Download Drug-scavenging Liposomes Attenuate the

Drug-scavenging Liposomes Attenuate the Cardiovascular
Toxicity of Overdosed Calcium Channel Blockers
Vincent Forster, Paola Luciani, Jean-Christophe Leroux*
Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich,
Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland
*
To whom correspondence should be addressed:
ETH Zurich
Wolfgang-Pauli-Str. 10, HCI H 301
8093 Zurich, Switzerland
Telephone: +41 (0)44 633 73 10
Fax: +41 (0)44 633 13 11
Email: [email protected]
NOTICE: this is the author’s version of a work that was accepted for publication in Biomaterials.
Changes resulting from the publishing process, such as peer review, editing, corrections, structural
formatting, and other quality control mechanisms may not be reflected in this document. Changes may
have been made to this work since it was submitted for publication. A definitive version was
subsequently
published
in
Biomaterials,
[VOL
33,
ISSUE
13,
2012]
DOI:
10.1016/j.biomaterials.2012.01.042.
The following are the supplementary data related to this article are freely available online at:
http://www.sciencedirect.com/science/article/pii/S0142961212000889
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Abstract
Calcium channel blocker (CCB) overdose is potentially lethal. Verapamil and diltiazem are
particularly prone to acute toxicity due to their dual effect on cardiac and vascular tissues. Unfortunately,
conventional decontamination measures are ineffective in accelerating blood clearance and, to date,
few efforts have been made to develop antidotes. To address the issue, injectable long-circulating
liposomes bearing a transmembrane pH gradient are proposed as efficient detoxifying agents of CCB
poisoning. By scavenging the drug in situ, these circulating nanocarriers can restrict its distribution in
tissues and hinder its pharmacological effect. In vitro, we showed that liposomes stability in serum and
their ability to sequester CCBs could be finely tuned by modulating their internal pH, surface charge,
and lipid bilayer structure. Subsequently, we verified their efficacy in reversing the cardiovascular effects
of verapamil in rats implanted with telemetric pressure/biopotential transmitters. In animals orally
intoxicated to verapamil, an intravenous injection of the liposomal antidote rapidly attenuated the
reduction in blood pressure. Areas under diastolic, systolic, and mean pressures curves were
significantly reduced by up to 60% and the time to hemodynamic recovery was shortened from 19 to
only 11 h. These findings confirm the protective effect of pH-gradient liposomes against cardiovascular
failure after CBB intoxication, and endorse their potential as efficient, versatile antidotes.
Keywords: blood pressure, calcium channel blocker, liposome, intoxication, drug uptake
Introduction
The number of acute drug intoxications is growing every year. Of the 1.2 million cases reported
in 2009 in North America, cardiovascular drugs ranked second in terms of mortality and fourth with
regard to morbidity [1]. Approved in the early 1980s, verapamil (VP) and diltiazem (DTZ) are firstgeneration calcium channel blockers (CCBs). Their pharmacological mechanism is based on blocking
cellular calcium import through slow, voltage-dependent, L-type channels. Producing simultaneous
negative inotropic and chronotropic effects in the heart, seconded by reduction of systemic vascular
resistance, they are used for the treatment of heart-rhythm disturbances (i.e., atrial fibrillation and
supraventricular tachyarrhythmia), angina, and hypertension [2]. Being safe at therapeutic doses,
several major factors render these first-generation CCBs potentially dangerous when taken in excess:
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i) their afore-mentioned cumulative effects make advanced cardiac life support (ACLS) particularly
complicated; ii) owing to their relatively short plasma half-lives (2-7 h), they are mostly dispensed as
sustained release preparations, which complicates gastrointestinal decontamination; iii) after rapid and
extensive hepatic metabolism, their major metabolites retain some activity. However, the first-pass
hepatic enzymes are rapidly saturated in intoxicated patients, increasing the bioavailability of the more
potent parent drugs. Hallmarks of overdose include bradycardia and hypotension for all CCB
poisonings, furthermore, VP and DTZ evoke, in some cases, heart conduction dysfunctions or even
complete AV block and cardiac arrest.
