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Keywords:

  • lipid emulsion;
  • clomipramine;
  • resuscitation;
  • pharmacokinetics

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Limitations
  7. Conclusions
  8. Acknowledgments
  9. References

Objectives:  Drug sequestration to an expanded plasma lipid phase has been proposed as a potential mechanism of action for lipid emulsions in lipophilic cardiotoxin overdose. The authors set out to document plasma and peritoneal diasylate clomipramine concentration after resuscitation with lipid emulsion in a rabbit model of clomipramine-induced hypotension.

Methods:  Twenty sedated mechanically ventilated New Zealand White rabbits were allocated to receive either 12 mL/kg 20% Intralipid or 12 mL/kg saline solution, following clomipramine infusion to 50% baseline mean arterial pressure (MAP). Hemodynamic parameters and serum clomipramine concentration were determined to 59 minutes. Peritoneal dialysis with 20% Intralipid or saline solution was evaluated for clomipramine concentration.

Results:  Mean arterial pressure was greater in lipid-treated animals as assessed by repeated-measures analysis of variance (F[1,14] = 6.84; p = 0.020). Lipid infusion was associated with elevated plasma clomipramine concentration and reduced initial volume of distribution (Vd; 5.7 [±1.6] L/kg lipid vs. 15.9 [±7.2] L/kg saline; p = 0.0001). Peritoneal diasylate clomipramine concentration was greater in lipid-treated animals (366.2 [±186.2] μg/L lipid vs. 37.7 [±13.8] μg/L saline; p = 0.002).

Conclusions:  Amelioration of clomipramine-induced hypotension with lipid infusion is associated with reduced initial Vd and elevated plasma clomipramine concentration consistent with intravascular drug–lipid sequestration. Concomitant peritoneal dialysis with lipid emulsion enhances clomipramine extraction.

Augmentation of conventional resuscitative efforts with lipid infusion has resulted in successful outcome in case reports of inadvertent local anesthetic toxicity1–4 and intentional combined bupropion/lamotrigine overdose.5 These follow pioneering work by Weinberg and others demonstrating efficacy for lipid emulsion in rat6 and dog7 models of bupivacaine-induced cardiovascular collapse. Lipid emulsion has furthermore been reported as effective in promoting recovery from verapamil8 and propranolol9 intoxication in animal models.

The exact mechanism of the antidotal action of lipid emulsions remains to be elucidated. Three potentially complementary explanations encompassing both pharmacokinetic and pharmacodynamic mechanisms have been forwarded. The first proposes preferential sequestration of lipophilic compounds into a newly created intravascular lipid compartment [sink]. Reestablishment of drug equilibrium in an expanded lipid phase results in lesser free drug concentration available to tissues.6,7 The second hypothesis suggests augmentation of cardiac performance through provision of preferential myocyte energy substrates, with bolus lipid administration serving to overcome pharmacologic impediments to free fatty acid oxidation.4,5,10 Finally, lipid emulsion may restore myocardial function by increasing intracellular calcium concentration.11

We have previously demonstrated accelerated recovery from clomipramine-induced hypotension, electrocardiogram (ECG) QRS prolongation, and profound cardiovascular collapse with infusion of lipid emulsion in whole rabbits.12 The beneficial effect observed with lipid infusion was postulated in part as secondary to substantial drug–lipid sequestration; however, no pharmacokinetic measures were undertaken. Greater understanding of the beneficial mechanism of action for lipid in this setting would serve to inform future study and guide clinical application.

