intravenous lipid emulsion for the treatment of permethrin toxicosis in...

INTRAVENOUS LIPID EMULSION FOR THE TREATMENT OF

PERMETHRIN TOXICOSIS IN CATS

by

Rachel Elizabeth Peacock

BVSc MVS

This thesis is presented for the degree of

Research Masters with Training (Veterinary Medical Science) of Murdoch University

2014

i

I declare that this thesis is my own account of my research and contains as its main

content work which has not previously been submitted for a degree at any tertiary

institution.


ii

INTRAVENOUS LIPID EMULSION IN THE TREATMENT OF

PERMETHRIN TOXICOSIS IN CATS


Murdoch University, 2014

Abstract

Over the last decade, a growing interest has emerged in the use of intravenous lipid

emulsions in the treatment of lipophilic drug toxicoses. Initial interest in the therapy

was prompted by its successful use in rats and dogs for reversing the life-threatening

cardiovascular effects of local anaesthetic overdoses. A postulated mechanism of

action of intravenous lipid emulsion in lipophilic drug toxicoses is lipid partitioning,

which is creation of an intravascular lipid compartment into which lipophilic drugs may

be bound and sequestered away from their sites of action. Permethrin is a highly

lipophilic insecticide which can cause significant morbidity and mortality in cats

through its neuroexcitatory effects. Permethrin is a common ingredient in flea

treatments marketed for dogs and accidental administration to cats is common. The

aims of this study were to (1) determine if a lipid emulsion added to permethrin-

containing feline plasma in vitro would lead to a decrease in plasma permethrin

concentration thus supporting a lipid sink effect, and (2) assess the clinical response to

intravenous lipid emulsion administration in cats with permethrin toxicosis. In the in

vitro study, addition of a lipid emulsion to permethrin-containing feline plasma led to a

significant reduction in plasma permethrin concentration within 30 minutes. In the

clinical trial, there was a significant difference in the distribution of clinical stages over

time between treatment groups, with cats receiving 20% intravenous lipid emulsion

having lower clinical stages earlier than cats receiving the saline control. The results of

these studies support the use of intravenous lipid emulsion in the treatment of

permethrin toxicosis in cats and the in vitro study supports intravascular lipid

partitioning as a mechanism of action. Future research is needed to confirm lipid

partitioning as a mechanism of action of intravenous lipid emulsion in vivo for

lipophilic drug toxicoses, determine the metabolic fate of lipid sequestered drugs and

iii

ascertain adverse effects of intravenous lipid emulsion at the doses recommended for

drug toxicoses.

iv

Acknowledgments

The author would like to thank Associate Professor Robert Trengove and Mr Bruce

Peebles from the Murdoch University Separation Science Laboratory for their

contribution in determining the permethrin concentrations of the samples for the in

vitro study reported in this thesis, and Dr Katrin Swindells from Western Australian

Veterinary Emergency and Specialty for her input into study design for the clinical trial.

v

TABLE OF CONTENTS

Contents Chapter 1: Use of intravenous lipid emulsion for lipophilic drug toxicoses excluding local anaesthetics .............................................................................................................. 1

Background ................................................................................................................... 1

Method .......................................................................................................................... 1

Results ........................................................................................................................... 3

Use of intravenous lipid emulsion in specific toxicoses ............................................ 3

Proposed mechanisms of action of intravenous lipid emulsion for drug toxicoses ................................................................................................................................. 35

Intravenous lipid emulsion formulations ................................................................ 40

Intravenous lipid emulsion dose ............................................................................. 41

Adverse effects of intravenous lipid emulsions ...................................................... 42

Conclusions ................................................................................................................. 46

References ................................................................................................................... 46

Chapter 2: Permethrin toxicosis in cats .......................................................................... 53

Chapter 3: Summary and hypothesis for present study ................................................. 57

Chapter 4: Sequestration of permethrin into lipid emulsion; an in vitro feline plasma model .............................................................................................................................. 58

Introduction ................................................................................................................ 58

Methods ...................................................................................................................... 59

Results ......................................................................................................................... 62

Discussion .................................................................................................................... 64

Conclusions ................................................................................................................. 66

Footnotes .................................................................................................................... 67

References ................................................................................................................... 67

Chapter 5: A randomized controlled clinical trial of an intravenous lipid emulsion as an adjunctive treatment for permethrin toxicosis in cats ................................................... 70

Introduction ................................................................................................................ 70

Materials and Methods ............................................................................................... 71

Results ......................................................................................................................... 76

Discussion .................................................................................................................... 80

Conclusion ................................................................................................................... 85

Footnotes .................................................................................................................... 85

References ................................................................................................................... 85

Chapter 6: Conclusion ..................................................................................................... 90

Appendix 1: Raw data for in vitro experiment ................................................................ 91

vi

Appendix 2: Raw data for validation of permethrin toxicosis clinical staging system ... 92

Appendix 3: Raw data for clinical trial ............................................................................ 93

Bibliography .................................................................................................................. 100

1

Chapter 1: Use of intravenous lipid emulsion for lipophilic drug

toxicoses excluding local anaesthetics

Background

Intravenous lipid emulsion (ILE) therapy for severe local anaesthetic systemic toxicities

has been endorsed by professional bodies in their practice guidelines.1,2 Initial interest

in the therapy was prompted after Weinberg et al (1998) demonstrated that both

pretreatment and resuscitation with ILE resulted in amelioration of bupivacaine-

induced cardiotoxicity in rats.3 The same author later confirmed these findings after

successful resuscitation of dogs from bupivacaine-induced cardiac arrest using an ILE.4

Later, Weinberg et al (2008) showed that ILE resulted in superior haemodynamic

recovery compared to epinephrine in a rat model of bupivacaine overdose.5 Resulting

from these studies, numerous human case reports have been published demonstrating

complete neurological recovery after ILE administration for cardiac arrest due to local

anaesthetic systemic toxicity following failure of conventional resuscitation efforts.6-8

The successful use of ILE for local anaesthetic systemic toxicity has resulted in research

and clinical application of ILE in toxicosis caused by other lipophilic drugs including

beta-adrenergic blockers,9-16 antidepressants,17-22 calcium channel blockers,23-33 and

barbiturates.34 Also included in the veterinary literature is the use of ILE for toxicoses

caused by insecticides including macrocyclic lactones,35-38 and permethrin.39,40

The purposes of this review are to assess evidence for 1) efficacy of ILE in the

treatment of non-local anaesthetic lipophilic drug toxicoses, 2) proposed mechanisms

of action of ILE therapy for this application, 3) a clinical dosing regime of ILE for

lipophilic drug toxicoses, and 4) the safety of ILE for lipophilic drug toxicoses. If ILE

therapy is an effective antidote for lipophilic drug toxicosis then the implied reduction

in morbidity and mortality in veterinary patients would be substantial.

Method

Literature relevant to the first purpose of the review was identified by searching the

following biomedical databases from their inception up to December 31, 2012:

2

Embase, ISI Web of Science, Ovid MEDLINE, PLoS Medicine, Proquest, PubMed, Scopus

and Toxnet. Search queries were adapted for each database but search terms were:

antidote, arrest, cardiac, drug, fat emulsion, Intralipid®, lipid emulsion, poison, rescue,

resuscitation, toxic, toxicity and toxin. Articles pertaining to local anaesthetic systemic

toxicosis or reported as abstracts were excluded. Editorials, letters and review articles

were excluded but their references were searched to identify other articles which may

be relevant to this review. Articles whose abstracts were not relevant to the purpose

of the review were excluded. Full text versions of the remaining articles were

retrieved. The references of all of these selected articles were searched for any

additional articles of relevance.

Articles pertaining to ILE treatment of a specific toxicosis were grouped into the

following categories: barbiturates, beta-adrenergic blockers, calcium channel blockers,

cyclic antidepressants, insecticides and other case reports. Within each of these

groups, articles were divided according to the study method into experimental studies,

animal case reports, human case reports or human clinical trials. Data recorded from

the experimental studies included the methodology of the study, population

characteristics, type and dose of ILE, timing of ILE administration in relation to toxin

exposure, other therapies administered and reported study outcomes. Data recorded

from case reports included toxic agent, patient signalment, type and dose of ILE,

timing of ILE administration in relation to toxin exposure, other therapies administered

and reported response to ILE. Critical review of each article was then performed and a

conclusion was made as to the efficacy of ILE in the treatment of toxicosis induced by

each group.

Additional literature relevant to the other three purposes of the review was found by

searching for the additional terms: adverse, calcium, myocyte, parenteral nutrition and

safety. Articles whose abstracts were not relevant to the purpose of the review were

excluded. Full text versions of the remaining articles were retrieved. The literature was

then evaluated for evidence to support current recommendations for the dose of ILE

for lipophilic drug toxicoses and its safety at such doses, and for evidence to support

the proposed mechanisms of action.

3

Results

The literature search yielded 467 potentially relevant citations. Assessment of the titles

and abstracts of these citations led to 427 articles being excluded leaving 40 articles

reviewed.

Use of intravenous lipid emulsion in specific toxicoses

Barbiturates

Experimental studies

One experimental study was identified. Russell & Westfall (1962) investigated the

effects of two different intravenous lipid emulsions on thiopental-induced anaesthesia

time in rats compared to a saline control group and found that both formulations

significantly reduced anaesthesia times (p<0.009 and p<0.034).34 Limitations of this

study include the small sample size, short observation period and lack of blinding. In

addition, the study also used a different ILE compared to recent toxicological studies

and does not reflect common commercially available ILE as it was made from

cottonseed and corn oils.

Animal case reports

No published case reports were identified describing ILE for the treatment of

barbiturate overdose in animals.

Human case reports


barbiturate overdose in people.

Human clinical trials

No published clinical trials were identified describing ILE for the treatment of

barbiturate overdose in people.

Conclusions

There is limited data to support the use of ILE in cases of barbiturate toxicosis.

4

Beta-adrenergic blockers

Tables 1 and 2 provide an overview of the literature available for ILE therapy in beta-

adrenergic blocker toxicosis.


Six experimental studies were identified, four of which investigated ILE for propranolol

toxicosis and one each for atenolol and metoprolol toxicosis. In a preliminary study by

Cave et al (2006) there was no effect on mortality in rats treated with ILE compared to

placebo for propranolol toxicosis.10 Amelioration of propranolol-induced QRS

prolongation did occur but there was no significant effect on heart rate. Similarly,

Bania et al (2006) showed that pretreatment with ILE in rats with propranolol-induced

bradycardia did not increase the heart rate.11 However, there was a significant increase

in mean arterial blood pressure in ILE pretreated rats compared to a saline control. A

later study by Harvey et al (2008) supported this result by showing a significant

increase in mean arterial blood pressure in rabbits receiving ILE compared to rabbits

receiving a saline control for propranolol toxicosis.11 Again, there was no significant

effect on heart rate. In the study by Harvey et al (2011) ILE was compared to a high

dose insulin and glucose protocol and there was no difference in mean arterial blood

pressure, heart rate or survival between groups.16 In the only model of atenolol

toxicosis retrieved, Cave & Harvey (2009) failed to demonstrate any effect of ILE on

blood pressure or heart rate in rabbits compared to a saline control.12 In the murine

metoprolol study, Browne et al (2010) did not detect a significant difference in pulse

rate or mean arterial blood pressure between an ILE treated group and a saline control

group.13

A limitation of all studies except for the study by Harvey et al (2011) was that

intravenous models were used, which may not reflect serum and tissue concentrations

that occurs during massive oral ingestion. Therefore, the effect of ILE on beta-

adrenergic blocker toxicosis induced by ingestion is still not known. All studies also had

small numbers of animals. In addition, all studies were performed over a short

duration of less than 60 minutes; therefore any reintoxication after lipid metabolism

may not have been apparent. None of the studies had methodology to assess for

adverse effects of ILE. There may have been some bias in the studies by Cave et al

5

(2006), Harvey et al (2008), Cave & Harvey (2009), Browne et al (2010) and Harvey et

al (2011) as these studies were not blinded; however, the endpoints were objective

measures that would decrease the potential for bias. The study by Harvey et al (2008)

did not use death as an end-point and it is possible that further beneficial effects

beyond those reported may have been seen if a greater severity of toxicosis was

induced. It is possible that in the studies by Cave et al (2006), Harvey et al (2008), Cave

et al (2009), Browne et al (2010) and Harvey et al (2011), the use of ketamine in the

anaesthetic regime may have augmented improvements seen in haemodynamic

performance. Bania et al (2006) used isoflurane as the anaesthetic.

Animal case reports

No published case reports were identified describing ILE for the treatment of beta

blocker toxicosis in animals.

Human case reports

Two peer-reviewed case reports were identified of people with beta-adrenergic

blocker toxicosis receiving ILE treatment.14,15 It is difficult to determine whether the

improvements in haemodynamic parameters after ILE treatment were due to its

effects or merely circumstantial. The case report by Stellpflug et al (2010) showed

deterioration in a patient to the point of pulseless electrical activity despite aggressive

conventional therapies. Cardiopulmonary and cerebral resuscitation was initiated for

eight minutes and a bolus of ILE was given. Within 30 seconds of ILE administration

there was return of spontaneous circulation with tachycardia and hypertension after

60 seconds. Requirements for vasopressor support reduced within 5 minutes of ILE. In

the case report by Jovic-Stosic et al (2011) ILE treatment was given as hypotension,

tachydysrrhythmia and seizures persisted for eight hours despite conventional

treatment. Within 10 minutes of starting the ILE the seizures stopped, and within 25

minutes the blood pressure normalised. In both of these case reports it is difficult to

determine the contribution of ILE treatment on patient status as numerous other

medications were being concurrently administered with changing doses depending on

patient response to treatment. In addition, neither case report confirmed beta-

adrenergic blocker toxicosis with blood analysis and the diagnosis was presumptive

based on history and clinical findings.

6

Human Clinical Trials

No published clinical trials were identified describing ILE for the treatment of beta

blocker toxicosis in people.

Conclusions

There is some evidence to support minor improvement in mean arterial blood pressure

with ILE treatment in experimental models of beta-adrenergic blocker toxicosis. It is

not established whether this treatment is superior to conventional therapies.

Treatment with ILE may be beneficial in the clinical setting of severe beta-adrenergic

blocker toxicosis where conventional therapies have failed.

7

Table 1: Summary of experimental trials using ILE for beta-adrenergic blocker toxicosis Trial Subject (n) Drug (IV

unless specified)

Treatment evaluated

Timing of ILE infusion

Length of follow-up of subjects

Outcomes measured (units of measure)

Results

Cave; 20069 Randomised, controlled laboratory study

Rats (10) Propranolol 0.4mg/kg/min until death

ILE 20% 16 mL/kg, over 4 min OR NS 0.9% 16 mL/kg, over 4 min

4 min pre-intoxication

Until death Mean survival time (min) HR reduction at 60% of control group LD50 (%) QRS width change at 60% of control group LD50 (%)

No difference in survival time: NS 18.75 (CI 14.25-23) vs ILE 47 (CI 4.5-89.75), p=0.15 No significant difference in HR: NS 10.3 (CI 2.0-18.5) vs ILE 0 (CI -9.3-9.3), p=0.06 Decreased QRS duration in ILE group: NS 17.3 (CI 7.2-23.2) vs ILE -0.9 (CI -4.7-2.9), p=0.016

Bania; 200610 Blinded, randomised controlled laboratory study

Rats (14) Propranolol 21mg/kg over 60 min



60 min MAP (mmHg) HR

Greater MAP in ILE group at 15 and 30 min: NS 80.9 vs ILE 111.6, p<0.014 (15 min) NS 71.7 vs ILE 102.2, p<0.034 (30 min) No difference in HR between groups: NS ≈ ILE, p>0.05 No further data given

Harvey; 200811 Randomised, controlled laboratory study

Rabbits (20)

Propranolol 4.2 mg/min until MAP 60% of baseline


At intoxication 15 min MAP median at 15 min (mmHg) HR

Greater MAP in ILE group: NS 53 (IQR 12.75) vs ILE 69 (IQR 17.5), p<0.029 No difference in HR between groups: NS ≈ ILE, p>0.05 No further data given

8

Table 1: Summary of experimental trials using ILE for beta-adrenergic blocker toxicosis Trial Subject (n) Drug (IV

unless specified)

Treatment evaluated




Results

Cave; 200912 Randomised, controlled laboratory study

Rabbits (20)

Atenolol 25mg/min until MAP 60% baseline

ILE 20% 6 mL/kg over 4 min OR NS 6 mL/kg over 4 min

At intoxication 15 min MAP (mmHg) HR

No difference in MAP between groups: NS 51 (IQR 34-63) vs ILE 42 (13-45), p>0.05 No difference in HR between groups: NS ≈ ILE, p=0.06

Browne; 201013 Randomised, controlled laboratory study

Rabbits (20)

Metoprolol 400mg/h until MAP 60% baseline

ILE 20% 6 mL/kg over 4 min OR NS 6 mL/kg over 4 min

At intoxication 15 min MAP (mmHg) Pulse rate

No difference in MAP between groups: NS 43 (IQR 50-70) vs ILE 56 (38-54), p=0.12 No difference between groups: NS 180 (IQR 160-196) vs ILE 186 (IQR 170-216), p=0.31


Rabbits (14)

Propranolol 40mg/kg enterally and 0.3 mg/kg IV bolus then 4mg/kg/h until MAP 50% baseline

ILE 20% 10 mL/kg over 5 min OR NS 7mL/kg over 5 min, Actrapid insulin 3 U/kg and 0.5 g/kg glucose (ING group)

At intoxication 60 min Survival HR MAP Rate pressure product (mmH.min-1)

No difference in survival between groups: ING 4/5 vs 2/5 ILE, p=0.524 No difference in HR between groups: ING ≈ ILE, p=0.06 No further data given No difference in MAP between groups: ING ≈ ILE, p=0.106 No further data given Greater at 60 min in the ING group: ING > ILE, p<0.05 No further data given

CI 95% confidence interval; HR heart rate; ILE intravenous lipid emulsion; IQR interquartile range; MAP mean arterial blood pressure; NS normal physiological saline 0.9% NaCl

9

Table 2: Summary of published case reports using ILE for beta-adrenergic blocker toxicosis Author; year Patient

signalment Clinical context

Drugs involved

Clinical signs prior to ILE ILE regimen Timing of ILE infusion

Other treatments Outcome

Stellpflug; 201014

48 y male, unknown weight

Intentional overdose

Cocaine unknown mg Diazepam unknown mg Nebivolol unknown mg Ethanol unknown mg

GCS 3 Generalised tonic clonic seizure HR 25 bpm Hypotension (60/30 mmHg) Dilated slow pupils SpO2 96% on 15 L/min O2 Then PEA

ILE 20% 100 mL bolus then 900 ml over 60 min

Estimated 4 h after ingestion After two cycles of CPR

Crystalloids Atropine Glucagon Insulin Glucose Lorazepam Phenytoin Isoprenaline External cardiac pacing Epinephrine Mechanical ventilation

Following ILE bolus: ROSC after 30 s Tachycardia and hypertension after 60 s Epinephrine requirements decreased within 5 min then stopped after 7 h ECG reverted to normal sinus rhythm

Jovic-Stosic; 201115

31 y female, unknown weight


Propranolol 3.6 g Ethanol unknown mg

Coma Hypotension (65/35 mmHg) Seizures SpO2 87%

ILE 20% 100 mL bolus then 400 mL over 20 min 1 h later a further 500ml ILE 20% was given over 50 min

Estimated 8 h after ingestion After persistent hypotension, tachydysrhythmia, and seizures despite treatment

NS Glucose Diazepam Atropine Glucagon Insulin Sodium bicarbonate Dopamine Mechanical ventilation

Following ILE bolus: Seizures stopped Blood pressure normal in 25 min, the decreased again after further 30 min Second ILE: Normal sinus rhythm and blood pressure 20 min after starting

HR heart rate; ILE intravenous lipid emulsion; NS normal physiological saline 0.9% NaCl; ROSC Return of spontaneous circulation

10

Calcium channel blockers

Tables 3 and 4 provide an overview of the literature available for ILE therapy in calcium

channel blocker toxicosis.


