Perivascular Renal Denervation (PVRDTM): Chemical Renal Denervation with Micro-Doses of Ethanol Using the PeregrineTM Renal Denervation Device

Fig. 13.1

Schematic drawings showing sequence of chemical denervation with EtOH. The upper left panel shows the anatomy of the mid-portion of the renal artery and shows no significant organs in the vicinity of the very localized EtOH delivery. The middle panel shows the device deployed with micro-dosing of EtOH targeted to the adventitial space (blue halos in right panel)

The key concepts and rationale for this methodology to create renal denervation are: (1) to deliver a very small volume of a highly potent neurolytic agent (EtOH) precisely to the target area in the adventitia and the peri-adventitia; (2) to deliver the agent with such tiny (micro) needles such that even with full systemic heparin treatment there would be essentially no peri-arterial bleeding risk after the needle entry through the intima and media and into the adventitia, (3) to use an agent such as EtOH that is lipophilic and agrascopic, such that simultaneous injection from three needles placed in one step, at 120° needle separation radially around the renal artery would reproducibly create circumferential spread of the neurolytic agent, and confined to the adventitial space, and allow circumferential sympathetic nerve kill with minimal effects upon the intima and media of the renal artery (nerve kill without renal artery vessel wall injury); (4) to determine the needle depth and doses required to get “deep” sympathetic nerve kill (nerve injury out to 10–12 mm deep to intimal surface), which may be crucial in achieving reproducible and efficient sympathetic denervation; (5) to determine whether or not there is predictable and dose-dependent sympathetic denervation, as judged by the drop in renal parenchymal norepinephrine levels in a porcine model; (6) to determine the safety as judged by short-term (2 weeks) and longer term (3 month) histopathological and angiographic studies in a porcine model; (7) to determine whether or not this technology could be safely applied in clinical cases and finally; (8) to determine whether or not this procedure could have the potential to create renal denervation in humans without the pain that is associated with “thermal” renal denervation using either RF or ultrasound techniques.

Pre-clinical Testing

Extensive pre-clinical testing has now been completed in order to evaluate the safety and efficacy of chemical neurolysis, via adventitial injection of very small doses of dehydrated EtOH to as a means to perform sympathetic denervation, in a porcine model [19].

A novel, three needle-based delivery device, (Peregrine System™, Ablative Solutions, Inc., Menlo Park, CA) was introduced via the femoral artery into renal arteries of adult swine using fluoroscopic guidance. The drug injection catheter is an endovascular delivery catheter that contains three distal needles housed inside of individual guide tubes, which are contained within the catheter. The catheter has a steerable, radio-opaque 2 cm fixed, floppy guide-wire at its distal end to minimize renal artery trauma and allow steerability, when needed, into appropriate branch vessels (Figs. 13.1 and 13.2a–c).

Fig. 13.2

Panel (a) shows the device handle that controls the tubes, needles and injection port. In (b), the device is deployed and centered in a porcine renal artery during contrast injection from the guiding catheter. Black arrow shows tip of guide tube against the intima and orange arrow shows tip of radio-opaque 0.008″ injection needle. Panel (c ) show injection of ~0.2 ml of dilute contrast demonstrating injection 100 % limited to the adventitial space. The black arrow shows the appearance of a small volume of dilute contrast that is angiographically apparent in the adventitial and peri-adventitial layer of the renal artery after the purposeful injection of 0.30 ml of dilute contrast through the deployed Peregrine needles. In Panel (d) immediate necropsy is shown after injecting 0.15 ml EtOH combined with methylene blue to define the circumferential and very defined longitudinal (black arrows) spread of EtOH in the tunica adventitia

The animal studies were conducted under the general principles of Good Laboratory Practice (GLP) regulations as set forth in 21 CFR 58. Animals were pre-medicated with 325 mg of aspirin and 75 mg of clopidogrel by mouth once daily for 2 days before the procedure. The animals were assigned to study groups at random, before the procedure began.

The animals were pre-medicated with intramuscular injection of telazol combined with atropine. When recumbent, animals were anesthetized with a mixture of isoflurane and oxygen delivered via facemask. When sufficiently anesthetized the animals were intubated and connected to a closed-circuit anesthesia system and maintained on isoflurane combined with oxygen. Blood was collected for evaluation of hematology (CBC) and serum chemistry. Urine was obtained via cystocentesis.

After the animals were prepared for sterile surgery, one femoral artery was accessed using the Seldinger technique, and a seven French introducer was placed. Intravenous heparin was given in all animals to achieve an ACT of >250 s. In all cases the right and left renal arteries of the pig were engaged using a seven French RDC guiding catheter. Prior to ethanol or saline injections, angiography of each renal artery was performed using iodixanol contrast diluted by 25 % with normal saline.

