The pre-administration of the anti-inflammatory drugs dexamethasone (DEX) and cortisone acetate reduces toxicity and enhances efficacy of anticancer agents in murine models and in human clinical trials (1–5). We previously reported on the formulation of the lipophilic dexamethasone palmitate ester (DEX-P) in nanoparticles (NPs) employing a microemulsion template engineering technique to achieve tumor-specific delivery of dexamethasone (6). The nanoparticles exhibited significantly enhanced stealth properties as indicated by reduced macrophage uptake and decreased adsorption of opsonin proteins in in vitro assays (6). Unexpectedly, preliminary biodistribution studies of NPs containing [3H]-DEX-P in tumor-bearing mice showed that the radiolabel was cleared from the circulation rapidly and exhibited high liver uptake. Our previous in vitro release studies demonstrated that rapid release of the radiolabel from the NPs was observed when 10% mouse plasma was used as the medium, while nominal release was observed in phosphate-buffered saline (PBS) buffer (6). Esterolysis of NP-associated DEX-P was presumed to be the main cause for the rapid drug release in plasma, as most of the released radioactivity was in the form of DEX and not DEX-P. High degradation rates of ester prodrugs in rodent plasma has been attributed to increased esterase activity, while only minimal degradation in human plasma has been observed (7–9). Based on our observation of the release of [3H]-DEX from NPs in mouse plasma, we studied the release of DEX from nanoparticles in various plasma sources as a guide for the design of future in vivo experiments

Howard, Melissa D.

Jay, Michael

Leggas, Markos

Lu, Xiuling

Potter, Philip M.

Rinehart, John J.

Talbert, Dominique R.