Six experimental studies were identified, five of which investigated ILE for verapamil

toxicosis and one for nifedipine toxicosis. Three laboratory studies of verapamil

toxicosis in rats and dogs showed improvements in survival times and/or survival rates

where measured in the ILE group compared to a saline control.23,24,27 The study by

Medlej et al (2008) showed this survival benefit even when ILE was used two hours

prior to the onset of verapamil toxicosis. Another model of verapamil toxicosis

reported by Bania et al (2008) showed no difference in survival in the ILE treated group

where conventional therapy for toxicosis was being administered as the control

group.25 One study investigated ILE dosing regimens in models of verapamil toxicosis.26

In this study, Perez et al (2008) showed increased survival times when a bolus of 18.6

mL/kg was used, compared to boluses of 0 mL/kg, 6.2 mL/kg, 12.4 mL/kg, 24.8 mL/kg

and 37.6 mL/kg. The single study investigating ILE in nifedipine toxicosis showed no

difference in survival times between the ILE group and saline control.29

Tebbutt et al (2006) showed higher heart rates in rats with verapamil toxicosis treated

with ILE compared to a saline control,23 but Bania et al (2007) in their dog model of

verapamil toxicosis did not detect any difference in heart rate between the two

groups.24 This difference may be due to the smaller sample size of the study by Bania

et al (2007), the use of a different animal model, the lower dose and dose rate of ILE

administered or the low survival in the control group, rendering meaningful

comparison of haemodynamic and respiratory parameters impossible. A later study

performed by Medlej et al (2008) which investigated the survival and haemodynamic

effects of pretreatment with ILE in a murine model of verapamil toxicosis showed no

difference in mean arterial pressure or base excess at any time point, and a significant

difference in heart rate only 30 minutes after the start of the verapamil infusion. The

methodology of this study was different to that of Tebbutt et al (2006), however it is

interesting that the haemodynamic benefits were minimal despite using higher ILE

doses than Tebbut et al (2006) albeit as a pretreatment. Only one study investigated

11

ILE treatment compared to conventional therapy with high dose insulin, and there was

no difference in haemodynamic parameters between the two groups.25 The single

study investigating ILE in nifedipine toxicosis showed no difference in haemodynamic

parameters between the ILE group and saline control.29

All studies were performed in heavily sedated or anaesthetised animals. In general, the

anaesthetic regime is not thought to have interacted with the results. A special

mention is given to the study by Tebbutt et al (2006) who used ketamine to sedate the

rats and allowed them to spontaneously ventilate.23 Ketamine has previously been

shown to increase blood verapamil concentrations in rabbits and it is possible that a

similar interaction occurs in rats.41 This raises the possibility that part of the conferred

benefit of ILE results from a synergistic effect of ILE and ketamine in lowering tissue

verapamil concentrations. This benefit may be due to a reduction in sedation-related

hypoventilation and was not specifically assessed in the study by Tebbutt et al (2006)

despite resting respiratory rates being documented and seven of the 30 rats having

arterial blood gas measurements performed. However, the LD50 of verapamil in rats

obtained in other experiments was similar to that of the control group in the study by

Tebbutt et al (2006) making any differences in survival unlikely to be due to the effects

of ketamine on verapamil concentration. Anaesthesia regimens in rats that utilise

ketamine have caused sudden death and this may still be a confounding factor

particularly with the small sample size in the study by Tebbutt et al (2006).42

A limitation of all of the studies was that an intravenous model was used that may not

reflect the serum and tissue concentrations reached during massive oral ingestion.

Therefore, the effect of ILE on calcium channel blocker toxicosis induced by ingestion is

not known. All studies also had a small sample size and treatments were not blinded.

In addition, all studies were performed over a short duration of less than four hours;

therefore any reintoxication after lipid metabolism may not have been apparent. None

of the studies had sufficient methodology to assess for adverse effects of ILE. The

study by Perez et al (2008) used an ILE dose of 37.6 mL/kg in one group of rats as a

representation of what may be considered a very high dose of ILE.26 Two of the five

rats died suddenly after infusion of ILE began, however the cause of death was not

determined and may not have been related to the ILE infusion.

12

Animal case reports


verapamil toxicosis in animals.

Human case reports

Five human case reports were identified, with four reporting ILE use for verapamil

overdose and one for amlodipine overdose. It is difficult to determine whether the

improvements in haemodynamic parameters after ILE treatment were due to its

effects or merely circumstantial. The case reports by Young et al (2009) and Franxman

et al (2011) showed a reduction in the need for vasopressor support within hours of

commencement of ILE, despite therapy for hypotension for 17.67 and 21.33 hours

respectively prior to ILE therapy.30,31 The case reports by Liang et al (2011) and French

et al (2011) showed a less dramatic response to ILE treatment, though the total dose

of verapamil ingested was higher in these two reports, compared to the earlier ones.

In all of these case reports it is difficult to determine the contribution of ILE treatment

on patient status as numerous other medications were being concurrently

administered with changing doses depending on patient response to treatment.32,33 In

addition, only the reports by Young et al (2009) and French et al (2011) confirmed

verapamil toxicosis via serum concentrations. The case report by French et al (2011) is

unique in that it did measure serum triglyceride and verapamil concentrations before

and after ILE treatment. West et al (2010) used ILE for a severe amlodipine overdose,

however any benefits that occurred were not described as the focus of the report was

an accidental overdose of ILE.43


No published clinical trials were identified describing ILE for the treatment of

verapamil toxicosis in people.

Conclusions

There is good evidence to support increased survival times and rates in experimental

models of verapamil toxicosis with ILE treatment. There is also some evidence that ILE

improves haemodynamic function in experimental models of verapamil toxicosis. It is

not established whether ILE treatment is superior to conventional therapies.

13

Treatment with ILE may be beneficial in the clinical setting of severe calcium-channel

blocker toxicosis where conventional therapies have failed.

14

Table 3: Summary of experimental trials using ILE for calcium channel blocker toxicosis Trial Subject (n) Drug (IV

unless specified)

Treatment evaluated




Results

Tebbut; 200623 Blinded randomised controlled laboratory study

Rats (30) Verapamil 37.5 mg/kg/h until death

ILE 20% 12.4 mL/kg, over 5 min OR NS 0.9% 12.4 mL/kg over 5 min

5 min after commencement of verapamil

Until death Survival time (min) Verapamil LD50 (mg/kg) Rate of drop in HR (bpm)

Increased survival time in ILE group: NS 24±9 vs ILE 44±21, p=0.003 Increased LD50 in ILE group: NS 13.6 (CI 12.2-15.0) vs ILE 25.7 (CI 24.7-26.7), p value not provided Reduction in verapamil induced bradycardia in ILE group: NS 6.8 (CI 8.3-5.2) vs ILE 10.7 (CI 12.6-8.9), p=0.001

Bania; 200724 Blinded randomised controlled laboratory study

Dogs (14) Verapamil 6 mg/kg/h until 50% drop in MAP, then 2 mg/kg/h



120 min Survival rate at 120 min (%) Mean survival time (min) MAP difference at 60 min (mmHg) HR at 60 min BE at 60 min (mEq/L)

Increased survival rate in ILE group: NS 14 (CI 0.5-53) vs ILE 100 (CI 59-100), p=0.01 Increased survival time in ILE group: NS 75 (CI 63-86) vs ILE >120 (CI >120), p=0.002 Greater MAP in ILE group: NS 10.2-53.1 lower ILE, p<0.05 No further data given No difference in HR between groups: NS ≈ ILE, p>0.05 No further data given Higher BE in ILE group: NS -16.7 vs ILE -12.8, p=0.03

15


unless specified)

Treatment evaluated




Results

Bania; 200825 Blinded randomised controlled laboratory study

Dogs (14) Verapamil 6 mg/kg/h until 50% drop in MAP, then variable rate to maintain MAP at 50% of baseline for 30 min

ILE (concentration not provided) 18.6 mL/kg, over 1 h OR Insulin (HIE) 60 U/h in equivalent volume over 1 h

30 min after MAP 50% of baseline

4 h Survival time (min) Haemodynamic parameters

No increase in survival time: HIE 187 (CI 173-202) vs ILE 191 (CI 167-214), p=0.78 No difference in haemodynamic parameters: HIE ≈ ILE for MAP, HR, CO, left ventricular pressure, pH or BE No further data given

Perez; 200826 Controlled dose-escalation laboratory study

Rats (30) Verapamil 15 mg/kg/h

ILE 20% bolus: 1. 0 mL/kg 2. 6.2 mL/kg 3. 12.4 mL/kg 4. 18.6 mL/kg 5. 24.8 mL/kg 6. 37.6 mL/kg


Until death Median survival time (min) MAP, HR and BE

Increasing ILE dose resulted in increased mean survival times: 1. 34 vs 2. 58 vs 3. 63 vs 4. 144 vs 5. 126 vs 6. 130, p value not provided Greater MAP, HR and BE in 24.8 mL/kg ILE group

Medlej; 200827 Controlled laboratory study

Rats (14) Verapamil 15 mg/kg/h

ILE 20% 18.6 mL/kg, over 30 min OR NS 0.9% 18.6 mL/kg, over 30 min

Pretreatment 2 h prior to verapamil infusion

Until death Survival time (min) MAP HR (bpm)

Increased survival time in ILE group: NS 39 (CI 31-47) vs ILE 53 (CI 43-63), p=0.03 No difference in MAP between groups: NS ≈ ILE, p>0.05 No further data given Increased HR at 30 min in ILE group: NS group HR lower than ILE by 53 (CI 0.8-105) at 30 min NS ≈ ILE at all other times, p>0.05 No further data given

16


unless specified)

Treatment evaluated




Results

Chu; 200929 Randomised controlled laboratory study

Rats (20) Nifedipine 30 mg/kg/h

ILE 20% 18.6 mL/kg, over 30 min OR NS 0.9% 18.6 mL/kg, over 30 min

5 min after commencement of nifedipine

2 h Mean survival time (min) MAP, HR and BE

No increase in survival time: NS 34 (CI 26.25-41.75) vs ILE 81 (CI 42.78-119.22), p=0.35 No difference in MAP, HR or BE between groups: NS ≈ ILE, p>0.05 No further data given

BE base excess; CI 95% confidence interval; HIE high dose insulin euglycemia; HR heart rate; ILE intravenous lipid emulsion; LD50 lethal dose 50; MAP mean arterial blood pressure; NS normal physiological saline 0.9% NaCl;

17

Table 4: Summary of published human case reports using ILE for calcium channel blocker toxicosis Author; year Patient


Drugs involved Clinical signs prior to ILE

ILE regimen Timing of ILE infusion


Young; 200930 32 y male, unknown weight


Verapamil 13.44 g Levothyroxine 1.125 mg Bupropion 4.8 g Zolpidem CR 200 mg Quetiapine unknown mg Clonazepam 22 mg Benazepril unknown mg

Stuporous HR 77 bpm Hypotension (96/42mmHg) Dilated slow pupils SpO2 90%

ILE 20% 100 mL over 20 min then 0.5 mL/kg/h for 23 h

Estimated 17.67 h after ingestion After persistent hypotension despite treatment

NS 9 L Calcium gluconate Noradrenaline Glucagon Intubation Mechanical ventilation

Following ILE bolus: Noradenaline infusion halved within 1 h, stopped within 8 h Glucagon stopped within 2 h Extubated the next day Neurologically intact

Franxman; 201131



Verapamil 4.08 g Dyspnoea Lethargy Diaphoresis Hypotension (76/41mmHg)

ILE 20% 100 mL over 20 min then 0.5 mL/kg/h for 8 h

Estimated 21.33 h after ingestion After persistent hypotension despite treatment

NS 2 L Noradrenaline Activated charcoal Calcium gluconate Glucagon Dopamine (noradrenaline stopped)

Following ILE bolus: Dopamine infusion halved within 4 h, stopped within 8 h

18






Liang; 201132 41 y female unknown weight


Verapamil 19.2 g Hypotension (SAP 50-60 mmHg) HR 50-60 3rd degree AV block Lethargy Developed anuric renal failure and refractory hypoxaemia

ILE 20% 100 mL over 20 min then 0.5 mL/kg/h for 3 d

On day 4 of hospitalisation – unknown period of time from ingestion After persistent hypotension despite treatment

Intravenous fluids Activated charcoal Calcium chloride Dopamine Isoproterenol Noradrenaline Adrenaline Transvenous pacing HIE Glucagon Vasopressin Mechanical ventilation Haemodialysis

Following ILE bolus: Noradrenaline infusion halved within 3 h Within 48 h, vasopressin stopped, minimal pressor support with adrenaline and noradrenaline, pacing stopped. Within 72 h pressors stopped, HIE stopped, positive pressure ventilation stopped, haemodialysis stopped.

19






French; 201133

41 y male unknown weight


Verapamil 6.3 g Hypotension (SAP 80 mmHg) Complete heart block Lethargy Hypoxia

ILE unknown% 2x 100 mL boluses, 500 mL over 30 min ILE unknown% 100 mL bolus, 150 mL over 15 min

Estimated 19 h after ingestion After persistent hypotension despite treatment Estimated 29 h after ingestion

Atropine Glucagon HIE Norepinephrine Dopamine Calcium gluconate/chloride Adrenaline Phenylephrine Transvenous pacing Vasopressin

Following first ILE doses: Improvement in blood pressure, no other data given Following second ILE dose: Haemodynamics improved, no other data given On day 2 dopamine and phenylephrine stopped On day 3 epinephrine and vasopressin stopped

West; 201043 71 y, female, unknown weight


Amlodipine 120 mg Hypotension (SAP 79 mmHg) Deteriorating mental status Oliguria

ILE 20% 2000 mL over 4h (accidental overdose)

Estimated 14 h after ingestion

Intravenous fluids Vasopressors (not specified) Packed red blood cells Calcium gluconate Phenylephrine Vasopressin HIE

Following ILE: Any effects were not reported

AV atrioventricular; HIE high dose insulin euglycemia; HR Heart rate; ILE intravenous lipid emulsion; LD50 lethal dose 50; NS normal physiological saline 0.9% NaCl; SAP systolic arterial pressure

20

Cyclic antidepressants

Tables 5 and 6 provide an overview of the literature available for ILE therapy in cyclic

antidepressant toxicosis.


Seven experimental studies were identified, three of which investigate ILE for

amitriptyline toxicosis, three for clomipramine toxicosis and one for imipramine

toxicosis. All are tricyclic antidepressants. In a preliminary study, Yoav et al (2002)

demonstrated a decreased mortality in rats from imipramine toxicosis by 80% in an ILE

treated group when compared with a saline control.18 Harvey & Cave (2007)

demonstrated accelerated recovery in rabbits from clomipramine-induced

hypotension when compared with both saline control and sodium bicarbonate.20 In the

same experiment, ILE decreased mortality from severe clomipramine toxicosis by 100%

when compared to sodium bicarbonate. The same study did not show any increase in

heart rate or electrocardiogram parameters between the three treatment groups. In a

subsequent experiment,20 the same investigators reproduced the accelerated recovery

from hypotension with ILE administration in a similar rabbit model of clomipramine

toxicosis. In addition, increases in serum clomipramine concentration in the ILE group

were observed in parallel with hemodynamic recovery. Bania & Chu (2006) failed to

show a statistically significant difference in survival or hemodynamic parameters with

ILE administration in a rat model of amitriptyline toxicosis.19 Similarly, Litonius et al

(2012) did not demonstrate a survival or haemodynamic benefit of ILE over Ringer’s

acetate in a porcine model of amitriptyline toxicosis despite significant increase in

plasma amitriptyline concentration in the ILE group.22

Limitations of these studies include the small sample sizes, short observation periods

and lack of blinding. Data from these studies should not be extrapolated to include all

cyclic antidepressants, as each drug will have different affinities for sodium channels,

different volumes of distribution and log P value. These differences affect their toxicity

in vivo and potentially the beneficial effects of ILE, if any exist. A limitation of the study

by Harvey & Cave (2007) is that a low dose of sodium bicarbonate was used and acid-

base balance was not monitored.20 Therefore, this therapy may not reflect what may

occur in clinical practice; higher dosing within a safe acid-base range may have

21

produced a more favourable outcome. In addition, both of the studies by Harvey and

Cave used a mixed anaesthetic regime and their interaction with ILE is unknown; in

particular that of ketamine which has been discussed previously.20,21 All studies used

the intravenous route to induce cyclic antidepressant toxicosis and this is not reflective

of the clinical scenario where enteral absorption normally occurs.

Two studies investigated the effect of ILE on human plasma levels of amitriptyline.

Minton et al (1987) asked four human volunteers to ingest 75 mg of amitriptyline daily

for 10 days with volunteers being randomized on day eight into either an ILE or saline

control group and on day 10 subjects crossed over.17 Mean plasma concentrations

were 13.8% higher in the ILE group but statistical significance was not reached

(p>0.05). More recently, Litonius et al (2012) demonstrated in a porcine model a 90%

higher total amitriptyline concentration in an ILE treated group compared to a Ringer’s

acetate control group (p<0.001).22

Animal case reports

No published case reports were identified describing ILE for the treatment of cyclic

antidepressant overdose in animals.

Human case reports

Two peer-reviewed case reports were identified describing ILE for the treatment of

cyclic antidepressant toxicosis in people. Engels & Davidow (2010) describe ILE use to

treat a case with vasopressor-refractory hypotension from amitriptyline toxicosis and

report a rapid decrease in the requirement for vasopressors after ILE administration.44

Boegevig et al (2011) describe the reversal of life threatening QRS complex widening

and prolonged QT interval following dosulepin overdose within 15 minutes of ILE

administration.45

Human clinical trials

No published clinical trials were identified describing ILE for the treatment of cyclic

antidepressant overdose in humans.

22

Conclusions

There is some evidence to support minor improvement in mean arterial blood pressure

and survival with ILE treatment in experimental models of tricyclic antidepressant

toxicosis. It is not established whether this treatment is superior to conventional

therapies. Treatment with ILE may be beneficial in the clinical setting of severe tricyclic

antidepressant toxicosis where conventional therapies have failed.

23

Table 5: Summary of experimental trials using ILE for cyclic antidepressant toxicosis Trial Subject (n) Drug (IV

unless specified)

Treatment evaluated




Results

Minton; 198717 Randomised, controlled, cross-over study

Human (4) Amitriptyline 72 mg/d PO for 10 days

ILE 20% 500 mL over 5 h OR NS 0.9% 500 mL over 5 h

Randomised on day 8 and cross-over on day 10

5 h Mean plasma amitriptyline concentration

No difference in mean concentration: NS ≈ ILE, p>0.05

Bania; 200619 Randomised controlled laboratory study

Rats (22) Amitriptyline 42 mg/kg over 60 min

ILE 20% 15 mL/kg, over 15 min OR NS 0.9% 15 mL/kg over 15 min


60 min Survival rate MAP (mmHg) dP/dt

No difference in survival rate between groups: NS ≈ ILE, p=0.21 No further data given Increased MAP at 45 min in ILE group: NS 37.8 vs ILE 53.1, p=0.047 NS ≈ ILE at all other times, p>0.05 No difference in dP/dt between groups: NS ≈ ILE, p>0.05 No further data given

Litonius; 201222

Randomised controlled laboratory study

Pigs (20) Amitriptyline 15 mg/kg over 15 min

ILE 20% 1.5 mL/kg for 1 min, then 0.25 ml/kg/min for 29 min OR Ringer’s acetate 1.5 mL/kg for 1 min, then 0.25 ml/kg/min for 29 min

Immediately after amitriptyline infusion

30 min Plasma amitriptyline concentration Survival (%) HR, MAP, CO

90% higher in ILE group, p<0.001 No difference between groups: Ringer’s acetate 20 vs ILE 50, p=0.35 No difference between groups: Ringer’s acetate ≈ ILE, p>0.05

24


unless specified)

Treatment evaluated




Results

Harvey; 200720 Part 1 Randomised controlled laboratory study

Rabbits (30)

Clomipramine 320 mg/kg/h until MAP 50% of baseline

ILE 20% 12 mL/kg, over 4 min OR NS 0.9% 12 mL/kg over 4 min OR NaHCO3 8.4% 3 mL/kg over 4 min

At toxicosis when MAP 50% of baseline

15 min Mean difference in MAP (mmHg) dP/dt HR QRS duration

Mean MAP difference lower in NS group: NS <ILE 19.5 (CI 10.5-28.9) Mean MAP difference lower in NaHCO3 group: NS <ILE 11.5 (CI 2.5-20.5) Greatest at 3 and 5 min in ILE group No difference in HR between groups: NS ≈ NaHCO3 ≈ ILE, p>0.05 No further data given No difference in QRS between groups: NS ≈ NaHCO3 ≈ ILE, p>0.05 No further data given

Harvey; 200720 Part 2 Randomised controlled laboratory study

Rabbits (8) Clomipramine 240 mg/kg/h until MAP 25 mmHg

ILE 20% 8 mL/kg, over 4 min OR NaHCO3 8.4% 2 mL/kg over 4 min

At toxicosis when MAP <25 mmHg or pulse pressure <1 mmHg

10 min Survival at 10 min after treatment (%)

Increase in survival in ILE group: NS 0 vs ILE 100, p=0.029


Rabbits (20)

Clomipramine 240 mg/h until MAP 50% of baseline

ILE 20% 12 mL/kg, over 4 min OR NS 0.9% 12 mL/kg over 4 min

At toxicosis when MAP 50% of baseline

59 min Greatest mean difference in MAP (mmHg) HR

9 min after ILE: 12.79 (CI 2.77-22.81) No difference in HR between groups: NS ≈ ILE, p>0.05 No further data given

25


unless specified)

Treatment evaluated




Results

Yoav; 200218 Randomised, controlled laboratory study

Rats (24) Imipramine 12.5 mg

ILE 10% 2.5 mL over unknown time OR NS 0.9% 2.5 mL over unknown time

With imipramine

24 h Mortality (%) Lower mortality in ILE group: NS 100 vs ILE 20

CO cardiac output; HR heart rate; ILE intravenous lipid emulsion; MAP mean arterial pressure; NS normal physiological saline 0.9% NaCl;

Table 6: Summary of published human case reports using ILE for cyclic antidepressant toxicosis Author; year Patient





Engels; 201044 27 y male, unknown weight


Amitriptyline 4.25 g GCS 3 Seizures Hypothermia Wide QRS Tachycardia

ILE 20% 100 mL bolus then 400 mL over 30 min

Unknown in relation to ingestion

Midazolam Sodium bicarbonate Intravenous fluids Intubation CPCR Epinephrine Norepinephrine

Following ILE: Epinephrine stopped within 1 h Norepinephrine stopped within 4 h

Boegevig; 201145



Dosulepin 5.25 g Seizures Wide QRS Increased QT interval

ILE 20% 100 mL over 5 min then 400 mL over 20 min


Activated charcoal Intravenous fluids Diazepam Intubation Assisted ventilation

Following ILE: Normal ECG with 20 min

GCS Glasgow coma scale; ILE intravenous lipid emulsion

26

Insecticides

Table 7 provides an overview of the literature available for ILE therapy in toxicosis

caused by insecticides.