The management of severe CCB poisoning remains challenging for emergency physicians.
Although conventional decontamination protocols may help in reducing gastric drug concentrations,
they are inappropriate when not applied early. Whereas induction of emesis is contraindicated because
of potential rapid degradation of patients’ state of health, gastric lavage and activated charcoal
administration need to be implemented within the first hour after ingestion to produce significant
improvements. Volunteer studies have shown that, after that time, drug absorption by porous charcoal
is decreased to values of questionable clinical importance [3]. It has been speculated that whole-bowel
irrigation with polyethylene glycol assists in CCB removal from the gastrointestinal tract, especially when
sustained release formulations are involved [4]. However, this aggressive intervention can be harmful
and its effectiveness is uncertain when patients are already hemodynamically unstable [5]. Finally,
accelerating drug removal with extracorporeal hemodialysis or hemoperfusion is also of limited value
due to the high volume of CCB distribution and their extensive adsorption onto proteins [2]. Despite the
availability and use of abundant supportive measures, cardiovascular collapse and death remain alltoo-frequent outcomes. Amongst the 123 lethal exposures associated with cardiovascular drugs
reported by the National Poison Data System in 2009, DTZ and VP alone were responsible for more
than 30% of the fatalities (20 and 18 cases, respectively). Even with intensive care and adequate ACLS,
the high lethality of CCB overdoses arises, in part, from the lack of decontamination algorithms to
reverse the effects of the already-absorbed drug.
Because most CCBs are highly absorbed after oral administration (>90%) [2], they should be
rapidly and efficiently sequestered to reverse their hemodynamic and myocardial actions. In this regard,
the administration of intravenous lipid emulsions (ILE) has emerged recently as a promising measure
in emergency medicine. Although originally developed for parenteral nutrition, ILE have been hijacked
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from their initial role to manage local anesthetic systemic toxicity. Their mechanism, although not yet
characterized, can be partially explained by the “lipid sink” theory, according to which the fat emulsion
provides an additional vascular compartment that draw tissue-bound hydrophobic toxins into plasma
where they are trapped within the transiently-created lipid phase (Figure 1) [6]. Growing evidence
supports this hypothesis, including numerous animal in vivo data showing that ILE can modify drug
pharmacokinetics and distribution and human case studies reporting successful resuscitations after ILE
perfusion [7-10]. However, as ILE have never been optimized for such use, several drawbacks have
become apparent. These include the necessity of emulsion administration at high doses because of
their fast clearance and low drug sequestration capacity, possible toxicity relapse after initial
improvement owing to discharge of the previously-sequestered drug [11], potentially injurious
complications when employed during hypoxia [12], and other risks of pancreatitis and
hypertriglyceridemia that have yet to be clarified [7,8,13].
Figure 1: Strategies for treating drug overdose with nanocarriers. Both ILE and pH-gradient liposomes can reduce
free drug concentrations in the body by acting as sinks for the toxin. Drug scavenging can be driven by a favorable
oil/water partition coefficient (Log P) in the case of fat emulsion (bottom middle), or enhanced by an additional
driving-force conferred by a difference in pH between the interior and exterior of the liposomes (pH int and pHext,
respectively; bottom right). Cint and Cext represent captured and external drug concentrations, respectively. Upon
sequestration, the toxin is redistributed from peripheral tissues to the blood compartment, bringing its concentration
below the toxic level (bottom left).
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In view of these limitations, long-circulating pH-gradient liposomes stand as potential
surrogates to ILE. Being more efficient than the latter in scavenging exogenous molecules, the
liposomes would rapidly limit drug interactions with critical organs and favor a progressive transport to
the liver where toxins and liposomes are metabolized. We have demonstrated recently that they could
be exploited, when adequately formulated, to extract drugs under ex vivo and in vivo conditions [14,15].
Their high stability and long circulation time, as well as the innocuous character of their main
constituents, make them promising candidates as injectable detoxifiers. In the present work, longcirculating pH-gradient liposomes were investigated to sequester DTZ and VP, and reverse their
cardiotoxic effect. Their detoxifying action was monitored for the first time in an unrestrained animal
model of oral VP intoxication.
