Pharmacodynamics and Cardiopulmonary Side Effects of CW002, a Cysteine-reversible Neuromuscular Blocking Drug in Dogs

• ❖ CW002, a nondepolarizing neuromuscular blocker, can be reversed by intravenous cysteine

• ❖ Many novel neuromuscular blockers cause histamine release or cardiovascular complications

• ❖ In dogs, CW002 did not consistently cause histamine release and reduced blood pressure and cardiac output only at 25 times or greater the ED⁹⁵dose for neuromuscular blockade

• ❖ Further development of this rapid acting and rapidly reversible neuromuscular blocker is warranted

RECENT data indicate that the nondepolarizing neuromuscular blocking drug gantacurium exhibits an ultra-short duration of action largely dependent on the rate at which endogenous cysteine binds to and inactivates the molecule.1–3The novel pharmacology of gantacurium has led to development of the nonhalogenated, symmetrical, benzylisoquinolinium fumarate diester compound CW002 (previously known as AV002), a drug that interacts more slowly with endogenous cysteine to produce a longer duration of action4but can be rapidly reversed by injection of exogenous cysteine, even in the absence of spontaneous muscle recovery.4,5Accordingly, CW002 may represent a neuromuscular blocking drug with a duration that can be easily tailored to clinical needs.

A limitation to the clinical use of many novel neuromuscular blocking drugs has been an unfavorable cardiopulmonary response profile manifest as histamine release, hypotension, either primary (ganglionic blockade) or secondary (reflex) tachycardia, and/or bronchospasm.6–8Previous investigation in dogs has demonstrated that gantacurium does not elicit any effect on peak inspiratory pressure or lung compliance but will induce histamine release and a decrease in arterial blood pressure when doses in excess of 25 times of that required to produce a 95% neuromuscular blockade (ED⁹⁵) are administered.9The current study was designed to characterize the pharmacodynamic profile of CW002 in dogs and to determine the effect of incrementally larger doses of CW002 on hemodynamics and ventilatory dynamics in the context of simultaneous changes in plasma histamine.

Eight adult beagles weighing between 9.0 and 13.0 kg were used for the study after approval of the protocol by the Animal Care and Use Committee of Weill Cornell Medical College (New York, NY). Each animal underwent two procedures: the first to estimate the ED⁹⁵for CW002 and the second, 5–7 days later, to evaluate cardiopulmonary side effects after administration of incrementally larger doses administered as multiples of the ED⁹⁵. The experimental protocol is summarized in figure 1.

The chemical structure of CW002 is shown in figure 2. Compound used for the study was synthesized by Cedarburg Pharmaceuticals (Grafton, WI) with a purity of 95%. CW002, in powder form, was dissolved in 0.9% saline to a concentration of 1 mg/ml.

After an overnight fast and sedation with subcutaneous acepromazine (0.1 mg/kg), anesthesia was induced with 5 mg/kg intravenous propofol, the trachea was intubated, and the lungs were ventilated with a mixture of 30% oxygen, 70% nitrous oxide, and 1.0–1.5% isoflurane. Inspiratory pressures, volumes, and flow rates were continuously monitored via  side-stream spirometry, and inspired and expired gas composition was assessed with infrared analysis (Datex Ultima, Helsinki, Finland). From ventilatory pressures and volumes, pulmonary compliance was calculated on a breath-to-breath basis. Arterial oxygen saturation was continuously measured using oximetry, minute ventilation was adjusted to maintain an end-tidal Pco²of 30–32 mmHg, and body temperature was maintained at approximately 37.5°C with a water-circulating heating blanket. After placement of electrocardiogram leads, the right femoral artery was cannulated with a 22-gauge catheter for measurement of systemic arterial pressure, and a suture loop was placed around a superficial segment of tendon attached to the left tibialis anterior muscle. The tendon loop was attached to a force transducer (FT10 C; Grass Instruments, Quincy, MA), preloaded with 50 g tension, and twitch responses elicited by supramaximal train-of-four electrical stimulation of the peroneal nerve were recorded every 12 s. After preparation, a 15- to 20-min stabilization period was allowed before initiating the protocol. During this time, end-tidal isoflurane concentration was allowed to equilibrate at approximately 1.3%, and consistency of twitch height was confirmed. Potency of CW002 for producing neuromuscular blockade in each animal was then assessed by bolus administration of multiple doses starting at 0.001 mg/kg. An interval of 60 min was allowed between doses with full recovery of twitch height and verification of normal train-of-four stimulation. For each animal, three doses producing between 2 and 99% block were fit to a log-logit model to estimate individual ED⁹⁵. Finally, to confirm cysteine reversibility, in four dogs, a dose reflecting 3 × ED⁹⁵was administered, followed 1 min later by injection of 50 mg/kg cysteine prepared as the l-cysteine base (Sigma Chemical Company, St. Louis, MO) dissolved in 0.9% saline to a concentration of 100 mg/kg (pH 5.5). After return of neuromuscular function, the animals were awakened, the trachea was extubated, and activity was monitored for clinical signs of recurrent muscle weakness over the next 2 h.

