Parenteral emulsions and liposomes to treat drug overdose

Advanced Drug Delivery Reviews 90 (2015) 12–23

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Parenteral emulsions and liposomes to treat drug overdose☆ Robert Damitz, Anuj Chauhan ⁎ Department of Chemical Engineering, University of Florida Gainesville, FL 32611, United States

a r t i c l e

i n f o

Article history: Received 14 January 2015 Received in revised form 2 June 2015 Accepted 10 June 2015 Available online 15 June 2015 Keywords: Detoxification Drug sequestration Lipid rescue Critical care Emulsions Liposomes

a b s t r a c t Drug overdoses from both pharmaceutical and recreational drugs are a major public health concern. Although some overdoses may be treated with specific antidotes, the most common treatment involves providing supportive care to allow the body to metabolize and excrete the toxicant. In many cases, supportive care is limiting, ineffective, and expensive. There is a clear medical need to improve the effectiveness of detoxification, in particular by developing more specific therapies or antidotes for these overdoses. Intravenous lipid emulsions (ILEs) have been investigated as a potential treatment for overdoses of local anesthetics and other hydrophobic drugs. While ILE therapy has been successful in several cases, its use beyond local anesthetic systemic toxicity is controversial and its mechanism of detoxification remains a subject of debate. ILEs were not originally developed to treat overdose, but clarifying the mechanisms of detoxification observed with ILE may allow us to design more effective future treatments. Liposomes are highly biocompatible and versatile formulations, thus it was a natural step to explore their use for drug overdose therapy as well. Several researchers have designed liposomes using a variety of approaches including surface charge, pH gradients, and inclusion of enzymes in the liposome core to optimize the formulations for detoxification of a specific drug or toxicant. The in vitro results for drug sequestration by liposomes are very promising and animal trials have in some cases shown comparable performance to ILE at reduced lipid dosing. This narrative review summarizes the current status and advances in the use of emulsions and liposomes for detoxification and also suggests several areas in which studies are needed for developing future therapies. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emulsions as detoxification agents . . . . . . . . . . . . . . . . . . . . . . 2.1. Current status of intravenous lipid emulsion detoxification or “lipid rescue” 2.2. Proposed mechanisms of action . . . . . . . . . . . . . . . . . . . . 2.3. Effects of protein binding and ionization on pharmacokinetic sequestration 2.4. Effect of lipid composition on ILE detoxification . . . . . . . . . . . . . 2.5. Other non-triglyceride formulations . . . . . . . . . . . . . . . . . . 3. Liposomes as detoxification agents . . . . . . . . . . . . . . . . . . . . . . 3.1. Liposomes with pH gradients . . . . . . . . . . . . . . . . . . . . . 3.2. Modified phospholipids for electrostatic uptake of charged drugs . . . . . 3.3. Liposomes containing sequestering agents, antidotes, and enzymes . . . . 4. Comparative studies of liposome and emulsion detoxification . . . . . . . . . . 5. Considerations for future detoxification therapies . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Current and Forthcoming Approaches for Systemic Detoxification”. ⁎ Corresponding author at: Chemical Engineering, University of Florida, Gainesville, FL 32611-6005, United States. Tel.: +1 352 392 9513. E-mail address: [email protected]fl.edu (A. Chauhan).

http://dx.doi.org/10.1016/j.addr.2015.06.004 0169-409X/© 2015 Elsevier B.V. All rights reserved.

Toxicity from illicit drugs, prescription medication overdoses, or exposure to hazardous chemicals is a major global health concern. Drug overdoses account for nearly 4.6 million emergency department visits [1] and over 80,000 preventable deaths [2] in the United States

R. Damitz, A. Chauhan / Advanced Drug Delivery Reviews 90 (2015) 12–23

each year. Overdoses may be treated by specific antidotes including naloxone and flumazenil, but it is difficult and expensive to develop antidotes for each of the commonly overdosed drugs. Gastric lavage and charcoal administration can minimize absorption into the bloodstream, but these techniques are only useful to limit enteral absorption and remain controversial. Gastric lavage has several risks including perforation of the GI tract or pharynx, aspiration pneumonia, hypoxia, or dehydration and is not effective more than an hour after toxicant ingestion [3,4]. Charcoal administration also becomes less effective more than an hour after ingestion [5] and is contraindicated for acidic or alkali toxicants. In many cases, the most common approach is to provide the support needed to maintain the patient's stability and relying on the endogenous ability to metabolize or excrete the toxicant. Several parenteral therapies are being investigated to alleviate or reverse the effects of overdose or toxicity. The general strategy involves injection of colloidal solutions which exhibit a high partitioning of the drug or toxicant. These colloids are thought to create a separate inert pharmacokinetic compartment which reduces the drug or toxicant concentration in free blood and affected tissues to help restore organ function and allow endogenous metabolism, excretion, or redistribution to occur more rapidly (Fig. 1). Emulsions and liposomes are the most common colloids investigated for toxicity therapy, but these therapies are still under investigation and are strongly debated in literature. Intravenous lipid emulsions (ILEs) or intravenous fat emulsions (IFEs) consist of nanometer-sized droplets of triglyceride oils in water stabilized by phospholipid surfactants. These emulsions were originally developed for total parenteral nutrition (TPN), but only recently were they first proposed as a detoxification therapy [6]. Despite the lack of controlled human trials [7], clinical guidelines have been disseminated by multiple anesthesia associations for treating overdoses of local anesthetics using the soybean oil-based formulation Intralipid® [8,9]. ILE usage still remains very controversial for detoxification from other drugs, particularly when the drug is being absorbed by the enteral route [7,10,11]. The mechanism (or mechanisms) responsible for recovery from toxicity is a complicated issue and remains undetermined [7,12–16]. The emulsions investigated for detoxification have been limited to TPN emulsions when in fact the scope of emulsion design is extremely broad. It is likely that emulsion formulations could be optimized for drug sequestration to improve the efficacy of emulsionbased detoxification.

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Liposomes are spherical lamellar particles comprised of phospholipid bilayers which encapsulate an inner aqueous vesicle. Liposomes are well-established as a drug delivery vehicle with several liposomebased formulations on market [17]. As a detoxification therapy, liposomes were first investigated as a carrier for chelating agents for heavy metal poisoning in 1973 [18]. However, only recently has the focus shifted to the liposomes themselves acting as detoxification agents. Liposomes can be modified in several ways to improve drug sequestration, and this paper summarizes several of the recent advances in liposome design for detoxification. Although not as established as emulsion therapy, liposomes may prove to be effective detoxification agents especially in cases where ILE is not effective. In this review, we summarize the current opinions and recent advances on the use of emulsions and liposomes in parenteral detoxification therapy. We also report on studies where emulsions and liposomes have been compared concurrently and present our considerations for future detoxification work. This narrative review aims to expand the breadth of formulations considered and encourages more rigorous evaluation of detoxification therapies. 2. Emulsions as detoxification agents 2.1. Current status of intravenous lipid emulsion detoxification or “lipid rescue” ILE (typically Intralipid®) is under investigation as a detoxification therapy for many lipophilic drugs and toxicants including local anesthetics, tricyclic antidepressants (TCAs), beta blockers, calcium channel blockers, and other illicit substances. While the multiple clinical reports of ILE therapy leading to successful detoxification are encouraging, the consensus is not universally positive due to the undetermined primary mechanism or mechanisms of action for detoxification [7, 12–15], concerns over the routes of intoxication [7,10], a lack of human trials [7], different partition coefficients of different drugs [13], and bias in the literature. The current status and opinion of ILE for detoxification therapy from various substances are summarized in Table 1. For a detailed investigation of studies and case reports, the authors direct readers to several recent reviews cited in this section [12, 15,19]. Although concerns exist over the lack of controlled human trials and the dominant mechanism of action, the opinion among researchers and

