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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 Forster et al. 2012 Accepted Author Manuscript 1 | 12 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: Forster et al. 2012 Accepted Author Manuscript 2 | 12 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 Forster et al. 2012 Accepted Author Manuscript 3 | 12 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). Forster et al. 2012 Accepted Author Manuscript 4 | 12 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). Forster et al. 2012 Accepted Author Manuscript 5 | 12 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 Forster et al. 2012 Accepted Author Manuscript 6 | 12 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, Forster et al. 2012 Accepted Author Manuscript 7 | 12 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 Forster et al. 2012 Accepted Author Manuscript 8 | 12 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 Forster et al. 2012 pH gradient (data not included). A CHOL-phospholipid Accepted Author Manuscript chimera (1,29 | 12 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). Forster et al. 2012 Accepted Author Manuscript 10 | 12 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 Forster et al. 2012 Accepted Author Manuscript 11 | 12 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. Forster et al. 2012 Accepted Author Manuscript 12 | 12 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. Forster et al. 2012 Accepted Author Manuscript 13 | 12 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 Forster et al. 2012 Accepted Author Manuscript 14 | 12 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 Forster et al. 2012 Accepted Author Manuscript 15 | 12 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 Accepted Author Manuscript 16 | 12 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. References [1] Bronstein AC, Spyker DA, Cantilena LR, Green JL, Rumack BH, Giffin SL. 2009 Annual Report of the American Association of Poison Control Centers’ National Poison Data System (NPDS): 27th Annual Report. Clin Toxicol 1997;48:979–1178. [2] Henry PD. Comparative pharmacology of calcium antagonists: nifedipine, verapamil and diltiazem. Am J Cardiol 1980;46:1047–1058. 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