This experiment was conducted to examine the proposed beneficial mechanism of action for lipid emulsion in lipophilic drug toxicity. Two related hypotheses were explored. First, we postulated elevated plasma drug concentration following infusion of lipid emulsion in lipophilic drug intoxication, consistent with drug sequestration to an expanded lipid phase. Second, we proposed augmented extraction of lipophilic drug from the resultant expanded plasma lipid phase with extracorporeal means of concomitant lipophilicity. Specifically, we sought to document pharmacokinetic parameters following resuscitation with intravenous (IV) lipid emulsion, and peritoneal dialysis with lipid emulsion, in a rabbit model of clomipramine-induced hypotension

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Limitations
  7. Conclusions
  8. Acknowledgments
  9. References

Study Design

The study was performed at the Ruakura Animal Research Facility, Hamilton, New Zealand. All study protocols were approved by the Ruakura Animal Ethics Committee. Animal care and husbandry were in accord with institutional ethical guidelines.

Animal Subjects

Adult New Zealand White rabbits of mixed gender and weighing between 2.0 and 2.7 kg were sedated with 50 mg/kg ketamine and 4 mg/kg xylazine by intramuscular injection and placed on a surgical board. Three-lead electrocardiography was instituted and venous catheterization of the marginal vein of the ear was performed. Anesthesia was continued with ketamine/xylazine infusion (10 and 1.5 mg/mL) at 1 mL/kg/hour. Arterial catheterization of the central artery of the contralateral ear was connected in standard fashion (pressure transducer, Edwards Lifesciences, Irvine, CA) to a Hewlett-Packard 78834A neonatal monitor (Hewlett-Packard, Palo Alto, CA). Tracheostomy was performed (truncated [5-cm] 3.5-mm endotracheal tube) and mechanical ventilation instituted with inspiratory flow rate of 0.25 L/second 100% oxygen at 25 breaths/minute via Nuffield series 200 pediatric ventilator (Penlon Ltd, Abington, England). Inspiratory:expiratory ratio was set at 1:2. Ventilatory adequacy was assessed by colorimetric capnography with maintenance of end-tidal carbon dioxide in the 2%–5% range. Vecuronium at 0.1 mg/kg was administered after establishment of mechanical ventilation and repeated as necessary to maintain paralysis. At the completion of the study protocol, all animals were euthanized with IV bolus injection of 3 mL pentobarbitone at 300 mg/mL.

Study Protocol

Clomipramine Infusion and Resuscitation Protocol.  On completion of surgical interventions, a period of stabilization (range = 5–7 minutes) was allowed. Clomipramine infusion was commenced at Time 0 at 240 mg/hr (8 mg/mL at 30 mL/hour) and continued to a target mean arterial pressure (MAP) of 50% baseline (median time 5 minutes). Study agents were then delivered by manual IV push over a period of 4 minutes (time = 5–9 minutes) timed by stop-clock. Control animals received 12 mL/kg 0.9% saline solution, and test animals 12 mL/kg 20% Intralipid based on our previous experiment.12 All solutions were warmed to 37°C prior to utilization.

Intraperitoneal Protocol.  At 35 minutes, a custom manufactured peritoneal dialysis catheter was introduced into the abdominal cavity under aseptic technique and positioned in the right iliac fossa. Peritoneal dialysis was instituted at 39 minutes with control animals receiving 10 mL/kg 0.9% saline solution and animals treated with IV lipid receiving 10 mL/kg 20% Intralipid over a 1-minute period. The abdomen was manually agitated following instillation, and at 5-minute intervals thereafter, to avoid pooling. Peritoneal diasylate was aspirated at 59 minutes, affording a 20-minute dwell interval.

Hemodynamic metrics (pulse rate, systolic and diastolic blood pressure, MAP) were collected at baseline, 25% MAP reduction, target MAP (50% baseline MAP), 7 and 9 minutes (representing midway and completion of IV rescue), and then at 2.5-minute intervals to 19 minutes and 5-minute intervals to 59 minutes. Blood samples (3 mL) were drawn via arterial catheter at 25% MAP reduction; target MAP; and 7, 9, 19, 39, and 59 minutes. Plasma was separated by centrifugation at 2,325 g for 10 minutes and refrigerated prior to analysis for clomipramine/desmethylclomipramine concentration. Peritoneal diasylate was likewise forwarded for clomipramine assay.