Fig. 1. Diagram illustrating different hypothesized methods of drug sequestration and their pharmacokinetic effects. Reprinted with permission from Bertrand N, Bouvet C, Moreau P, Leroux J-C. Transmembrane pH-Gradient Liposomes to Treat Cardiovascular Drug Intoxication. ACS Nano. 2010 Dec 28;4(12):7552–8. Copyright 2010 American Chemical Society.

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Table 1 List of drugs for which intravenous lipid emulsion is under investigation and current recommendations. Relevant references are included. Drug or toxicant type

Examples

Current recommendations

Local anesthetics

Bupivacaine, ropivacaine, and mepivacaine Amitriptyline, imipramine, dosulepin, and doxepin Propranolol, carvedilol, and nebivolol Verapamil and diltiazem Flecainide and propafenone Parathion and malathion Haloperidol

Accepted for systemic toxicity [8,9]. Only if immediate threat to life and other therapies are ineffective [7]. Can exacerbate enteral absorption [10,11].

Tricyclic antidepressants Beta blockers Calcium channel blockers Anti-arrhythmics Organophosphates Other drugs

physicians alike [7–9,13–16,19] suggest that ILE be used to treat local anesthetic systemic toxicity (LAST). Local anesthetics are unique in that they are typically administered intravenously in a clinical setting thus accidental overdose cases are typically detected early with fewer unknowns. Other drugs which cause systemic toxicity may be absorbed through other routes with more complicated pharmacokinetics (PK). ILE may actually enhance absorption if administered early after an oral overdose only exacerbating toxicity. For example, toxicity was worsened when ILE was given to rodents early after oral overdose of amitriptyline [11]. These findings have led to more scrutiny over ILE usage in case reports [7], and authors have presented guidelines suggesting where ILE detoxification would be useful [10]. ILE is currently only recommended when no response is seen to standard resuscitation therapies for lipophilic tricyclic antidepressants [10], beta blockers [20], calcium channel blockers [21], and other illicit substances [22].

and flow rate directly combat reduced circulation or cardiac arrest caused by systemic toxicity. Improved circulation can also aid pharmacokinetic redistribution of toxicants. A secondary cardiotonic effect also may occur by increasing myocardium calcium levels [15]. Fettiplace et al. recently demonstrated that pharmacokinetic sequestration alone cannot explain recovery from LAST in rats [26]. Rats were dosed with toxic concentrations of bupivacaine before receiving ILE treatment. Pharmacodynamic (PD) properties were measured and compared with several pharmacokinetic–pharmacodynamic (PK/PD) models for bupivacaine accounting for various mechanisms of ILE action. The best model agreement was achieved when a sequestering and cardiotonic effect was included [26]. The same authors extended this work in greater detail also accounting for accelerated metabolic effects with ILE therapy [27]. Again, only the combined cardiotonic and sequestration PD/PK model could achieve agreement with the PD observations in rats dosed with bupivacaine and treated with ILE. Although increased metabolism was observed with ILE, it did not have a significant effect on tissue concentrations. Pre-treating rats with metabolism inhibitors prior to bupivacaine toxicity and ILE therapy had no effect on recovery [27]. These studies demonstrate that ILE provides multiple but not necessarily independent mechanisms to assist in recovery from overdose. The many drugs and toxicants for which ILE detoxification has been proposed have many different chemical properties and pharmacokinetic profiles. It is unlikely that there is a dominant mechanism or mechanisms for all cases. Certainly pharmacokinetic arguments alone are insufficient to validate ILE as a proposed detoxification therapy for a given drug or toxicant [16]. Additional detailed investigations of the influence of all potential mechanisms (such as Fettiplace et al. [26,27]) for each drug or toxicant considered are encouraged. 2.3. Effects of protein binding and ionization on pharmacokinetic sequestration

2.2. Proposed mechanisms of action The mechanism or mechanisms responsible for ILE detoxification remain a subject of extensive study and discussion. For the purposes of this review, we introduce the proposed mechanisms of action and summarize the current opinions in the field. For detailed investigations on ILE detoxification mechanisms, the authors direct readers to informative recent reviews [7,13–15]. The most prominent theory is the so called “lipid sink” or pharmacokinetic sequestration (PKS) argument. Lipid emulsions are comprised of triglyceride oils which have high affinity for lipophilic drugs or toxicants. Injection of these emulsions effectively creates an inert lipidrich pharmacokinetic compartment within the bloodstream which draws lipophilic drugs or toxicants away from tissues and free blood to establish a new equilibrium. Reduced tissue concentrations help to restore organ function and allow the body to safely metabolize and eliminate the drug or toxicant. PKS has been demonstrated in animals for several different lipophilic drugs [7] and observed in human clinical studies for amitriptyline [23] and bupivacaine [24]. However, caution must be exercised as PKS will not always lead to reduced toxicity. The lipid-rich compartment provided by ILE can actually increase enteral absorption of lipophilic drugs or toxicants through the GI tract which may increase the severity of toxicity [11,25]. ILE also provides nourishment to cells and tissues which may contribute to recovery of organ function during toxicity. These emulsions originally derived for parenteral nutrition contain an oil phase of triglycerides or three fatty acids bound to a glycerol backbone. Fatty acids are metabolized endogenously to form ATP by cellular mitochondria. Fatty acids also have direct effects on sodium and calcium channels which can specifically inhibit the antagonism of local anesthetics on sodium channels or inhibit the action of calcium channel blockers [7]. A direct cardiotonic effect from ILE administration has also been observed in experimental models [7]. The increased blood pressure

In order for a drug or toxicant to be effectively sequestered by a detoxification therapy, it must partition favorably into the injected phase over the tissues. Many drugs and toxicants also adsorb to proteins present in the blood. In some instances, the amount bound to protein can be very high. Thus, in order for a detoxification therapy to have a substantial pharmacokinetic effect, the total mass of drug or toxicant which can be sequestered by the emulsion should be greater than or equal to the amount dissolved in blood plasma plus bound to blood proteins and cells. It should however be noted that improved pharmacokinetic sequestration may not necessarily lead to improved clinical effect. The connection between pharmacokinetic redistribution and detoxification remains unresolved. Nonetheless, an example of pharmacokinetic sequestration taking place is represented in Eq. (1). ðK oil þ K s SÞf oil V b cfree K eff ;oil f oil ¼ ≥1 V b cfree þ K p f p V b cfree 1 þ Kp fp