The Peregrine™ device was advanced into the left or right renal artery via the guiding catheter. Once the operational section of the device was positioned within the target site in the mid-portion of the renal artery, the three guide tubes are deployed spatially at 120°, one to another (see Figs. 13.1 and 13.2b). The tubes were simultaneously deployed up against the intimal surface (Figs. 13.1 and 13.2b), using the advancement mechanism in the control handle (Fig. 13.2a). These atraumatic tubes have radiopaque distal tip markers such that one can clearly define the position of the tubes, particularly when contrast is injected via the guiding catheter (Fig. 13.2b).

Once deployed, the three tubes serve to reproducibly “center” the device within the renal artery. The 0.008″ needles that reside within the distal tip of the tubes are advanced to a depth of 3.5 ± 0.25 mm deep to the intima (i.e., beyond the tip of the guide tube). This function is also performed via the specialized handle, which allows simultaneous advancement of the three injection needles. These tiny needles are made radiopaque, so that they can be easily seen under fluoroscopy. Although not part of the clinical protocol, in animals dilute contrast can be injected once the needles are positioned to confirm placement relatively deep in the adventitial space (Fig. 13.2c).

It should be noted that these needles are the equivalent of a ~30 gauge needle so that they can be safely advanced through the renal arterial wall without causing bleeding. Prior to conducting this study we confirmed that needles of this size could be repeatedly advanced through the wall of the renal artery of pigs that had been pre-treated with high doses of heparin (ACTs 300–600), with no detectable bleeding at the needle puncture sites. This is a key observation relevant to the safety of this approach.

Once the tubes and needles are deployed it is easy to confirm, fluoroscopically, that the needle tips are well outside the luminal space and ~2.5–3.0 mm deep to the media and the external elastic lamina in a normal porcine renal artery (Fig. 13.2b). This corresponds to an injection depth that approximates the border between the renal artery adventitia and peri-adventitia, and which corresponds to a depth to the middle of the renal sympathetic nerve field, as defined in pressure-fixed human histopathological studies by Virmani et al. [18].

The successful deployment of the tubes and needles was confirmed by angiography. EtOH or saline (sham) fluid was then administered, using a 1.0 ml luer-lock syringe attached to the proximal injection lumen at the handle of the catheter. This injection lumen is in fluid continuity with the distal end of all three needles. The injection is performed over 15–20 s.

Three volumes of EtOH were used in this study: 0.15 ml/artery (n = 3 pigs/6 arteries), 0.30 ml/artery (n = 3 pigs/6 arteries) and 0.60 ml/artery (n = 3 pigs/6 arteries). A procedural control group was also studied using the injection of 0.4 ml of saline/artery (n = 3). This was a “sham” arm to control for nonspecific effects that might be caused by mechanical injury from either the guide tubes or the needles, and/or any non-specific effects of fluid delivery. Once the treatment agent was injected, the dead-space of the catheter was flushed with a very small volume of normal saline. After treatment of the first renal artery the device was removed from the animal, inspected and flushed. The contra-lateral renal artery was then engaged and the same fluid injection sequence was performed in the contralateral renal artery. After the treatment of the second renal artery, the animals were recovered and housed for restudy and sacrifice at 2 weeks post-intervention. The animals were treated with aspirin 162 mg/day for 7 days after intervention.

The circumferential spread of EtOH was evaluated in separate experiments by combining 0.125 ml of EtOH with 0.25 ml of methylene blue (stain). The volume of 0.15 ml was then injected under fluoroscopic guidance. Immediate necropsy was performed and demonstrated reproducible and circumferential spread of the 0.15 ml of the EtOH/methylene blue mixture (Fig. 13.2d). Histopathology was also used to evaluate circumferential spread of alcohol by having the pathologist evaluate and document the location (in terms of circumference) of any noted neuritis and neurolysis. The histopathological examination showed extensive and circumferential nerve injury at the 0.15, 0.30 and 0.60 ml EtOH injection volumes.

Safety and effectiveness of the device were evaluated. The efficacy of denervation was determined by measurement of renal parenchymal norepinephrine (NE) levels (analyzed by HPLC, with electrochemical detection), and using histopathologic evaluation of the peri-renal nerves at the end of the 2-week survival period (Fig. 13.3). Safety was evaluated by histopathologic evaluation of the renal artery and kidney as well as evaluation of clinical pathology. Blood and urine were collected in all animals treated with the device for evaluation of systemic and renal health at baseline and at the time of sacrifice.

