Liposome preparation and validation

We prepared liposomes in 20 mL batches in our facility in Wellington New Zealand one week prior to the experiment using the thin film method, as previously described [15]. In brief, 100 mg of pure lipids in molar ratios 54% dipalmitoylphosphatidylcholine [DPPC]; 45% cholesterol and 1% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] [DSPEmPEG 2000] (Merck, Darmstadt, Germany) were dissolved in 20 mL 99% ethanol/1% methanol (Pure Nature, Auckland, New Zealand). The organic solvent was evaporated off in a rotating spherical flask suspended in a water bath at 40 °C while a stream of pure nitrogen was passed over the solution, until a thin film formed on walls of the flask. Once removed from the water bath, the stream of nitrogen was continued for another 2 h to ensure that the thin film was completely dried.

We prepared a solution of 250 mM citric acid by adding 9.6 gm of citric acid (Pure Nature, Auckland, New Zealand) to 200 ml sterile water (Fresenius Kabi, Bad Homburg, Germany). pH of this solution was measured at 2.2. 20 mL of this solution was added to each thin film and the flask was then rotated in a bath ultrasound at 40 kHz until all visible film was suspended. The flask was then placed upright in the ultrasound bath for 10 further minutes to ensure all lipid was fully suspended.

Liposome size was measured using a Malvern mastersizer 3000 (Malvern Panalytical, Malvern, England).

In vivo experimentLocation

We undertook this experiment in the Small Animals Colony of the Ruakura Agresearch facility in Hamilton, New Zealand.

Ethical approval

All animal protocols for this experiment were approved by the Agresearch Ruakura Animal Ethics Committee. The project approval number was 2339.

Animals

Animals were kept in single gender enclosures with no chance of pregnancy. Twelve-hour light–dark cycles (lights on/off at 07:00/19:00 h) and climate control were maintained. We used 20 female Sprague–Dawley rats for this experiment. Four were used for initial dose ranging experiments and 16 for the full study protocol.

Amitriptyline dose ranging experiments

In order to create a steady state model we added amitriptyline to the drinking water of 4 rats for 7 days. This duration was chosen as it represents greater than four half-lives of amitriptyline in the rat, the time taken to reach a presumptive steady state [16, 17]. At the end of seven days exposure we took a tail vein blood sample under isoflurane anaesthesia. Animals were returned to a group enclosure on recovery. There was a washout period of at least 2 weeks amitriptyline free before repeating at a higher dose if needed. Before a higher dose of amitriptyline was used water intake during the dosing period was analysed, and dose was not subsequently increased if there was a 10% or greater reduction in water intake from baseline. The tail vein blood sample was sent for amitriptyline concentration measurement. Once we identified a dosing regimen that permitted study and did not reduce water intake (estimated 50 mg/kg/day amitriptyline), the 4 animals used in this phase were euthanized according to institutional protocols.

Dialysis experiment

We used 16 animals aged 24–28 weeks in this phase of the experiment. At commencement of the study protocol, animals were sedated with ketamine at 50 mg/kg (Mayne Pharma Ltd., Auckland, New Zealand), and xylazine at 4 mg/kg (Bayer HealthCare, Leverkusen, Germany) via single intraperitoneal injection. We then placed animals on a warming board at 38.5 °C. An intravenous (IV) cannula was placed in the tail vein using a needle over catheter technique (24 G × 0.75 in Insyte, BD, Switzerland). 1% lignocaine was instilled in the anterior neck and following dissection a tracheostomy tube (14 G × 1.77 in Insyte, BD, Switzerland) was placed under direct vision. Mechanical ventilation using a small animal ventilator (Inspira ASV, Harvard Apparatus, USA) was then undertaken with 100% oxygen as the driving gas admixed with 2% isoflurane (Merial, Auckland, New Zealand) via a vaporizer (Somnosuite Small Animal Anesthesia System, Kent Scientific Corporation, Torrington, USA) to maintain anaesthesia.