Materials and Methods
Preparation of pH-gradient liposomes
Large, unilamellar vesicles composed of different lipids and internal buffers were formulated as
described in Supplementary Data. Except for St, Da, and Cp (definitions are given in Figure 2A), which
were purchased from Avanti Polar Lipids (Alabaster, AL), all lipids were kind gifts from Lipoid GmbH
(Ludwigshafen, Germany). Typically, liposomes composed of egg phosphatidylcholine, dipalmitoyl
phosphatidylcholine,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (EPC, DPPC, and DSPE-PEG, respectively), and cholesterol (CHOL; Sigma-Aldrich, St.
Louis, MO) were prepared by the film hydration/extrusion method [16]. Lipids and CHOL were dissolved
in chloroform which was subsequently removed under continuous nitrogen flow, and kept under vacuum
for >12 h. The lipid film was hydrated with citrate buffer (250 mM, pH 2 if not otherwise stated), and the
transmembrane pH gradient was established after elution on PD MidiTrapTM G-25 columns (GE
Healthcare, Buckinghamshire, UK) equilibrated with isotonic 20 mM HEPES-buffered saline (HBS) at
pH 7.4. For in vivo studies, the pH gradient was established by dialysis (>12 h) against un-buffered
sodium chloride (150 mM) in 100-kDa Float-A-Lyzer® devices (Spectrum Labs, Rancho Dominguez,
CA). Typical liposome size and polydispersity indexes were 135 nm and 0.05, respectively (Table S1),
as measured by dynamic light scattering (DelsaNano C, Beckman Coulter, Krefeld, Germany).
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Stability of transmembrane pH gradient
Liposomes, prepared as described above with citrate hydration buffer (pH 4) containing 5 mM
of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS; Invitrogen, Carlsbad, CA), were incubated at 37°C in
presence or absence of serum proteins (HBS with 50% v/v fetal bovine serum, FBS; Invitrogen). Their
internal pH was measured by fluorescence spectroscopy (Cary Elipse, Varian, Mulgrave, Australia)
according to a dual-wavelength ratiometric method [17] as further detailed in Supplementary Data.
In vitro drug uptake kinetics
VP and DTZ (Sigma-Aldrich) uptake kinetics were monitored in 50% FBS in side-by-side
diffusion cells (PermGear, Hellertown, PA) at 37°C. The donor compartment (liposome-free) was
separated from the receiver compartment (containing liposomes) by a polycarbonate membrane with
100-nm pores. Both units contained 0.5 mM of drug so that its uptake by the vesicles in the receiver
compartment was directly related to its reduction in the other side of the membrane. The drug-to-lipid
molar ratio (calculated on total system volume) was set to 0.3 for all experiments. Aliquots of 100 µL
were sampled from the donor compartment 3, 30 min, 1, 2, 4, 8, and 23 h after injection of pH-gradient
liposomes in the receiver compartment. VP and DTZ were then extracted from the withdrawn aliquots
and quantified by high-performance liquid chromatography as detailed in Supplementary Data. The
drug capture capacities (CC) of the formulations were quantified by equation 1, where C stands for
concentration.
(1)
Transmitter implantation
All animal experiments were performed in accordance with procedures and protocols approved
by the cantonal veterinary authorities (Kantonales Veterinäramt Zürich, license number 2009082).
Twenty-four male Sprague-Dawley rats (190 to 230 g; Charles River Laboratories, Sulzfeld, Germany)
were implanted with a telemetric transducer (TL11M2-C50-PXT, Data Sciences International, DSI, St.
Paul, MN) to monitor electrocardiography (ECG), arterial blood pressure, body temperature (BT), and
activity (body movements). Analgesia was provided by the administration of buprenorphine (0.05 mg/kg
subcutaneously) given 1 h pre- and 4 h postoperatively. The transmitters were placed in the peritoneal
cavity under aseptic conditions and anesthesia with isoflurane inhalation (1.5 to 2%, adequacy
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monitored by limb withdrawal response to toe pinching) according to the manufacturer’s instructions.
