The authors have declared that no competing interests exist.
Conceived and designed the experiments: IT JTB. Performed the experiments: PV FMM. Analyzed the data: PV IT MTS BDW. Contributed reagents/materials/analysis tools: IT MTS BDW. Wrote the paper: PV FMM. Supervised overall project: IT. Supervised animal experiments: MTS BDW. Critically reviewed the manuscript: IT MTS JTB BDW.
To enhance the drug-like properties of the endogenous opioid peptide endomorphin-1 (
Centrally acting opioids, such as morphine, are the most frequently used analgesic agents for the treatment of severe nociceptive pain but they also produce a range of unpleasant adverse effects including constipation, analgesic tolerance and cardio-respiratory depression
Previously, we showed that incorporation of lipoamino acids (LAAs), which combine the structural features of lipids and amino acids, into the structure of short peptides can improve the metabolic stability and membrane permeability of endomorphins
All endomorphin analogs,
Compound
Compound | cAMP, IC50 (nM) |
||||
|
5.0±1.2 | 2.70±0.9 | 0.2±0.02 | 1.1±0.2 | 14.0±6.8 |
2.3±0.36 |
1.0±0.34 |
(1.1±0.09) |
(9.4±0.43) |
||
|
24.1±3.6 | 4.2±1.5 | 2.5±0.3 | 0.06±0.01 | 0.3±0.02 |
|
77.4±7.1 |
ND | 90.1±12.2 |
25.7±1.3 |
31.4±1.45 |
|
96.3±8.3* | 41.4±3.3* | 12.4±4.7* | 0.47±0.12 | 1.30±0.22 |
The apparent permeability,
The
The IC50 values (nM) were estimated from dose-response curves shown in
These values are obtained from the same experiment. ND, not determined. Each value represents mean ± SEM. *
Opioid binding affinity and
Unlike compound
Binding affinity of compound A)
Dose-response curves for the inhibitory effects of compounds A)
To investigate if the stabilization of endomorphin-1 by LAA conjugation resulted in active analogs following systemic administration, the comparative antinociceptive effects of parent peptide
Paw withdrawal threshold (PWT) versus time curves for single bolus i.v. doses of compounds A)
Analogs
The highest doses of compounds
Single i.v. bolus doses of compounds
The anti-allodynic effects of compound
The anti-allodynic effects of single i.v. bolus doses of compound
The effects of the opioid receptor agonist
A) Lack of inhibitory effect of
Development of tolerance to the pain-relieving activity of compound
Five consecutive i.v. bolus doses of compound
Similar to the parent compounds
Endomorphins like other small peptides are degraded rapidly by membrane-bound exo- and endopeptidases in the blood
It was shown that introduction of Dmt into the structure of endomorphin-1 derivatives not only enhanced MOP receptor binding affinity, as reported previously
Morphine produces antinociception
A common side-effect produced by MOP-receptor agonists is constipation, which occurs because of a reduction in stool hydration and delayed GI transit
Our findings showed that less tolerance developed to the analgesic effect of compound
We have successfully designed and developed two systemically active lipid-modified derivatives of endomorphin-1 with promising potential for the treatment of neuropathic pain. Lipid derivatives
This research was approved by and conducted in compliance with the guidelines of The University of Queensland Animal Ethics Committee (AEC#CIPDD/366/09/NHMRC).
Dimethylformamide (DMF), trifluoroacetic acid (TFA) and piperidine of peptide synthesis grade were purchased from Merck Biosciences (Kilsyth, VIC, Australia), and acetonitrile suitable for HPLC was purchased from RCI Labscan Ltd. (Bangkok, Thailand). Fmoc-protected amino acids and Rink amide MBHA resin (100–200 mesh) with a loading of 0.4–0.8 mmol/g were obtained from Novabiochem (Melbourne, VIC, Australia) or Mimotopes (Clayton, VIC, Australia), apart from Fmoc-dimethyltyrosine (Fmoc-Dmt) which was sourced from Chiramer, Inc. (USA). Cell culture reagents were purchased either from Sigma Aldrich or Gibco (VIC, Australia). Other chemicals were purchased from Sigma Aldrich (VIC, Australia). Boc-C10-LAA was synthesized according to a published procedure
Peptides were assembled on Rink amide MBHA resin following the in situ neutralization protocol for Fmoc chemistry
Upon completion of the peptide sequence, the resin was washed with DMF, dichloromethane and methanol, and dried under vacuum over night. The peptide was cleaved from the resin using a mixture of TFA (95%), triisopropyl silane (2.5%) and water (2.5%). After 2 hours, the solvent was removed and the peptide precipitated using cold diethyl ether. The resulting solid was lyophilized from acetonitrile, water and TFA (50∶50∶0.1).