After induction of anesthesia and preparation as described earlier for determination of ED⁹⁵, an additional arterial catheter was inserted for blood sampling. In six of eight dogs, a 5.0-French pulmonary artery catheter was also positioned via  the right external jugular vein, and a sternotomy was performed. In these animals, an electromagnetic flow probe (Carolina Medical Instruments, King, SC) was placed around the ascending aorta for continuous blood flow measurement and a conductance/micromanometer catheter (Millar Medical Instruments, Dallas, TX) was inserted into the left ventricle (LV) via  an apical stab for continuous measurement of pressure and volume. Offset of the volume signal (parallel conductance) was determined by injection of 5 ml 3% saline via  the pulmonary artery catheter, and stroke volume measurements were cross-calibrated with the electromagnetic flow probe. During the procedure, estimated fluid deficits were replaced with lactated Ringer's solution, which was subsequently maintained at a rate of 5–7 ml · kg⁻¹· h⁻¹throughout the experiment.

After a 15-min stabilization period, a CW002 dose reflecting 3 × ED⁹⁵for that animal was administered as an intravenous bolus. After complete spontaneous recovery of twitch height and a normal train-of-four response, thus allowing for comparison to the time course for cysteine reversal of the same dose in the initial experiment, the cardiopulmonary side effects of incrementally larger boluses of CW002 were determined by first injecting 6.25 × ED⁹⁵and then doubling the dose every 15 min up to a dose of at least 100 × ED⁹⁵. During the incremental CW002 dosing, arterial blood samples were obtained for histamine analysis (immunoassay kit, Immunotech International, Marseille, France) before and 1 min after each dose, and hemodynamic effects were assessed from peak changes that occurred during the 3 min after drug injection. Hemodynamic data were recorded in both analog and digital format. From direct measurements, LV ejection fraction was calculated, the first derivative of LV pressure increase (dP/dt) was normalized to end-diastolic volume as a load-corrected index of LV contractility, and the time constant of isovolumic relaxation (τ) was calculated with the assumption of a nonzero asymptote as an index of lusitropy.

For the 3 × ED⁹⁵dose of CW002 in each dog, the time to 5, 25, 75, and 95% recovery of first twitch was determined, with the 95% value regarded as duration of neuromuscular blockade. To verify cysteine reversibility, the time to 95% recovery of first twitch after 3 × ED⁹⁵CW002 doses given alone or followed after 1 min by cysteine administration (n = 4) was compared by paired t  test. Alterations in baseline (preinjection) cardiopulmonary variables over time along with changes from baseline after each CW002 dose (postinjection) were assessed by two-way ANOVA for repeated measures and the Holm-Sidak test when appropriate. Correlation between changes in plasma histamine concentration and changes in blood pressure was assessed by linear regression analysis. Data are presented as mean ± SD, with a P  value of less than or equal to 0.05 considered significant.

