.0 mL CH2Cl2, 0.25 mL Et3SiH, 0.25 mL H2O, and 0.05 mL anisole per 1.0 g peptide resin) for 2 h at RT. After cleavage was complete, the solutions were filtered to remove the cleaved resin, and the resulting solution was concentrated to an oil in vacuo. After concentration, cold Et2O was poured over the peptide solutions for precipitation. The crude peptides were centrifuged, dried, and purified via preparative reverse-phase high performance liquid chromatography (HPLC) with a linear gradient of 5-80% CH3CN:0.1% aqueous TFA to yield the pure reduced compounds. These samples were cyclized with a solution of 3.0 equiv of K3Fe(CN)6 at pH 8.5 for roughly 16 h using a high-dilution, reverse-addition protocol. Then, these solutions were acidified to pH 4.0, anion-exchanged with Amberlite IRL-80 exchange resin, filtered, and lyophilized. The crude cyclic material was repurified with a preparative as before. CTAP: 63 mg (yield 8.7%), purity >99% at 280 nm, retention time (Rt): 10.55 min (gradient 5-80% / 15min). Calc. C51H69N13O11S2 Mw: 1103.5, Found electrospray ionization (ESI) [M+H]+ 1104.6. gCTAP5: 47 mg (yield 6.4%), purity >99% at 280 nm, Rt: 10.07-min (gradient 5-80% / 15 min). Calc. C64H90N16O20S2 Mw:1466.6, Found ESI [M+H]+ 1467.3.
Animals for LID model and Tail Flick experiments
Male Sprague-Dawley rats (n=8 for LID study: 250 g and n=8 for Tail Flick: 200 g; Harlan, Indianapolis, IN) were used and housed in a temperature and humidity-controlled room with 12-hour light/dark cycles with food and water available ad libitum. All animals were treated as approved by the Institutional Animal Care and Use Committee at the University of Arizona and in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Number of animals used and their suffering were minimized.
The animals (n=8) were gently restrained and nociception was administered by dipping the distal third of the tail in a 52°C water bath. Latency to tail-flick was recorded as the time required for the tail to withdraw from the bath, with a cutoff of 10-seconds to prevent tissue damage. Prior to administering compounds, three measurements of tail-flick latency were recorded with 2-min intervals between tests to establish control latency. The antinociceptive effect of intraperitoneal (i.p.) morphine (10 mg/kg) was determined for each animal every 15 min with measurements of tail-flick latency between 15-90 min post-injection. The individual abilities of CTAP and gCTAP5 (0.1, 0.5 and 1 mg/kg, i.p.) to antagonize the antinociceptive effect of morphine were then tested at 48 h intervals. The m-opioid receptor antagonists were administered 10-min after morphine in order to coincide with its peak effect. Latency time and maximum possible effect (%MPE) were calculated at 45 min post m-opioid receptor antagonists injection. %MPE=(post injection latency-baseline latency)/[cutoff (10 sec)-baseline latency]x100.
The unilateral 6-hydroxydopamine (6-OHDA)-lesion rat PD model
Injection of 20 mg 6-OHDA (5.0 mg/ml in 0.9% sterilized saline with 0.02% ascorbic acid; Sigma, St Louis, MO) in 2 locations in the medial forebrain bundle (MFB) , as published [11,12]. The rate of the injection was 0.5 ml per min using a Stoelting microinjector (Stoelting Co., Wood Dale, IL). Rats were pretreated 30 min prior with 12.5 mg/kg desipramine hydrochloride (Sigma, St Louis, MO) given i.p. to prevent damage to noradrenergic neurons.
Induction of LID in unilateral lesioned rats
1) Two weeks after surgery the unilateral 6-OHDA-lesioned rats were injected with D-amphetamine (5.0 mg/kg, i.p. injection; Sigma) to induce asymmetrical dopamine release. The number of ipsiversive rotations during 1-minute intervals, every 5 min were counted for a total of 60 min after the injection. 2) Rats with ≥4 rotations/minute were selected and were daily treated with 7 mg/kg L-DOPA (with 15 mg/kg benserazide, both i.p.; Sigma) for 3 weeks to establish LID, and the 6 rats that developed stable LID were included in the LID study.
Behavioral analysis in the LID rat model
L-DOPA-induced abnormal involuntary movements (AIMs) were scored by an experimentally blinded investigator according to an established protocol [11,12], with a ‘within subjects cross over design’ to have a vehicle control for every drug tested. For each drug animals were randomized to either receive vehicle or drug on one testing day, and switched 3-4 days later, so that each drug has a separate vehicle control. For quantification of the severity of the AIMs, rats were observed individually in their standard cages every 20th minute at 20-80 minutes after an injection of L-DOPA, and were classified as described [11,12]. The sum of limb, axial, and orolingual (LAO) AIMs and the sum of locomotor AIMs scores per testing session were used for statistical analyses.
Euthanasia and brain tissue harvest
Rats were sacrificed after the last dose of drug with carbon dioxide. For a quantitative measure of the extent of the PD-lesion for n=6 animals the whole brains were extracted, striatal tissue was prepped and frozen at -80oC. To validate the lesions post hoc quantitative dopamine (DA) measurements in striatal tissue was then conducted with HPLC-EC, as published [11,12].
Statistical analysis was performed using GraphPad Prism 8.1 software (GraphPad Software, Inc., La Jolla, CA). Repeated measures one-way ANOVA with Tukey post hoc tests was used for the tail-flick time point raw MPE data. For the DA analysis a two-tailed t-test of the raw data was conducted. Non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons post hoc tests was used to compare the effect of treatment on LAO- and locomotor AIMs. The null hypothesis was rejected when p < 0.05.