Clomipramine/Desmethylclomipramine Biochemical Analysis.  Total plasma clomipramine and desmethylclomipramine concentrations were determined by high-performance gas chromatography with mass selection following initial extraction into heptane-isoamyl alcohol at basic pH, back extraction into an acidic aqueous solution, and then reextraction into toluene-isoamyl alcohol according to the method described by Sioufi et al.13 This methodology has proven accurate in determining clomipramine concentrations in tissues of high adiposity/lipemia.14 Trimipramine was furthermore incorporated prior to initial extraction as an internal standard.

Pharmacokinetic Analysis.  Individual clomipramine plasma concentration–time curves were analyzed by the noncompartmental method. Each curve was initially evaluated by computerized curve stripping techniques, followed by nonlinear least squares regression using PK Solutions 2.0 software (Summit Research Services, Montrose, CO) to obtain kinetic parameters (initial volume of distribution [Vd], mean resistance time [MRT], elimination half-life [t½], and clearance [CL]).

Data Analysis

Power analysis was based on the results of our previous work in rabbits and sought a difference in MAP equivalent to 1 standard deviation (SD) at baseline (equal to 1.5 previously documented SDs at toxicity12) at 5 minutes postresuscitation. This yielded a sample size of n = 10 animals in each group, with power set at 80% and significance criteria set at 0.05. A physiologic rather than pharmacokinetic endpoint was used in sample size estimation, given both anticipated exponential relationships between drug concentrations and effect and the study goal to examine drug kinetics contemporaneously with hemodynamic recovery. Employing a pharmacologic parameter may have additionally resulted in a Type II error on physiologic variables.

Statistical analysis of all variables was performed with SPSS for Windows (Version 10.0, SPSS, Chicago, IL). Hemodynamic parameters and plasma clomipramine concentration were evaluated as prospective a priori endpoints. Rate of change in MAP (computed over the preceding monitoring interval) and increment in MAP following peritoneal dialysis (MAP 44 min less MAP 39 min) were examined as post hoc variables of interest. Comparison between lipid and saline groups of sequential hemodynamic metrics was conducted using two-way repeated-measures analysis of variance. The distribution of quantitative variables was examined to detect significant departure from normality by the Shapiro-Wilks test. Two-tailed Student’s t-test was used to determine statistical significance between groups. The critical p-value retained for significance was 0.05.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Limitations
  7. Conclusions
  8. Acknowledgments
  9. References

No difference was observed in baseline hemodynamic parameters (pulse rate, systolic and diastolic blood pressures, MAP) for the two groups. MAP was greater after resuscitation in the lipid-treated group (F[1,14] = 6.84; p = 0.02; Figure 1). The greatest MAP differential was observed immediately following lipid administration (mean difference [±SD] MAP 10.53 mm Hg, 95% confidence interval [CI] for difference = 1.78 to 19.28 mm Hg; and 12.79 mm Hg, 95% CI difference = 2.77 to 22.81 mm Hg at 7 and 9 minutes, respectively). Rate of change in MAP was greater in the Intralipid-treated group at 7 minutes (12.3 [±4.4] mm Hg/min Intralipid vs. 6.17 [±2.6] mm Hg/min saline; p = 0.006) only. A trend toward greater MAP increment following peritoneal diasylate administration was observed (mean difference ∂MAP 13.07 mm Hg; 95% CI difference = −4.96 to 31.1 mm Hg; p = 0.083). No statistically significant difference in pulse rate was observed between groups.

image

Figure 1.  Mean arterial pressure (MAP) vs. time: Intralipid- and saline-treated groupings. Data presented as mean ± SE.