In individual animals, estimated ED⁹⁵ranged from 0.006–0.012 mg/kg. Pooled dose response data subjected to log-logit analysis are shown in figure 3and demonstrate an ED⁹⁵of 0.009 mg/kg, with a 95% CI of 0.008–0.11. On average, 3 × ED⁹⁵CW002 had an onset of 2.6 ± 0.9 min and a duration of 47 ± 9 min. A bolus dose of 50 mg/kg cysteine administered 1 min after injection of 3 × ED⁹⁵CW002 rapidly facilitated the return of neuromuscular function, with 95% twitch recovery evident within 3.7 ± 0.6 min.

Throughout the incremental bolus dosing protocol, end-tidal carbon dioxide, isoflurane concentration, and body temperature remained constant. As shown in figure 4, there were no differences among any of the preinjection baseline values for heart rate, mean arterial pressure (MAP), mean pulmonary arterial pressure, or cardiac output. For MAP, CW002 produced a small but significant reduction using repeated-measures analysis at 25 × ED^95.This effect became progressively larger with subsequent doses. Dose-related reductions were also evident in heart rate and cardiac output starting at 50 × ED⁹⁵and mean pulmonary arterial pressure at 100 × ED⁹⁵. Similarly, systemic vascular resistance decreased with increasing CW002 dose (fig. 4), but there were no attendant changes in pulmonary vascular resistance, peak inspiratory pressure, or lung compliance.

The cardiac responses to incremental doses of CW002 are shown in figure 5. There were no changes in preinjection baseline values during the dosing interval. The drug did not significantly alter LV end-diastolic volume, stroke volume, or ejection fraction at any dose but did decrease dP/dt and dP/dt normalized to end-diastolic volume, consistent with reduced inotropy and increased τ, indicative of negative lusitropy, at doses of 50 × ED⁹⁵and higher. However, although consistent, changes in inotropy and lusitropy were relatively small.

In that ED⁹⁵exhibited a 2-fold range, there was a substantial variation in the absolute CW002 dose in milligrams per kilogram administered for each multiple of ED⁹⁵. This allowed for plotting the relationships between a wide range of doses and changes in MAP and plasma histamine. As shown in figure 6, regression of the CW002 dose–MAP response relationship estimates that 0.84 mg/kg will produce a 20% decrease in MAP, yielding a safety ratio for this endpoint in the isoflurane-anesthetized dog of 0.84 mg/kg or 93 × ED⁹⁵. In contrast to the direct relationship between the dose and a decrease in MAP, a similar pattern was not evident for histamine release. As depicted in figure 7, over the dose range examined, CW002 elicited an increase in plasma histamine for only two dogs. As such, a safety ratio for histamine release could not be determined. Consistent with the nonuniformity of histamine release is the lack of correlation between changes in MAP and plasma histamine for most dogs (fig. 7). In contrast, there was a correlation between blood pressure change and the increase in plasma histamine concentration in the two dogs that did exhibit release.

CW002 is a nonhalogenated, symmetrical, benzylisoquinolinium fumarate diester compound with a duration of action largely dependent on the rate at which endogenous cysteine adducts to the molecule.4,5Preliminary data indicate that CW002 (previously known as AV002) produces neuromuscular blockade of intermediate duration in monkeys and cats.4The current study demonstrates that CW002 is also a potent neuromuscular blocking drug in dogs (ED⁹⁵, 0.009 mg/kg), with 3 × ED⁹⁵having an onset and duration of 2.6 ± 0.9 and 47 ± 9 min, respectively. Consistent with molecular inactivation by cysteine, the duration of CW002 can be markedly shortened by intravenous injection of cysteine, with 50 mg/kg administered 1 min after a 3 × ED⁹⁵dose decreasing duration to 3.7 ± 0.6 min.

In that clinical use of many novel neuromuscular blocking drugs has been precluded by an unfavorable cardiopulmonary response profile,6–8the current study was designed to define the effect of incrementally larger doses of CW002 on hemodynamics, ventilation pressures, and pulmonary compliance. Our data indicate that when given at supratherapeutic doses, CW002 produces dose-related effects on hemodynamics, particularly systemic blood pressure. By using a repeated-measures statistical model, even small changes can be significant when they occur consistently from animal to animal, and this was evident from the fact that an average decrease in MAP of only 4 mmHg after administration of 25 × ED⁹⁵was statistically significant. Nonetheless, even at doses as high as 100 × ED⁹⁵, the cardiovascular responses to CW002 are relatively modest, reflecting, on average, changes less than or equal to 20%. Furthermore, there was no evidence of bronchoconstriction at any dose.