ð1Þ

The amount of drug or toxicant which can be sequestered by a given therapy is measured by its partition coefficient. For an emulsion, the partition coefficient is the concentration ratio of the drug or toxicant dissolved in the oil phase over the aqueous phase at equilibrium, Koil. We also assume that the drug can adsorb on the emulsion surface with Ks representing the concentration ratio of the drug on the particle surface to the free drug in plasma. Kp is the equilibrium partition coefficient of a given drug between protein and the aqueous phase, Vb is the total volume of blood, fi are volume fractions of oil or protein in the blood, S is the specific surface area per oil volume, and cfree is the serum concentration of drug. The above equation neglects blood cell binding for simplicity. Drug adsorption and absorption can be lumped together into an effective partition coefficient Keff,oil = Koil + KsS, where the specific surface area S can be related to the particle radius, R, as S = 3/R. Typically, the fraction of drug bound to protein is reported

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in percent (%P), thus Kp can be calculated from the reported data as shown in Eq. (2). 1  1 −1 %P



Kp ¼ fp

ð2Þ

Alternatively, the minimum oil/water partition coefficient required for lipid sequestration to be substantial is given below in Eq. (3). K eff ;min ¼

1 þ Kp f p f oil

ð3Þ

Each pharmaceutical compound and metabolite will have different degrees of plasma protein binding and partition coefficients for a given sequestration agent. Table 2 lists protein binding and minimum oil/water partition coefficients required for PKS to be substantial for several drugs which are under investigation for ILE detoxification. Table 2 also includes octanol/water partition coefficient, or log P, values for each drug. It should however be noted that the octanol/water partition coefficient will differ from the actual partitioning of drug between blood serum and lipid emulsion. Several candidate drugs for ILE detoxification are also predominantly charged at physiological pH. The lipid solubility of charged compounds is typically negligible. Since the solubility of the charged drugs would be negligible in oil, it would appear that ILE will not effectively sequester such drugs. However, those drugs could potentially partition on the surface through electrostatic interactions with charged phospholipid surfactants in the emulsion. Intralipid® 20% was reported to have a zeta potential between −45 and −40 mV indicating the emulsion does have significant surface charge [28]. While the zeta potential increases after exposure to electrolytes such as those present in the blood, the partial surface charge of ILE could be sufficient to sequester a high concentration of charged drug on the emulsion surface. Additionally, the charged portion of ionized drugs could interact with the surface through electrostatics, while still interacting with the oily interior thereby maintaining sequestration. To our knowledge, binding of charged drugs to the surface of ILE droplets has not been explored even though it may be relevant in determining the role of PKS in the detoxification of ionized drugs. Recently, investigators have suggested the usage of the distribution coefficient, or log D, to better predict actual equilibrium behavior of drugs [29]. The distribution coefficient accounts for the un-ionized and ionized forms of a drug in both octanol and water phases. While this ratio is more difficult to measure and depends on

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the pH of the system evaluated, it may be a better predictor of PKS for the many drugs being studied. Data for log D are still scarce in the literature, but we suggest that further studies consider the log D values at biological pH to evaluate PKS for a given drug. French et al. recently measured the amount of drug sequestered by Intralipid® from human serum in vitro for several drugs and compared the results to various tabulated data for these drugs. Reported values of protein binding and percent ionized at pH 7.4 were not statistically significant in predicting the experimental results. The only statistically significant factors were the reported log P and the pharmacokinetic volume of distribution for each drug [30]. French et al. also compared their findings with actual human case reports with mixed results which may demonstrate the importance of other mechanisms involved in ILE detoxification and the complications in translating in vitro measurements to in vivo clinical findings [30]. Nonetheless, their observation that the amount of drug sequestered by Intralipid® can be predicted by the drug's log P and volume of distribution values alone may have powerful implications. It is possible that reported that information on drug distribution or partitioning and protein binding can be used to assess a drug's ability to be sequestered by ILE. However, a greater understanding of the effect of drug ionization and protein binding is needed to make definitive statements about the pharmacokinetic role of ILE. 2.4. Effect of lipid composition on ILE detoxification Only limited attention has been given to the emulsion formulation in the broader discussion of emulsion detoxification to date. The type of oil in an emulsion can have a profound impact on a drug or toxicant's equilibrium. Emulsions studied for detoxification have been mostly limited to triglyceride oil formulations used for total parenteral nutrition (TPN). TPN emulsions may contain varying types and concentrations of triglyceride oils (Table 3) [31]. Changes in the fatty acid chain length or amount of unsaturation in these triglycerides may alter drug partitioning or change the response to other mechanisms proposed for detoxification. Recent assessments of lipid type on drug detoxification are summarized below. Mazoit et al. compared the binding capacity of several local anesthetic drugs from buffered saline to Intralipid® (pure long-chain triglycerides, LCTs) and Lipofundin® (50/50 mixture of LCTs and mediumchain triglycerides, MCTs) in vitro [32]. All local anesthetics were found to partition more favorably into Intralipid® than Lipofundin® in all conditions observed. This was attributed to the greater lipophilicity of the LCTs in Intralipid® than the 50/50 mixture of MCTs and LCTs in

Table 2 List of critical properties for pharmacokinetic sequestration of several drugs grouped by drug type. Drug class

Drug

pKa

% Ionized at pH 7.4

% Protein bound

Minimum log10Koil

Log P (oct/water)

Predicted effect of lipid sequestration

Local anesthetics

Bupivacaine Lidocaine Mepivacaine Ropivacaine Atenolol Propranolol Carvedilol Nebivolol Verapamil Diltiazem Amlodipine Amitriptyline Clomipramine Imipramine Dosulepin Diazepam Zolpidem Oxycodone

8.1 7.8 7.7 8.07 9.6 9.42 8.74 8.9 8.92 8.18 9.45 9.4 9.2 9.4 8.5 3.4 6.2 8.21

83.37 71.53 66.61 82.39 0.63 0.95 4.37 3.07 2.93 14.23 0.88 99.01 98.44 99.01 92.64 0.01 94.06 86.59

95 60–80 75 94 6–16 N90 98 98 90 70–80 97.5 96 97–98 86 84 98.5 92.5 45

3.00 2.22 2.30 2.92 1.75 2.70 3.40 3.40 2.70 2.30 3.30 3.10 3.30 2.55 2.49 3.52 2.82 1.96

3.41 2.44 1.95 2.9 0.16 3.48 4.19 2.44 3.79 2.8 3 4.92 5.19 4.8 4.49 2.82 1.2 1.04

Marginal Marginal Poor Marginal Very poor Marginal Marginal Very poor Very good Marginal Poor Very good Very good Excellent Excellent Poor Very poor Poor

Beta blockers

Calcium channel blockers

Tricyclic antidepressants

Hypnotics Analgesics

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Table 3 List of lipid composition and concentration of all emulsions available for total parenteral nutrition. Trade name(s)