Purification of the crude peptides was carried out on a Vydac C18 column (22×250 mm) at a flow rate of 10 mL/min with a 70 minute gradient of 40% to 80% solvent B (0.1% TFA in acetonitrile/water 9∶1; solvent A: 0.1%TFA in water). The collected fractions were analyzed by mass spectrometry and analytical HPLC on a Vydac C18 column (4.6×250 mm, 5 µm) at a flow rate of 1 mL/min and a gradient of 0% B to 100% B over 30 minutes. Pure fractions were combined and lyophilized. Purity of peptides was confirmed by analysis on a different HPLC column (C4, 4.6×250 mm, 5 µm) and high resolution mass spectrometry.
Compound
Compound
Compound
Compound
Cell lines were obtained from the American Type Culture Collection (Rockville, USA). Human colorectal adenocarcinoma (Caco-2) cells were maintained in T-75 flasks with culture medium consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% non-essential amino acids. Flasks were kept in an incubator at 95% humidity and 37°C, in an atmosphere of 5% CO2. The culture medium was changed every second day. Upon reaching 80% confluence, cells were subcloned using 0.25% trypsin–EDTA (ethylenediaminetetraacetic acid). Experiments were performed using cells with passage numbers 55–75. Human neuroblastoma (SH-SY5Y) cells were maintained in DMEM:Hams F12 medium supplemented with 10% FBS, 1% glutamine, 1% non-essential amino acids and 1000 U/mL Streptomycin/Penicillin. Cells were subcloned in the same manner as Caco-2 cells, and passage numbers 10–20 were used in the assays.
A suspension of 5×104 Caco-2 cells in 100 µL culture medium was added to the wells of a 96-well cell culture plate. Culture medium supplemented with 1% penicillin/streptomycin (100 U/mL) was changed every second day.
After 21–28 days, the cells were washed with 0.2% EDTA (2×100 µL) and Hank's Balanced Salt Solution containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HBSS-Hepes, pH 7.4; 3×100 µL). HBSS–Hepes buffer was added to each well (100 µL), and the cells were disrupted by two one-second pulses with a Sonics Vibracell ultrasonic processor set at 60% amplitude at 130 W. The cell debris was removed by centrifugation (2000 rpm, 5 minutes) and the protein content of three wells was determined using the Bio-Rad protein assay kit, by comparison with a standard curve generated from known concentrations of bovine serum albumin. The protein content of the cell homogenate was adjusted to 0.6–0.9 mg/mL in a clean 96-well plate.
The peptides were dissolved in HBSS–Hepes buffer containing 1% DMSO (dimethyl sulfoxide), and each peptide solution (200 µM, 100 µL) was added to three wells and the plate was incubated at 37°C in a shaker at 400 rpm. At pre-selected time points (5, 10, 15, 20, 30, 40, 50, 60, and 120 min), samples (10 µL) were collected and enzymatic digestion was stopped by addition of 5 µL TFA. These samples were then diluted with water (75.5 µl) and DMSO (9.5 µL) and analyzed by LC-MS to determine the concentration of peptide remaining in the solution.
LC-MS was carried out on a Luna Phenomenex C18 column (2.0×50 mm, 5 µm) with a gradient of 100% A (0.1% formic acid in water) to 100% B (0.1% formic acid in acetonitrile/water 9∶1) over 5.5 minutes at a flow rate of 0.3 mL/min. The mass spectrometer was set to selective ion monitoring in the positive ion electrospray mode. A standard curve was generated from standards containing known peptide concentrations, and used to determine the peptide concentration in the samples.
A suspension of 105 Caco-2 cells (100 µL) in culture medium supplemented with 1% penicillin/streptomycin (100 U/mL) was pipetted into polycarbonate cell culture inserts (pore size 0.4 µm, 6.5 mm diameter, Transwell®) in a 24-well plate, and the same medium was also added to the basolateral chamber (0.6 mL). Medium in both chambers was changed every second day.