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Clomipramine dosing and estimated pharmacokinetic parameters utilizing the noncompartmental method are shown in Table 1. Individual plots of plasma clomipramine concentration versus time for saline- and lipid-treated animals are presented in Figures 2 and 3, respectively. All plasma desmethylclomipramine levels, performed concurrently with clomipramine assay, were less than or equal to 15 μg/L (the lowest limit of assay sensitivity) and therefore reported as equal. Clomipramine concentration in peritoneal diasylate and clomipramine extraction ratio [peritoneal diasylate clomipramine concentration:plasma clomipramine concentration before dialysis] were greater in the lipid-treated group (Table 2).

Table 1.   Plasma Noncompartmental Pharmacokinetics of Clomipramine following Infusion of 0.9% Saline Solution or 20% Intralipid in Rabbits
 Saline (n = 10) Lipid (n = 10) p-Value
  1. Data are presented as mean ± SD.

  2. CL = clearance; MRT = mean resistance time; t½ = elimination half-life; Vd = volume of distribution.

Gender (male:female)6:45:50.653
Weight (g)2294 ± 191 2308 ± 271 0.902
Clomipramine dosing (mg/kg) 9.5 ± 3.7 8.8 ± 4.0 0.693
Vd (L/kg) 15.9 ± 7.2 5.7 ± 1.6 0.0001
MRT (min)40.1 ± 16.8 20.3 ± 9.6 0.005
t½ (min)39.4 ± 17.0 18.5 ± 7.8 0.003
CL (L/min/kg) 0.283 ± 0.057 0.250 ± 0.132 0.489
image

Figure 2.  Individual clomipramine vs. time plots: saline group.

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image

Figure 3.  Individual clomipramine vs. time plots: Intralipid group.

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Table 2.   Peritoneal Diasylate Clomipramine Concentration and Extraction Ratio
 Saline (n = 10) Lipid (n = 10) p-Value
  1. Data presented mean ± SD.

  2. ER = extraction ratio; PD = peritoneal diasylate.

Plasma clomipramine (μg/L) T39min 211.5 ± 72.1 196.5 ± 191.0 0.840
PD clomipramine (μg/L) 37.7 ± 13.8 366.2 ± 186.2 0.002
ER clomipramine0.20 ± 0.007 3.19 ± 2.38 0.009

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Limitations
  7. Conclusions
  8. Acknowledgments
  9. References

In this rabbit model, infusion of lipid emulsion resulted in more rapid and complete resolution of clomipramine-induced hypotension compared to saline control. Improved hemodynamic metrics were associated with reduced initial volume of clomipramine distribution and elevated total plasma clomipramine concentration. These findings support the forwarded hypothesis of clomipramine partitioning into triglyceride droplets of Intralipid circulating in plasma, thereby decreasing tissue drug concentration and manifest toxicity.

Previous investigators have demonstrated altered pharmacokinetic parameters when drugs of high lipophilicity are formulated in lipid carriers or coinfused with lipid emulsions.15,16 Similarly, propofol, a highly lipophilic anesthetic, exhibits greater Vdcentral and Vdsteady state and dissimilar evanescence when delivered in a lipid-free carrier,17 indicating that formulation can profoundly influence clinical response characteristics. In contrast, our results indicate altered clomipramine pharmacokinetics favoring substantial drug–lipid sequestration when lipid is infused after the point of attaining toxicity. The associated hemodynamic improvement suggests a direct role for lipid in amelioration of myocardial clomipramine intoxication.

The function of lipid in augmenting myocardial clomipramine washout has not been directly addressed in our study. This would require repeated myocardial biopsy or sampling of coronary sinus effluent and was not possible with the model employed. Results from previous work demonstrating normalization of clomipramine-induced ECG QRS prolongation with lipid application12 are, however, consistent with enhanced myocardial clomipramine elimination. Additional support for drug–lipid sequestration may be drawn from in vitro experiments showing preferential movement of the similarly lipophilic agents bupivacaine18 and amitriptyline19 toward the lipid phase when added to plasma treated with lipid emulsion. Additionally, enhanced myocardial bupivacaine18 and amitryptyline20 washout has been demonstrated from isolated rat hearts undergoing lipid augmented and phospholipid-based nanovesicle perfusion, respectively. Serum bupropion levels have furthermore been shown to correlate with triglyceride elevation following lipid injection in a patient resuscitated from combined enteric bupropion/lamotrigine overdose, suggesting intravascular drug sequestration in the clinical scenario of self-poisoning.5