Lipid composition

Available lipid concentrations

Intralipid, Lipoven, and Liposyn III Structolipid Lipofundin, Medialipide, and Vasolipid Clinoleic Omegaven Lipoplus SMOFLipid

100% soybean oil (LCT) 64% soybean oil (LCT) and 36% coconut oil (MCT) 50% soybean oil (LCT) and 50% coconut oil (MCT) 20% soybean oil (LCT) and 80% olive oil (LCT) 100% fish oil (unsat. LCT) 40% soybean oil (LCT), 50% coconut oil (MCT), and 10% fish oil (unsat. LCT) 30% soybean oil and 25% olive oil (LCTs), 30% coconut oil (MCT), and 15% fish oil (unsat. LCT)

10%, 20%, and 30% 20% 10%, 20% 20% 10% 20% 20%

Lipofundin® [33]. Ruan et al. demonstrated improved extraction of three local anesthetics with MCT/LCT emulsions over pure LCT emulsions from human plasma in vitro [34]. The authors explained the discrepancy by the different media (buffered saline vs. human serum) from which lipid extraction was reported. The study with human serum presents a more complicated equilibrium as triglyceride oils and drugs may both be binding to blood proteins. Both of these studies reported that drugs with greater log P values were sequestered to a greater extent by both emulsions. Again, it should be noted that experimental partition coefficients differ significantly from reported octanol/water partition coefficients since the lipid compounds are different from octanol. Each drug and oil type will have its own unique solubility equilibrium which theoretically will yield different PKS in vivo. In an isolated rat aorta model, Ok et al. reported reduced vasodilation when isolated rat aortas exposed to toxic concentrations of local anesthetics were infused with MCT/LCT or LCT emulsions. The concentration–response of ILE infusion was strongest for the most lipophilic anesthetics with a decreasing response for bupivacaine (log P = 3.41), ropivacaine (log P = 2.90), and lidocaine (log P = 2.44). ILE infusion did not cause a statistically significant response over control for mepivacaine (log P = 1.95). Although only statistically different for bupivacaine, mean MCT/LCT emulsion concentration–response values were higher than LCT emulsions for all drugs. These results suggest that MCT/LCT emulsions may be more effective at sequestering lipophilic local anesthetics and improving recovery from toxicity [35]. Animal models of ILE recovery from local anesthetic toxicity have not definitively established a superior formulation. Candela et al. measured PK and electrophysiologic effects from infusion of pure LCT and MCT/LCT emulsions in a model of bupivacaine toxicity in piglets. Directly after ILE infusion, mean plasma bupivacaine concentrations were lower with both emulsions compared to control. Although no statistically significant difference was reported between the three cases, mean plasma concentrations were slightly lower with MCT/LCT emulsion treated piglets than LCT. The greatest difference however was in the pharmacodynamics. Lipid emulsions effectively reversed bupivacaine's effect on several cardiac parameters. Both emulsions reported similar recovery, but MCT/LCT emulsion was more effective at restoration of left ventricle pressure than pure LCT emulsion [36]. Li et al. observed pure LCT emulsions mitigated toxicity of bupivacaine in rats slightly better than MCT/LCT emulsions [37]. Bupivacaine concentrations in arterial blood plasma and cardiac tissue were statistically different between emulsion formulations, but both emulsions reduced bupivacaine concentration considerably from plasma and arterial tissue rapidly after treatment. No difference between the two emulsion formulations was observed on myocardial energetic measurements. Bonfim et al. reported favorable recovery from ropivacaine toxicity in large pigs treated with pure LCT and MCT/LCT emulsions. No statistically significant difference in PD parameters was observed between the two emulsion formulations with the exception of pulmonary vascular resistance index which increased with MCT/LCT over LCT. PK values were not measured [38]. Udelsmann and Melo recently reported similar results from bupivacaine toxicity in large pigs. LCT and MCT/LCT emulsions were administered to favorably reverse toxicity. No statistically

significant difference between formulations was observed on PD parameters, but PK values were not measured [39]. No comparative evaluations of detoxification between lipid types are reported clinically. These limited reports suggest that differences in the ILE formulation may have a significant effect on PKS and recovery from toxicity. However, detailed investigations are needed to evaluate each formulation's effect on the multiple mechanisms proposed for ILE detoxification. 2.5. Other non-triglyceride formulations All of the above discussion of emulsion detoxification has been limited to formulations developed for TPN. All TPN emulsions contain triglyceride oils in the nanoemulsion size range (between 100 and 1000 nm droplet diameter). However, the scope of emulsion design is extremely broad. Emulsions using oils not derived from triglycerides have not been widely investigated as a detoxification agent despite far greater potential for sequestration and uptake over LCTs. Other formulations to consider are microemulsions (droplet diameter b 100 nm) which are thermodynamically stable and can offer improved shelf stability. Microemulsions also have much greater surface area to volume ratios than larger emulsions. Varshney et al. investigated emulsion detoxification by characterizing drug uptake in vitro from a novel microemulsion formulation. Microemulsions of ethyl butyrate stabilized with Pluronic and fatty acid sodium salts were demonstrated to double the amount of bupivacaine extracted from normal saline by Intralipid® at identical oil concentrations [40]. While optimizing the proposed microemulsion for maximal drug uptake, several observations led to the conclusion that bupivacaine uptake likely involves adsorption at the emulsion surface. For example, the surfactant type and concentration had a profound effect on the amount of drug extracted. Also, the fatty acid chain length of the cosurfactant affected the amount of drug extracted by the emulsion. Damitz and Chauhan recently reported large differences in partitioning of the general anesthetic drug propofol in different oil compounds. Of the five oils investigated, soybean oil (LCT) actually had the lowest partition coefficient while ethyl butyrate showed a 4fold increase in partition coefficient [41]. These oils were investigated as excipients for an improved propofol delivery vehicle, but the same partitioning principles can generally be applied to detoxification. Novel emulsions with different oil types have not been extensively studied for detoxification in vivo. Non-triglyceride formulations may also not have strong cardiotonic effects which have been reported as important to recovery from toxicity. Although only anecdotal inferences can be made on extending this improved partitioning argument to sequestering lipophilic drugs in vivo, it is clear only a very narrow scope of the broad field of emulsion design has been studied for detoxification. 3. Liposomes as detoxification agents Liposomes are a versatile platform which can be modified in several ways to sequester drugs or toxicants. The hydrophilic interior of