No published experimental studies were identified describing ILE for the treatment of

toxicosis caused by insecticides.

Animal case reports

Six animal case reports were identified of toxicosis caused by insecticides being treated

with ILE. In all of these case reports it is difficult to determine the contribution of ILE

treatment on patient status as numerous other medications were being concurrently

administered with changing doses depending on patient response to treatment.

In the first published case report, Crandell et al (2009) report a tenuous link between

clinical improvement in a puppy with moxidectin toxicosis and the use of ILE.35 They

report that mechanical ventilation was discontinued within two hours of the first ILE

dose, however it appears that respiratory parameters were improving and that

weaning from the ventilator was imminent prior to ILE administration. The second ILE

dose in this case report was given due to redevelopment of seizures, however, at the

end of ILE administration a diazepam infusion was started and clinical signs of seizures

resolved within 30 minutes and the patient was ambulatory but ataxic. It is not

possible to say that these clinical changes were due exclusively to ILE administration.

The dog was asymptomatic when discharged home after two days, a time frame which

was shorter than other reports of moxidectin toxicosis, which were between 3-10

days.46-49

Three case reports describe ILE use for ivermectin toxicosis. The case series by Wright

et al (2011) did not document any clinical improvement after ILE treatment for

ivermectin toxicosis in three dogs homozygous for the ABCB1-1Δ gene mutation.36 The

authors proposed that the absence of response to ILE in these dogs may have been

due to a failure to significantly decrease the brain ivermectin concentration. The

authors did, however, document an increase in serum ivermectin concentration in all

27

dogs after the administration of ILE, supporting drug sequestration into an

intravascular lipid compartment as a mechanism of action of ILE in lipophilic drug

toxicoses. A later case report by Clarke et al (2011) claimed successful ILE treatment of

a Border collie with ivermectin toxicosis.37 Clinical improvement in the dog was seen

six hours after the first ILE dose and one and a half hours after the second dose 12

hours later. It is difficult to conclude that the ILE was directly responsible for any

clinical improvement in this case. The authors did document a modest increase in

serum ivermectin concentration after each ILE infusion. Of all the case reports

documenting the use of ILE for ivermectin toxicosis, the report by Bruenisholz et al

(2012) demonstrates the closest temporal relationship between ILE administration and

clinical improvement.38 The report also demonstrated a considerable increase in

plasma triglyceride concentrations after ILE infusion and a corresponding increase in

plasma ivermectin concentrations.

Two case series were identified describing the use of ILE for permethrin toxicosis in

cats. The first published report by Brückner & Schwedes (2012) showed a reduction in

pentobarbital requirements in two cats after lipid infusion but no other discernible

changes in patient condition.39 Haworth & Smart (2012) reported a subjective

improvement in clinical signs in three cats with permethrin toxicosis treated with ILE,

however the cases were less severe than those reported by Brückner et al (2012)

making the two case series difficult to compare.40 Neither case series measured serum

permethrin concentrations. It is not possible to determine whether the improvements

in clinical signs after ILE treatment in these cases were due to its effects or merely

circumstantial.

Human case reports

No published case reports were identified in people describing ILE for the treatment of



No published clinical trials in people were identified describing ILE for the treatment of


28

Conclusions

There is limited data to support the use of ILE in the treatment of toxicosis caused by

the macrocyclic lactones, moxidectin and ivermectin, or for permethrin.

29

Table 7: Summary of published animal case reports using ILE for toxicosis caused by insecticides

Author; year Patient signalment

Clinical context

Clinical signs prior to ILE

ILE regimen Timing of ILE infusion Other treatments Outcome

Crandell; 200935

16 week Female JRT 3.2 kg

Exposure to horses who had received 2% moxidectin

Seizures Bradycardia Hypotension (102/34 mmHg) Coma Tachypnoea SpO2 88% on room air

ILE 20% 6.5 mL bolus then 12 mL/h for 2 h ILE 48 mL over 30 min

First dose estimated 10 h after exposure due to: Persistent bradycardia and inability to wean off ventilator Second dose estimated 25 h after exposure due to: Seizuring

NS 16 mL/h Mechanical ventilation Atropine Glycopyrrolate Diazepam Supplemental oxygen Activated charcoal

Following first ILE: Mechanical ventilation stopped After 11 h extubated Following second ILE: After 30 min ambulatory, chewing bandages After 2 h oxygen support stopped After 6 h eating

Wright; 201136

1 y Male neuter Miniature Australian shepherd 8.0 kg

Exposure to horses who had received ivermectin

Coma Hypoventilation

ILE 20% 1.5 mL/kg then 0.25 mL/kg/h for 14 h

Up to 3 d after exposure and 23 h after onset of clinical signs due to persistent coma and needed for mechanical ventilation

IVFT Gastric lavage Activated charcoal Mechanical ventilation NG feeding tube Parenteral nutrition

Following ILE: No change in patient condition

2 y Female spey Australian shepherd 18.2 kg

Exposure to horses who had received ivermectin

Recumbency Stupor

ILE 20% 1.5 mL/kg then 15 mL/kg over 30 min

Estimated 6 h after exposure

IVFT Flumazenil

Following ILE: Minimal transient improvement in patient condition

30



Clinical context



5 y Female Miniature Australian Shepherd 7.6 kg

Administration of 165 μg/kg ivermectin orally for ectoparasites

Recumbency Stupor

ILE 20% 1.5 mL/kg then 15 mL/kg over 30 min


IVFT Oxygen supplementation Ampicillin Enrofloxacin Amoxycillin-clavulanic acid Doxycycline

Following ILE: No improvement in patient condition

Clarke; 201137

2 y Female spey Border collie 17.8 kg

Accidental ingestion of 6 mg/kg ivermectin equine dewormer

Hyperthermia (40.3oC) Obtundation Bilateral mydriasis with absent PLRs Muscle tremors Recumbent

ILE 20% 1.5 mL/kg then 15 mL/kg over 60 min ILE 20% 1.5 mL/kg then 15 mL/kg over 60 min

First dose estimated 8 h after exposure due to severity of clinical signs, dose of ivermectin ingested and breed predisposition Second dose estimated 20 h after exposure due to continued clinical signs

IVFT Following first ILE: After 6 h dog was ambulatory and more responsive and dazzle reflex returned Following second ILE: After 1.5 h dog was alert, tremors reduced, weak PLRs

Bruenisholz; 201238

11 m Male Shetland Pony 26 kg

Accidental overdose of 5.4 mg/kg ivermectin equine dewormer

Recumbent Coma Tachycardia Hypothermia

ILE 20% 1.5 mL/kg then 7.5 mL/kg over 30 min ILE 20% 1.5 mL/kg then 7.5 mL/kg over 30 min

First dose 71 h after exposure due to no clinical improvement Second dose 90 h after exposure due to no clinical improvement

IVFT Glucose Sarmazenil Cefquinome Flunixin meglumine

Following first ILE: Slight improvement Following second ILE: By end of infusion conscious, sternally recumbent and able to hold head, started to eat. Able to stand within 1 h with assistance

31



Clinical context



Brückner; 201239

3 y Female spey Maine Coon 4.7 kg

Administration of 2 mL of 74.4% permethrin for ectoparasites

Lateral recumbency Generalised tremor Seizures Salivation

ILE 20% 2 mL/kg then 16 mL/kg over 4 h ILE 20% 2 mL/kg then 16 mL/kg over 4 h

First dose estimated 24 h after exposure due to high doses of drugs required to control clinical signs Second dose estimated 36 h after exposure due to no clinical improvement

IVFT Diazepam Washing Propofol Phenobarbital Frusemide Pentobarbital

Following first ILE: By end of infusion pentobarbital CRI reduced by ⅔ Following second dose: By end of infusion pentobarbital CRI reduced by ½

9 y Male neuter Cat 3.9 kg

Administration of 2 mL of 74.4% permethrin for ectoparasites

Lateral recumbency Coma Generalised tremor Salivation

ILE 20% 2 mL/kg then 16 mL/kg over 4 h


IVFT Diazepam Washing Propofol Pentobarbital

Following ILE: After 2 h pentobarbital CRI reduced by ½

Haworth; 2012

40

9 m Female spey DSH 3.8 kg

Administration of 842 mg/kg permethrin for ectoparasites

Severe muscle fasciculations

ILE 20% 1.5 mL/kg over 30 min then 11.25 mL/kg over 45 min

Estimated 72 h post exposure

IVFT Propofol Methocarbamol Washing Intubation

Following ILE: After 1.5 h extubation occurred, ambulatory and grooming

2 y Female spey DSH 4.2 kg


Moderate to severe muscle tremors

ILE 20% 1.5 mL/kg over 90 min then 8.5 mL/kg over 30 min


IVFT Methocarbamol Washing

Following ILE: Marked improvement in tremors

1 y Female DLH 6.0 kg


Moderate tremors Ataxia Hyperaesthesia

ILE 20% 15 mL/kg over 60 min


IVFT Methocarbamol Washing

Following ILE: All clinical signs resolved over the next few hours

CRI continuous rate infusion; DSH domestic shorthair; DLH domestic longhair; ILE intravenous lipid emulsion; IVFT intravenous fluid therapy; JRT Jack Russell Terrier; NS normal physiological saline 0.9% NaCl; PLRs pupillary light reflexes

32

Other case reports Five further human case reports documenting ILE for the treatment of other lipophilic

drug toxicoses were identified. The details of the case reports are summarised in Table

8.

The first case report documenting ILE use in a non-local anaesthetic drug toxicosis was

reported by Sirianni et al (2008) where a girl suffered cardiopulmonary arrest after an

intentional overdose of bupropion, a nontricyclic antidepressant, and lamotrigine, an

anti-seizure sodium channel blocker.50 Cardiopulmonary resuscitation was attempted

unsuccessfully for 70 minutes before a 100 mL bolus of 20% ILE was administered.

Within one minute there was a return of spontaneous circulation and the patient

ultimately was discharged with minor neurological impairment. Later, Livshits et al

(2011) reported a less dramatic response to ILE in a case of bupropion toxicoses,

however the case was less severe than that reported by Sirianni et al (2008).51 Nogar et

al (2011) report ILE use in a case of severe lamotrigine toxicosis, however, its use was

not the focus of their case report and no changes were specifically reported after its

use.52 Castanares-Zapatero et al (2012) also report ILE use for a person with

lamotrigine overdose, but did report an improvement in cardiac conduction as a result

of an ILE bolus.53 Finn et al (2009) reported improvement in Glascow coma score in a

patient with quetiapine and sertraline toxicoses concurrent with the administration of

ILE.54

Conclusions

It is highly likely that there is an over representation of perceived favourable outcomes

which have been reported, biasing the value of these case reports. All case reports had

other therapies administered concurrently, making it difficult to determine the

contribution of the ILE to patient status. Experimental research is required to assess

the effects of ILE in these drug toxicoses.

33

Table 8: Summary of other published human case reports using ILE for drug toxicosis Author; year Patient





Sirianni; 200850

17 y female, 55 kg


Bupropion 7.95 g Lamotrigine 4 g

Seizure PEA

ILE 20% 100 mL bolus

Estimated 11 h after ingestion After persistent CPA for 70 min despite CPCR

Intravenous fluids Naloxone Intubation Mechanical ventilation CPCR Epinephrine Magnesium Sodium bicarbonate Calcium chloride Norepinephrine

Following ILE bolus: Palpable pulse Sinus rhythm return in 15 min Pressors reduced within 15 min

Livshits; 201151

51 y, female, unknown weight


Diphenhydramine unknown mg Bupropion unknown mg

Seizure Hypotension (SAP 80 mmHg) Wide QRS complex

ILE 20% 1.5 mL/kg bolus x2, then 15 mL/kg over 1 h


Intravenous fluids Sodium bicarbonate Calcium gluconate Glucagon Dopamine Norepinephrine Intubation Haemodialysis

Following ILE: Normotensive within 30 mins Pressor support reduced within 16 h

Nogar; 201152 48 y, female, unknown weight


Lamotrigine 7.5 g Seizure Coma Wide QRS Pulseless

ILE 20% 360 mL Estimated 4 h after ingestion

Intubated Rocuronium Lorazepam Sodium bicarbonate Lidocaine Amiodarone Calcium chloride CPCR Phenobarbital Propofol Vercuronium Fosphenytoin

Following ILE: Effects were not detailed

34

Table 8: Summary of other published human case reports using ILE for drug toxicosis Author; year Patient





Castanares-Zapatero; 201253

50 y, female, unknown weight


Lamotrigine 3.5 g GCS 7 QRS wide LBBB

ILE 20% 1.5 mL/kg bolus, then 30 mL/kg/h for 10 h

Estimated 5h after ingestion

Intubation Mechanical ventilation Magnesium Sodium bicarbonate

Following ILE bolus: Narrowing of QRS and return to sinus rhythm within a few minutes

Finn; 200954 61 y, Male, unknown weight


Quetiapine 4.3 g Sertraline 3.1 g Benzodiazepine unknown g

GCS 3 Hypotension (88/64 mmHg) Hypothermia

ILE 20% 1.5 mL/kg bolus, then 8 mL/kg over 1 h


Intubation Mechanical ventilation Intravenous fluid Flumazenil

Following ILE: GCS 9 Extubated

CPA cardiopulmonary arrest; CPCR cardiopulmonary-cerebral resuscitation; GCS Glascow coma scale; ILE intravenous lipid emulsion; LBBB left bundle branch block; PEA pulseless electrical activity; SAP systolic arterial pressure

35

Proposed mechanisms of action of intravenous lipid emulsion for drug toxicoses

Experimental and clinical studies support three postulated mechanisms of action of ILE

in the treatment of lipophilic drug toxicoses: 1) lipid partitioning, 2) myocardial

mitochondrial recovery, and 3) direct cardiac inotropy.

Intravascular lipid partitioning

The simplest mechanism proposed is the creation of an intravascular lipid sink, which

establishes a new pharmacokinetic equilibrium. In this mechanism, infusion of an ILE

leads to an expanded lipid compartment in the plasma into which lipophilic drugs may

be bound thus reducing free drug concentration. In addition, the drug may bind in

sufficient quantity to result in movement of drug from target tissues into the

intravascular compartment, thus reversing toxicosis. The sequestered drug might then

be shunted to organs that metabolise or sequester lipids which may reduce toxicity

through a reduced volume of distribution or enhanced metabolism of the lipophilic

drug. Infused ILE follows similar metabolic pathways as endogenous chylomicrons,

being metabolised within skeletal muscle, splanchnic viscera, myocardium,

subcutaneous tissues and the liver.55 However, the metabolic fate of the sequestered

drug is unknown, and there is a possibility that as metabolism of the ILE occurs, the

drug will be released allowing toxicosis to reoccur.

There is good evidence to support lipid partitioning as a mechanism of action. In an in

vitro study by Weinberg et al (1998) the authors report a 12:1 lipid to plasma ratio of

bupivacaine concentration, after lipid was added to rat plasma containing

bupivacaine.3 In the same study, rats were pretreated with ILE or a saline control and

then infused with bupivacaine until asystole occurred. At asystole, plasma bupivacaine

concentration was measured. Rats infused with a 30% ILE had a significantly (p<0.05)

higher plasma bupivacaine concentration than control rats. This study supports

sequestration of bupivacaine into the intravascular space, however, the study design

does not specifically demonstrate that there was movement of bupivacaine from

cardiac myocytes into the plasma. In a later study, Weinberg et al (2006) perfused

isolated rat hearts with a buffer solution containing bupivacaine, and showed that

myocardial bupivacaine concentration was significantly (p<0.0016) lower in hearts that

had subsequently been perfused with an ILE, compared to a buffer control.56 In

36

addition, the vascular effluent concentration of bupivacaine was significantly (p<0.008)

higher in the ILE treated group compared to the control.

In a preliminary study by Krieglstein et al (1974) either ILE or xylitol was added to

rabbit blood, which contained chlorpromazine.57 A significant (p<0.05) decrease in the

fraction of free chlorpromazine was seen in the ILE group and no change was seen in

the xylitol group. An in vitro study by Fallon and Chauhan (2006) showed that the

addition of lipid vesicles to bovine serum containing amitriptyline reduced the free

drug concentration by a factor of 3.5, and the authors speculated that drug

sequestration was due to both electrostatic interactions as well as hydrophobic

effects.58 Dhanikula et al (2007) showed that the addition of lipid vesicles to protein

free and albumin-containing buffers containing amitriptyline led to 98.1% uptake of

the drug into the vesicles.59 In addition, it was demonstrated that a lower internal pH

of the vesicles led to greater drug uptake. Mazoit et al (2009) used an in vitro

experiment with buffer solution containing bupivacaine, levobupivacaine and

ropivacaine to demonstrate high binding capacity of ILE for these drugs.60 The study

also showed that a long-chain triglyceride ILE had a higher binding capacity than a

medium-chain/long-chain triglyceride ILE. Also, that lowering the pH of the buffer

solution decreased the binding capacity of both emulsions. French et al (2011)

explored lipid partitioning further and examined the in vitro reduction of free drug

concentration of 11 drugs in serum following the addition of an ILE.61 Using a multiple

linear regression model, they concluded that the binding of drugs to the ILE could be

predicted by combining the lipid partition constant with the volume of distribution,

since together these values accounted for 88% of the variation in the decrease in

serum drug concentration after the addition of an ILE. In a unique in vitro model,

Samuels et al (2012) assessed the efficacy of lipid partitioning by measuring the

suppression of methaemoglobin formation in blood, after the addition of an ILE, by

drugs of varying lipophilicity that are known to cause methaemoglobin formation.62

Only the most lipophilic drug had significant (p<0.001) suppression of methaemoglobin

formation, supporting lipid partitioning as a mechanism of action.

In a rabbit model of clomipramine toxicosis, Harvey et al (2009) demonstrated a

significant (p=0.0001) increase in plasma clomipramine concentration in lipid treated

37

rabbits compare to a saline control, consistent with intravascular lipid sequestration.21

In an isolated rat heart model, Zausig et al (2009) showed that ILE shortened

haemodynamic parameter recovery times in bupivacaine perfused hearts, compared

to ropivacaine and mepivacaine perfused hearts.63 The authors concluded that this

difference was because of the lower lipophilicity of the latter two drugs compared to

bupivacaine, thus supporting the idea of lipid partitioning of drugs after ILE

administration. Further evidence for lipid partitioning is given by Niiya et al (2010)

whose porcine model of amiodarone-induced hypotension demonstrated increased

plasma amiodarone concentrations in pigs infused with ILE compared to Ringer’s

acetate solution.64 Furthermore, when blood samples were ultracentrifugated, there

was a significantly higher amiodarone concentration in the lipid phase, compared to

the plasma aqueous phase, in ILE treated pigs. Also, amiodarone concentrations in the

plasma aqueous phase of ILE treated pigs were significantly lower than those of the

control pigs. In another porcine model, Litonius et al (2012) showed that in pigs with

amitriptyline toxicosis, an ILE infusion prevented a decrease in plasma total

amitriptyline concentration, resulting in a 90% higher (p<0.001) total concentration

and significantly (p=0.014) lower free fraction of plasma amitriptyline, compared with

the control group.22

Numerous case reports infer lipid partitioning of drugs has occurred after ILE

administration. Sirriani et al (2008) demonstrated an increase in serum bupropion

concentration and triglyceride concentration after an ILE infusion in a patient with

cardiovascular collapse from a bupropion and lamotrigine overdose.50 No change in

the serum concentration of lamotrigine was noted and this observation could be

explained by the high lipophilicity of bupropion compared to the low lipophilicity of

lamotrigine. Litz et al (2008) described a patient with mepivacaine toxicosis whose

plasma mepivacaine concentration decreased after ILE infusion.65 The authors suggest

that increased distribution or partitioning into alternate lipid stores was the likely

cause for this change; however, it is unclear whether the mepivacaine concentration

was from lipid free plasma or not, making it difficult to speculate on the cause of the

decrease. French et al (2011) described a patient with verapamil toxicosis who

received an ILE infusion.33 Serum verapamil concentrations measured from the

aqueous phase decreased following ILE infusion, which was concurrent with increased

38

triglyceride levels. A dog with ivermectin toxicosis reported by Clarke et al (2011) had

modest increases in serum ivermectin concentrations immediately after an ILE

infusion.37 These case reports support the conclusions of experimental studies; that

lipophilic drugs can partition into an expanded plasma lipid phase created by an ILE

infusion. However, confirmation of lipid partitioning does not always imply an

improvement in clinical signs of toxicosis in vivo.

Myocardial mitochondrial recovery

Under normal aerobic conditions, fatty acids are the primary substrate for myocardial

adenosine triphosphate (ATP) production. Local anaesthetics, in particular

bupivacaine, may impair the production of ATP through inhibition of carnitine

acyltransferase, which is essential for the transport of fatty acids across the inner

mitochondrial membrane.66 Therefore, a proposed mechanism of action of ILE in drug

toxicosis is reversal of cardiodepressant effects from altered myocardial fatty acid

transport. Tricyclic antidepressants and verapamil have also been shown to alter

mitochondrial oxidative phosphorylation,67,68 so this proposed mechanism of action

may extend to these drugs. An ILE may overcome this inhibition of fatty acid transport

through mass action thus improving myocardial performance in these toxicoses as

critical energy substrates are restored.