We placed a carotid arterial line under direct vision using a catheter over needle technique, tying off the hub of the catheter and the artery a small distance cephalad to insertion (24 G × 0.75 in Insyte, BD, Switzerland). The arterial catheter was used for blood sampling and to monitor arterial pressure and heart rate via the arterial pressure waveform (BP Amp, AD Instruments, Bella Vista, Australia). Anaesthesia, mechanical ventilation and blood pressure monitoring were continued for the duration of the experiment. Haemodynamic metrics were recorded on a standardized template at predetermined time points in the experiment. We monitored depth of anaesthesia at 5 min intervals using paw squeeze and corneal reflex.

At the completion of instrumentation there was a stabilization period of 5 min for each animal. After this a baseline blood sample was taken to measure amitriptyline concentration (nominally at 0 min, the start of the experiment). All blood samples taken during the experiment were 0.5  mL in volume. Animals were randomized into 2 groups of 8 to receive either saline control or intravenous lipid emulsion. The saline control animals received 5ml of 0.9% saline (Baxter Pharmaceuticals, Toongabbie, Australia) as a single slow intravenous injection at the start of the experiment. The lipid emulsion animals received 1ml of 20% Intralipid® (Fresenius Kabi, Bad Homburg, Germany) at the start of the experiment followed by an infusion at 0.07 mL per minute. We used this dose of lipid emulsion as we calculated it would maintain 1% lipaemia in a 300-g rat with an assumed blood volume of 20mL and a half-life of lipid droplets of 10 min [18]. The difference in dosing for the two intravenous agents was based on an expected difference in intravascular half-life [19].

A second blood sample was taken 10 min after the first blood sample.

We then centrifuged 10 × 1 mL tubes of 5 mg/mL pH gradient DPPC/cholesterol liposome suspension at 17,500 G for 10 min to form a liposome pellet at the base of the test tube. At 46 min post the first blood sample we aliquoted off remaining citrate and resuspended liposomes in Hemosol (Baxter, Deerfield, Illinois, USA). We then added resuspended liposomes to final volume of 20 mL, to make a dialysate containing 50 mg of pH gradient DPPC/cholesterol liposomes suspended in 20 mL of Hemosol.

As this protocol ran with a single operator the pre dialysis blood sample was taken 5 min prior to commencement of dialysis (T45) for amitriptyline concentration. We regarded this as the blood concentration at the commencement of dialysis for analysis.

At 50 min we injected 20 ml of the dialysate described above into the peritoneum. The dialysate was manually agitated at one-minute intervals until the end of the experiment.

At 60 min we euthanized all animals by injection of pentobarbitone in the tail vein. This resulted in rapid demise with universal cessation of the circulation as assessed by the arterial line trace within seconds.

After demise, we sampled blood and peritoneal fluid for amitriptyline concentration measurement.

Average blood amitriptyline concentration during dialysis was taken as the average of the blood concentrations at 45 and 60 min.

ERest

In previous work we have estimated peritoneal blood flow over the dwell and divided the amount extracted into dialysate by the estimated amount of amitriptyline which perfused the peritoneal membrane during the dwell [10, 11]. We defined this as the estimated extraction ration (ERest). In this experiment we estimated peritoneal blood flow at 2 mL/min [20]. A 10-min dwell time with a 20 mL dialysate volume meant ERest was calculated to be the ratio of dialysate to average blood concentration during peritoneal dwell.

Amitriptyline concentration measurement

Amitriptyline concentrations were measured using a liquid chromatography–mass spectrometry method (Sciex 3200 MSD Aglient 1200 series LS, Sciex [Redwood City, USA]) with isotope matched internal standards. The whole blood sample was prepared by a protein crash with acetonitrile, followed by centrifugation after which an aliquot was presented to the instrument for analysis. A four-point matrix-matched standard curve was used to calibrate the analyser for each sequence with two levels of quality control.

Statistical analysis

We powered this experiment for a 50% increase in blood and peritoneal dialysate concentration of amitriptyline with the intravenous lipid emulsion using Student’s t test. We used a mean blood concentration 200 nmol/L and standard deviation of 50 nmol/L in powering the study [21]. With a standard deviation of 50nmol/L and a true difference of 100 nmol/L, we achieved > 80% power that an effect would be observed on blood concentrations with 8 subjects in each group.

The ratio of standard deviation to expected effect size is the same for peritoneal dialysate as for blood [7], giving the same power with 8 subjects.

Data were analysed using GraphPad Prism®. Mann–Whitney, one-way ANOVA and two-way repeat measures ANOVA were used as appropriate.