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liposomes can be formulated at a modified pH compared to the surrounding medium. Phospholipids which make up the hydrophobic liposome membrane can also be modified with surface charges to electrostatically attract drug compounds. Polyethylene glycol (or other similar polymers) can be attached to these phospholipids to improve biocompatibility and increase the circulation time of these liposomes in vivo. Liposomes can also be used to carry metabolic enzymes or sequestering agents for specific drugs or toxicants to reduce toxic effects. Liposomes have not been investigated clinically for drug overdose therapy, but several authors have demonstrated the viability of liposomal detoxification in vitro and in animal models. A brief summary of these investigative studies with relevant parameters is included in Table 4 [42–59]. 3.1. Liposomes with pH gradients The solubility of various drugs in aqueous solutions depends strongly on the pH due to equilibrium ionization. The uncharged forms of many drugs are hydrophobic, and ionization of such drugs significantly increases the water solubility. Based on this principle, several liposome detoxification therapies have been investigated which contain an aqueous interior at a modified pH at which the target drug is ionized. The difference in interior pH creates a significant concentration gradient for the uncharged drug to diffuse across the membrane into the interior of liposomes and sequester drug from the bloodstream. Mayer et al. first explored these acidic gradient liposomes in 1999 in order to reduce toxicity and extend the efficacy of the anticancer drug doxorubicin. Mice were treated with liposome formulations of various internal pH prior to and after intravenous administration of doxorubicin. While neutral and acidic liposomes both reduced the free drug concentrations, a greater reduction was observed with acidic liposomes. Rats pre- and post-treated with liposomes demonstrated reduced plasma concentrations and reduced toxicity while maintaining drug efficacy [60]. While the motivation for Mayer et al.'s work was maintaining antitumor properties of doxorubicin while reducing systemic toxicity, recent studies from Dr. Leroux's group have adapted pH gradient liposomes for detoxification of several drugs [42–46]. Dhanikula et al. measured the uptake of the lipophilic antipsychotic drug haloperidol from saline and blood serum in vitro with multilayered liposomes (spherulites) [42]. They observed far greater uptake of the drug in spherulites with acidic internal pH compared to those with no pH gradient. Although the amount of drug sequestered by spherulites was significantly less in blood serum over saline due to haloperidol's strong binding to blood proteins [42], this study was the first demonstration of pH gradient liposomal particles for detoxification therapy. One year later, the same authors measured the effects of liposomes with and without pH gradients on amitriptyline [43]. The authors first demonstrated greatly enhanced uptake of amitriptyline with acidic liposomes in vitro. Despite amitriptyline's strong protein binding, 97% of drug was still sequestered from a 3% albumin solution with acidic liposomes. The authors then measured the PD effects of liposomes with and without pH gradients on isolated rat hearts dosed with toxic concentrations of amitriptyline. Neutral pH liposomes had no statistically significant difference over control, but liposomes with internal pH of 3.0 showed a marked improvement in organ recovery from toxicity [43]. Bertrand et al. also demonstrated the viability of acidic liposomes on sequestering the calcium channel blocker, diltiazem [44]. Acidic liposomes (pH 3.0) sequestered significantly more drug than neutral liposomes and Intralipid® in vitro. The amount of drug sequestered decreased from all therapies when blood proteins were included due to diltiazem protein binding. The authors also demonstrated improved PK and PD from rats pretreated with acidic liposomes before administration of toxic concentrations of diltiazem. Significantly increased plasma concentrations of the drug and its primary metabolite were

17

observed with the group treated with acidic liposomes [44]. Forster et al. extended this work using acidic liposomes to sequester verapamil in addition to diltiazem [45]. Acidic gradient liposomes captured 13 times the amount of verapamil captured by neutral liposomes in vitro. The amount of verapamil sequestered by acidic liposomes was not significantly lower in the presence of blood proteins. The authors then observed the detoxifying effects of acidic liposomes in vivo at 1–3 h following delivery of toxic concentrations of verapamil by gavage to rats. Two different concentrations of acidic liposomes were used due to the lack of established protocols for liposome therapy. Both liposome concentrations showed statistical improvement of cardiac measurements over saline control [45]. Forster et al. recently evaluated the effects of adding pH gradient liposomes to peritoneal dialysis fluid [46]. Larger size liposomes (850 nm) were used for this study because smaller liposomes were observed to enter the circulation. Liposome-supported peritoneal dialysis (LSPD) extracted far more ammonia than traditional peritoneal dialysis fluid in vitro and in rats. Dialysate concentrations of several other drugs including propranolol, amitriptyline, haloperidol, and phenobarbital were enhanced with LSPD from orally-dosed rats. LSPD had favorable PD effects over traditional dialysis fluids for several toxic doses in rats [46]. pH gradient assisted liposomal detoxification is a promising therapy for several drugs and toxicants which has been demonstrated in animal models. However, liposomal detoxification therapies have yet to be established clinically and whether they are more or less effective than ILE is still undetermined. Additional issues that may need exploration include the stability of the liposomal pH gradient on the shelf and after injection. 3.2. Modified phospholipids for electrostatic uptake of charged drugs Fallon and Chauhan first demonstrated amplified uptake of amitriptyline with negatively surface-charged liposomes in vitro in 2006 [47]. Amitriptyline primarily exists in its positively charged acidic form at biological pH (pKa = 9.4). The authors explored the effect of solution pH on drug uptake and found the uptake efficiency of liposomes increases as the percentage of uncharged amitriptyline increases. This observation led to a suggested uptake mechanism of partitioning of the lipophilic uncharged drug into the lipid bilayer. Sequestration of drug was still present even at lower pH indicating the presence of electrostatic interactions between the negatively-charged liposomes and the positively-charged drug [47]. Howell and Chauhan elaborated on this idea of electrostatic sequestration by charged liposomes [48]. They observed blood proteins competitively bind to the negatively-charged liposomes as well to reduce their effectiveness for drug uptake. Despite the competitive inhibition of charged sites on the liposome surface, high amounts of amitriptyline and nortriptyline, the primary metabolite of amitriptyline, were still sequestered by charged liposomes in vitro. Increasing the ratio of charged to uncharged phospholipids increased the amount of drug sequestered since the effective surface charge of the liposomes became more negative [48]. The same authors further investigated this to conclude that electrostatic effects are important to primarily attract charged drugs at biological pH to the liposome surface, but experimental data suggest that the drug also sequesters into the lipophilic bilayer of the liposome (Fig. 2) [49]. The partitioning into the bilayer was demonstrated by an increased leakage of calcein from the liposomes with drug sequestration, suggesting an increased permeability of the bilayer due to the drug entry. The mass of drug that can bind to the liposomes due to the electrical double layer was calculated to be significantly smaller than the experimentally measured amounts. This phenomenon was demonstrated in vitro with tricyclic antidepressants (amitriptyline, imipramine, and dosulepin) and the local anesthetic bupivacaine alike. TCAs were observed to have greater binding efficiencies than bupivacaine experimentally. Based on these results, it was

18 Table 4 List of drugs and toxicants for which liposomal detoxification therapies are under investigation to treat overdose or exposure. The specific type of liposome treatment is noted with as much data as available. Summaries of several recent investigations are included with references. Drug or toxicant

Type of colloid treatment

Lipid composition

Dosing

Exp. conditions

Effect

Ref.