Supporting this theory, a study by Van De Velde (1996) showed that dogs infused with

ILE after undergoing a 10 minute episode of regional myocardial ischaemia had

improved cardiac contractility compared to dogs receiving saline.69 In addition, this

beneficial effect was lost if ILE treated dogs were pretreated with oxfenicine, which is a

drug that inhibits carnitine acyltransferase and thus fatty acid oxidation. The authors

concluded that this beneficial effect was due to enhanced free fatty acid availability

and oxidation in cardiac myocytes. This is in contrast to another study investigating ILE

therapy for verapamil toxicoses, where oxfenicine did not block the beneficial effects

of ILE in rats with induced verapamil toxicosis.70 Later, Stehr et al (2007) demonstrated

in perfused isolated rat hearts,71 that an ILE delivered at a concentration too low to

significantly change the bupivacaine concentration in the effluent was able to reverse

bupivacaine-induced cardiac dysfunction. This study therefore delineates that a

beneficial effect of ILE may be due solely to reversal of free fatty acid blockade in

myocytes and not because of the effects of lipid partitioning. Further evidence of

39

mitochondrial recovery comes from Partownavid et al (2012) who demonstrated in

rats that fatty-acid oxidation is required for successful rescue of bupivacaine-induced

cardiotoxicity with an ILE.72 In addition, the same study showed that this rescue action

was associated with inhibition of mitochondrial permeability transition pore opening, a

step in cell death, which is a mechanism of mitochondrial depolarization in bupivacaine

toxicosis. There were no studies identified that specifically measure myocardial cellular

energy during toxicoses and ILE infusion.

Direct cardiac inotropy

Lipid metabolites have been shown to accumulate in the ischaemic myocardium,

causing an increase in intramyocyte calcium concentration.73 In the ischaemic

myocardium, this is normally a prelude to cell death, however in the non-ischaemic

drug-depressed myocardium, additional intracellular calcium may have an inotropic

effect.74 An ILE may also attenuate reperfusion injury in ischaemic myocardium

through its inhibition of mitochondrial permeability transition pore opening, despite

rising intracellular calcium concentration, which normally causes the pore to open

leading to cell death.75 Therefore, the provision of additional lipid metabolites, such as

through the administration of ILE, may restore myocardial function in cases of

cardiotoxicosis. This may be particularly true of toxicosis caused by calcium channel

blockers though there is limited direct evidence for this mechanism.

No studies were found that assessed intramyocyte calcium levels during lipophilic drug

toxicosis alone, or with ILE therapy. Therefore, this mechanism of action is purely

speculative.

Conclusions

Not all proposed mechanisms of action of ILE will be relevant to every lipophilic drug

toxicosis and, prior to ILE administration, consideration must be given to the

mechanism of toxicosis and chemical properties of the drug. The mechanism of

intravascular lipid partitioning is well supported by experimental studies across a range

of drugs with diverse mechanisms of toxicosis; however, these observations do not

imply an in vivo effectiveness of reversing clinical signs of toxicosis. There is good

evidence to support ILE reversal of myocardial fatty acid transport blockade from local

anaesthetic toxicosis, but not for other lipophilic drugs. The mechanism of direct

40

cardiac inotropy through increased intramyocyte calcium concentration is hypothetical

at this time and further research is required to assess its relevance to lipophilic drug

toxicosis and subsequent ILE therapy.

Intravenous lipid emulsion formulations

Formulations of ILE are generally composed of medium-chain triglycerides, long-chain

triglycerides, or a combination of both. The emulsions have a mean droplet size of 0.1-

1.0 μm, though some research investigating lipophilic drug toxicoses have used

emulsions with a droplet size <0.1 μm. The most common ILE available in Australia

consists of concentrations between 10-30% of long-chain triglycerides derived from

soybean oil combined with egg phospholipids and glycerol. The major long-chain

triglyceride fatty acid components are linoleic, oleic, palmitic, linolenic and stearic.

Though soybean oil is most commonly used as the major source of triglycerides, other

source are used in other formulations including safflower oil, olive oil and fish oil. The

20% ILE called Intralipid®, which has been studied the most in the application of

lipophilic drug toxicoses, typically contains 20% soybean oil, 1.2% egg phospholipids

and 2.25% glycerin in water,76 which is similar to other 20% ILE formulations. The

osmolality of a 20% ILE is approximately 350 mOsm/kg. Chylomicrons in ILE are cleared

from the blood in a manner similar to endogenous chylomicrons.55 The half-life in

blood of ILE chylomicrons has been reported to be between 5.34 and 6.51 minutes.77

There are very few articles that attempt to determine the optimal properties of ILE for

lipophilic drug toxicoses. An in vitro study by Fallon & Chauhan (2006) demonstrated

that negatively charged triglycerides were more effective at sequestering the lipophilic

drug amitriptyline that those with a neutral charge.58 However, the particle size of the

emulsion used was significantly smaller than those found in lipid emulsions

commercially available for patient use. Dhanikula et al (2007) showed a lower internal

pH of lipid vesicles led to greater amitriptyline uptake in an in vitro model.59 Mazoit et

al (2009) showed in an in vitro experiment that a long-chain triglyceride ILE had a

higher binding capacity for three local anaesthetics than a medium-chain/long-chain

triglyceride ILE.60 In an intact rat model, Li et al (2011) showed that medium-

chain/long-chain triglyceride ILE and long-chain triglyceride ILE were no different in

their ability to reverse severe bupivacaine overdose.78 However, this study also

41

showed that significantly more rats receiving the medium-chain/long-chain triglyceride

ILE subsequently developed intractable cardiac arrest. A later in vitro study by Ruan et

al (2012), using human serum spiked with local anaesthetics, showed that adding a

medium-chain/long-chain triglyceride ILE extracted the lipophilic drugs to a greater

extent than did adding a long-chain triglyceride ILE.79

Conclusions

The optimal formulation of ILE for lipophilic drug toxicosis is yet to be determined. A

20% long-chain triglyceride lipid emulsion has been most widely studied and is readily

commercially available. As with all experimental studies, the in vitro ability of an ILE to

sequester a drug will not necessarily translate into effectiveness at reversing signs of

toxicosis in vivo.

Intravenous lipid emulsion dose

Current professional body guidelines for treating severe local anaesthetic systemic

toxicity recommend a 1.5 mL/kg bolus of a 20% ILE followed by an infusion of 15

mL/kg/h for up to 50 minutes.1,2 These guidelines have been applied to a range of

other lipophilic drug toxicoses, however the optimal dose of ILE for toxicological

emergencies has not been well researched.

A study by Perez et al (2008) investigated the optimal dose of ILE for reversing signs of

verapamil toxicosis in rats, and found that a dose of 18.6 mL/kg resulted in the

greatest improvement in the primary outcomes of survival rate and survival time.26

Increasing the dose further to 24.8 mL/kg did not improve survival statistics but did

lead to greater improvements in the secondary outcomes of mean arterial blood

pressure, heart rate and base excess. Increasing the dose to 37.6 mL/kg conferred no

benefit to primary or secondary outcomes compared to the aforementioned doses.

This study administered these doses relatively quickly (37.6 mL/kg over 15 min)

compared to current recommendations. Perez et al (2009) continued their research by

investigating the benefits of infusion rates of ILE on rats with severe verapamil

toxicosis.28 They administered an ILE at a dose of 18.6 mL/kg over 15, 30, 45, or 60

minutes. The greatest survival times were seen in rats receiving the infusion over 30

minutes or less. Experimental studies reported in this review use infusion doses

42

between 6.0-18.6 mL/kg over variable infusion times. It is quite possible that these

infusion volumes are conservative and higher doses should be trialled. Hiller et al

(2010) demonstrated the LD50 of a 20% soy-based ILE in rats to be 67.8 mL/kg,80 which

is a dose much higher than those used in previous experimental studies, suggesting

there is a wide safety margin for further increases in ILE dose for lipophilic drug

toxicoses. It is possible that the lack of clinical response to ILE in experimental studies

may be due to ineffective dosing of the ILE.

Repeated ILE boluses have also been advocated in both human and veterinary

publications though the recommendations are not consistent.1,2.81 Bolus dosing of ILE

may help to reverse life-threatening cardiovascular effects of certain drug intoxications

faster than what can be achieved by a continuous infusion alone. However, based on

the short half-life in blood of ILE chylomicrons of less than 7 minutes a bolus is unlikely

to significantly affect the size of the lipid sink achieved with a continuous rate infusion.

Therefore, it is questionable whether a bolus is required before the start on an ILE

effusion in drug toxicoses which do not have life-threatening cardiovascular effects.

Conclusions

The current recommendations for ILE infusion volumes and infusion rates may be too

conservative to optimise clinical response to an ILE or to demonstrate a benefit in

experimental trials. Further research is needed to determine optimal dosing regimes in

lipophilic drug toxicoses which appear to respond to ILE therapy. The use of a bolus

prior to starting an ILE infusion may not be necessary in drug toxicoses where life-

threatening cardiovascular collapse is not present, but the use of a bolus is not

expected to be detrimental where its administration does not delay the onset of ILE

therapy.

Adverse effects of intravenous lipid emulsions

The safety of ILE in the treatment of lipophilic drug toxicoses has not been established

and information must be extrapolated from studies on ILE use for parenteral nutrition.

The main differences between the two applications are the dose and duration of

infusion, with higher doses for shorter durations being used for treatment of toxicoses.

43

Package inserts provided with the ILE outline the contraindications and possible

adverse events. Contraindications include hyperlipidaemia, severe hepatic insufficiency

and allergy to eggs or soy protein.76,82 Another contraindication could be myocardial

infarction as fatty acids increase intracellular calcium concentration and may promote

cell death.73

Safety of ILE administration is mostly related to stability of the emulsion and plasma

clearance in susceptible patients. Unstable emulsions are more likely to cause an acute

adverse event within 10-20 minutes of starting an effusion. In people, an acute adverse

event to ILE is characterised by chest or back pain, headache, diaphoresis, nausea,

vomiting, hyperthermia and dyspnoea, which are signs consistent with an

anaphylactoid reaction.76,82 Sub-acute reactions in people, commonly referred to as fat

overload syndrome, may be observed after several days of infusion and are associated

with thrombocytopenia, leucopaenia, fever, epigastric pain, hepatomegaly,

splenomegaly, jaundice, transient increases in liver enzymes and pancreatitis.76,82-84

Sub-acute reactions are more common where high infusion rates or prolonged

duration (>6 weeks) of ILE are used and/or when ILE is administered to patients with

reduced plasma clearance mechanisms.

Concerns in the literature have been raised as to the effects of ILE on the liver and

lungs, when used for toxicological emergencies. In a rat model, Driscoll et al (2006)

showed that the oxidative stress after infusion of an unstable ILE was mostly

associated with the liver and coincided with increased serum AST concentration.85

Studies have also demonstrated reduced reticuloendothelial function of the liver after

ILE infusion.85,86 In a guinea pig model, Driscoll et al (2005) showed that administration

of an unstable ILE led to significant oxidative stress in lung tissue.87 Venus et al (1989)

showed that an ILE infused to patients with adult respiratory distress syndrome (ARDS)

significantly reduced the PaO2:FiO2 ratio, and increased alveolar to arterial oxygen

gradient, mean pulmonary arterial pressure, pulmonary vascular resistance and

pulmonary venous admixture.88 These effects were confirmed by Hwang et al (1990)

who showed that in patients with ARDS, ILE decreased PaO2:FiO2 ratios and increased

alveolar to arterial oxygen gradients, while there was no effect in patients with

infectious pulmonary disease or chronic obstructive pulmonary disease.89 A more

44

recent study by Lekka et al (2004) monitoring cardiopulmonary function in placebo and

ILE treated people, with and without ARDS, found that people with ARDS treated with

ILE had decreased oxygenation parameters, reduced pulmonary compliance and

increase pulmonary vascular resistance.90 By performing bronchoalveolar lavage

before and after treatment they were able to show that ILE treatment in ARDS patients

led to an increase in the lavage fluid of total protein, phospholipid and platelet-

activating factor concentrations, and increased phospholipase activity, and

neutrophils. The authors speculated that ILE infusion used in ARDS patients stimulated

release of inflammatory mediators that can cause oedema, further inflammation and

surfactant alterations. These studies show that ILE can have effects on pulmonary

function and its use in people and animals with abnormal respiratory function should

be considered with caution. A case reported by Sirianni et al (2008) described acute

lung injury in a person after treatment with an ILE for unresponsive cardiopulmonary

arrest, secondary to lamotrigine and bupropion toxicosis.50 It is unknown whether the

acute lung injury was a direct effect of the ILE or because of prolonged

cardiopulmonary arrest or the preceding seizure activity.

Fat embolism following ILE infusion has been reported in people. Barson et al (1978)

reported four infants with histological evidence of fat embolism.91 In a case series,

Hessov et al (1979) showed, at post-mortem, accumulations of white clotted material

in the heart with a fatty acid composition similar to the infused ILE.92 Similar

accumulations were found in small vessels of the lungs, cerebrum and kidneys. The

authors hypothesised that fibrin may have precipitated in the hyperlipaemic plasma.

Schröder et al (1984) later demonstrated that intravascular fat emboli present in the

lungs of 22 infants were all post-mortem artefact.93 It is therefore not definitively

established whether ILE can lead to fat emboli.

Neurological complications have been reported in fat overload syndrome which

include weakness, focal and generalised seizures, and altered mentation. Histological

evaluation of two children that died in such circumstances showed cerebral

endothelial and intravascular lipid deposition leading to areas of necrosis in the brain,

and also spleen, liver, kidney and lymph nodes.83

45

Microbiological contamination of the ILE may also occur with inappropriate handling or

intravenous catheter placement, with phlebitis and/or bacteraemia resulting. In

addition, ILE infusion has been associated with reduced immune function.

Nordenström et al (1979) showed that infusion of ILE to healthy individuals lead to

impairment of the chemotactic and random migration of leukocytes which was

correlated to the dose of ILE and the degree of hypertriglyceridaemia.94 They also

showed in vitro, that an ILE impaired leucocyte motility. A more recent study by Kang

& Yang (2008) demonstrated reduced neutrophil function in dogs with an ILE infusion

delivering 200% of the animal’s basal energy requirements in 2 hours.95 In both studies

normal leukocytes function was restored within 24 hours of ending the infusion. It is

unlikely, given the short duration of ILE administration for drug toxicosis, that these

effects on immune function would have any significant clinical effect. However,

caution perhaps should be exercised when considering ILE administration for drug

toxicoses in animals with existing immune compromise.

Only one adverse event was reported in the literature reviewed pertaining to ILE use

for non-local anaesthetic lipophilic drug toxicosis. In the study by Niiya et al (2010) pigs

treated with ILE for amiodarone toxicosis developed skin redness and mottling, and in

two pigs with the most marked skin changes, hypoxaemia was noted.64 In one human

case report, a dose of lipid five times that intended (2 L as opposed to 400 mL) lead to

no appreciable change in haemodynamic function in a 71 year-old female with

amlodipine toxicosis.43 The only abnormality noted was lipaemic serum and artifactual

serum biochemistry values. These alterations in serum biochemistry values were also

confirmed in rats by Hiller et al (2010),80 and hyperlipidaemia was observed in two

dogs treated with ILE for ivermectin toxicosis in a case series by Wright et al (2011).36

Hiller et al (2010) also established an LD50 of a 20% soy-based ILE in rats of 67.8 mL/kg,

which is a dose much higher that proposed for the treatment of lipophilic

intoxications.80

Other theoretical adverse events that may occur using ILE for lipophilic drug toxicosis

include increased enteric absorption of an ingested lipophilic drug due to the

expanded intravascular lipid compartment, and the possibility for reintoxication if the

drug diffuses out of the ILE or is released upon its metabolism.

46

Conclusions

Adverse events of ILE for lipophilic drug toxicosis has not been studied and research is

need to ascertain its safety for this application. Information available to date would

suggest that ILE are safe, when administered correctly and where contraindications to

their administration do not exist, even at the doses recommended for lipophilic drug

toxicosis. However, close monitoring during the infusion and in the hours thereafter is

recommended.

Conclusions

There is some evidence to support the use of ILE in severe toxicosis caused by beta-

adrenergic blockers, calcium channel blockers and cyclic antidepressants where

conventional therapies have failed, however further experimental research is needed.

The optimal formulation and dose of ILE for lipophilic drug toxicoses have not been

established. Until further research is performed, the current recommendation of a 20%

long-chain triglyceride lipid emulsion administered as a 1.5 mL/kg bolus followed by a

15 mL/kg infusion over 30-60 minutes seems reasonable. This dose may prove to be

too conservative and the infusion time too long to maximise the benefit of ILE and

altering these administration parameters in refractory cases of lipophilic drug toxicoses

could be justified. The administration of ILE at the recommended dose appears safe

however further research is need to specifically assess for adverse effects of this higher

dose of ILE compared to its more commonplace application in parenteral nutrition

formulations.

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two dogs. J Small Anim Pract 2006;47:620-624.

49. Gallagher AE, Grant DC, Noftsinger MN. Coma and respiratory failure due to moxidectin

intoxication in a dog. J Vet Emerg Crit Care 2008;18:81-85.

50. Sirianni AJ, Osterhoudt KC, Calello DP, et al. Use of lipid emulsion in the resuscitation of a patient

with prolonged cardiovascular collapse after overdose of bupropion and lamotrigine. Ann Emerg

Med 2008;51:412-415.

51. Livshits Z, Feng Q, Chowdhury F, et al. Life-threatening bupropion ingestion: is there a role for

intravenous fat emulsion? Basic Clin Pharmacol Toxicol 2011;109:418-422.

52. Nogar JN, Minns AB, Savaser DJ, Ly BT. Severe sodium channel blockade and cardiovascular

collapse due to a massive lamotrigine overdose. Clin Toxicol 2011:49:854-857.

53. Castanares-Zapatero D, Wittebole X, Huberlant V, et al. Lipid emulsion as rescue therapy in

lamotrigine overdose. J Emerg Med 2012;42:48-51.

54. Finn SD, Uncles DR, Willers J, et al. Early treatment of a quetiapine and sertraline overdose with

Intralipid. Anaesthesia 2009;64:191-194.

55. Olivecrona G, Olivecrona T. Clearance of artificial triacylglycerol particles. Curr Opin Clin Nutr

Metab Care 1998;1:143-151.

56. Weinberg GL, Ripper R, Murphy P, et al. Lipid infusion accelerates removal of bupivacaine and

recovery from bupivacaine toxicity in the isolated rat heart. Reg Anesth Pain Med 2006;31:296-

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57. Krieglstein J, Meffert A, Niemeyer D. Influence of emulsified fat on chlorpromazine availability in

rabbit blood. Experientia 1974;30:924-926.

50

58. Fallon MS, Chauhan A. Sequestration of amitriptyline by liposomes. J Colloid Interf Sci 2006;300:7-

19.

59. Dhanikula AB, Lamontagne D, Leroux JC. Rescue of amitriptyline-intoxicated hearts with nanosized

vesicles. Cardiovasc Res 2007;74:480-486.

60. Mazoit JX, Le Guen R, Beloeil H, Benhamou D. Binding of long-lasting local anesthetics to lipid

emulsions. Anesthesiology 2009;110:380-386.

61. French D, Smollin C, Ruan W, et al. Partition constant and volume of distribution as predictors of

clinical efficacy of lipid rescue for toxicological emergencies. Clin Toxicol 2011;49:801-809.

62. Samuels TL, Willers JW, Uncles DR, et al. In vitro suppression of drug-induced methaemoglobin

formation by Intralipid® in whole human blood: observations relevant to the ‘lipid sink theory’.

Anaesthesia 2012;67:23-32.

63. Zausig YA, Zink W, Kell M, et al. Lipid emulsion improves recovery from bupivacaine-induced

cardiac arrest, but not from ropivacaine- or mepivacaine-induced cardiac arrest. Anesth Analg

2009;109:1323-1326.

64. Niiya T, Litonius E, Petaja I, Neuvonen PJ, Rosenberg PH. Intravenous lipid emulsion sequesters

amiodarone in plasma and eliminates its hypotensive action in pigs. Ann Emerg Med 2010;56:402-

8.e2.

65. Litz RJ, Roessel T, Heller AR, Stehr SN. Reversal of central nervous system and cardiac toxicity after

local anesthetic intoxication by lipid emulsion injection. Anesth Analg 2008;106:1575-1577.

66. Weinberg G, Palmer J, VadeBoncouer T, et al. Bupivacaine inhibits acylcarnitine exchange in cardiac

mitochondria. Anesthesiology 2000;92:523-528.

67. Weinbach E, Costa J, Nelson B, et al. Effects of tricyclic antidepressant drugs on energy-linked

reactions in mitochondria. Biochem Pharmacol 1986;35:1445-1451.

68. Kline J, Leonova E, Williams T, et al. Myocardial metabolism during graded intraportal verapamil

infusion in awake dogs. Cardiovasc Pharmacol 1996;27:719-726.

69. Van de Velde M, Wouters PF, Rolf N, et al. Long-chain triglycerides improve recovery from

myocardial stunning in conscious dogs. Cardiovasc Res 1996;32:1008-1015.

70. Bania T, Chu J, Lyon T, et al. The role of cardiac free fatty acid metabolism in verapamil toxicity

treated with intravenous fat emulsions. Acad Emerg Med 2007;14:S196-197.

71. Stehr SN, Ziegeler JC, Pexa A, et al. The effects of lipid infusion on myocardial function and

bioenergetics in L-bupivacaine toxicity in the isolated rat heart. Anesth Analg 2007;104:186-192.