After 21–28 days, the integrity of the tight junctions and the monolayers was assessed by measuring the transepithelial electrical resistance (TEER) using the Millicell-ERS epithelial volt-ohmmeter system (Millipore Corporation). Wells that gave TEER values of 1500–4280 (Ωcm2) were used for the experiment. Minor TEER value differences of ±500 (Ωcm2) were detected after the assay, indicating that none of the peptides were toxic to the cells.
After the initial TEER test, the Caco-2 cell monolayers were washed with HBSS-Hepes buffer (3×) and then incubated with the same buffer at 37°C. After 30 minutes the buffer was removed from the apical chamber and 100 µL aliquots of peptides, dissolved to a concentration of 200 µM in HBSS-Hepes containing 1% DMSO, were added to at least three wells. As a further test of the Caco-2 cell monolayer integrity, radiolabelled [14C]-D-mannitol (1.80 µCi) in HBSS-Hepes buffer was added to three wells. The plates were incubated in a shaker set to 400 rpm at 37°C, and samples (0.4 mL) were collected from the basolateral chamber at 30, 90, 120 and 150 minutes and replaced with the same volume of buffer. At the end of the experiment, 50 µL were collected from the apical chamber of each well. LC-MS quantification of the peptides was performed as described in Caco-2 cell homogenate stability assay. The radioactivity of the [14C]-D-mannitol samples was quantified by liquid scintillation counting (Liquid Scintillation Systems, BECKMAN LS3801, USA), and the apparent permeability (
Vr volume of the receiver chamber (mL)
A surface area of the cell monolayers
C0 initial concentration in the donor chamber (M or dpm/mL)
Fresh human plasma was obtained from healthy and consenting volunteers (ethics approval number: 2006000950). The peptides were dissolved in PBS containing 5% DMSO (1 mg/mL), and 300 µL aliquots were mixed with 300 µL of pre-warmed plasma and incubated at 37°C. At selected time points (5, 10, 20, 30, 40, 60, 90, and 120 min), samples (50 µL) were collected and plasma proteins precipitated by addition of acetonitrile (75 µL). After centrifugation (10,000 rpm, 10 minutes), the peptide content of the supernatant was analyzed by HPLC with a Vydac C18 column (4.6×250 mm, 5 µm) at a flow rate of 1 mL/min and a gradient of 100% A (0.1% TFA in water) to 100% B (0.1% TFA in acetonitrile/water 9∶1) over 30 minutes.
A suspension of SH-SY5Ycells (105 cells per well) was seeded into 24-well plates and incubated overnight. The cells were washed with PBS and pre-incubated with 300 µL binding buffer (50 mM Tris buffer, pH 7.4, 2% BSA). A mixture of [3H]-DAMGO (100 µL, 15 nM), DPDPE (100 µL, 1 µM) and 100 µL aliquots of each peptide in a range of concentrations (0.1 nM to 100 µM) was added to each well and the plate was incubated at 37°C. An excess of [3H]-DAMGO (1 µM) was used to determine non-specific binding and buffer (100 µL) was measured as the blank. After 60 minutes, the cells were washed with PBS and 1 M NaOH (0.5 mL) was used to lyse the cells. The radioactivity of the cell suspension was measured by scintillation counting (Liquid Scintillation Systems, BECKMAN LS3801, USA). Data analysis was performed using the one-site competition binding model (GraphPad Prism™ (v5.0) software).
SH-SY5Y cells, which express MOP and DOP receptors, were seeded (105 cells/mL, 100 µL) in a 96-well plate and incubated overnight to reach 80% confluence. Culture medium supplemented with 30 µM forskolin was used make peptide solution (100 nM) and aliquots (100 µL) of each peptide were added to three wells. After incubation at 37°C for 30 minutes, the culture medium was removed and lysis buffer from a cAMP Biotrak EIA kit (Amersham, Buckinghamshire, UK) was added, followed by an anti-cAMP antibody. After incubation at 4°C for 2 hours, a cAMP peroxidase conjugate was added and the plate incubated for another hour at 4°C. Then, the cell lysate was incubated with a 3,3′5,5′-tetramethylbenzidine (TMB) substrate provided by the Biotrak EIA kit and finally 1 µM sulfuric acid was added. Absorbance of each well was determined using a plate reader at 450 nm and compared with a cAMP standard curve (concentration 0–3.2 pmol). Data was represented as a percentage of the maximum cAMP response to forskolin, shown as a mean (±SEM) of three separate experiments each performed in triplicate (GraphPad Prism™ (v5.0) software).