Briefly, laparotomy was performed, and the tip of the pressure catheter (1 cm) was inserted into the
abdominal aorta distal to the renal arteries. A drop of Histoacryl ® adhesive (B. Braun, Tuttlingen,
Germany) was applied at the insertion site to fix the catheter and seal the juncture. Two ECG electrodes
were tunneled subcutaneously until preset locations [18], and the device was eventually sutured to the
abdominal wall while closing the midline incision. During the recovery period of 1 week, the animals
were housed individually with appropriate care and given analgesics if needed (buprenorphine, 0.05
mg/kg subcutaneously).
Experimental design
After recovery from surgery, the animals were pair-housed with access to food and water and
remained in environmentally-enriched cages equipped with receivers (RPC-1, DSI) throughout the data
collection period. Each animal was subjected to maximum 3 consecutive, randomly-distributed
experiments, after which they were euthanized by cardiac puncture performed under isoflurane
anesthesia (2%). Between each experiment, the rats were allowed a washout period of 1 week to
ensure complete balancing of blood volume and clearance of the drug and detoxification systems. They
were weighed prior to each experiment. After at least 2 h of baseline recording, the animals were
gavaged per os (p.o.) with 50 mg/kg of VP in 10% polysorbate 80 (Sigma-Aldrich) (tp.o.=0), followed 1
or 3 h later with an intravenous (i.v.) injection of either pH-gradient liposomes (150 or 250 mg/kg), ILE
(Intralipid® 10%, 250 mg/kg), or an equal volume (2.3 mL/kg) of normal saline via the lateral tail vein
(ti.v.). The administered doses of VP matched its the mean toxic ingestion reported in a one-year clinical
evaluation of CCB overdoses (i.e., ~50 mg/kg) [19]. Interestingly, the latter dose corresponds to half of
the median oral lethal dose in rats (LD 50, 110 mg/kg). Liposomes and ILE were injected based on the
currently-recommended protocol for ILE therapy of drug poisoning (i.e., 300 mg/kg i.v. bolus followed
by slow infusion) [7,8]. To verify that the vesicles were void of hemodynamic effects, i.v. administration
of liposomes vs. saline was compared, in animals previously gavaged with water (Figure S1).
Telemetric data collection and analysis
Recordings of arterial pressure, ECG, BT, and activity were sampled for 20 s every min by
Dataquest A.R.T. 4.3 acquisition software (DSI). Central aortic mean, systolic and diastolic pressures
(MAP, SP, and DP, respectively), and heart rate (HR) were derived from blood pressure signals,
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whereas myocardial contractility was estimated indirectly from the time between the Q wave of the ECG
and the A point (onset of upstroke) of the blood pressure waveform (Q-A interval, QAI) [18]. Data
collection started at least 2.5 h prior to intoxication, providing basal measures (portion of data not
included) to which the physiological recordings were normalized to quantify the percentage of change
vs. baseline. Areas under the curves (AUCs) of the recorded parameters were calculated for 24 h after
tp.o. by OriginPro 8.5 (OriginLab Corporation, Northampton, MA). Stress artifacts caused by animal
handling during oral administration (between 0 and 1 h) and i.v. injection (between 1 and 1.5 or 3 and
3.5 h for ti.v.=1 and 3 h, respectively) were excluded from area calculation. Recovery time following
intoxication was set as the time when the AUC of MAP reached its minimal value (Figure S2).
Statistical analysis
In vivo data were analyzed by nonparametric statistical methods with OriginPro. For multiple
groups, Kruskal-Wallis 1-way ANOVA on the rank test was followed by post-hoc pair-wise comparison
with Dunn’s test. Data originating from only 2 groups were analyzed with the Mann-Whitney test. A
value of p≤0.05 was considered significant.