Haloperidol, docetaxel, and paclitaxel

200:30 or 200:50 (w/w) ratio of soy PC to cholesterol (CHOL)

NA

In vitro

200:50 (w/w) soy PC to CHOL

Diltiazem

Acidic pH gradient liposomes

58:38:4 (mol) ratio of Egg PC:CHOL: DSPE-PEG, 2.5 mM total lipid

Appx 0.2 mg lipids total dosing heart 275 mg lipid/kg

Ex vivo, rat hearts In vitro and in vivo, rats

Verapamil

Acidic pH gradient liposomes

50:45:5 (mol) of DPPC:CHOL:DSPE-PEG

150 and 250 mg lipid/kg

In vivo, rats

Amitriptyline

Negative surface charged liposomes

NA

In vitro

Amitriptyline, imipramine, dosulepin, and bupivacaine Bupivacaine

Negative surface charged liposomes Compared ILE and negatively charged liposomes

50:50 (mol) of DMPC:DOPG, 4 mg/mL lipid loading. 95:5 (mol) of DOPG:DPPE-PEG at 1.44 mg/mL lipid loading Pure DOPG or 95:5 DMPG:DPPE-PEG (mol) at 0.72 or 1.44 mg/mL lipid loading 80:20 (mol) POPC:POPG to either 7.8 or 15.6 mM lipid loading

NA

In vitro In vivo, pigs

Amiodarone, ketamine, and amitriptyline Clomipramine

Compared ILE and negatively charged liposomes Compared ILE and charged liposomes Rhodanese enzyme loaded liposomes

1.5 mL solution/kg initial bolus + 0.25 mL solution/kg-min for 29 mins NA

pH 3.0 gradient liposomes absorbed up to 75% haloperidol, neutral pH albumin loaded liposomes absorbed 94% docetaxel and 91% paclitaxel pH gradient liposomes demonstrated improved recovery over those with no pH gradient pH 3.0 gradient liposomes increased drug uptake over ILE and neutral liposomes in vitro. Pretreatment with acidic liposomes improved drug pharmacokinetic properties over saline control N90% capture efficiency with optimal formulation. Improved recovery with acidic liposomes over ILE and control. Similar effects with two different lipid dosings. Liposome uptake more efficient with less charged drugs or liposome surface becomes more charged. Blood proteins competitively bind to liposome. Excellent sequestration of drug. TCAs bind more strongly than local anesthetic due to structural differences. Similar PK/PD data obtained from recovery of toxic exposure to bupivacaine.

[41]

Amitriptyline

Acidic pH gradient spherulite liposomes, encapsulated albumin Acidic pH gradient spherulites

[50]

94:4 (mol) DOPG:DPPE-PEG to approximately 8 mg/mL lipid loading 82.7:9.2:5.1:3 (mol) DOPC or POPC:CHOL: PEG-PE:DOTAP to 10 mg/mL lipid. Loaded with 0.25 mg/mL Rh and 30 mM DTO 56.9:38:5.1 molar ratio POPC:CHOL:PEG-PE

3 mL/kg liposome solution at 8 mg/mL (0.8%) Appx 10 mg encapsulated Rh/kg

In vivo, rabbits

NA

In vitro

at biological pH, stronger drug–liposome interactions observed than drug–ILE for each drug studied Comparable recovery demonstrated for ILE and liposomes, liposome lipid dosing appx 4% of ILE dosing High degrees of enzyme encapsulation and stability of liposome formulations. Survivability of mice given up to 15x LD50 of KCN Up to 90% enzyme capture efficiency demonstrated. Enzyme-loaded liposomes characterized and stability of formulations for over 3 weeks demonstrated. Up to 35% antidote capture efficiency with pure DPPC liposomes. Treatment with antidote-encapsulated liposomes more effective at treating overdose than free antidote administration. Incr. bioavailability with orally-delivered loaded liposomes over free chelating agents. Bioavailability comparable to IV injected free chelating agents. Characterized loaded liposomes and demonstrated to effectively sequester and detect mercury. Improved PK from loaded liposomes in rats. Improved systemic removal of radionucleotides from loaded liposomes.

In vitro

In vitro, in vivo mice

Organo-phosphates

Hydrolyzing phosphotriesterase enzyme loaded liposomes

Acetaminophen

Antidote loaded neutral liposomes

Pure DPPC liposomes

320 mg lipid/kg and 25 mg antidote/kg

In vivo, rats

60

Chelating agent loaded liposomes

Soy lecithin liposomes loaded with 8.5% chelating agent

1 mmol/kg oral or 0.28 mmol/kg IV of chelating agent

In vivo, rats

Chelating agent loaded liposomes Chelating agent loaded liposomes

DOPE and PEG-PE liposomes loaded to 50 mM fluorescein with chelating agent 60:30:10 (mol) of DOPC:CHOL:PG and 64:30:6 DOPC:CHOL:DSPE-PEG at 100 mM lipid loading

NA

In vitro

Various chelating agent doses

In vivo, rats

Co

Mercury Plutonium

[43]

[44,45]

[46,47]

[48] [49]

[51] [52]

[53]

[54]

[55]

[56] [57,58]

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Cyanide

80:20 (mol) POPC:POPG

[42]

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Fig. 2. Plausible locations for two different classifications of drug to bind to anionic-charged liposomes. (A) Shows the potential binding site of a tricyclic antidepressant (TCA) where the uncharged portion of the drug aligns within the lipid bilayer and the positively charged head groups align with the negatively charged phospholipid head and aqueous bulk. (B) Shows the potential binding site of a local anesthetic where two uncharged portions align within the lipid bilayer and positively charged groups interact with the negatively charged phospholipids. Reprinted with permission from Howell BA, Chauhan A. Interaction of Cationic Drugs with Liposomes. Langmuir. 2009 Oct 20;25(20):12,056–65. Copyright 2009 American Chemical Society.

proposed that the drug interacts both with the surface through the electrostatic interactions and also with the bilayer through hydrophobic interactions (Fig. 2). The dual interactions lead to very high interaction energies, resulting in excellent sequestration of the drug, with as high as 99% bupivacaine bound to the liposomes in human plasma containing 1.44 mg lipid/mL. The differences in uptake efficiency were attributed to structural differences in the lipophilic and hydrophilic groups between TCAs and bupivacaine. These differences were further observed experimentally by measuring the amount of entrapped dye released from the interior of liposomes after binding of different drugs. Greater amounts of drug absorbed in the liposome bilayer would cause more dye leakage which was indeed observed experimentally [49]. Demonstration of the detoxifying capabilities of surface charged liposomes has been mostly limited to in vitro work, but the results are encouraging, especially when considering drugs which primarily exist in their charged form at biological pH. Compelling evidence for a proposed uptake mechanism has been presented, but validation of

this through further work is recommended. Surface charged liposomes are a promising therapy for several drugs such as tricyclic antidepressants for which ILE therapy remains controversial. 3.3. Liposomes containing sequestering agents, antidotes, and enzymes Liposomes have long been used as drug delivery vehicles [17,61,62] and several liposome-based pharmaceutical formulations are available today including AmBisome®, Doxil®, Visudyne®, and Marqibo®. The first suggestion of liposomes as a detoxification agent occurred in 1973 where chelating agents were included in liposomes for sequestration of plutonium [18]. Since then, liposomes have been proposed and evaluated as a vehicle to contain several other sequestering agents, antidotes, or metabolizing enzymes. Budai et al. characterized in vitro liposomes containing hydrolyzing phosphotriesterase, an enzyme which amplifies metabolism of organophosphates [54]. Stability and enzyme loading were demonstrated for