Adult male Specific Pathogen Free (SPF) Sprague-Dawley (SD) rats weighing between 160–200 g were purchased from The University of Queensland Biological Resources breeding facility. They were housed in groups of 3 with free access to food and water in a light- and temperature-controlled room (light on 06:00–18:00 h; 22.2±0.2°C) and 51.6–65.2% humidity. Prior to initiation of experimental procedures, rats were acclimatized for at least a 3-day period.
Chronic constriction injury (CCI) of the sciatic nerve in the rat was used as a model of neuropathic pain and was induced according to the method of Bennett and Xie
Paw withdrawal thresholds (PWT's), expressed in grams, were measured using a set of calibrated Von Frey filaments (Touch Test- North Coast, 800-821-9319). Animals were acclimatized for at least 15 min in wire mesh metabolic cages (20×20×17 cm). Von Frey filaments which delivered forces in the range 2–20 g at 2 g intervals were used according to the up-down method
Test items were formulated in 10% DMSO (Sigma-Aldrich) and 20% Cremophor (Sigma-Aldrich) in water and were administered as i.v. bolus doses via tail-vein injection. Morphine sulphate (Hospira, Melbourne Australia) was used as the positive control and vehicle as the negative control in this study. In accordance with animal ethics guidelines for reduction of the number of animals used in experiments, the PWT values for the negative and positive controls shown here have also been included in another manuscript (P. Varamini et al. 2012, submitted). Test and control items were administered to groups of CCI-rats according to a ‘washout’ protocol such that each rat received 3–4 single i.v. bolus doses in a rising dose sequence, of one test item or vehicle with each dose separated by a 2–3 day washout period. On each dosing occasion, Von Frey PWTs were determined in the ipsilateral and contralateral (non-operated side) hindpaws immediately prior to test item administration (baseline PWT values) and at the following times post-dosing: 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, and 3 h.
Groups of CCI-rats (
Assessment of constipation inducing properties:
Castor oil-induced diarrhea test in rats
The ability of test items to inhibit castor-oil induced diarrhea as an index of constipation was assessed. Prior to the start of experimentation, drug-naive rats weighing 200–250 g were fasted for 16 h with free access to water. Individually caged rats were acclimatized for 1 h without access to food or water prior to drug or vehicle administration. Compound
Charcoal test
The charcoal test was assessed in groups of drug-naive rats to measure gastrointestinal (GI) transit. Prior to experimentation, rats (200–250 g) were fasted for 16 h with free access to water. Single i.v. bolus doses of compound
At 14 days after CCI surgery, rats (350–400 g) were randomly assigned to one of four treatment groups and administered twice-daily i.v. bolus doses of one test item or vehicle for 5 consecutive doses. Group 1 CCI-rats received compound
To obtain ΔPWT values, the PWT versus time data were normalized by subtracting pre-dosing baseline PWT values for each individual rat as follows:
All data are expressed as mean (± SEM). One-way ANOVA was used to compare normalized ΔPWT AUC values of each LAA-conjugate between the ipsilateral and contralateral hindpaws in addition to GI propulsion and castor-oil induced diarrhea inhibition produced by compound
Synthesis of endomorphin-1 (1) and its derivatives (2–4). (i) 20% piperidine/DMF, 0.5 h (ii) AA (1. Fmoc-Phe-OH 2. Fmoc-Trp(Boc)-OH 3. Fmoc-Pro-OH 4. Fmoc-Tyr(tBu)-OH or Fmoc-Dmt-OH), HBTU, DIPEA, DMF, rt, 1.5 h (iii) HBTU, DIPEA, DMF, rt, 1.5 h (iv) TFA, triisopropylsilane, H2O, rt, 2 h.
(TIF)
We thank Ms. Sharareh Eskandari for her assistance in performing membrane permeability and metabolic stability experiments for compound