The rapidity of hemodynamic recovery observed with lipid infusion in both laboratory and clinical reports nevertheless appears inconsistent with a solely pharmacokinetic mechanism of action, which inherently requires mass extraction of highly protein-bound drug from the myocardium. Potential synergistic mechanisms, including overcoming pharmacologic impediments to mitochondrial free fatty acid oxidation,4,5,10 and/or indirect elevation in myocyte calcium concentration,11 may be functioning in concert to effect observed improvements in cardiovascular performance.

We undertook peritoneal dialysis with lipid emulsion to explore the potential for enhancement of clomipramine elimination given the relative lack of efficacy of alternative extracorporeal techniques (hemodialysis, hemoperfusion). Our preliminary findings of increased clomipramine extraction in this context may portend development of lipid-based dialysis modalities for compounds not amenable to these standard techniques. The observed trend toward increased MAP increment with peritoneal instillation of lipid merits additional consideration. Intralipid may have proven noxious to viscera, provoking increased sympathetic outflow or splanchnic vasoconstriction with subsequent blood pressure elevation. Alternatively, peritoneal absorption of lipid21 may serve to augment similar mechanisms of action of IV bolus injection.

Limitations

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Limitations
  7. Conclusions
  8. Acknowledgments
  9. References

The main limitations of this study are inherent in the model of toxicity utilized. Bolus clomipramine infusion confers a dissimilar toxicity profile to enteric poisoning and, as such, extrapolation of our findings to the clinical scenario of tricyclic antidepressant overdose must proceed with some caution. Moreover, the limited duration of monitoring, albeit used to demonstrate the interval of greatest hemodynamic benefit, fails to allow for determination of whole body pharmacokinetic parameters. We have furthermore been unable to quantify any potential interaction between ketamine/xylazine utilized as sedation and the infused lipid, which may have contributed to improvement in hemodynamic metrics. Similar sedation regimes have, however, been used in animal models9,12 with minimal observed confounding.

Peritoneal dialysis with lipid emulsion was conducted in concert with IV lipid only. A more complete evaluation would require a crossover study design. The unanticipated trend toward elevation in MAP associated with peritoneal lipid instillation furthermore compounds interpretation of MAP beyond 39 minutes. Prior to this time point, MAP trajectory favored equilibration. Clearly further study is required to determine the duration of the beneficial effect of lipid and the utility of bolus versus bolus plus continual infusion administration protocols.

There is a growing body of experimental and clinical data supporting infusion of lipid emulsions in lipophilic drug toxidromes and drug-induced cardiac arrest, in addition to those of local anesthetics. The results of the present study support toxin–lipid sequestration as one potential mechanism for the observed benefits. Further study is indicated to explore postulated mechanisms of actions of lipid, optimal formulations, and dosing regimens.

Conclusions

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Limitations
  7. Conclusions
  8. Acknowledgments
  9. References

In this rabbit model, amelioration of clomipramine-induced hypotension with lipid infusion was associated with reduced Vd and elevated plasma clomipramine concentration, suggesting significant drug–lipid sequestration. Additional beneficial mechanisms of action are not excluded. Peritoneal dialysis with lipid emulsion resulted in increased clomipramine extraction, suggesting potential for therapeutic efficacy in lipophilic drug poisoning.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Limitations
  7. Conclusions
  8. Acknowledgments
  9. References

The authors thank the Waikato Medical Research Foundation for funding the present study and Mr. Ric Broadhurst for assistance in animal manipulations.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Limitations
  7. Conclusions
  8. Acknowledgments
  9. References