72. Partownavid P, Umar S, Li J, et al. Fatty-acid oxidation and calcium homeostasis are involved in the

rescue of bupivacaine-induced cardiotoxicity by lipid emulsion in rats. Crit Care Med 2012;40:2431-

2437.

73. De Villiers M, Lochner A. Mitochondrial Ca2+ fluxes: role of free fatty acids, acyl-CoA and

acylcarnitine. Biochem Biophys Acta 1986;876:309.

74. Huang J, Xian H, Bacaner M. Long-chain fatty acids activate calcium channels in ventricular

myocytes. Proc Natl Acad Sci USA 1992;89:6452-6456.

75. Rahman S, Li J, Bopassa JC, et al. Phosphorylation of GSK-3B mediates intralipid-induced

cardioprotection against ischemia/reperfusion injury. Anesthesiology 2011;115:242-253.

51

76. 20% Intralipid® [package insert]. Baxter Healthcare Corporation, Deerfield, IL; 2006.

77. Park Y, Damron BD, Miles JM, Harris WS. Measurement of human chylomicron triglyceride

clearance with a labeled commercial lipid emulsion. Lipids 2001;36:115-120.

78. Li Z, Xia Y, Dong X, et al. Lipid resuscitation of bupivacaine toxicity: Long-chain triglyceride emulsion

provides benefits over long- and medium-chain triglyceride emulsion. Anesthesiology

2011;115:1219-1228.

79. Ruan W, French D, Wong A, et al. A mixed (long- and medium-chain) triglyceride lipid emulsion

extracts local anesthetic from human serum in vitro more effectively than a long-chain emulsion.


80. Hiller DB, Di Gregorio G, Kelly K, et al. Safety of high volume lipid emulsion infusion: a first

approximation of LD50 in rats. Reg Anesth Pain Med 2010;35:140-144.

81. Fernandez AL, Lee JA, Rahilly L, et al. The use of intravenous lipid emulsion as an antidote in

veterinary toxicology. J Vet Emerg Crit Care 2011;21:309-320.

82. 20%, Ivelip [package insert]. Baxter Healthcare Ltd, Auckland, NZ; 2001.

83. Schulz PE, Weiner SP, Haber LM, et al. Neurological complications from fat emulsion therapy. Ann

Neurol 1994;35:628-630.

84. Kasi VS, Estrada CA, Wiese W. Association of pancreatitis with administration of contrast medium

and intravenous lipid emulsion in a patient with the acquired immunodeficiency syndrome. South

Med J 2003;96:66-69.

85. Driscoll DF, Ling PR, Bistrian BR. Pathological consequences to reticuloendothelial system organs

following infusion of unstable all-in-one mixtures in rats. Clin Nutr 2006;25:842-850.

86. Jensen GL, Mascioli EA, Seidner DL, et al. Parenteral infusion of long- and medium-chain

triglycerides and reticuloendothelial system function in man. J Parenter Enteral Nutr 1990;14:467-

471.

87. Driscoll DF, Ling PR, Quist WC, Bistrian BR. Pathological consequences from the infusion of unstable

lipid emulsion admixtures in guinea pigs. Clin Nutr 2005;24:105-113.

88. Venus B, Smith RA, Patel C, Sandoval E. Hemodynamic and gas exchange alterations during

intralipid infusion in patients with adult respiratory distress syndrome. Chest 1989;95:1278-1281.

89. Hwang TL, Huang SL, Chen MF. Effects of intravenous fat emulsion on respiratory failure. Chest

1990;97:934-938.

90. Lekka ME, Liokatis, Nathanail C, et al. The impact of intravenous fat emulsion administration in

acute lung injury. Am J Respir Crit Care Med 2004;169:638-644.

91. Barson AJ, Chistwick ML, Doig CM. Fat embolism in infancy after intravenous fat emulsions. Arch

Dis Child 1978;53:218-223.

92. Hessov I, Melsen F, Haug A. Postmortem findings in three patients treated with intravenous fat

emulsions. Arch Surg 1979;114:66-68.

93. Schröder H, Paust H, Schmidt R. Pulmonary fat embolism after intralipid therapy--a post-mortem

artefact? Light and electron microscopic investigations in low-birth-weight infants. Acta Paediatr

Scand 1984;73:461-464.

52

94. Nordenström J, Jarstrand C, Wiernik A. Decreased chemotactic and random migration of

leukocytes during Intralipid infusion. Am J Clin Nutr 1979;32:2416-2422.

95. Kang JH, Yang MP. Effect of short-term infusion with soybean oil-based lipid emulsion on

phagocytic responses of canine peripheral blood polymorphonuclear neutrophilic leukocytes. J Vet

Intern Med 2008;22:1116-1173.

53

Chapter 2: Permethrin toxicosis in cats Permethrin, a type I pyrethroid insecticide, is an active ingredient in many over-the-

counter “spot-on” flea treatments available for pets. Permethrin toxicosis in cats has

been reported subsequent to the application of these products.1-3 Reported clinical

signs of toxicosis include seizures, convulsions, muscle fasciculations, tremors,

twitching, shaking, ataxia, hyperaesthesia, hyperthermia, mydriasis, temporary

blindness, vomiting and ptyalism.4-6 These predominately neuroexcitatory signs are

due to its mechanism of action, which makes it effective as an insecticide.

The predominant effect of permethrin is modulation of the gating kinetics of individual

voltage-dependent sodium channels, resulting in repetitive discharge.7-10 Permethrin

slows the closing of the sodium channel m-gate allowing some sodium influx to

continue, resulting in a long tail current after the primary action potential.11 The tail

current causes elevation of the after-potential to threshold potential, resulting in

repetitive discharging of excitable cells.8,11,12 Only a very small percentage, as low as

0.62%, of sodium channels need be modified by a pyrethroid to result in neural

dysfunction.13-15 Other proposed mechanisms of neuroexcitation include changes in

the (Ca2++Mg2+)-ATPase pump and the voltage-dependent chloride channels, in

particular Maxi chloride channels. The importance of these other mechanisms has not

been fully determined.16-18

Mammals have decreased sensitivity to permethrin despite the mechanisms of action

being common between insects and mammals. There are two factors pertinent to the

selective toxicity of permethrin. The first is rate of detoxification, which is faster in

mammals due to their body temperature generally being 10oC higher than insects.15 In

addition, the chance of detoxification occurring before reaching the target site

decreases with small body size making insects more susceptible.15 The second is

intrinsic nerve sensitivity, which is higher in insects. This is because their sodium

channels do not recover as quickly from depolarization as mammalian sodium

channels and their low body temperature enhances the magnitude of permethrin’s

effect on sodium influx.8,9,15,19

54

Of the domestic mammals, cats are particularly sensitive to permethrin toxicosis for

reasons unknown. Variable dermal absorption of permethrin has been demonstrated

across a number of mammalian species.20,21 Although this has not been investigated in

cats, they have been shown to have higher dermal absorption of other drugs,22 and

therefore increased systemic absorption of permethrin may be one cause for their

increased susceptibility to permethrin toxicosis. Permethrin is metabolised by ester

hydrolysis and oxidation by liver microsomal enzymes.23,24 The hydrolytic enzymes that

degrade permethrin esters may have a slower rate of hydrolysis in cats compared with

other species and thus may also be a cause of increased susceptibility to toxicosis in

cats.

Clinical diagnosis of permethrin toxicosis in cats is based on exposure history and

consistent clinical signs. Studies have shown that permethrin and its metabolites can

be detected in human blood and urine,25,26 however, no studies have reported blood

permethrin concentrations in cats with permethrin toxicosis, or correlated such

concentrations with clinical signs. In addition, the technology for such assays is not

widely available. Therefore, at this time, measurement of the concentration of

permethrin, or its metabolites in biological samples, is not routinely performed to

confirm exposure or assess the severity of permethrin toxicosis in cats in the clinical

setting.

Treatment is supportive and there is no antidote to toxicosis. Dermal contamination is

the most common route of intoxication in cats and clipping the area of application,

followed by bathing in warm water with a detergent is effective as a decontamination

measure.21 Drugs to control neuroexcitatory signs are often required and intravenous

drugs reported to be used include methocarbamol, diazepam, pentobarbital and

propofol.4-6 Intravenous fluids are often needed to maintain intravascular volume and

hydration and to provide diuresis where myoglobinuria is present. Attention to nursing

care is also mandatory, especially when animals are recumbent, and oxygen

supplementation, with or without endotracheal intubation, may be required in such

cases.

55

Reported mortality rates are variable, ranging from 2.4% to 16.9%.3-5 Substantial

morbidity is associated with clinical signs and can last days to weeks, with reported

hospitalisation periods up to eleven days.5,6 Permethrin is highly lipophilic with a log P

of 6.1 at 20oC.27 Therefore, intravenous lipid emulsion may be an effective antidote in

the treatment of permethrin toxicoses in cats leading to a reduction in morbidity and

mortality.

References

1. Richardson, JA. Permethrin spot-on toxicosis in cats. J Vet Emerg Crit Care 2000;10:103-106.

2. Linnett, P-J. Permethrin toxicosis in cats. Aust Vet J 2008;86:32-35.

3. Malik R, Ward MP, Seavers A, et al. Permethrin spot-on intoxication of cats: literature review and

survey of veterinary practitioners in Australia. J Feline Med Surg 2010;12:5-14.

4. Sutton NM, Bates N, Campbell A. Clinical effects and outcome of feline permethrin spot-on

poisonings reported to the Veterinary Poisons Information Service (VPIS), London. J Fel Med Surg

2007;9:335-339.

5. Dymond NL, Swift IM. Permethrin toxicity in cats: a retrospective study of 20 cases. Aust Vet J

2008;86:219-223.

6. Boland LA, Angles JM. Feline permethrin toxicity: retrospective study of 42 cases. J Fel Med Surg

2010;12:61-71.

7. Vijverberg HPM, van den Bercken J. Action of pyrethroid insecticides on the vertebrate nervous

system. Neuropathol Appl Neurobiol 1982;8:421-440.

8. Vijverberg HP, van der Zalm, van den Bercken J. Similar mode of action of pyrethroids and DDT on

sodium channel gating in myelinated nerves. Nature 1982;295:601-603.

9. Vijverberg HPM, de Weille JR. The interaction of pyrethroids with voltage dependent sodium

channels. Neurotoxicology 1985;6:23-34.

10. Lombert A, Mourre C, Lazdunski M. Interaction of insecticides of the pyrethroid family with specific

binding sites on the voltage-dependent sodium channel from mammalian brain. Brain Res

1988;459:44-53.

11. Narahashi T. Nerve membrane ionic channels as the primary target of pyrethroids. Neurotoxicology

1985;6:3-22.

12. Vijverberg HPM, van den Bercken J. Neurotoxicological effects and the mode of action of

pyrethroid insecticides. Crit Rev Toxicol 1990;21:105-126.

13. Lund AE, Narahashi T. Dose-dependent interaction of the pyrethroid isomers with sodium channels

of squid giant axon membranes. Neurotoxicology 1982;3:11-24.

14. Tatebayashi H, Narahashi T.Differential mechanism of action of the pyrethroid tetramethrin on

tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels. J Pharmacol Exp 1994;270:595-

603.

56

15. Song JH, Narahashi T. Modulation of Sodium Channels of Rat Cerebellar Purkinje Neurons by the

Pyrethroid Tetramethrin. J Pharmacol Exp Ther 1996;277:445-453.

16. Lawrence LJ, Casida JE. Stereospecific action of pyrethroid insecticides on the gamma-aminobutyric

acid receptor-ionophore complex. Science 1983;221:1399-1401.

17. Bloomquist JR, Adams PM, Soderlund DM. Inhibition of gamma-aminobutyric acid-stimulated

chloride flux in mouse brain vesicles by polychlorocycloalkane and pyrethroid insecticides.

Neurotoxicology 1986;7:11-20.

18. Michelangeli J, Ronson MJ, East JM, et al. Fluorescence and kinetic studies of the interactions of

pyrethroids on the (Ca2+Mg2+)-ATPase. Biochem Biophys Acta 1990;1028:58-66.

19. Chinn K, Narahashi, T. Temperature-dependent subconducting states and kinetics of deltamethrin-

modified sodium channels of neuroblastoma cells. Pflugers Arch 1989;43:571-579.

20. Sidon EW, Moody RP, Franklin CA. Percutaneous absorption of cis- and transpermethrin in rhesus

monkeys and rats: anatomic site and interspecies variation. J Toxicol Env Health 1988;23:207-216.

21. Hughes MF, Edwards BC. In vitro dermal absorption of pyrethroid pesticides in human and rat skin.

Toxicol Appl Pharm 2010;246:29-37.

22. Sarasola P, Jernigan AD, Walker DK, et al. Pharmacokinetics of selamectin following intravenous,

oral and topical administration in cats and dogs. J Vet Pharmacol Therap 2002;25:265-272.

23. Ross MK, Borazjani A, Edwards CC, Potter PM. Hydrolytic metabolism of pyrethroids by human and

other mammalian carboxylesterases. Biochem Pharmacol 2006;71:657-669.

24. Nakamura Y, Sugihara K, Sone T, et al. The in vitro metabolism of a pyrethroid, permethrin, and its

hydrolysis products in rats. Toxicology 2007;235:176-184.

25. Ogata-Kawata H, Matsuda M, Onda N, et al. Direct analysis of permethrins in human blood by SPE-

GS/MS. Chromatography 2007;28:119-124.

26. Lin CH, Yan CT, Kumar PV, Li HP, Jen JF. Determination of pyrethroid metabolites in human urine

using liquid phase microextraction coupled in-syringe derivatization followed by gas

chromatography/electron capture detection. Anal Bioanal Chem 2011;401:927-937.

27. Tomlin CDS. The Pesticide Manual: A World Compendium. 14th ed. British Crop Production Council;

2006, pp. 813-814.

57

Chapter 3: Summary and hypothesis for present study

The literature supports the view that an ILE could be beneficial across a range of

lipophilic drug toxicosis. The literature also supports intravascular lipid partitioning as a

common mechanism of action of ILE in the treatment of lipophilic drug toxicosis.

Intravenous lipid emulsion for this application appears to be safe. Permethrin is a

highly lipophilic insecticide that causes significant morbidity and mortality in cats

through its neuroexcitatory effects. Before recommending ILE treatment routinely in

cats with permethrin toxicosis, evidence for its efficacy needs to be established.

The aims of this study were to (1) determine if a lipid emulsion added to feline plasma

containing permethrin in vitro would lead to a decrease in plasma permethrin

concentration thus supporting lipid partitioning as a potential mechanism of action,

and (2) assess the clinical response to intravenous lipid emulsion administration in cats

with permethrin toxicosis.

The research hypothesis was that (1) the addition of a lipid emulsion to feline plasma

containing permethrin would significantly decrease the plasma permethrin

concentration once the lipid supernatant was removed, (2) the clinical stages of

permethrin toxicosis in ILE-treated cats would improve earlier compared to cats

receiving a control.

58

Chapter 4: Sequestration of permethrin into lipid emulsion; an in vitro feline plasma model

Introduction

Permethrin is a pyrethroid insecticide and is the active ingredient in a number of over-

the-counter flea treatments for dogs. Permethrin is generally considered to have low

toxicity in mammals.1,2 However, permethrin toxicosis is well documented in cats and

the reasons for the susceptibility of this species are not known.3-11 Reports from the

United Kingdom and the United States of America indicate that permethrin is one of

the most common causes of intoxication in cats,7,12 with a reported mortality rate as

high as 16.9% (127/750) with an additional 5.2% (39/75) of cats euthanised.11

Interest in intravenous lipid emulsion (ILE) therapy has been stimulated by success in

reversing the life-threatening cardiovascular effects of local anaesthetic overdose in

rats and dogs.13,14 Since then, more rapid resolution of clinical signs or augmentation

of cardiopulmonary resuscitation efforts have been reported after ILE administration

for a range of other drug toxicoses including permethrin.15,16

One possible mechanism of action of ILE therapy in lipophilic drug toxicoses is the

formation of a lipid sink. The ILE is thought to create an intravascular lipid

compartment into which lipophilic drugs are sequestered from their sites of action

until they are metabolised.17 The lipid sink theory has been supported by in vivo

reports of increases in plasma drug concentrations after ILE infusion, along with clinical

improvement of toxicosis in humans and animals.13,18-21 In addition, in vitro

experiments measuring drug concentrations in plasma containing LE have shown a

significant increase in drug concentration in the lipid phase of the solution, compared

to the plasma, or a reduction in the free drug concentration in the plasma.13,22,23

Permethrin is a highly lipophilic drug and there have been subjective reports of an

improvement in clinical signs in cats with permethrin toxicosis after treatment with an

ILE.15,16 It is unknown if this improvement may be due to sequestration of permethrin

within the lipid compartment, consistent with the lipid sink theory. The aim of this

study was to determine whether a LE added to feline plasma containing permethrin

would decrease the plasma permethrin concentration. Our research hypothesis was

59

that the addition of a LE to feline plasma containing permethrin would significantly

decrease the plasma permethrin concentration once the lipid supernatant was

removed.

Methods

Sample preparation

Twelve units of feline fresh frozen plasma were obtained from the institutions feline

blood bank. Permission for use was granted from the institution’s Animal Ethics

Committee. The plasma was warmed to 37oC and then pooled in a glass conical flask.

The plasma was vortexed by an orbital shakera at 215 rpm for 5 minutes at 37oC and

then divided into three equal volumes to perform the following experiment in

triplicate.

A 450 μL aliquot of plasma was removed to serve as a blank matrix. Permethrinb with

an isomer ratio of 25% cis- and 75% trans- was added to the remaining plasma to a

concentration of 125 μg/mL. The solution was then vortexed by an orbital shakera at

215 rpm for 5 minutes at 37oC. A 3 mL aliquot was removed to serve as a control

solution to measure for permethrin degradation over time and an aliquot of 450 μL

was removed from this solution to confirm the permethrin concentration prior to

adding the LE.

A 20% LEc was added to the feline plasma + permethrin solution at a volume

equivalent to 5.9% v/v (time 0 hours). The solution was agitated continuously by an

orbital shakera at 215 rpm at 37oC for the duration of the experiment. At time 0.5, 1, 3

and 6 hours after the LE was added, samples were removed from the solution and

subjected to ultracentrifugationd at 22,000g at 37oC for 10 minutes to separate the

plasma from the LE. Plasma samples were collected for permethrin quantification and

the remaining plasma was discarded leaving a film of lipid supernatant that was also

collected. All samples were placed into individual screw-cap amber glass vials and were

immediately stored at -80oC until analysis.

Working solutions preparation

Stock solutions of 10 μg/mL high purity13C6 labeled cis- and trans- permethrine were

used as internal standards and were prepared to a working solution concentration of

60

500 ηg/mL with a cis:trans ratio of 1:1 using 3% toluenef and acetonitrileg.

Perchloropentacyclodecaneh was used as an instrumental standard alongside each

sample injection and was diluted in acetonitrile to a working solution concentration of

5 μg/mL.

Modified QuEChERS extraction

Plasma samples were defrosted and allowed to warm to a room temperature of 22oC.

A 120 μL aliquot from each plasma sample was added to a 15 mL labelled centrifuge

tube containing 3 mL of ultrapure wateri. The centrifuge tubes were capped and

vortexed by hand for 30 seconds. After completion of the mixing, 3 mL of 13C6 labelled

permethrin 10μg/mL working solution was added to the tubes, the tubes were capped

and vortexed by hand for 30 seconds. The mixture was then added to 15 mL labelled

centrifuge tubes containing pre-packaged dry extraction reagentsj of 2.0 g anhydrous

magnesium sulphate, 0.5 g sodium chloride, 0.25 g disodium

hydrogencitratesesquihydrate and 0.5 g trisodium citrate dehydrate. The tubes were

capped and agitated by hand for 10 s and were then placed on ice until cool. The tubes

were then shakenk for 60 seconds and then centrifugedl at 5,251 g for 5 minutes. After

centrifugation, 1 mL of crude acetonitrile extract containing permethrin was removed

from each centrifuge tube and transferred to the 2 mL labelled centrifuge tubes

containing pre-packaged clean-up reagentsj of 150 mg anhydrous magnesium sulphate

anhydrous, 150 mg clean-up n-2 aminoethyl, 50 mg endcappedoctadecyl. The

centrifuge tubes were capped, vortexed by hand for 10 seconds and were then placed

on ice until cool. Once cool the centrifuge tubes were vortexed by hand for 30 seconds

and microfugedm at 9,000 g for 2 minutes. From each tube a 450 μL aliquot of

supernatant was transferred to 2 mL labelled screw-cap amber glass vials. As a

performance standard, 50 μL of perchloropentacyclodecane working solution was

added to each vial and vortexed by hand for 30 seconds. Aliquots of 100 μL from each

vial were transferred to labelled amber injection vials.

Gas chromatography

Samples were analysed with a gas chromatographn (GC) with autosamplero. Injections

were performed using 10 μL autosampler syringesp and an injectorq using inlet liners

for split injectionsr. The autosampler was set to perform five injection acetonitrile

61

syringe washes, one 2 μL sample injection and 10 post-injection acetonitrile syringe

washes. The injector used liquid CO2 coolant (enabled at 290oC) and was programmed

to hold the temperature at 270oC for 2 minutes and then ramp at 100oC/minute to

300oC where it was held for 5.5 minutes. The split vent was closed for 1.5 minutes and

then switched to split mode with a ratio of 100:1 for 3.5 minutes and then 20:1 until

the end of the run. The carrier gas was ultrahigh purity helium with a constant flow

rate of 1.1 mL/min. A 10 m integrated columns was coupled to a 30 m, 0.25 mm i.d.,

0.25 μm film thickness analytical columnt. The GC oven was initially set at 80oC for 3

minutes and then ramped at 40oC/minute to 300oC where it was held for 3.5 minutes.