Results
Stability of the transmembrane pH gradient
Liposomes containing an acidic internal compartment were initially developed to actively
encapsulate basic drugs with high yields [20,21]. This “remote-loading” technique involves exposing the
drug molecules to preformed liposomes that possess a trans-bilayer ion gradient. Drug redistribution
occurs such that uncharged molecules diffuse into the vesicles, become protonated, and subsequently
accumulate within the core because of inhibited re-permeation (Figure 1). When employed as antidotes,
liposomes should keep their pH gradient while they circulate in the bloodstream to efficiently take up
the distributed drug. This implies that the lipid membrane should, in the presence of plasma
components, retain the aqueous content of the vesicle to ensure its acidity while at the same time being
highly permeable to the overdosed drug.
Liposomes composed of EPC rapidly lost their pH gradient, even in protein-free buffer (pH 7.4)
(Figure S3). As reported previously [21], vesicle stability could be substantially increased by substituting
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EPC (gel-to-liquid phase transition temperature (Tm) ≈–5°C) with the high-phase transition lipid DPPC
(Tm=41°C), and by addition of the membrane fluidity modulator CHOL. Numerous studies have indeed
shown that CHOL plays a role in phospholipid packing, stabilizing the bilayer, reducing its permeability,
and increasing the retention of solutes in serum [22,23]. Liposomes prepared with DPPC/CHOL/DSPEPEG were stable for approximately 3 days (internal pH ≤5.0) in HEPES buffer. The same trend was
observed in the presence of 50% serum (Figure S4), i.e., reduction of proton flux upon DPPC and CHOL
incorporation in the bilayer. Vesicles containing 45 mol% CHOL and either EPC or DPPC, initially
prepared with pH 4 citrate buffer, maintained an internal pH between 5 and 6 for at least 6 h. These 2
formulations were assessed in vitro for their capacity to extract DTZ and VP from serum-containing
buffer and further optimized to maximize drug uptake (vide infra).
Capture capacity of pH-gradient liposomes
Figure 2B compares the AUCs of drug uptake kinetics calculated in the first 8 h (Figure 3, gray
area), for 22 formulations. The graph demonstrates that under identical conditions (internal citrate buffer
pH 3, 200 mM), DPPC afforded the liposomes a 1.8-fold larger AUC than the formerly-tested EPC
formulation [15]. The highest CC was achieved with the formulation composed of DPPC/CHOL/DSPEPEG (50:45:5 mol%, referred to as D50C45P5) and an isotonic internal citrate buffer with a
concentration of 250 mM and a pH of 2.0. The AUC of this formulation was 13-fold higher than that
possessing no transmembrane pH gradient and surpassed the ILE by a factor of 20. As reported
previously with EPC liposomes [15], increasing the ionic concentration above 250 mM resulted in
decreased uptake capacity, explained by hypertonic internal osmotic pressure, which destabilizes the
vesicles. All other attempts to further increase drug uptake proved to be unsuccessful. These included
the substitution of DPPC: (i) by an anionic lipid (DMPG) to promote electrostatic interactions with DTZ,
(ii) by phospholipids with lower (DMPC, Tm=23°C) or higher phase transition temperatures (DSPC,
Tm=54°C and DMPE, Tm=49°C), (iii) by sphingomyelin, which is reported to increase liposome stability
towards oxidation and enzymatic degradation [24,25], as well as (iv) by replacement of the negativelycharged steric stabilizer DSPE-PEG by its neutral ceramide counterpart (defined as Cp in Figure 2A)
and, (v) by the use of an internal ammonium sulfate buffer instead of sodium citrate. Moreover, crosslinked liposomal formulations prepared with diacetylene-polymerizable lipid [26], exhibited poor uptake
(CC <0.1 µmol drug / µmol lipid, Table S1), mainly because of their inability to stably maintain the
transmembrane
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not
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A
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chimera
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dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, St), which has previously been shown to form
liposomes extremely resistant to content release in serum [27], was also tested. Good drug
sequestration was observed with liposomes composed of DPPC/St/CHOL/DSPE-PEG (35:40:20:5
mol%), but it still did not outperform the DPPC-based optimal formulation.