In contrast to gantacurium, another drug inactivated by cysteine adduction, the cardiovascular response to high doses of CW002 does not seem to be primarily mediated by histamine release; although dose-related changes in MAP occurred in all animals, increases in plasma histamine concentration did not. In addition, although high-dose gantacurium has little effect on heart rate, cardiac output, or myocardial contractility in beagles,10high-dose CW002 decreased these variables. Accordingly, although the mechanisms behind the hemodynamic effects of CW002 remain unclear, the relationship between changes in blood pressure, blood flow, and cardiac performance at high doses suggests that the response is multifactorial.

Although our data clearly show that the duration of CW002 can be markedly shortened by administration of intravenous cysteine, the study provides little information regarding the cysteine–CW002 interaction or metabolism of the resulting adduction product. Preliminary data from ongoing pharmacokinetic and toxicology studies indicate that at pH 7.4 and temperature of 37°C, cysteine interacts nonenzymatically with CW002 to produce an adduction product (NB 1043–10) that is 60-fold less potent than the parent molecule as a neuromuscular blocking drug.4,10Formation of the adduct rapidly occurs in the presence of a slight excess of cysteine in vitro  and seems to be relatively unstable under physiologic conditions, undergoing alkaline hydrolysis to two charged, monoquaternary fragments that are probably excreted via  urine and bile (John J. Savarese, M.D., Department of Anesthesiology, Weill Medical College of Cornell University, New York, NY, May, 2009, unpublished toxicokinetic data). Importantly, cysteine adduction permanently changes CW002 such that there is no potential for cysteine to dissociate and “reactivate” the molecule. These in vitro  findings are consistent with in vivo  observations from the current study, indicating no return of neuromuscular blockade after cysteine reversal of CW002.

Results of the study need to be interpreted in the context of several limitations. First, CW002 is roughly five times more potent in dogs than in nonhuman primates,4an animal model that may be more appropriate for predicting potency in human. Although the precise reason for this difference in potency is unclear, it is probably not a reflection of species differences in the availability of endogenous cysteine given that the potency of gantacurium is similar in both animal models.2,10Nonetheless, the fact that a considerably larger dose of CW002 is required to produce the same effect in nonhuman primates raises the question of whether incremental dosing with multiples of the ED⁹⁵in this animal model would reveal more pronounced cardiovascular effects and histamine release. Accordingly, extrapolation of findings from the current study to predict patient responses to CW002 should be performed cautiously, particularly in light of the supervening clinical influences of advanced age, cardiopulmonary morbidity, and concomitant pharmacotherapy. Second, aspects of the experimental design could have influenced the conclusions of the study. For example, whether the pharmacodynamics of CW002 can be influenced by anesthetic technique remains unknown. Furthermore, given that repeated stimulation of mast cells can lead to depletion of histamine stores, it is possible that high doses of CW002 given as a first dose would have produced a greater increase in plasma histamine in animals that were “releasers” and potentially a demonstrable increase in those that were found to be “nonreleasers” in response to incremental dosing. Finally, although the study confirms that CW002 is reversible by cysteine in dogs, it does not specifically address dose–response relationships or cysteine side effects. Based on previous investigation,10a dose of 50 mg/kg was chosen for the current study, because this amount was maximally effective in nonhuman primates. However, preliminary data suggest that lower doses are also effective in the dogs5and may be more clinically attractive given that high-dose cysteine has been reported to elicit a sustained pressor response in dogs and adverse central nervous system effects in rodents.10 

In summary, our data indicate that CW002 is a potent, cysteine-reversible neuromuscular blocking drug with no apparent cardiopulmonary effects when administered at clinically relevant doses in male beagles. At high doses (> 25 × ED⁹⁵), the compound can produce modest, transient, hemodynamic change that is not associated with any alteration in respiratory mechanics or consistent histamine release. These data suggest that CW002 may represent a worthwhile target for clinical development.