The transfer line temperature was 250oC.

Mass spectrometry

The GC was coupled with a triple quadrupole mass spectrometeru. The source

temperature was 200oC and the manifold temperature was 37oC. The collision gas used

in the second quadrupole was argon at 1.5 Torr. The retention times of chemicals of

interest were: perchloropentacyclodecane 10.37 minutes, 13C6cis-permethrin 10.47

minutes, 13C6 trans-permethrin 10.55 minutes, cis-permethrin 10.47 minutes and

trans-permethrin 10.55 minutes. The transitions and optimum collision energies of the

chemicals of interest were: perchloropentacyclodecane 274-237(15), 274-239(15),

272-237(20); 13C6cis-permethrin 189-159(25), 189-121(30),189-132(30),189-144(30);

13C6 trans-permethrin 189-159(25), 189-121(30),189-132(30),189-144(30); cis-

permethrin 183-165(15), 183-153(10); and trans-permethrin 183-165(15), 183-153(10).

Permethrin concentrations

Chromatographic peak areas of the samples, labelled standards and

perchloropentacyclodecane were exported into an Excel spreadsheet template from

the instrument data filesv. Quantification of permethrin was performed by comparing

chromatographic peak areas of labeled permethrin to unlabelled permethrin. This peak

area ratio and the known concentration of labelled permethrin isomers added to the

samples prior to extraction were used to determine the concentration of cis- and

trans-permethrin in the samples. The concentrations of cis- and trans- permethrin

were summated to calculate the total permethrin concentration in the plasma at each

time point. The concentration of cis- and trans- permethrin at the 0 hour time point

62

was estimated using the measured concentrations prior to LE addition and calculating

the expected dilution by the volume of LE.

Statistical analysis

The total permethrin concentration in the plasma was the response of interest and

was tested for normality using the Shapiro-Wilk statistic with the null hypothesis of

normality rejected at p<0.05. Since the data followed a normal distribution, a one-way

ANOVA for repeated measures was used to test for an effect of time on permethrin

concentration. Where there was a significant effect of time at p<0.05, comparisons

across time points were made using Tukey’s adjustment for multiple comparisons, with

the adjusted p<0.05 used to determine significance. A similar analysis was also

performed on the control solution samples taken to monitor degradation of

permethrin over time. Data was summarised as mean +/- SD. Statistics were

performed using a commercial available statistical software programw.

Results

Appendix 1 contains the raw data for the chromatographic peak areas of the

compounds on interest.

Instrumental precision

The coefficient of variation for the peak area of the perchloropentacyclodecane signal

as an instrumental standard was 9.85%. The coefficient of variation for the peak area

of the 13C6 cis-permethrin and 13C6 trans-permethrin signals were 10.02% and 12.91%

respectively.

Permethrin concentrations

The blank matrix, before any addition of permethrin, had a mean total permethrin

concentration of 1.53 μg/mL (SD ± 0.17 μg/mL). There was a significant change in the

total permethrin concentration in the control solution over time (P<0.001), with

fluctuation over time but no pattern of change (Table 1, Figure 1).

63

Table 1 - Concentration of total permethrin in feline control plasma over time.

Time (h) Mean (μg/mL) SD (μg/mL) Lower confidence

limit (μg/mL)

Upper confidence

limit (μg/mL)

0 124.7p 3.8 115.2 134.1

0.5 138.6q 1.9 133.9 143.3

1 126.6p 2.7 119.8 133.3 3 126.8p 0.6 125.4 128.1

6 138.6q 0.7 136.9 140.2 Means with the same letter are not significantly different (p>0.05)

Figure 1 - Concentration of total permethrin in feline plasma after addition of a lipid

emulsion (LE). The control solution had no addition of LE. Points are mean ± standard

deviation error bars. Means with the same letter are not significantly different

(p>0.05).

64

In regards to the plasma with LE added, there was a significant change in the total

permethrin concentration in the plasma over time (p<0.001). There was an immediate

significant (P<0.05) decrease after the addition of LE and then removal of the lipid

supernatant at 0.5 hours (Table 2, Figure 1). The permethrin concentration remained

significantly decreased until the 3 hour time point after which it significantly increased

at 6 hours.

Discussion

In this study there was a significant decrease in the concentration of permethrin in

plasma after the addition of a 20% LE. This supports the lipid sink theory of lipophilic

drug sequestration after use of ILE in feline permethrin toxicosis. Theoretically,

sequestration of a drug into a newly created intravenous lipid compartment through

the administration of an ILE should reduce the amount of free drug available to cause

toxicosis. Other in vitro studies have reported similar findings to this study using

different lipophilic drugs including chlorpromazine,22 local anaesthetics,13,23,24 and

verapamil.23

An attempt to quantify the concentration of permethrin in the lipid supernatant was

made using the method described. However, there was significant matrix interaction

with the compounds of interest that was non-linear. Further research and

development to quantify the permethrin concentration in the lipid layer would have

been required, but was not pursued due to the significant reduction in plasma

permethrin concentration demonstrated after the addition of the LE.

Table 2 - Concentration of total permethrin in feline plasma over time after addition of a lipid emulsion.

Time (h) Mean (μg/mL) SD (μg/mL) Lower confidence

limit (μg/mL)

Upper confidence

limit (μg/mL)

0 124.7a 3.8 115.2 134.1

0.5 22.4b 0.6 20.0 23.8

1 18.7b 0.7 16.0 20.4

3 17.6b 0.7 15.8 19.5 6 31.3c 0.5 30.0 32.6 Means with the same letter are not significantly different (p>0.05)

65

The plasma permethrin concentration dropped immediately after the addition of the

lipid product and remained decreased but with a slight, yet significant increase at the

six hour time point. The temperature of the solution may have increased over time as

it was continuously agitated and this may have altered the equilibrium of permethrin

molecules between the lipid and plasma phases. Alternatively, it is possible that over

time some of the lipid molecules degraded releasing permethrin into the plasma. The

plasma permethrin concentration at six hours was still well below the initial time 0

hour concentration.

There were small but significant fluctuations in the total permethrin concentration in

the control solution over time. Although the change was significant, this variation was

within the range of instrumental precision. It is possible that an increase in the

temperature of the solution over time may have altered permethrin kinetics, or the

change may reflect a sampling or measurement error. These fluctuations, while noted,

were minimal compared to the dramatic changes seen after the addition of the lipid

product.

The blank plasma had small amounts of permethrin quantified. As the detection limits

of the analysis are at the low ηg/mL level, this value represents a real background in

the plasma. It is possible that the cats which donated blood for the institutions feline

blood bank had been treated with permethrin and this was either not recorded in the

database or was not remembered by an owner. Alternatively, this may be a matrix

effect, which is known to be problematic in pesticide residue analysis using gas

chromatography-mass spectrometry.25 Labelled internal standards were used in this

study to account for matrix effects.

This study was designed to investigate the lipid sink theory, which provides

justification for using an ILE to treat permethrin toxicosis. Limitations are expected in

applying the results of an in vitro investigation to in vivo conditions. The concentration

of permethrin used in this study may be higher than what would be expected in vivo.

The concentration was calculated based on the amount of permethrin in a topical

permethrin product for large dogs, the volume of distribution of permethrin in the

rat,26 and the known dermal bioavailability in cats of selamectin, which is much higher

in cats than other species.27 However, while the concentration used here cannot be

66

easily translated to clinical scenario, the reduction in plasma permethrin concentration

with the addition of a LE is clearly demonstrated.

The volume of 20% LE added to plasma was estimated based on available data,

including the ILE half-life and volume of distribution in rats and an infusion rate of 15

mL/kg/h,28,29 to approximate a lipid concentration that may be achieved in vivo. This

may be an over estimation of the true lipid concentration achieved in vivo, based on

current dosing recommendations. One study suggested that the concentration of

intact lipid droplets in plasma during rapid ILE infusion could not be expected to be

greater than 1% even with large volumes.24 Other in vitro studies have added LE to

plasma in amounts varying from 1-20% v/v of the solution.22,23 It is possible that if

lower in vivo concentrations are achieved with ILE administration compared to the

concentration used in this study that the drug sequestration in vivo may not be as

marked. However, the objective of this study was to simply demonstrate a lipid sink

effect when adding LE to plasma containing permethrin. Further studies using in vivo

models are warranted in order to investigate any dose-dependent effect.

The in vivo effectiveness of the lipid sink mechanism may not be as simple to

demonstrate. In vitro investigations cannot replicate the effects of the endothelium,

sites of drug action or effects of metabolism of the ILE on serum drug concentration. A

number of in vivo studies and case reports in people have shown increased serum drug

concentrations after the administration of ILE.18,19,22,23 This suggests that a new

intravascular lipid compartment can alter a drug’s pharmacokinetic equilibrium leading

to movement of a drug from its site of action into the intravascular compartment thus

reducing toxicosis.18-21 Further evidence supporting the lipid sink mechanism includes

the observation that ILE decreased tissue bupivacaine content by 70% in isolated

murine heart preparations,30 and that ILE significantly suppressed methaemoglobin

formation by a highly lipophilic drug but did not suppress maethaemoglobin formation

caused by less lipid soluble drugs.31

Conclusions

A LE sequesters permethrin in pooled feline plasma. This supports the theory that a LE

injected intravenously can sequester lipophilic drugs in vivo. This may explain how an

67

ILE ameliorates clinical signs in animals and people with lipophilic drug toxicosis,

including permethrin toxicosis in cats.

Footnotes

aOrbit Shaker, Labline Instruments Inc, Melrose Park, USA.

bDermcare-Vet Pty Ltd, Springwood, Australia.

c20% Ivelip®, Baxter Healthcare, Old Toongabbie, Australia.

dAvanti® J-25i with JA 25.50 fixed angle rotor, Beckman Coulter Inc, Brea, USA.

eCambridge Isotopic Laboratories Inc, Andover, USA.

fLab-Scan, Pathumwan, Thailand.

gThermo Fisher Scientific, Scoresby, Australia.

hMirex®,Ultra Scientific Inc, N.Kingston, USA.

iOptima® LC/MS Water, Thermo Fisher Scientific, Scoresby, Australia.

jQuEChERS, UCT Inc, Bristol, USA.

kSK-400, Fast and Fluid Management BV, Unanderra, Australia.

lAllegra® X-15R, Beckman Coulter, Brea, USA.

mIEC Micromaxmicrocentrifuge, Thermo Fisher Scientific, Scoresby, Australia.

nBruker 450, BrukerBioSciences Pty Ltd, Preston, Australia.

oBruker CP-8400, BrukerBioSciences Pty Ltd, Preston, Australia.

pSGE Analytical Science Pty Ltd, Ringwood, Australia.

qBruker PTV 1079, BrukerBioSciences Pty Ltd, Preston, Australia.

rRestekTM Split Precision® Liners with deactivated wool, Thermo Fisher Scientific,

Scoresby, Australia.

sIntegra-Guard®, Thermo Fisher Scientific, Scoresby, Australia.

tRestek Rxi®-5Sil MS, Thermo Fisher Scientific, Scoresby, Australia.

uSCION TQ™ GC-MS, BrukerBioSciences Pty Ltd, Preston, Australia.

vBruker MS Workstation 8 Chemical Analysis software, BrukerBioSciences Pty Ltd,

Preston, Australia

wSAS 9.3 SAS Institute, Cary, USA

References

1. Vais H, Williamson MS, Devonshire AL, Usherwood PNR. The molecular interactions of pyrethroid

insecticides with insect and mammalian sodium channels. Pest Manag Sci 2001;57:877-888.

68

2. Salgado VL, Herman MD, Narahashi T. Interactions of the pyrethroid fenvalerate with nerve

membrane sodium channels: temperature dependence and mechanism of depolarisation.


3. Mount ME, Moller G, Cook J. Clinical illness associated with a commercial tick and flea product in

dogs and cats. Vet Hum Toxicol 1991;33:19-27.

4. Meyer EK. Toxicosis in cats erroneously treated with 45% to 65% permethrin products. J Am Vet

Med Assoc 1999;215:198-203.

5. Richardson JA. Permethrin spot-on toxicosis in cats. J Vet Emerg Crit Care 2000;10:103-106.

6. Bottcher IC, Schenk HC, Tipold A. Permethrin intoxication in ten cats - retrospective evaluation.

Tierarztl Prax 2006;34:185-190.


poisonings reported to the Veterinary Poisons Information Services (VPIS), London. J Feline Med

Surg 2007;9:335-340.

8. Linnett P-J. Permethrin toxicosis in cats. Aust Vet J 2008;86:32-35.


2008;86:219-223.

10. Boland LA, Angles JM. Feline permethrin toxicity: retrospective study of 42 cases. J Feline Med Surg

2010;12:61-71.

11. Malik R, Ward MP, Seavers A, et al. Permethrin spot-on intoxication of cats: literature review and

survey of veterinary practitioners in Australia. J Feline Med Surg 2010;12:5-14.

12. Merola V, Dunayer E. The 10 most common toxicoses in cats. Vet Med 2006;101:339-342.




14. Weinberg G, Ripper R, Feinstein D, Hoffman W. Lipid emulsion infusion rescues dogs from

bupivacaine-induced cardiac toxicity. Region Anesth Pain M 2003;28:198-202.


intravenous lipid administration. Tierarztliche Praxis Kleintiere 2012;40:129-134.


toxicosis. J Vet Emerg Crit Care 2012;22:697-702.

17. Weinberg GL. Lipid emulsion infusion: resuscitation for local anesthetic and other drug overdose.




2009;16:151-156.


amiodarone in plasma and eliminates its hypotensive action in pigs. Ann Emerg Med 2010;56:402-

8.e2.



69

21. Litonius E, Niiya T, Neuvonen PJ, Rosenberg PH. No antidotal effect of intravenous lipid emulsion in


Pharmacol 2012;110:378-383.

22. Krieglstein J, Meffert A, Niemeyer D. Influence of emulsified fat on chlorpromazine availability in

rabbit blood. Experientia 1974;30:924-926.


clinical efficacy of lipid rescue for toxicological emergencies. Clin Toxicol 2011;49:801-809.


emulsions. Anesthesiology 2009;110:380-386.

25. Hajslova J, Zrostlikova J. Matrix effects in (ultra)trace analysis of pesticide residues in food and

biotic matrices. J Chromatogr A 2003;1000:181-197.

26. Anadón A, Martinez-Larrańaga MR, Diaz MJ, Bringas P: Toxicokinetics of permethrin in the rat.


27. Sarasola P, Jernigan AD, Walker DK, et al. Pharmacokinetics of selamectin following intravenous,

oral and topical administration in cats and dogs. J Vet Pharmacol Therap 2002;25:265-272.

28. Hultin M, Carneheim C, Rosenqvist K, Olivecrona T. Intravenous lipid emulsions: removal

mechanisms as compared to chylomicrons. J Lipid Res 1995;36:2174-2184.


veterinary toxicology. J Vet Emerg Crit Care 2011;21:309-320.


recovery from bupivacaine toxicity in the isolated rat heart. Region Anesth Pain M 2006;31:296-

303.




70

Chapter 5: A randomized controlled clinical trial of an intravenous lipid emulsion as an adjunctive treatment for permethrin toxicosis in cats

Introduction

Intravenous lipid emulsion (ILE) therapy has been shown to improve signs of toxicosis

in people and animals with lipid-soluble drug toxicoses. Initial interest in the therapy

was prompted after Weinberg et al demonstrated that both pre-emptive treatment

and resuscitation with ILE resulted in amelioration of bupivacaine-induced

cardiotoxicity in rats.1 Intravenous lipid emulsions have since been included in

treatment guidelines for local anaesthetic systemic toxicosis.2,3 An increase in the rate

of resolution of clinical signs, or augmentation of cardiopulmonary resuscitation

efforts, has also been reported after ILE administration in experimental models of

other lipophilic drug toxicoses including beta blockers,4 antidepressants,5,6,7 calcium

channel blockersa,8,9 and thiopentone.10 Numerous human and animal case reports

have reported apparent beneficial effects of ILE in clinical toxicosis due to beta

blockersb, antidepressants,11,12 antipsychotics,13,14 calcium channel blockers,15 sodium

channel blockersc, antimalarialsc, baclofen16 and insecticides.17-20 However, these case

reports need to be interpreted with caution as blood drug levels were not confirmed

and response to ILE treatment was based on subjective clinical assessment.

The mechanisms of action of ILE in the treatment of lipophilic drug toxicoses have not

been fully elucidated. One proposed mechanism of action is the formation of an

intravascular lipid sink where lipid-soluble drugs are sequestered away from their sites

of toxicity and are then redistributed to biologically inert sites.21-23 Ex vivo and in vitro

experimental models have been successfully used to demonstrate this theory.23-26 In

addition, increases in the blood concentration of lipophilic drugs after ILE

administration have been reported in live animal experiments and in human and

veterinary case reports, suggesting that lipid partitioning of lipophilic drugs does occur

in vivo.7,18,27,28 However, confirmation of lipid partitioning does not necessarily

translate into an improvement in clinical signs of toxicosis in vivo.

71

Permethrin is a lipid-soluble insecticide common in spot-on flea treatments for dogs,

and permethrin-associated toxicosis in cats exposed to these products is frequently

reported.29,30 Permethrin binds to the voltage-gated sodium channels of myelinated

nerves, slowing their closure and resulting in repetitive neuronal discharge.31,32 Clinical

signs of permethrin toxicosis are therefore neuroexcitatory in nature and can cause

substantial morbidity and mortality.29,33-36 Mortality rates have been reported to be

between 2.4-16.9%.30,33,36 In one study, it was reported that 5.2% of cats with

permethrin toxicosis were euthanized due to owner financial constraints.30

Diagnosis of permethrin toxicosis in cats is based on exposure history and consistent

clinical signs. Studies have shown that permethrin and its metabolites can be detected

in human blood and urine,37,38 however no studies have reported blood permethrin

concentrations in cats with permethrin toxicosis, or correlated such concentrations

with clinical signs. In addition, the technology for such assays is not widely available.

Therefore, at this time, measurement of the concentration of permethrin or its

metabolites in biological samples is not routinely performed to confirm exposure or

assess the severity of permethrin toxicosis in cats in the clinical setting.

There are two published case reports describing a total of five cats with permethrin

toxicosis where ILE was used as an adjunctive treatment.19,39 Despite its clinical use, no

prospective clinical trials have been published to support its efficacy. The aim of this

study was to assess for any clinical benefit of ILE for permethrin toxicosis in cats by

comparing the progression of clinical signs before and after treatment with ILE

compared to a saline control. To accomplish this objective, a clinical staging system

was developed and validated in order to standardize the assessment of clinical signs in

cats with permethrin toxicosis. Our hypothesis was that clinical stages of permethrin

toxicosis in ILE-treated cats would improve earlier compared to cats receiving a saline

control.

Materials and Methods

The study had approval from the university Animal Ethics Committee and a license for

the use of animals for scientific procedures was obtained in each state where required.

72

Clinical Staging System

Design

A clinical staging system was designed using the dichotomous outcomes “yes” and

“no” to questions based on abnormalities found on physical examination (Figure 1).

The order of the observations was based on expert clinical opinion of the anticipated

deterioration or improvement of common clinical signs in cats with permethrin

toxicosis. The clinical staging system had six stages, ranging from Stage A for cats that

had no abnormalities detected to Stage F for cats with grand mal seizures.

Figure 1 – Clinical staging system to assess cats with permethrin toxicosis.

Validation

Thirteen cats with permethrin toxicosis were video recorded for 1-3 min. Each clinical

stage, as defined by the staging system, was represented at least once. The series of

73

video footage was then shown to five veterinarians and five veterinary nurses

individually (Trial 1), with each cat shown in a random order to each observer, as

determined by random number generationd. Each observer assigned a clinical stage to

each of the cats shown in the video footage. One month later, the same observers

were shown the same footage of each cat in another random order (Trial 2) and each

observer assigned a clinical stage to each of the cats viewed.

Statistical analysis – clinical staging

The stage for each video was assessed for both intra-viewer variability (repeatability),

controlling for video, and inter-viewer variability (reliability), controlling for trial. A

Cochran-Mantel-Haenszel test of conditional independence was used to test for

association; with failure to reject the null hypothesis of independence indicative of no

intra- or inter-viewer variability. A p≤0.05 was used as criteria for determination of

association. All analyses were performed with a commercially available statistical

software packagee.

Clinical Trial

Design

The clinical trial was prospective, multicenter, randomized and controlled, involving

one university veterinary teaching hospital and 12 private veterinary emergency

hospitals across four states within Australia. Client-owned cats were enrolled between

March 2011 and June 2012.The clinical trial was planned based on a power of 80% and

an effect size of 1 (difference in means/standard deviation). That is, it was estimated

that control cats would take twice as long to recover with a variance of 100%. With

significance at 0.05, a total sample size of 130 was estimated for this result. Since this

estimate was very conservative, and for ethical reasons, an interim statistical analysis

was planned to be performed after 30 cats had been enrolled in the clinical trial to

determine if either a significant beneficial effect was detected or unforeseen adverse

events were occurring.

A box of study materials was delivered to each veterinary hospital. Each box contained

six treatment envelopes and a folder that contained study information for the client,

client consent forms and information for the attending veterinarians. Each of the six

74

treatment envelopes contained a study method and a pre-randomized treatment of

either a 500ml bag of 0.9% sodium chloridef, which served as the control treatment, or

a 500ml bag of a 20% ILEg as the intervention treatment. Pre-randomization was

performed by random number generationd. Veterinarians were not blinded to the

treatment.

