Pre-clinical investigation of liquid sirolimus for local drug delivery

Percutaneous interventional therapies such as drug-eluting stents (DESs) and drug-coated balloons (DCBs) are widely used to treat peripheral artery disease (PAD). Paclitaxel has been the predominant antiproliferative coating but safety concerns have prompted exploration of sirolimus (rapamycin) as an alternative. Mechanistically, sirolimus forms a complex with FKBP12 to inhibit mTOR, arresting the cell cycle at G1-S (cytostatic), whereas paclitaxel is cytotoxic via microtubule stabilization. Prior uses of sirolimus in PAD have been as solid coatings on stents/balloons; liquid sirolimus delivery has not been demonstrated. This study asks whether liquid sirolimus can be locally delivered into the arterial wall and retained under physiological flow using liquid drug delivery devices in an ex vivo porcine artery bioreactor.

Study design: Pilot, ex vivo benchtop study using harvested porcine carotid arteries in a closed-loop pulsatile bioreactor mimicking physiological flow and pressure. Artery segments (5–8 cm) were thawed, prepared, and mounted in housings; flow and pressure were monitored via an ultrasonic flow meter and catheter pressure transducer, with a signal generator and gear pump controlling hemodynamics. Culture medium was DMEM (low glucose) supplemented with L-glutamine, sodium pyruvate, pyridoxine, 10% FBS, and antibiotics. Device comparison: Four liquid drug delivery (LDD) devices were tested for penetration depth using a fluorescent paclitaxel analog (Flutax-1) at 0.1 mg/ml in a 1:1 saline:iohexol solution (DMSO stock). Devices and delivery parameters: - Bullfrog micro-infusion device: microneedle deployment via low-pressure balloon inflation (2 atm) to inject drug into wall. - ClearWay RX catheter: microporous low-pressure balloon infusion at 4 atm. - Occlusion Perfusion Catheter (OPC): proximal/distal occlusion balloons inflated to nominal (4 atm). Liquid delivered to achieve chamber pressure 0.2–0.4 atm, with in/out-flow ports; built-in fiber optic pressure sensor. - TAPAS: proximal/distal occlusion balloons with adjustable treatment zone; infusion through lumen at 2 atm. Delivery time for all devices: 120 s. After treatment, arterial segments were processed for histology and fluorescence microscopy. Histology and imaging: 5-mm perfused segments embedded in OCT; 12-µm cross-sections cut on cryostat; mounted and imaged on Echo Revolve microscope (FITC filter). Depth of penetration measured from internal elastic lamina to maximum signal, normalized to medial thickness. Liquid sirolimus/paclitaxel delivery: Solid sirolimus and paclitaxel were dissolved in ethanol at 6 mg/ml; a commercial liquid paclitaxel (6 mg/ml in polyoxyethylated castor oil) was also used. Each drug was diluted to 2.4 mg/ml with saline and iohexol (2:1:2 by volume). Based on penetration testing, the OPC was selected for pharmacokinetic (PK) studies. Drugs were delivered for 2 min at 0.2–0.4 atm chamber pressure. After device removal, arteries were exposed to pulsatile flow. Tissues were harvested at 1 h and 24 h for PK analysis. For comparison, a sirolimus-coated balloon (SELUTION SLR, 1 µg/mm²) was deployed (balloon-to-artery ratio 1.1:1; 60 s inflation) with 1-h tissue harvest. Pharmacokinetics: Arterial segments were stored at −80°C and quantified for sirolimus and paclitaxel by validated HPLC–ESI–LC-MS/MS at an external bioanalytical lab (iC42 Clinical Research and Development). Statistics: Data reported as mean ± SD. Device comparisons: one-way ANOVA with Tukey post hoc. Tissue drug concentrations: one-way and two-factor ANOVA with replication (GraphPad Prism 9). Significance threshold p ≤ 0.05.

- Penetration depth (Flutax-1; n=3/device): OPC achieved the deepest average penetration into the medial wall: 233.65 ± 160.80 µm; TAPAS 127.22 ± 67.76 µm; ClearWay 117.55 ± 76.87 µm; Bullfrog 2.12 ± 3.78 µm. Trend toward differences between devices (p = 0.098); trend for OPC > Bullfrog (Tukey p = 0.069). - Liquid sirolimus delivery with OPC under flow: Arterial tissue retained 5.17 ± 4.48 ng/mg at 1 h and 0.78 ± 0.55 ng/mg at 24 h (−84.86%, p = 0.10). - Comparators with OPC: Dissolved paclitaxel 18.80 ± 25.61 ng/mg (1 h) and 1.77 ± 2.41 ng/mg (24 h; −90.6%, p = 0.23). Liquid paclitaxel 9.87 ± 6.28 ng/mg (1 h) and 3.06 ± 2.50 ng/mg (24 h; −69.02%, p = 0.09). No significant differences among groups at either time (p = 0.48 at 1 h; p = 0.33 at 24 h). - Versus sirolimus-coated balloon (SCB): At 1 h, liquid sirolimus levels exceeded those from a SCB (SELUTION SLR) (5.17 ± 4.48 ng/mg vs 0.0106 ± 0.002 ng/mg; p = 0.1096).

This study demonstrates feasibility of delivering liquid sirolimus directly to the arterial medial layer using a liquid delivery catheter. Among four tested LDD devices, the Occlusion Perfusion Catheter (OPC) provided the greatest penetration depth of a fluorescent model drug, likely due to its design enabling isolated, pressurized delivery and real-time pressure monitoring within the treatment chamber. Pharmacokinetic analyses under physiologically relevant flow showed that liquid sirolimus can be retained within arterial tissue for at least 24 hours, with early concentrations above the commonly cited therapeutic threshold (~1 ng/mg) and comparable to liquid and dissolved paclitaxel delivery via the same device. Notably, liquid sirolimus achieved considerably higher 1-hour tissue levels than a commercially available sirolimus-coated balloon in the ex vivo system, highlighting a potential advantage of pressurized liquid delivery for rapid intramural uptake and reduced particulate embolization risk. Given concerns about paclitaxel safety in PAD and mechanistic advantages of sirolimus (cytostatic, anti-inflammatory), liquid sirolimus delivery may offer an alternative local therapy platform. Future optimization (e.g., excipients to enhance lipophilicity and retention) may further improve pharmacokinetics and therapeutic efficacy.

This work provides the first evidence that liquid sirolimus can be locally delivered into the arterial wall using an LDD catheter. The OPC achieved the deepest drug penetration among four evaluated devices. Liquid sirolimus delivered with the OPC produced 1-hour tissue concentrations similar to liquid/dissolved paclitaxel and markedly higher than a sirolimus-coated balloon in the ex vivo model. Liquid sirolimus delivery may represent an innovative approach for PAD treatment amid safety concerns with paclitaxel-coated devices. Further studies should evaluate excipient strategies to enhance sirolimus retention and test efficacy in inhibiting neointimal growth and restenosis in vivo.

- Ex vivo benchtop model with healthy porcine carotid arteries lacking disease features (intimal thickening, fibrosis, calcification), branching, and bifurcations typical of clinical PAD, which may affect penetration and diffusion. - Absence of whole blood in the circuit may influence delivery and retention, particularly in longer-duration studies. - Small sample sizes for device comparison (n=3/device) limited statistical power; several findings showed trends but did not reach significance. - Only short-term pharmacokinetics (1 h and 24 h) were assessed; longer-term retention and biological efficacy were not evaluated.