Figure 1: Strategies for treating drug overdose with nanocarriers. Both ILE and pH-gradient liposomes can reduce
free drug concentrations in the body by acting as sinks for the toxin. Drug scavenging can be driven by a favorable
oil/water partition coefficient (Log P) in the case of fat emulsion (bottom middle), or enhanced by an additional
driving-force conferred by a difference in pH between the interior and exterior of the liposomes (pH int and pHext,
respectively; bottom right). Cint and Cext represent captured and external drug concentrations, respectively. Upon
sequestration, the toxin is redistributed from peripheral tissues to the blood compartment, bringing its concentration
below the toxic level (bottom left).
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Figure 3 illustrates the comparative uptake kinetics of VP and DTZ by D50C45P5 liposomes in
50% FBS. Both drugs were tested independently or in combination. Despite their different pKas (8.9 vs.
7.7, respectively) and extent of binding to plasma proteins (VP ~90% vs. DTZ ~75%) [6], they could be
sequestered at high levels with comparable efficiencies (>90%, Table S1). Indeed, uptake in the
presence of serum was not significantly lower than in protein-free buffer (Figure S5). These data confirm
the remarkable extraction capacity of the optimized transmembrane pH-gradient formulation under
physiologically-relevant conditions. Interestingly, VP and DTZ were also taken up conjointly in a similar
fashion (drug capture efficiencies >65%) when both drugs were incubated together with the liposomes,
indicating that several basic drugs can be sequestered simultaneously. This is a promising finding
considering that half of fatal drug intoxications imply overexposure to more than 1 compound [1].
Figure 3: Uptake kinetics of VP and DTZ by pH-gradient liposomes (D50C45P5). The concentration of VP and
DTZ was set at 0.5 mM when used alone or at 0.25 mM when co-incubated in the same medium (combination).
The gray area illustrates an example of AUC compared in Figure 2B. Means±SD, n=3-6.
Effect of pH-gradient liposomes on hemodynamic parameters
The optimal formulation D50C45P5 was then employed to rescue rats that were orally
intoxicated with VP (Figure 4A). Oral VP intake at 50 mg/kg induced a 20% drop in MAP, DP and SP in
less than 10 min (Figure 4B). As opposed to what has been observed during severe human exposures
to CCBs, no significant decline in HR was noted in comparison to the control group (water p.o.). In
agreement with previous reports [28,29], a decrease in myocardial contractility (reflected by an increase
in the QAI) could be seen after VP administration (Figure S1). Intoxication was not severe enough to
produce any measurable changes in the ECG profile. Parameters, such as QRS duration, the interval
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between Q and T waves, or the height of the R wave, were derived from ECG but no differences
between resting and intoxication periods were observed. Of all measured parameters, blood pressure
was the most affected in terms of magnitude and duration. Indeed, in the control group, MAP returned
to its pre-intoxication value after 19 h (Figure 4B). However, after liposome administration (250 mg/kg)
1 h post-intoxication, the animals recovered their normal MAP in only 11 h. Recovery time was also
significantly improved in rats receiving a lower liposome dose (150 mg/kg, 12 h) compared to the ILEtreated group (250 mg/kg, 16 h) (Figure 5).
Figure 4: (A) Temporal schematization of the in vivo experimental design. (B) Impact of pH-gradient liposomes on
the hypotensive effect of VP (50 mg/kg, p.o.). Liposomes, administered 1 h post-intoxication (ti.v.), considerably
reduced MAP recovery time. The gray area indicates the AUC assessed for comparison between groups in the
following figures. For clarity, the data were smoothed with the Savitzky-Golay algorithm and SD only showed every
5 points.
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Figure 5: Recovery times to basal blood pressure after VP intoxication (50 mg/kg p.o.) and treatment (t i.v.=1 h) with
normal saline, ILE, or liposomes. Means±SD, n≥6, *p≤0.05.