Inclusion and exclusion criteria

Cats were included in the study if they had direct application of a ‘spot-on’ permethrin

product by their owner, had clinical signs of permethrin toxicosis at presentation with

a clinical stage C, D, E or F, and their owner had signed a client consent form. Cats were

excluded from the study if they had already received treatment for their toxicosis prior

to presentation or were clinical stage A or B at presentation as treatment may not

have been indicated. Additional exclusion criteria included obesity (body condition

score ≥7 out of 9) or a previous diagnosis of diabetes mellitus, cardiac or renal disease,

in order to minimize potential risks associated with fluid therapy or ILE therapy.

Treatment

Initial emergency stabilisation was performed using intravenous methocarbamol to

effect for tremors and intravenous diazepam to effect for seizures. Recommendations

for initial doses were 40 mg/kg for methocarbamol and 0.5-1 mg/kg for diazepam.

Dermal decontamination was then performed, which included clipping of the patch of

fur where the permethrin product had been applied followed by bathing of the cat in

tepid water with a dishwashing liquid detergent.

After bathing, 15 mL/kg of the randomized treatment was administered over 60

minutes, which is the equivalent of 0.25 mL/kg/min for 60 minutes. The use of

additional open-label drugs or fluid therapy was permitted at the discretion of the

clinician and details were recorded.

Assessment and monitoring

The time from application of the permethrin product to presentation was recorded.

Clinical stage, heart rate, respiratory rate and rectal temperature were recorded at

presentation (t=Pre), after bathing (t=Bath), immediately after the randomized

75

treatment was administered (t=0h), then every three hours for 12 hours, followed by

every six hours for the duration of the hospitalised period. The time from presentation

to clinical stage B (or A if improvement was faster, whichever was reached first) was

then calculated, as the authors agreed that this would be the stage at which discharge

home could reasonably occur. Adverse events detected on physical examination were

recorded during the infusion and at each time point from t=0 h. Clinicians were

specifically asked to monitor for clinical signs of nausea, abdominal pain,

hypersensitivity reaction or volume overload. Outcome was recorded as death,

euthanasia, discharge to primary care veterinarian or discharge home. The length of

hospitalisation was recorded.

Statistical Analysis – Clinical trial

The clinical stages assigned at each time point were treated as ordinal categorical data

and were summarized as relative frequencies within each treatment group, stratified

at each time point. The distribution of clinical stage categories stratified over time was

compared across treatment groups using the Friedman's test for repeated categorical

data. All other categorical data that described the characteristics of the treatment

groups was summarized as relative frequencies across groups. Differences in the

relative frequencies of these categorical variables were compared across treatment

groups using a Fisher's exact test. All categorical tests were performed using a two-

sided hypothesis with significance declared at p≤0.05.

Numerical data was tested for normality based on the Shapiro-Wilk test with the null-

hypothesis of normality rejected at p≤0.05. Non-normal numerical data was

summarized as median and quartiles and normal numerical data was summarised as

mean and 95% confidence intervals (CI). Selected non-normal numerical data was

compared across treatment groups using the Wilcoxon rank sum test (WR) and normal

numerical data was compared across treatment groups using a t-test (TT) adjusting for

equal or unequal variances. A two-sided hypothesis was tested with significance

declared at p≤0.05.

All analyses were performed with a commercially available statistical software

packagee.

76

Results

Clinical Staging

Appendix 2 contains the raw data for viewer scores for each trial.

Visual assessment of the scores showed almost perfect agreement (repeatability) of

scores across videos regardless of viewer and almost perfect reliability across viewers.

In Trial 1 there was discrepancy for two viewers for video 5 (stage B), one viewer for

video 6 (stage C) and one viewer in video 10 (stage B). In Trial 2 there was discrepancy

for two viewers for video 5 (stage B) and two viewers for video 6 (stage B). One viewer

had the same discrepancy in both trials for videos 5 and 6.

There was no association of trial by video, controlling for viewer (p=1.0), indicative of

no significant intra-viewer variability, and hence excellent repeatability. There was no

association of viewer by video, controlling for trial (p=1.0) indicative of no significant

inter-viewer variability, and hence excellent reliability.

Clinical Trial

Analysis of data was performed after 36 cats had been entered into the trial. At this

time, a significant benefit of the ILE was detected and the trial was stopped. Details of

the 36 cats included in the trial are given below. Appendix 3 contains the raw data for

population characteristics, assigned clinical stages for each time point, details of other

treatments administered, adverse events and outcome.

Population characteristics

Two of the 36 cats were excluded from data analysis as they had received treatment

from their local veterinarian prior to enrolment. Fourteen cats received the control

treatment and 20 cats received the ILE treatment. Cats were enrolled from nine of the

13 veterinary hospitals participating in the study.

There was no significant difference in age, frequency of sex, frequency of breed or

weight between the control and ILE-treated cats (Table 1). The median time from

application of the permethrin product to presentation for the control and ILE-treated

77

cats were not significantly different (p=0.180), with a median time of 14 h (1st quartile

7.0 h, 3rd quartile 24.0 h) and 13.5 h (1st quartile 3.0 h, 3rd quartile 18.0 h), respectively.

The relative frequency of clinical stages at presentation of the control and ILE -treated

cats were not significantly different (p=0.684, Table 2).

Table 1. Age, sex, breed and weight of 34 cats with permethrin toxicosis randomized

to receive 0.9% saline or intravenous lipid emulsion (ILE) treatment.

Control (n=14) ILE (n=20) p

Age, median years

(1stquartile, 3rdquartile)a

3.3

(1.3,4.5)

4.0

(1.4,5.1) 0.324

Sex, frequencyb

0.487 Male 7 7

Female 7 13

Breed, frequencyb

0.132

DSH 13 12

DMH 0 1

DLH 0 5

Other 1 2

Weight, median kg


4.2

(3.7,4.8)

4.5

(3.5,4.9) 0.931

aanalysis with Wilcoxon rank sum test; banalysis with Fisher’s exact test

Table 2. Relative frequency (n) of clinical stages at presentation in 34 cats with

permethrin toxicosis randomized to receive 0.9% saline or intravenous lipid emulsion

(ILE) treatmenta.

Clinical stage Control (n=14) ILE (n=20) p

C 0.36 (5) 0.45 (9)

0.684 D 0.29 (4) 0.10 (2)

E 0.21 (3) 0.25 (5)

F 0.14 (2) 0.20 (4)

aanalysis with Fisher’s exact test

78

Effect of ILE treatment on clinical stages

There was a significant difference (p<0.001) in the distribution of relative frequencies

(Figure 2) of clinical stages over time between control cats and ILE-treated cats, with

cats receiving a 20% ILE displaying lower clinical stages earlier.

Figure 2 - Relative frequencies of clinical stages at each time point in cats with

permethrin toxicosis randomized to receive 0.9% saline (control) or intravenous lipid

emulsion (ILE) treatment. The distribution of relative frequencies between the two

groups was significantly different (p<0.001).

79

There was a significant difference (p=0.006) between control and ILE-treated cats in

the time from presentation to achievement of clinical stage B (or A), with a mean time

of 16.2 h (95% CI 9.1-23.3 h) and 5.5 h (95% CI 1.6-9.5 h), respectively. There was no

significant difference (p=0.087) between control and ILE-treated cats in duration of

hospitalisation, with a mean time of 27.5 h (95% CI 18.8-36.2 h) and 19.4 h (95% CI

14.1-24.8 h), respectively.

No cats had worsening of their clinical stage while in hospital.

Other treatments administered

A range of drugs were administered to the study population. There was no significant

difference in the dose of methocarbamol (p=0.187 WR), diazepam (p=0.820 WR) or

intravenous fluid (p=0.650 WR) administered to control or ILE-treated cats (Table 3).

Alphaxalone was administered intravenously to one control cat (5.0 mg/kg) and three

ILE-treated cats (1.25, 1.85 and 12.36 mg/kg). Butorphanol was administered

intravenously to one control cat (0.39mg/kg) and two ILE-treated cats (0.20 and 0.25

mg/kg). Midazolam was administered intravenously as a continuous rate infusion to

two control cats (total doses 2.99 mg/kg over 8 h and 5.63 mg/kg over 22 h).

Acepromazine (0.06 mg/kg), medetomidine (4.0 μg/kg), phenobarbitone (2.0 mg/kg)

and propofol (7.41 mg/kg) were administered intravenously to one cat each among the

ILE-treated cats.

Table 3. Median dose of drugs administered to 34 cats with permethrin toxicosis

randomized to receive 0.9% saline or intravenous lipid emulsion (ILE) treatment.

Control (n=14) ILE (n=20) p

Methocarbamol, mg/kg


111.4 (14)

(80.0,171.9)

63.9 (20)

(46.3,131.8) 0.187

Diazepam, mg/kg


0.9 (7)

(0.7,2.0)

0.8 (10)

(0.6,2.6) 0.820

Intravenous fluid therapy, mL/kg


110.4 (6)

(55.9,165.9)

143.0 (10)

(39.0,320.0) 0.650

aanalysis with Wilcoxon rank sum test

80

Adverse effects

No adverse events as assessed by a physical examination were recorded for any

control cats. One of the ILE-treated cats had signs of pruritus of the right side of its

face that began 10 h after the end of the ILE infusion. Chlorpheniramine (0.30 mg/kg,

intramuscularly) was administered and the signs resolved over the following eight

hours.

Outcome

All cats survived to discharge. One control and two ILE-treated cats were discharged to

their referring veterinarians and later discharged home. One of these ILE-treated cats

re-presented to a veterinary emergency hospital the same night for reoccurrence of

permethrin toxicosis, after having been clinically normal at its referring veterinarian

throughout the day. Follow-up information was available for all cats for between five

to 19 months after treatment. No cats had presented to a veterinarian for clinical

disease of any kind in that time frame. Eight cats had presented for vaccination and

were reported as clinically normal at that visit.

Discussion

The results of this study demonstrate that the clinical signs of permethrin toxicosis in

cats treated with ILE improved earlier compared to cats receiving a saline control. By

decreasing the severity of clinical signs earlier in the course of treatment,

administration of an ILE reduced the morbidity of cats in this study. This result

supports the use of ILE as an adjunctive therapy in the treatment of this toxicosis. The

clinical trial was stopped after what was intended to be an interim statistical analysis

as a significant benefit was detected and the research team did not want to deprive

owners the option of an efficacious treatment for their cat.

A difference in hospitalisation time between the control and ILE-treated cats was not

detected in this study despite the demonstrated earlier improvement in clinical signs

of ILE-treated cats. This highlights the variable nature of relatively short hospitalisation

times, since the duration is dependent on factors other than clinical status of the

animal, including availability of owners to collect their cat. A significant difference may

have become apparent if more cats had been enrolled. However, it was more

81

important to the authors to determine when a cat reached a point at which it may be

stable enough for discharge (Stage B) and the clinical staging system was useful in this

regard. Using this analysis a significant difference was detected between control and

ILE-treated cats.

The clinical staging system validated for this study showed excellent repeatability and

reliability. The system was simple to use for both veterinarians and nursing staff as it

included easily-recognizable clinical signs. A limitation of this system is the possible

effect of drugs administered by the clinicians, and timing of those drugs, in relation to

the assignment of clinical stages. Treatment with diazepam or methocarbamol

immediately before the assessment of clinical stage in a cat that was seizuring may

reduce the stage assigned. Conversely, such treatment in an ambulatory cat with

tremors may cause recumbency and thus increase the stage assigned. However, as

there was no difference in the drugs or drug doses used between groups and there

was a large number of clinical stage assessments performed in this study, any

confounding effects due to the timing of drug administration are considered minimal.

Intravenous lipid emulsion therapy should not be the first line treatment for

permethrin toxicosis. Instead, immediate stabilisation of neuroexcitatory signs is

required using drugs such as methocarbamol. Mephenesin and its derivative

methocarbamol have been shown in rats to reduce the clinical signs and mortality of

permethrin and other pyrethroid toxicoses.40-42 Methocarbamol was the first line

treatment for muscle tremors in this study as it is regarded in the authors’ experience

to be the most efficacious drug for this clinical sign. Although commonly used by the

authors, the efficacy of intravenous methocarbamol for permethrin toxicoses in cats

has not been fully evaluated and it is not available in many countries. The use of

intravenous methocarbamol in this study may be why this study reported shorter

hospitalisation times compared to other published reports.33,36 Because of the

empirical clinical efficacy of methocarbamol in the treatment of permethrin toxicosis,

it was considered appropriate that treatment with ILE or saline in this study be

administered after methocarbamol. There was no difference in the dose of

methocarbamol used in the cats of either group and hence it is unlikely to have had an

effect on the results.

82

In this study, veterinarians were at liberty to use any supportive treatment thought

best for their case, however suggestions regarding emergency stabilisation and

treatment options were provided. This did not appear to be a confounding factor in

the statistical analysis, with the two groups having no significant difference between

them in regard to the dose of methocarbamol, diazepam or intravenous fluids

administered. All other treatments were used to control neuroexcitatory signs and

were unable to be evaluated due to the low numbers of cats in each group receiving

them. The ILE-treated cats did have more of these other drugs than the control cats

and it is possible that this may have contributed to the overall improvement in the

clinical signs seen in this group. However, there were more cats in the ILE-treated

group that had a severe clinical stage at presentation (Stage E or F), albeit not

significantly, and therefore additional drugs may have been required to stabilise these

cats.

The lipid sink theory is one of the proposed mechanisms of action for the increased

rate of resolution of clinical signs of lipophilic drug toxicosis.21-23 Drugs are considered

lipophilic if their octanol/water partition coefficient (log P) is > 1.0. Permethrin has a

log P of 6.1 at 20oC and therefore is considered highly lipophilic.42 However, the log P

value alone cannot predict the likely response to ILE administration in an animal with

toxicosis since other factors such as pH, temperature and a drug’s volume of

distribution influence ILE sequestration of a drug.23,25,43

The creation of a lipid sink may affect other drugs administered concurrently with ILE.

In regards to this study, it is possible that higher doses of methocarbamol may have

been required to control clinical signs in the ILE-treated cats. However, there was no

significant difference in the dose of methocarbamol between the control and ILE-

treated cats, which may be due to the comparatively low log P of methocarbamol (0.5

at 25oC).44 Similarly, higher doses of diazepam were not used in ILE-treated cats

compared to control cats despite the log P value of diazepam being higher (3.0 at 25oC)

than that of methocarbamol.44

83

An optimal dosing regime of ILE for lipophilic drug toxicosis has not been established. A

commonly reported dose of a 20% ILE is a bolus of 1.5 mL/kg followed by a continuous

rate infusion of 0.25 mL/kg/min for 30-60 minutes.45,46 A bolus was not used in this

study; however a bolus may be of benefit in toxicoses producing life-threatening

cardiotoxicity such as with local anaesthetic overdose. In such cases, ILE may overcome

the toxic effects of impairment of fatty acid transport into the myocardium.47

Intravenous lipid emulsions may also improve myocardial function by increasing intra-

myocyte calcium concentration in drug toxicoses such as calcium channel blocker

overdose.48-49 Permethrin does not have significant cardiotoxic effects and therefore

an ILE bolus for cats in this study was considered unnecessary. Also, given the short

half-life of exogenous lipids,50-52 it is likely that bolus therapy would not significantly

affect the size of the intravascular lipid sink created, therefore a continuous rate

infusion was considered to be sufficient. Further investigation is required to determine

the optimal dosing of ILE in lipophilic drug toxicoses.

Adverse events associated with the administration of ILE were only assessed based on

physical examination in this study, as permission for non-routine monitoring of the

cats was not sought from owners. Only one possible adverse event was reported for a

cat that developed facial pruritis. The cat had also received a number of other drugs

before and after the ILE was administered, therefore it is difficult to attribute this

observation directly to ILE administration. Adverse effects reported in people

administered ILE as a component of parenteral nutrition are rare and include

anaphylaxis, fever, vomiting, tachypnea, dyspnea, acute lung injury, phlebitis, fat

embolism and hyperlipidemia.51-55 Adverse events that have been reported after ILE

administration for drug toxicoses include gross hyperlipidemia, mottling of the skin in a

group of pigs, and mild phlebitis.16,27 Experimental studies to date have not been

specifically designed to assess for ILE-related adverse events at doses used for drug

toxicoses.6,17-20,56-61 This study was not powered to investigate adverse events, nor

designed to assess for all possible adverse events. A stage IV clinical trial is necessary

to specifically address the incidence of adverse events and clinicians should remain

vigilant in monitoring for adverse events when administering ILE.

84

The ILE-treated cat that returned for re-evaluation of worsening tremors had been

discharged to its regular veterinarian for monitoring throughout the day. It did not

receive any additional medications, was assessed as clinically normal and was

subsequently discharged after 7 hours. During the period at home the cat had been

grooming herself, and had been lying on a bed that she and the family dogs had used

after having the permethrin applied to them. It is highly plausible that the

reoccurrence of this cat’s clinical signs was due to re-exposure to permethrin. Release

of permethrin from a lipid sink during its metabolism cannot be excluded, but is

considered less likely given the time from ILE administration to the reoccurrence of

clinical signs, and the at-home conditions in the interim period.

No animals died in this study, which is in contrast to the study by Malik et al where

16.9% of cats died and 5% were euthanized.30 Other studies have shown lower

proportions of deaths or euthanasia,33,35,36 however the samples sizes for these

retrospective studies were much smaller than in Malik et al’s study. The zero mortality

in this study may be a factor of a higher standard of care available from 24-hour

emergency hospitals, a general improvement in the standard of care since data

collection in the other studies or the effect of bias, as animals presenting to 24-hour

emergency hospitals may be more likely to have committed owners that are financially

able to provide for their care.

This study was not blinded, which may have created bias in clinical stage assignment.

Blinding may have been performed by using opaque fluid bags and giving sets or by

having a person administer the treatment who was not involved in the assessment of

the cat. A further limitation of the study was that blood permethrin concentration was

not measured before or after ILE treatment. This information may have helped

characterise the lipid sink as the mechanism of action in permethrin toxicoses,

however, this was not an objective of this study. In addition, measurement of blood

permethrin concentration in each cat would have confirmed exposure to permethrin.

It would have been interesting to determine the relationship between clinical stages

and blood permethrin concentrations and this may be an area for future research.

85

Conclusion

The clinical staging system developed was repeatable and reliable and was a useful

tool to standardize the clinical assessment of cats in this study. In the clinical trial, the

clinical stages of permethrin toxicosis in ILE-treated cats improved earlier compared to

control cats. Clinically, this means that the signs of permethrin toxicosis in ILE-treated

cats improved earlier compared to the control cats making ILE a useful adjunctive

therapy in the treatment of this toxicosis in cats. Conducting a phase IV clinical trial,

where the administration of ILE is monitored over a large cohort of cats, will be

important to capture any adverse effects.

Footnotes

aBania T, Chu J, Lyon T, Yoon JT. The role of cardiac free fatty acid metabolism in

verapamil toxicity treated with intravenous fat emulsions [abstract]. Acad Emerg Med

2007;14(S1):S196-197.

bCarr D, Boone A, Hoffman RS, et al.Successful resuscitation of a carvedilol overdose

using intravenous fat emulsion [abstract]. Clin Toxicol 2009;47(7):727.

cHurley WT, Hanalon P. Lipid emulsion as an antidote at the Washington Poison

Center; use in carbamazepine, flecanide, hydrochloroquine, bupivacaine, and

buproprion [abstract]. Clin Toxicol 2009;47(7):729-730.

dMicrosoft Excel® 2010, Microsoft Incorporated, Redmond, WS.

eSAS® 9.3, SAS Institute, Cony, NC.

f0.9% sodium chloride, Baxter Healthcare, Old Toongabbie, Australia.

gIvelip©, Baxter Healthcare, Old Toongabbie, Australia.

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12. Carr D, Boone A, Hoffman RS, et al. Successful resuscitation of a doxepin overdose using

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15. Young AC, Velez LI, Kleinschmidt KC. Intravenous fat emulsion therapy for intentional sustained-

release verapamil overdose. Resuscitation 2009;80(5):591-593.

16. Bates N, Chatterton J, Robbins C, et al. Lipid infusion in the management of poisoning: a report of 6

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407-411.

21. Krieglstein J, Meffert A, Niemeyer DH. Influence of emulsified fat on chlorpromazine availability in

rabbit blood. Experientia 1974;30(8):924-926.

22. Straathof DJ, Driessen O, Meijer JW, et al. Influence of Intralipid infusion on elimination of

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30. Malik R, Ward MP, Seavers A, et al. Permethrin spot-on intoxication of cats Literature review and

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43. Leo AHC, Hansch C, Elkins D. Partition coefficients and their uses. Chem Rev 1971;71(6):525-616.

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47. Weinberg G, Palmer J, VadeBoncouer T, et al. Bupivacaine inhibits acylcarnitine exchange in cardiac

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48. Bacaner M, Hoey M, Ikenouchi H, Berry W. Inotropic and chronotropic actions by fatty acids which

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49. Huang J, Xian H, Bacaner M. Long-chain fatty acids activate calcium channels in ventricular

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50. Cotter R, Martis L, Cosmas F, et al. Comparison of the elimination and metabolism of 10%

Travamulsion and 10% Intralipid lipid emulsion in the dog. J Parenter Enteral Nutr 1984;8(2):140-

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51. Hultin M, Carneheim C, Rosenqvist K, Olivecrona T. Intravenous lipid emulsions: removal

mechanisms as compared to chylomicrons. J Lipid Res 1995;36(10):2174-2184.