Fig. 13.3

Bar graph showing the dose-response effect of adventitial EtOH delivery upon renal parenchymal norepinephrine level at 14 ± 3 days. There is a marked and dose-dependent reduction of NE levels versus both naive control animals and sham control animals injected with saline. Mean NE reduction was 54 % with 0.15 ml; 78 % with 0.30 ml and 88 % using 0.60 ml/artery. Standard deviation (SD) for each data set as shown. P values as shown

The animals were survived for 14 ± 3 days after treatment. At the end of the study period the animals were anesthetized and angiography of the treated right and left renal arteries was obtained to evaluate vessel patency and to look for any luminal narrowing compared to baseline angiography. Four additional animals were studied with follow-up with angiography and pathology at 3 months after ethanol denervation using 300 μL of EtOH (Figs. 13.4 and 13.5).

Fig. 13.4

Angiographic pictures and norepinephrine data from two pigs (upper and lower panel sets) at 3 month follow-up after 0.30 ml injection of EtOH in adventitial space by Peregrine^TM device. Left panels show renal angiogram prior to treatment. Middle angiogram shows Peregrine^TM device deployed during EtOH delivery and right angiographic panels show 3-month results with no evidence of any stenosis. In both of these kidneys there was an 88 % drop in renal parenchymal norepinephrine relative to untreated control kidneys (far right panels)

Fig. 13.5

Histopathology (H & E) of renal denervation at 30 days with 0.30 ml EtOH injection. The renal artery (intima and media) appears intact without evidence of injury or inflammation (black circle). The reaction to the EtOH appears quite limited to the adventitial layers. There is severe damage to the deep renal nerve bundles with vacuolization, nerve fiber disruption, and fibrosis of the perineural structures at a depth of 4–14 mm deep to the intima (blue hatched and red hatched boxes and magnified view from red box)

After angiographic follow-up at 14 days and at 3 months, a necropsy was performed. The renal arteries and kidneys were harvested for histopathological evaluation. Gross pathology to examine the status of the renal arteries was performed to look for renal artery abnormalities such as aneurysms, perforations, dissections, hematoma, etc., as well as inspection of the surrounding tissues for any abnormalities. The renal arteries and kidneys were harvested, retaining the peri-adventitial tissue around the artery. The renal artery tissue was embedded in paraffin using standard techniques. Tissue was stained with H&E. Multiple sites from each renal artery segment were labeled and sent for (blinded) microscopic evaluation by a board certified veterinary pathologist.

In all kidneys, four samples were obtained from random locations at each of the proximal, mid and distal regions of each kidney for a total of 12 samples/kidney. The tissue samples were weighed, placed in cryovials and flash frozen by immersion into dry ice. The frozen samples were then stored at −70 °C. They were sent in dry ice to an independent laboratory for (blinded) measurement of renal parenchymal norepinephrine levels.

Renal norepinephrine concentrations in the treated animals from this study were also compared to values from naïve control animals of the same age and species (n = 7) with renal tissue sampling performed in an identical fashion to the treated animals.

The safety of ethanol injection was also assessed in a separate nephrotoxicology study (n = 4). After deep engagement of the renal arteries 0.6 ml of EtOH was injected directly in both the right and left renal arteries (1.2 ml total EtOH/animal), over 20–30 s to replicate the timing of injection into the adventitial space when therapeutic EtOH neurolysis was performed. These animals had serial measurement of serum BUN, creatinine, electrolytes and body weight at days 1, 7 and at 30 days after the injection. Histopathological evaluation of the renal parenchyma was performed in all such treated kidneys at 30 days to look for any evidence of renal injury.

Additional, longer-term safety evaluation was obtained with angiographic follow-up at 90 days, in four additional animals (n = 8 arteries) treated with 300 μl (0.30 ml) EtOH. These studies were performed to look for any evidence of late renal artery stenosis (Fig. 13.4).

For statistical analysis, between-group comparisons were made using a Wilcoxon rank-sum test, performed in R (Version 2.14.1, Vienna, Austria). Data are shown in graphs as mean ± SD. A p value of <0.05 was considered significant.

Device success was defined as successful injection of the designated fluid without serious adverse events. The device was used successfully in all 16 animals and 32 renal arteries. Procedure time, measured from the advancement of the device into the renal artery, followed by deployment of needles, injection, and withdrawal back into the guiding catheter averaged approximately 90 s for each renal artery (range – 55–140 s). A small hematoma at the femoral access site was recorded in one animal. There was no other study-related morbidity or mortality.

At 2-weeks after ethanol-mediated renal denervation measurements of renal tissue NE showed an essentially linear dose response (R² = 0.95) between the EtOH volume delivered and the reduction of the renal parenchymal NE level (Fig. 13.3). The mean renal NE reductions were 54, 78 and 88 % at doses of 0.15 ml/artery, 0.30 ml/artery and 0.60 ml/artery, respectively (p < 0.0001 vs. combined controls; Fig. 13.3). The other statistical comparisons are shown in Fig. 13.3 and demonstrate a statistically significant reduction (p < 0.05) in renal parenchymal NE at all three doses, vs. sham controls.

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