Figure 6 outlines the AUCs of recorded parameters in saline, ILE, low- and high-dose liposome
groups. In the high-dose liposome group, the total hypotensive effect of VP was reduced significantly
by 2.5-fold and 2-fold (for MAP and SP, respectively), compared to saline-treated animals. The effect
on DP was even greater, with a 4-fold reduction in pressure drop vs. the saline group. In the low-dose
liposome group (150 mg/kg), a significant beneficial effect was also demonstrated for MAP and DP, but
not for SP. Control ILE treatment (250 mg/kg), which is currently used clinically, did not significantly
alleviate the decrease in blood pressure caused by VP, confirming its lower ability to sequester CCBs
[15]. Interestingly, the protective effect of liposomes was even noted when the vesicles were
administered 3 h after intoxication (Figure 7). For instance, the liposomal antidote produced significant
2-fold reductions in the AUCs of both MAP and SP vs. the saline control group.
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Figure 6: Influence of an i.v. injection of saline, ILE or pH-gradient liposomes on the pharmacological activity of
VP (50 mg/kg, p.o.). The drug was administered 1 h prior to the i.v. injection. Significant reductions of the drop in
MAP, SP and DP can be seen after treatment with liposomes. Means±SD, *p≤0.05.
Figure 7: Influence of an i.v. injection of saline or pH-gradient liposomes on the pharmacological activity of VP (50
mg/kg, p.o.). The drug was administered 3 h prior to the i.v. injection. Means±SD, *p≤0.05.
Discussion
The development of universal antidotes would constitute an important breakthrough in medical
sciences. Unfortunately, creating such cures with favorable characteristics and low toxicity is technically
challenging and far too costly [30]. For this reason, great hopes have been placed on ILE. However,
while they are about to be considered as gold standard therapy, they lack reliable efficiency and evoke
a number of side-effects. Hence, it is of paramount importance to question the mechanism of this
therapy and fill the gap with potential improvements. While these oil-in-water emulsions can only rely
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on a favorable Log P, the liposome-based therapy designed in this work is assisted by a different sink
hypothesis, taking advantage not only of the drugs’ lipophilic nature but also their basic character, to
enhance their sequestration. This finely-tuned antidote is capable of sequestering drugs in vivo with
much greater efficiency than ILE.
The CC of pH-gradient liposomes can, in principle, be modulated by several parameters, such
as internal pH, surface charge of vesicles, and membrane structure [20,31]. In the present work, the
nature of the intraliposomal buffer and its pH were found to influence drug uptake most significantly. A
large pH gradient across the vesicle membrane (i.e., low internal pH) was required to guarantee high
encapsulation efficiency. Liposome stability in the presence of plasma components was conferred by
the incorporation of 45 mol% of CHOL in the phospholipid bilayer. The highest drug capture was
achieved with DPPC-based liposomes containing a highly concentrated sodium citrate buffer at pH 2.
In vitro, the afore-mentioned liposomes could stably entrap VP and DTZ in their acidic interior
at high concentrations for more than 8 h (Figure 3). The final concentration inside the liposomes for
both drugs was >310 mM (assuming an internal liposome volume of 0.9 µL/µmole) [32] which, in the
case of VP, is 150-fold superior to its water solubility at pH 7.5. This high internal solubility can be
attributed to the protonation of VP and to the formation of a cationic dimer at low pH [33]. This efficient
encapsulation mainly relies on the vesicles’ active pumping mechanism conferred by its transmembrane
proton gradient. Partition in the lipid bilayer, while important during the diffusion process, seems to play
a secondary role in the overall uptake mechanism, as demonstrated by low drug capture by liposomes
prepared without pH gradient (Figure 2B). Likewise, the dispersed oil droplets of ILE, which only
sequester the drug by a favorable Log P, trapped only 9% of the VP captured by pH-gradient-bearing
liposomes (Table S1).