52. Park Y, Damron BD, Miles JM, Harris WS. Measurement of human chylomicron triglyceride

clearance with a labeled commercial lipid emulsion. Lipids 2001;36(2):115-120.

53. Hessov I, Melsen F, Haug A. Postmortem findings in three patients treated with intravenous fat

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54. Schulz PE, Weiner SP, Haber LM, et al. Neurlogical complications from fat emulsion therapy. Ann

Neurol 1994;35(5):628-630.

55. Driscoll DF. Lipid injectible emulsions: pharmacopeial and safety issues. Pharm Res

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56. Spence AG. Lipid reversal of central nervous system symptoms of bupivacaine toxicity.

Anesthesiology 2007;107(3):516-517.

57. Marwick PC, Levin AI, Coetzee. Recurrence of cardiotoxicity after lipid rescue from bupivacaine-

induced cardiac arrest. Anesth Analg 2009;108(4):1344-1346.

58. Weinberg G, Di Gregorto G, Hiller D, et al. Reversal of haloperidol-induced cardiac arrest by using

lipid emulsion. Ann Intern Med 2009;150(10):737-738.

59. DiGregorio G, Schwartz D, Ripper et al. Lipid emulsion is superior to vasopressin in a rodent model

of resuscitation from toxin induced cardiac arrest. Crit Care Med 2009;37(3):993-999.

89

60. Zausig YA, Zink W, Kell M, et al. Lipid emulsion improves recovery from bupivacaine-induced

cardiac arrest, but not from ropivacaine- or mepivacaine-induced cardiac arrest. Anesth Analg

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61. O'Brien TQ, Clark-Price S, Evans EE, Di Fazio R, McMichael M. Infusion of a lipid emulsion to treat

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90

Chapter 6: Conclusion The literature supports the view that an ILE could be beneficial across a range of

lipophilic drug toxicosis. The literature also supports intravascular lipid partitioning as a

common mechanism of action of ILE in the treatment of lipophilic drug toxicosis.

Intravenous lipid emulsion for this application appears to be safe, though further

research is needed to ascertain the adverse effects of ILE at the doses recommended

for drug toxicoses. Also the metabolic fate of the ILE and lipid sequestered drugs needs

to be further characterised.

In the in vitro study reported here, addition of a lipid emulsion to permethrin-

containing feline plasma led to a significant reduction in plasma permethrin

concentration within 30 minutes. The study supports intravascular lipid partitioning as

a mechanism of action of ILE administration for permethrin toxicosis in cats. Future

research is needed to confirm intravascular lipid partitioning as a mechanism of action

of ILE in vivo for permethrin toxicosis in cats. Future studies investigating lipid

partitioning in lipophilic drug toxicoses should aim to invest in the research and

development to measure the drug of interest in the lipid supernatant, which was not

achieved in the research reported here. Also, an in vitro model that uses tissue

samples and allows measurement of tissue drug concentrations before and after

perfusion with an ILE, and changes in drug concentration of the effluent, could

experimentally support this mechanism of action.

In the clinical trial reported here, the clinical stages of permethrin toxicosis in ILE-

treated cats improved earlier compared to control cats. Clinically, this means that the

signs of permethrin toxicosis in ILE-treated cats improved earlier compared to the

control cats making ILE a useful adjunctive therapy in the treatment of this toxicosis in

cats. Future studies should aim to serially measure plasma drug concentrations before

and after ILE infusion, and correlate any changes in concentration to changes in clinical

status.

91

Appendix 1: Raw data for in vitro experiment

Mass spectrometry chromatographic peak areas

Solution Mirex Cis-permethrin Trans-permethrin

Plasma + Permethrin Control (1) 196423 195073 144358



Time 0 h (1) 195363 162692 100643

Time 0 h (2) 211654 189791 105074

Time 0 h (3) 216127 187234 106505

30 min Control (1) 195236 136290 91349

30 min Control (2) 239763 150637 92423

30 min Control (3) 236830 153290 92632

30 min Plasma + Permethrin (1) 246598 138066 111916



1 h Control (1) 206938 176165 101387

1 h Control (2) 212454 183188 107287

1 h Control (3) 221456 184846 115242

1 h Plasma + Permethrin (1) 201604 168966 121015



3 h Control (1) 201945 165746 102358

3 h Control (2) 212647 178992 110081

3 h Control (3) 180526 182067 107339




6 h Control (1) 211375 176927 108039

6 h Control (2) 215798 178526 108578

6 h Control (3) 225651 184672 113670




92

Appendix 2: Raw data for validation of permethrin toxicosis clinical staging system

Trial 1

Viewer Video

1 (F)

Video 2

(C)

Video 3

(E)

Video 4

(C)

Video 5

(B)

Video 6

(C)

Video 7

(C)

Video 8

(D)

Video 9

(A)

Video 10 (B)

Video 11 (A)

Video 12 (D)

Video 13 (E)

Vet 1 F C E C B C C D A B A D E





Nurse 1 F C E C B C C D A B A D E

Nurse 2 F C E C B C C D A A A D E

Nurse 3 F C E C A C C D A B A D E

Nurse 4 F C E C A B C D A B A D E


Trial 2

Viewer Video

1 (F)

Video 2

(C)

Video 3

(E)

Video 4

(C)

Video 5

(B)

Video 6

(C)

Video 7

(C)

Video 8

(D)

Video 9

(A)

Video 10 (B)

Video 11 (A)

Video 12 (D)

Video 13 (E)

Actual F C E C B C C D A B A D E


Vet 2 F C E C B B C D A B A D E

Vet 3 F C E C A C C D A B A D E






Nurse 4 F C E C A B C D A B A D E


93

Appendix 3: Raw data for clinical trial

Package number

Signalment Randomisation Reactions to Lipid/

Saline Outcome

Age (y)

Sex Breed Weight

(kg) Lipid/ Saline

Repeated Reaction Treatment

Time (h)

Discharge

0 4.0 MN DSH 3.9 Lipid No None 27.0 Home

1 0.17 ME DLH 1.2 Lipid No None 21.75 Home

2 0.75 FN DSH 3.5 Lipid No None 10.0 Home

3 0.75 ME DSH 4.0 Saline No None 8.0 Home

4 2.5 MN DSH 3.5 Saline No None 14.0 Home

96 1.0 FE DLH 6.0 Lipid No None 9.5 Home

6 4.5 FN DSH 3.7 Saline No None 28.0 Home

7 11.0 MN DSH 4.5 Lipid No None 12.0 RV


11 12.0 MN DLH 4.9 Lipid No None 24.0 Home

12 4.0 FN BritBlue 5.6 Saline No None 34.0 Home

13 4.0 FE DSH 2.9 Saline No None 19.25 Home

14 3.0 FN DMH 4.1 Lipid No None 21.0 Home



25 0.2 FE DSH 1.4 Lipid No None 12.0 Home

31 1.25 MN DSH 4.2 Saline No None 42.0 RV



37 6.0 FE DSH 4.7 Lipid No None 10.5 RV


46 2.0 MN DLH 6.3 Lipid No None 20.0 Home

53 2.0 FE DSH 4.8 Saline No None 9.67 Home

54 5.25 FE DSH 3.3 Lipid No None 6.25 Home

69 3.0 FN DSH 3.5 Lipid No Hyper 32.0 Home

71 5.0 FN DLH 4.5 Lipid No None 26.0 Home






56 7.0 FN Birman 4.0 Lipid No None 9.0 Home


35 1.5 FE Cross 4.9 Lipid No None 11.0 Home

BritBlue – British blue, DLH – domestic long-haired cat, DLH – domestic medium-haired cat, DSH – domestic short-haired cat, FE – female entire, FN – female neuter, Hyper – hypersensitivity reaction, ME – male entire, MN – male neuter, RV – referring veterinarian

94

Package Number

Other Treatments*

Treatment 1

Total Dose Treatment

1 (mg)

Dose Treatment 1 (mg/kg)

Treatment 2


2 (mg)


Treatment 3


3 (mg)


0 Meth 700 179.49 IVFT 352 90.26 . . .

1 Meth 200 166.67 . . . . . .

2 Meth 320 91.43 IVFT 143 40.86 . . .

3 Meth 320 80.00 IVFT 180 45.00 . . .

4 Meth 280 80.00 IVFT 234 66.86 Diaz 3 0.86

96 Meth 240 40.00 IVFT 204 34.00 . . .

6 Meth 600 162.16 Diaz 2.5 0.68 . . .

7 Meth 700 155.56 IVFT 384 85.33 Diaz 12.5 2.78

8 Meth 330 72.37 IVFT 320 70.18 Diaz 2.5 0.55

11 Meth 250 51.02 IVFT 260 53.06 Pheno 9.8 2.00

12 Meth 448 80.00 Clav 250 44.64 . . .

13 Meth 300 103.45 Diaz 2.5 0.86 . . .

14 Meth 200 49.38 Diaz 1.25 0.31 But 1 0.25

19 Meth 200 39.22 Diaz 1.25 0.25 . . .

20 Meth 220 40.00 . . . . . .

25 Meth 48 35.04 . . . . . .

31 Meth 1550 373.49 Diaz 15 4.15 But 1.6 0.39

32 Meth 360 107.14 . . . . . .

33 Meth 166.4 40.00 . . . . . .

37 Meth 260 55.32 Diaz 4 0.85 . . .

45 Meth 660 171.88 Diaz 7.5 1.95 Alph 19.5 5.08

46 Meth 680 107.94 Diaz 9 1.43 . . .

53 Meth 950 197.92 IVFT 136 28.33 . . .

54 Meth 144 43.18 IVFT 28 8.40 . . .

69 Meth 595 172.46 Diaz 2.5 0.72 IVFT 85.9 24.90

71 Meth 175 38.89 IVFT N/A . . . .

72 Meth 1600 296.30 Diaz 20 3.70 Alph 10 1.85

16 Meth 381.6 119.25 IVFT 630 196.88 . . .

17 Meth 228 50.00 Diaz 2.28 0.50 . . .

70 Meth 920 219.05 Diaz 4 0.95 IVFT 567 135

39 Meth 240 50.00 Diaz 12.5 2.60 Alph 6 1.25

56 Meth 300 75.00 IVFT 39 9.75 . . .

74 Meth 626 133.19 IVFT 432 91.91 . . .

35 Meth 197 39.88 Diaz 4 0.81 . . .

Alph – Alphaxalone, But – butorphanol, Clav – clavulanic acid-amoxycillin, Diaz – diazepam, IVFT – intravenous fluid therapy, Meth – methocarbamol, N/A – not available, Pheno – phenobarbitone, Prop – propofol *IVFT is in mL for total dose and mL/kg for dose

95

Package Number

Other Treatments*

Treatment 4


4 (mg)


Treatment 5


5 (mg)


Treatment 6


6 (mg)


0 . . . . . . . . .

1 . . . . . . . . .

2 . . . . . . . . .

3 . . . . . . . . .

4 . . . . . . . . .

96 . . . . . . . . .

6 . . . . . . . . .

7 Alph 56.50 12.56 . . . . . .

8 . . . . . . . . .

11 . . . . . . . . .

12 . . . . . . . . .

13 . . . . . . . . .

14 . . . . . . . . .

19 . . . . . . . . .

20 . . . . . . . . .

25 . . . . . . . . .

31 Mid 12.40 2.99 . . . . . .

32 . . . . . . . . .

33 . . . . . . . . .

37 . . . . . . . . .

45 Mid 21.60 5.63 IVFT 495 128.91 . . .

46 . . . . . . . . .

53 . . . . . . . . .

54 . . . . . . . . .

69 . . . . . . . . .

71 . . . . . . . . .

72 Prop 40.00 7.41 ACP 0.324 0.06 IVFT 453 90.6

16 . . . . . . . . .

17 . . . . . . . . .

70 . . . . . . . . .

39 Med 19.20 4 But 0.96 0.20 IVFT 135 103.8

56 . . . . . . . . .

74 . . . . . . . . .

35 . . . . . . . . .

ACP – acepromazine, Alph – Alphaxalone, But – butorphanol, IVFT – intravenous fluid therapy, Med – medetomidine, Mid – midazolam, Prop – propofol *IVFT is in mL for total dose and mL/kg for dose

96

Package Number

Clinical Stage (A-F)

Presentation

After Bath

After Lipid/ Saline

3 h 6 h 9 h 12 h 18 h 24 h 30 h 36 h 42 h 48 h

0 C C C C C C C B B . . . .

1 C C E D B B B A . . . . .

2 E D B A A . . . . . . . .

3 E C C C C . . B . . . . .

4 D D C C C A A . . . . . .

96 C C C A A . . . . . . . .

6 C C C C C C C B B . . . .

7 E E E D B . B . . A . . .

8 F E D B B B B . . . . . .

11 C C B B B A A A . . . . .

12 C C C C B B A . . . . . .

13 E E D E C C C B . . . . .

14 E E C B A A N/A A . . . . .

19 F D C A A . . . . . . . .

20 C C B A A . . . . . . . .

25 D D C B A . . . . . . . .

31 D D D D D D D D D D D B B

32 C C B B B . . . . . . . .

33 C C B B B A A A . . . . .

37 D D B B B B . . . . . . .

45 E E E E E E E E D C B A A

46 C C B B B B B . . . . . .

53 C C C B B . . . . . . . .

54 C C A A . . . . . . . . .

69 C C C C B A A A A A . . .

71 C C B B A A . . . . . . .

72 F E C D C C C C B B B B .

16 D D D C C C C B B A A . .

17 F E B B B B B B . . . . .

70 F E E D D D C C B A . . .

39 E E E D D D C C C B A . .

56 F D D C A . . . . . . . .

74 D D D C C C C C B A A . .

35 E D C B A . . . . . . . .

N/A – not available

97

Package Number

Heart Rate (beats/min)

Presentation

After Bath

After Lipid/ Saline

3 h 6 h 9 h 12 h 18 h 24 h 30 h 36 h 42 h 48 h

0 200 . 124 130 140 160 212 140 160 . . . .

1 240 . 210 200 176 . 180 170 . . . . .

2 240 . 150 N/A 180 . . . . . . . .

3 220 . 180 N/A 200 . . . . . . . .

4 240 . 240 180 184 180 180 . . . . . .

96 180 . 180 180 N/A . . . . . . . .

6 200 240 240 240 200 . 240 200 210 . . . .

7 200 200 128 200 180 . . . . . . . .

8 180 . 180 180 160 . 160 . . . . . .

11 180 . 192 192 180 168 200 200 . . . . .

12 200 . 240 240 188 180 168 . . . . . .

13 200 . 230 192 224 120 216 204 . . . . .

14 192 . 196 196 196 166 N/A 200 . . . . .

19 130 . 210 184 200 . . . . . . . .

20 240 204 204 162 192 . . . . . . . .

25 240 220 200 180 180 . . . . . . . .

31 200 . 180 180 120 . 162 220 210 200 240 . .

32 230 . 130 190 128 . . . . . . . .

33 160 160 160 162 160 164 180 144 . . . . .

37 180 . 180 188 200 168 . . . . . . .

45 250 200 180 144 140 160 140 192 200 N/A N/A N/A N/A

46 180 . 190 180 168 160 168 . . . . . .

53 240 240 220 240 240 . . . . . . . .

54 240 192 192 240 . . . . . . . . .

69 176 . 180 164 212 192 220 180 200 152 . . .

71 240 . 210 220 204 204 . . . . . . .

72 200 . 130 200 180 . 164 140 204 200 140 120 .

16 180 144 140 144 121 152 140 152 160 192 216 . .

17 260 . 200 220 220 . 220 204 . . . . .

70 280 . 280 220 220 200 200 200 180 180 . . .

39 180 . 160 178 200 . 200 138 170 160 160 . .

56 240 . 168 192 192 . . . . . . . .

74 220 . 208 N/A 208 240 212 240 204 180 220 . .

35 220 . 220 216 220 . . . . . . . .


98

Package Number

Respiratory Rate (breaths/min)

Presentation

After Bath

After Lipid/ Saline

3 h 6 h 9 h 12 h 18 h 24 h 30 h 36 h 42 h 48 h

0 100 . 60 40 80 80 94 40 44 . . . .

1 60 . 48 80 44 . 36 45 . . . . .

2 N/A 32 68 N/A 60 . . . . . . . .

3 N/A 40 52 N/A 48 . . . . . . . .

4 40 . 20 20 40 44 32 . . . . . .

96 40 . 44 40 N/A . . . . . . . .

6 38 . 60 120 114 . 100 60 42 . . . .

7 28 28 20 36 36 . . . . . . . .

8 60 . 40 48 40 . 36 . . . . . .

11 40 . 44 40 40 60 68 56 . . . . .

12 60 . 60 60 48 20 48 . . . . . .

13 34 . 40 24 22 20 20 48 . . . . .

14 44 . 60 60 60 52 N/A 32 . . . . .

19 36 . 90 68 80 . . . . . . . .

20 72 30 30 96 112 . . . . . . . .

25 44 44 44 N/A 32 . . . . . . . .

31 100 . 80 80 24 . 36 40 30 32 30 . .

32 60 60 60 48 80 . . . . . . . .

33 32 . 30 48 32 60 40 48 . . . . .

37 24 . 28 22 24 32 . . . . . . .

45 60 . 20 40 28 20 24 36 32 N/A N/A N/A N/A

46 40 . 44 38 60 32 40 . . . . . .

53 48 48 36 24 36 . . . . . . . .

54 40 36 N/A 60 . . . . . . . . .

69 36 . 60 74 120 68 60 50 76 80 . . .

71 40 . 42 40 36 36 . . . . . . .

72 60 . N/A 40 28 . 32 32 N/A 44 28 24 .

16 36 40 36 20 64 44 52 24 76 48 84 . .

17 60 . 48 28 28 . 32 44 . . . . .

70 N/A . 64 58 54 48 48 40 28 28 . . .

39 28 . 30 30 32 . 36 24 24 20 28 . .

56 48 . 48 48 48 . . . . . . . .

74 60 . N/A N/A 60 64 60 64 60 60 N/A . .

35 56 . 42 48 42 . . . . . . . .


99

Package Number

Rectal Temperature (oC)

Presentation

After Bath

After Lipid/ Saline

3 h 6 h 9 h 12 h 18 h 24 h 30 h 36 h 42 h 48 h

0 38.9 36.2 36.1 37.0 37.4 37.7 38.0 38.1 38.1 . . . .

1 39.2 39.2 37.8 35.7 38.1 . 38.2 38.5 . . . . .

2 37.0 36.1 37.4 N/A 38.1 . . . . . . . .

3 39.1 38.0 38.6 N/A 38.1 . . . . . . . .

4 38.8 37.3 37.0 37.6 38.5 39.3 39.8 . . . . . .

96 38.6 37.3 37.2 N/A N/A . . . . . . . .

6 38.5 37.3 38.2 38.2 38.2 . 39.0 39.2 38.3 . . . .

7 39.1 35.6 35.3 37.5 38.8 . . . . . . . .

8 40.1 35.9 36.5 36.8 38.3 . 38.5 . . . . . .

11 37.7 36.9 36.4 37.7 38.0 38.1 37.6 N/A . . . . .

12 39.6 37.8 37.8 37.8 38.6 38.7 38.8 . . . . . .

13 39.1 38.1 36.7 38.3 38.4 38.5 38.7 38.8 . . . . .

14 38.6 36.9 38.7 38.7 39.0 38.7 N/A 38.7 . . . . .

19 38.8 38.3 38.8 38.4 38.7 . . . . . . . .

20 37.8 37.9 37.7 38.0 38.0 . . . . . . . .

25 39.1 39.3 38.8 38.6 38.5 . . . . . . . .

31 39.0 39.0 33.0 33.0 36.1 . 38.1 36.1 36.9 37.1 38.5 . .

32 37.6 37.6 37.5 37.9 38.3 . . . . . . . .

33 38.3 36.3 38.3 38.3 38.1 40.8 39.4 39.2 . . . . .

37 38.4 37.9 38.0 38.0 38.0 N/A . . . . . . .

45 40.9 36.9 34.0 38.0 38.4 38.7 37.7 37.7 39.0 N/A N/A N/A N/A

46 40.0 39.5 38.0 38.4 N/A 39.0 37.8 . . . . . .

53 39.2 39.1 38.2 38.4 38.6 . . . . . . . .

54 38.3 38.0 38.1 38.2 . . . . . . . . .

69 38.6 36.8 37.8 37.8 38.3 37.9 38.0 37.6 37.8 38.5 . . .

71 38.3 39.0 38.6 38.1 38.3 38.0 . . . . . . .

72 39.7 36.1 36.1 36.8 39.4 . 38.4 37.8 37.7 N/A 38.2 38.1 .

16 36.8 35.8 35.9 36.5 37.4 37.8 37.7 37.8 38.4 37.6 38.1 . .

17 39.5 37.3 38.0 38.3 38.3 . 38.3 38.7 . . . . .

70 39.2 37.9 37.8 38.1 38.3 38.5 38.6 38.5 38.5 38.4 . . .

39 41.0 39.4 38.0 36.0 37.8 . 39.9 40.6 . 38.8 37.8 . .

56 38.9 36.6 37.6 37.9 N/A . . . . . . . .

74 39.2 39.0 39.2 39.0 39.6 40.1 39.2 38.3 38.3 38.8 N/A . .

35 37.0 36.9 36.9 37.0 37.9 . . . . . . . .


100

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