20

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over three weeks suggesting the viability of liposomes as a carrier and delivery vehicle of organophosphate antidotes. Petrikovics et al. demonstrated improved encapsulation and stability of liposomes loaded with rhodanese, a sulfurtransferase enzyme used to metabolize cyanide. The toxic effects of cyanide exposure were rapidly reduced by treating mice with rhodanese-loaded liposomes [53]. For further discussion, the authors direct readers to a recent review article by Szilasi et al. which summarizes the development of liposomal carriers of enzymes to treat organophosphate and cyanide toxicity [63]. Liposomes are also being investigated to carry drug-specific antidotes. In 2013, Alipour et al. reduced hepatotoxic effects caused by acetaminophen overdose in rats by treating with N-acetylcysteineloaded liposomes [55]. N-acetylcysteine is the preferred antidote for acetaminophen toxicity, but multiple doses are typically required due to the short half-life of the antidote. The authors found that antidoteloaded liposomes were more effective at preventing toxic effects than an equal amount of free antidote treatment. This was partially attributed to a slower release profile from antidote-loaded liposomes [55]. In 2010, Levitskaia et al. demonstrated increased oral bioavailability of chelating agents when loaded into liposomes and improved sequestration of heavy metals from tissues [56]. Mice were dosed intravenously with 60Co and each group was given a different chelating agent therapy. Liposome-encapsulated chelating agents were given orally, and the non-encapsulated chelating agents were given both orally and intravenously. The increased bioavailability of the liposomeencapsulated chelating agents reduced 60Co tissue levels by 12–43% compared to non-encapsulated agents given orally, while the results were comparable between liposome-encapsulated chelating agents given orally and non-encapsulated agents given intravenously [56]. Yigit demonstrated loading of a meso-DMSA chelating agent into liposomes for detecting and sequestering mercury from tissues [57]. The loaded liposomes were characterized in vitro and demonstrated on human HeLa cells exposed to different concentrations of mercury. Phan et al. determined PK data of DTPA-loaded liposomes in rats for plutonium chelation therapy. Sustained release of the liposomes loaded with chelation agent was demonstrated [58]. The same authors then reported successful and improved decorporation, or systemic radionucleotide removal, of plutonium in a rat model using the chelating agent-loaded liposomes [59]. The flexibility of liposomes as carriers of sequestering agents, antidotes, and enzymes has thus been demonstrated in the lab and in animal models, and these promising results may translate to clinical therapies in the near future. However, there are some concerns regarding the processing and shelf life of liposomal systems which may need further exploration. Liposome based systems have been commercialized for drug delivery applications so it should be feasible to address the manufacturing issues, particularly if liposomes are proven to be superior to ILE therapies. 4. Comparative studies of liposome and emulsion detoxification Both ILE and liposomes have been investigated as detoxification therapies for several lipophilic drugs and toxicants. Although greater volume of work has been performed on ILE detoxification, some recent reports have suggested that tailored liposomes may be more effective therapies. Below we summarize and discuss several recent reports where these two therapies were compared. Bertrand et al. compared the capture capacity of several liposome formulations with Intralipid® 20% from solutions of diltiazem in vitro [44]. The capture capacity was calculated as the moles of diltiazem captured by the colloid divided by the moles of phospholipid in the formulation. In buffer, liposomes with internal pH of 3.0 had 40 times the capture capacity of Intralipid®. Neutral liposomes and Intralipid® had comparable capture capacities. In the presence of blood proteins in plasma solutions, the capture capacity of both acidic liposomes and Intralipid® decreased approximately 33% indicating that protein

binding has a reducing effect on drug uptake. These results demonstrate that acidic pH liposomes are more effective at sequestering diltiazem per molar phospholipid content [44]. Forster et al. demonstrated over 20 times the capture capacity of verapamil and diltiazem by pH 2.0 liposomes over Intralipid® 10% in vitro [45]. The detoxifying capabilities of the two colloidal therapies were then compared in vivo with rats orally dosed with toxic levels of verapamil. Either Intralipid® (at 250 mg/kg) or pH gradient liposomes (two concentrations: 150 and 250 mg/kg) were administered 1 h following verapamil, and PD parameters were recorded. ILE and both liposome concentrations demonstrated improved PD over saline control, but liposome therapies had lower mean values than even ILE. Only liposome-treated rats had statistically different times to recovery than control. Although the concentration of Intralipid® used was half that of the recommended dosing for ILE [8,9], improved recovery was observed even at lower doses of liposome [45]. Litonius et al. performed a comparative study on the relative efficacy of bupivacaine detoxification with Intralipid® and neutral pH liposomes in a swine model [50]. The lipids used in the liposomes were both negatively charged at biological pH. Toxic doses of bupivacaine were administered to pigs and either therapy was given once toxic conditions were reached. Liposome dosing was determined based on equivalent phospholipid loading to Intralipid® per the current ILE detoxification guidelines, and PK and hemodynamic properties were measured. However, all liposome therapies led to cardiovascular collapse in the pigs used prompting a reevaluation of the liposome formulation. This was believed to be caused by an increased complement activation-related pseudoallergy (CARPA) in pigs [64]. Although the liposome toxicity issues were seemingly resolved, no statistical difference in PK or hemodynamic values was observed between the two therapies [50]. These results are in agreement with previous in vitro work where neutral pH liposomes have comparable sequestration to ILE. Lokajova et al. explored the relative interactions of several pharmaceuticals (amiodarone, ketamine, and amitriptyline) with two lipid emulsions and one neutral pH liposome using capillary electrophoresis [51]. Intralipid® and ClinOleic® were the two ILEs used, while the liposome was an 80 PC:20 PG (mol%). Interaction of all drugs with the liposomal formulation was stronger than either lipid emulsion at biological pH [51]. The stronger drug–liposome interactions suggest that even neutral pH liposomes may have favorable drug sequestration capabilities. Cave et al. investigated differences in ILE and liposome detoxification therapy with toxic doses of clomipramine in a rabbit model [52]. After being dosed with clomipramine to toxic conditions, complete survival was observed with treatment of tailored neutral pH liposomes and ILE alike while poor survival (40%) was shown for the control sodium bicarbonate treatment. PK data was not collected from this study, but mean arterial pressure (MAP) was greatest in the ILE therapy receiving group and slightly lower in the liposome group. While the recovery of MAP was comparable for both test groups, the total lipid administered in the liposome group was only 4% of the total lipid administered in the ILE group [52]. These results suggest that both liposomes and ILE can be effective at resuscitation following clomipramine toxicity, but liposomes may be more effective at equivalent lipid dosing. While considerably less investigated than ILE, the stronger interactions observed between drugs and liposomes and comparable recovery profiles in animals from toxicity of several drugs suggest that liposomes may be just as effective as ILE for detoxification. The design versatility of liposomes (pH gradients, surface charges, encapsulated antidotes) also may make liposomes more attractive for future detoxification therapies. However, liposomes have several drawbacks such as being linked to elevated immune response after injection [64]. Liposomes may also not provide the additional cardiotonic effects demonstrated by ILE. We recommend additional emulsion and liposome detoxification comparative studies be performed for the many commonly-overdosed drugs.