PubMed

Carolina Digital Repository

Brief/Technical NoteNanoparticles Containing Anti-inflammatory Agents as ChemotherapyAdjuvants II: Role of Plasma Esterases in Drug ReleaseXiuling Lu,1 Melissa D. Howard,2 Dominique R. Talbert,2 John J. Rinehart,3 Philip M. Potter,4Michael Jay,1 and Markos Leggas2,5Received 10 October 2008; accepted 19 January 2009; published online 19 February 2009KEY WORDS: carboxylesterase; dexamethasone palmitate; esterolysis; release; solid lipid nanoparticle.INTRODUCTIONThe pre-administration of the anti-inflammatory drugsdexamethasone (DEX) and cortisone acetate reduces toxicityand enhances efficacy of anticancer agents in murine modelsand in human clinical trials (1–5). We previously reported onthe formulation of the lipophilic dexamethasone palmitateester (DEX-P) in nanoparticles (NPs) employing a micro-emulsion template engineering technique to achieve tumor-specific delivery of dexamethasone (6). The nanoparticlesexhibited significantly enhanced stealth properties as indicatedby reduced macrophage uptake and decreased adsorption ofopsonin proteins in in vitro assays (6). Unexpectedly, prelimi-nary biodistribution studies of NPs containing [3H]-DEX-P intumor-bearing mice showed that the radiolabel was clearedfrom the circulation rapidly and exhibited high liver uptake. Ourprevious in vitro release studies demonstrated that rapid releaseof the radiolabel from the NPs was observed when 10% mouseplasma was used as the medium, while nominal release wasobserved in phosphate-buffered saline (PBS) buffer (6).Esterolysis of NP-associated DEX-P was presumed to be themain cause for the rapid drug release in plasma, as most of thereleased radioactivity was in the form of DEX and not DEX-P.High degradation rates of ester prodrugs in rodent plasma hasbeen attributed to increased esterase activity, while onlyminimal degradation in human plasma has been observed (7–9). Based on our observation of the release of [3H]-DEX fromNPs in mouse plasma, we studied the release of DEX fromnanoparticles in various plasma sources as a guide for the designof future in vivo experiments.MATERIALS AND METHODSMaterialsTritiated dexamethasone-([6,7-3H(N)]; specific activity=35–50 Ci/mmol) was purchased from American RadiolabeledChemicals (Saint Louis, MO, USA). Bis(p-nitrophenyl)phosphate (BNPP) was purchased from Sigma-Aldrich (St.Louis, MO, USA). Mouse and human plasma containing Naheparin were purchased from Innovative Research (Novi, MI,USA). Plasma from nu/nu mice and Sprague–Dawley rats wereobtained from animals housed at the University of Kentuckyand from carboxylesterase-deficient mice (Es1e(−/−)/SCID)maintained at St. Jude Children’s Research Hospital.Preparation of Radiolabeled NanoparticlesThe procedure to prepare radiolabeled DEX-P and themethod to incorporate it into solid lipid nanoparticles by thenanotemplate engineering approach have been previouslydescribed (6). Briefly, [3H]-DEX-P was synthesized by thereaction of [3H]-DEX and palmitoyl chloride. Nanoparticleswere derived from a microemulsion comprising stearyl alcohol(1.6 mg/mL), [3H]-DEX-P (20% of the weight of the stearylalcohol), polysorbate 60 (0.4mg/mL), Brij78® (2.8mg/mL), andPEG6000 MS (3 mg/mL) that was prepared by adding warmPBS buffer (pH 7.4, 70°C) to themeltedmixture of oil, drug, andsurfactants. After stirring in a 70°C water bath for 1 h, the warmmicroemulsion was cooled to 25°C, resulting in the formation ofnanoparticles. Nanoparticle suspensions were diluted 1:30 (v/v)with filtered PBS buffer (0.22 μm filter, Nalgene International)and then incubated at 37°C for 0–20 h. Particle size distributionswere measured at 37°C by photon correlation spectroscopyusing a Coulter N4 Plus Submicron Particle Sizer (BeckmanCoulter Corporation, Miami, FL, USA), n=3.Release of Drug from Nanoparticles in Various MediaThe media used in the in vitro [3H] release study includedPBS buffer, plasma from nu/nu and carboxylesterase-deficient1550-7416/09/0100-0120/0 # 2009 American Association of Pharmaceutical Scientists 120The AAPS Journal, Vol. 11, No. 1, March 2009 (# 2009)DOI: 10.1208/s12248-009-9086-31 Division of Molecular Pharmaceutics, University of North Carolina,Chapel Hill, North Carolina 27599-3560, USA.2 Department of Pharmaceutical Sciences, College of Pharmacy,University of Kentucky, Lexington, Kentucky 40536-0082, USA.3 Department of Medicine, University of Kentucky, Lexington,Kentucky 40536-0093, USA.4 Department of Molecular Pharmacology, St. Jude Children’sResearch Hospital, Memphis, Tennessee 38105, USA.5 To whom correspondence should be addressed. (e-mail: mark.leggas@uky.edu)mice, rats, and humans. Human tumor (A549) homogenategrown in nu/nu mice and A549 cell lysate from cells grown invitro were also used. Heat-denatured plasma (70°C for 30 min)or BNPP-treated plasma were used as controls. A suspensionof NPs containing [3H]-DEX-Pwas added to 10%media (dilutedwith PBS buffer) at a ratio of 1:14 (v/v) and incubated at 37°C forup to 24 h. Ultrafiltration (Millipore Ultracel YM-100) was usedto separate the particles from the medium, and the radioactivitythat was released into the filtrate was quantified by liquidscintillation counting. Thin-layer chromatography (TLC) wasused to quantify the amount of [3H]-DEX-P and [3H]-DEX thathad been released into the medium.Statistical AnalysisData are presented as mean±standard deviation. Groupswere compared using analysis of variance, one-way or two-way tests as appropriate, with SigmaStat 3.11 software(Systat, San Jose, CA, USA). Differences were consideredstatistically significant when P<0.05, and the Holm–Sidakmethod was used to perform pair-wise multiple comparisonson significant effects and interactions.RESULTS AND