To date, very few in vivo studies have examined the impact of injecting empty pH-gradient
liposomes on the pharmacokinetics of low-molecular weight, weakly-basic drugs. Employing
doxorubicin as a model drug, Mayer et al. demonstrated that long-circulating EPC-based liposomes
could remotely capture the anticancer agent in the blood circulation, leading to altered tissue distribution
and increased drug plasma levels [34]. This study showed that a stable transmembrane pH gradient
could be maintained in the blood circulation and therefore provided indirect evidence that endogenous
ionizable compounds were not trapped in the liposomal core to a significant extent. More recently, we
showed that long-circulating liposomes (50% of injected dose circulating after 12 h) could efficiently
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sequester DTZ and especially its main active metabolite (deacetyl-diltiazem) in situ after i.v.
administration [15]. Interestingly, by increasing DTZ concentrations in the blood pool, the liposomes
were found to augment DTZ exposure to blood esterases, making the newly-formed metabolite readily
available for capture by circulating liposomes. While the latter study revealed the potential of pHgradient liposomes to sequester circulating/distributed CCBs, it did not represent a clinically-relevant
drug poisoning situation since DTZ was given i.v. and the antidote injected prior to drug exposure. In
contrast, in the present in vivo model, the animals were overdosed with VP via the oral route and treated
up to 3 h after intoxication.
The blood pressure recordings charted in Figure 4B, together with their derived AUCs (Figure
6), clearly demonstrate that the toxic effects of VP are readily reversible upon injection of pH-gradient
vesicles. The most striking outcome was the rapidity with which the treatment induced measurable
improvement of blood pressure depression. In less than 2 h after pH-gradient liposome injection, a
substantial increase in MAP could be detected, reducing the time needed for hemodynamic recovery
by 40-45% vs. the control group (Figure 4B). The more rapid recovery achieved with liposomes can be
related to their pH-gradient driving force, cornerstone of the system. It allowed extensive drug extraction
from intoxicated tissues with efficient and stable confinement of the drug in the blood pool.
The fast and efficient CC of pH-gradient liposomes could also be a key asset in overcoming the
major pitfalls observed in resuscitation with ILE. Recently, Marwick et al. reported a relapse of
cardiovascular toxicity 40 min after the successful lipid resuscitation of a bupivacaine-poisoned patient
[11]. This pharmacokinetic-related concern, explained by secondary drug release and recirculation, is
very unlikely to occur with pH-gradient liposomes. To alleviate the problem, extensive ILE infusion
amounts (up to a total of 1 L) have been suggested. However, a high dose of triglycerides injected in
the bloodstream is an additional concern that could mitigate enthusiasm surrounding the use of ILE for
detoxification. Indeed, interference with the immune system, pulmonary, and hepatic functions is a
potential risk associated with extensive ILE infusion [13].
In the future, pH-gradient liposomes may stand out as an alternative antidote to ILE. In a clinical
scenario, their superior plasma circulation time would decrease the need for repeated bolus injections
or high-dose infusions. Recently, Pütz et al. presented a novel double-filtration plasmapheresis method
capable of removing liposomes from human blood [35]. Combining this filtration technique with the pH-
Forster et al. 2012
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gradient liposomal system could represent an opportunity to further potentiate the detoxification effect
by quickly removing the sequestered drug from the body.
Conclusions
Our results demonstrate that pH-gradient liposomes constitute a promising therapy of severe
CCB-based intoxications. This study proved their unparalleled ability to reverse CCB cardiotoxicity, in
an in vivo model of oral poisoning. Being largely more competent in capturing CCBs than ILE, these
nano-scavengers could be injected at a lower dose to decontaminate the blood and peripheral organs.
Their innocuous character would ease rapid clinical translation as an adjunct to the usual ACLS
algorithms, to facilitate the resuscitation and recovery of hemodynamically-compromised patients. The
presented therapy seems to be an adequate answer to the quest for universal antidotes. In vitro, pHgradient liposomes can sequester virtually any low-molecular weight, weakly-basic agent making this
approach potentially applicable to a broad range of other drugs, such as sedatives, antipsychotics,
antidepressants and opioids.
Acknowledgments
This work was supported by the Swiss National Science Foundation [grant number
31003A_124882]. The authors wish to thank Dr. T.C. Weber for his help and advices in surgical
procedures and M.A. Gauthier, N. Bertrand, and N. Preiswerk for their critical reading of the manuscript.
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