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5. Considerations for future detoxification therapies The ultimate goal of any emergency detoxification therapy is to rapidly and effectively reduce drug concentrations below toxic levels in affected tissues. Several other factors should be considered and understood quantitatively to predict the dynamics of detoxification using any colloidal system. In addition to the amount of drug sequestered by a colloidal therapy, the time scale of sequestration is important as well. A recent study by Damitz and Chauhan demonstrated very rapid release of a drugcontaining emulsion after injection into excess plasma in vitro [65]. The release of drug from nano-scale emulsion droplets was observed to be driven primarily by solubility with complete release of drug occurring in less than 2 s. The authors also demonstrated that the presence of surfactants at the colloid interface did not impede the release of drug, thus it is reasonable that surfactants and phospholipids present on the colloid interface will not impede the adsorption or absorption of drugs after parenteral injection. Using a different experimental approach, Salmela and Washington also demonstrated rapid drug release from emulsion-based formulations. Complete release of drug from a 1 micron diameter emulsion in under 0.5 s was observed after dilution with buffer at various ratios [66]. Fallon and Chauhan also explored the dynamics of amitriptyline binding to liposomes and concluded that the time scales are rapid [47]. These studies show how nano-scale particles including emulsions and liposomes will rapidly equilibrate after introduction to a new system. Time scales for equilibrium may be significantly shorter than the time required for other relevant steps such as drug diffusion through tissues or clearance. Consequentially, nano-scale colloidal detoxification therapies which principally sequester drug through adsorption or absorption may be more effective than therapies which slowly consume drug through reaction by avoiding a potentially rate limiting reaction step. The total amount of drug uptake by colloids after injection depends on interactions of the drug with the colloid and also on drug–protein interactions as clearly evident from Eq. (1). Both the surface and the bulk of the colloids can be optimized to increase the effective partition coefficient. In an emulsion for example, surfactants can be selected to impart surface charge to the emulsion and/or oils can be selected which have increased partition coefficients for the drug to improve sequestration. Improved drug sequestration has also been demonstrated for drugs that are not ionized at physiological pH by altering the internal pH of liposome particles [42–45]. The colloid loading in a given detoxification formulation can also be altered to increase the overall binding capacity. In vitro measurements of the effective partition coefficients could be compared to the estimates from Eq. (3) or more detailed models available in literature [29,67,68] to determine whether proposed colloids are promising candidates for detoxification therapy. There are several other processes whose rates can affect the overall detoxification dynamics. Sequestration of drug by the colloids reduces the free drug concentration. Drug with then diffuse out of some fast equilibrating tissues such as the heart and the lungs to maintain equilibrium. Other tissues may continue to slowly release drug back into the circulation even after the detoxification therapy has been removed from circulation. Very small colloidal particles such as microemulsions (b 100 nm diameter) could potentially partition into the tissues by diffusing through fenestrated capillaries such as those in the liver and spleen which would accelerate removal of the colloids from systemic circulation. Ideally, absorption of colloids containing sequestered drug into the liver, spleen, or kidneys would decrease the amount of drug in the body and accelerate recovery from overdose. Even neglecting liver metabolism, the colloids would degrade over time due to the slow partitioning of the colloid components into the tissues. In 2004, Fallon et al. created a physiological-based pharmacokinetic (PBPK) model for drug detoxification using nanoparticles such as emulsions or liposomes [69]. This generalized model divided the

21

colloids into three classes: 1. particles that remain only in the blood compartment due to their size being larger than the capillary diameter, 2. particles that can pass through the capillaries but cannot partition into the tissues, and 3. particles that can transport across the capillaries into the tissues. The overall detoxification dynamics depend strongly on the type of colloid in addition to the drug binding capacity of the colloids. At present, there is very limited data for most of these physiological transport processes which makes it difficult to fully rationalize the in vivo data and/or rationally design optimal detoxification systems. Measuring or at least estimating each of these rates and then incorporating all the relevant mechanisms into a PBPK model would be a useful approach to design overdose therapies for various drugs such as the work done by Kuo and Akpa [70] and Fettiplace et al. [26,27]. While it would be useful to design formulations that can treat overdoses for at least a class of drugs, it would be unrealistic to expect any therapy to be applicable to all drugs. In the future we can hopefully move toward a range of options each targeted and optimized for a given drug or toxicant.

6. Conclusions While intravenous lipid emulsion (ILE) has been accepted as a detoxification therapy for overdose of local anesthetics, it remains controversial for several other drugs and toxicants including tricyclic antidepressants, calcium channel blockers, and beta blockers. Currently, ILE is only recommended as a late therapeutic option when other detoxification therapies have proven ineffective. The mechanism or mechanisms responsible for detoxification with ILE are still unresolved, although recent studies show pharmacokinetic sequestration (PKS), increased cardiotonic activity, ion channel regulation, and an additional energy source for cells and tissues may all contribute to recovery from toxicity. Different amounts of lipid partitioning, protein binding, and ionization at biological pH with different drugs likely affect the amount of PKS provided by ILE. These factors suggest that ILE cannot be a universal therapy for detoxification. Other emulsions comprised of different oils (developed for TPN or otherwise) have not been extensively studied despite greater amounts of PKS possible. Novel emulsion formulations including microemulsions should also be investigated as detoxification therapies. Liposomes also serve as a versatile colloidal platform for detoxification therapies. Novel liposomes have been designed with acidic or basic interiors to enhance encapsulation of drugs into their ionized forms. Selection of phospholipids with tailored surface charges can also optimize the amount of drug sequestered by liposomes. Efficacy of liposomes containing specific antidotes, enzymes, or chelating agents has been shown in animal models. However, the elevated immune responses caused after some liposome injections warrants caution in formulation design and particle size. Liposomes also lack the clinical familiarity of ILE. Several drug overdoses investigated with liposome therapy overlap with those used in ILE therapy. Comparative studies of ILE and liposome detoxification, while limited, have yielded important implications on the relative efficacy of each therapy. While it is unlikely that liposomes cause a cardiotonic or elevated metabolic effect, increased sequestration may be sufficient to provide recovery after toxicity. Future work would benefit greatly to consider both emulsion and liposomal detoxification options. It is unlikely that a universal detoxification therapy can be designed for all drugs and toxicants; the most effective treatments will likely differ with the given overdose or exposure situation. However, the expanding volume of work on emulsion or liposomal detoxification suggests that more effective therapies can be developed for the most prevalent toxicities. Detailed understanding of the mechanisms and transport of colloidal therapies is critical to optimize formulations and dosing strategies to achieve the best possible therapies.

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