DISCUSSIONThe size stability of [3H]-DEX-P loaded NPs was initiallyassessed in PBS buffer at 37°C to rule out the release of drugfrom the NPs due to particle degradation. As seen in Fig. 1, thesize of the particles remained constant during the first 3 h inwhich NP particle size was measured. A slight but notstatistically significant increase in the average particle sizefrom 125.7 (±16.3) nm to 164.7 (±24.3) nm was observed after20 h of incubation at 37°C.At all times, the suspension remainedclear with no evidence of particle aggregation or flocculation.These studies confirmed our previous results in which we used[14C]-stearyl alcohol, a primary matrix component, todemonstrate that the particles remained intact (6).Here, initial release studies showed that only 7.3±0.4%release of [3H] from NPs was detected after 7 days when PBSbuffer was used as the release media. In contrast, when theradiolabeled NPs were suspended in 10% mouse plasma, aburst release (5.0±0.3%) of the radiolabel was observed, andafter 3 h, approximately 55.7±0.4% of the radiolabel wasdetected in the filtrate. TLC analysis demonstrated that mostof the radioactivity (3H) in the retentate (i.e., in the NPs) waspresent as intact DEX-P, while analysis of the filtratesrevealed that 100% of the radioactivity was present as DEXindicating that de-esterification of DEX-P had occurred.The palmitate ester ofDEX is nearly insoluble in water andhas been reported to be an amphipathic molecule that can insertinto phospholipid monolayers (10). As such, during thepreparation of the NPs, DEX-P is expected to align itself inthe microemulsion precursor with the DEX group directedtoward the aqueous phase and the hydrophobic palmitate chainembedded in the oil phase. After cooling to form the NPs, thisorientation may allow plasma esterases easy access to the esterbond of DEX-P resulting in the release of DEX, which has awater solubility of 100 μg/mL, from the nanoparticle matrix.We anticipated that the addition of polyethylene glycol(PEG) derivatives to the NP formulations might impededegradation of DEX-P by shielding the molecule fromexposure to enzymes. While increased addition of PEG6000MS to NP formulations was able to reduce macrophageuptake in vitro (6), the addition of this PEGylating agent andothers, including DSPE-PEG5000, did not change the releaseprofile of radioactivity when the [3H]-DEX-NPs were incubatedin 10%mouse plasma. The results showed that incorporation ofamphipathic esters into PEGylated nanoparticles may notnecessarily protect the compound from enzymatic degradation.This has implications for studies conducted in mice that employvarious nanoparticle carriers containing esters, includingPEGylating agents bound to nanocarriers via ester linkages.Results from preliminary in vivo studies revealed adifference in the disposition of the radiolabeled drug inmouse and rat. Thus, additional studies measuring the releaseof radioactivity from [3H]-DEX-NPs in 10% heparinizedplasma from different species were conducted (Fig. 2). Thetime-dependent release over 24 h is depicted in the figurewith denatured 10% mouse plasma as the control, showingnegligible release (2.8±0.1%) after 24 h. Following 24 h ofFig. 1. [3H]-DEX-P nanoparticle size following incubation in PBS at37°C. Each value represents the mean±SD (n=3)Fig. 2. Release of [3H] from nanoparticles containing [3H]-DEX-P in10% plasma from different sources and in 10% human tumorxenograft homogenate. Each value represents the mean±SD (n=3)121Nanoparticles as Chemotherapy Adjuvantsincubation, extensive release was observed fromNPs suspendedin nu/nu mouse plasma (97.7±3.2%) and rat plasma (47.2±0.2%). In contrast, much lower release (22.0±1.0%) wasobserved when the NPs were suspended in plasma obtainedfrom carboxylesterase-deficient mice (Es1e(−/−)/SCID).Multiple comparisons versus the denatured plasma controlgroup were done using the Holm–Sidak method. The releaseof the radiolabel increased with time in mouse, rat, and Es1e(−/−)/SCID mouse plasma and was significantly different fromthat of the control group (P<0.001). The role of esterases in therelease of DEX from the nanoparticles was confirmed as mouseplasma containing the esterase inhibitor BNPP (1 mM) showedlow release (4.1±0.3%). Interestingly, nominal release (1.6±0.1%) was observed from NPs suspended in human plasma,showing no significant difference from the denatured plasmacontrol group. NPs incubated in a human tumor homogenateshowed a much greater extent of release than human plasma.Subsequently, to confirm that DEX-P hydrolysis in humantumor xenograft homogenate was not the result of any residualmouse plasma activity, we prepared cell lysates from the sametumor type (A549) grown in vitro. Extensive release was alsoobserved in 10% tumor cell lysate (88.9±5.5%) after 24 h.Carboxylesterase and butyrylcholinesterase are two majoresterases known to degrade prodrugs (11), and their activities ina variety of animal species have been thoroughly characterized(9). The release of radiolabeled drug from the NPs in 10%plasma from a carboxylesterase-deficient mouse was significant-ly lower than that observed in mouse and rat plasma, althoughan increase in the release of the radiolabel was observed withtime. It has been reported that liver homogenates have greateresterase-specific activities compared to plasma in both rodentsand humans (9). In our study, minimal drug release from theNPs was observed in human plasma, but DEX-P might be de-esterified in human liver because of its high esterase activity.Previous studies by us and others on opsonin protein adsorptionand macrophage uptake suggest that PEGylated nanoparticlescan avoid rapid uptake by the reticuloendothelial systemleading to increased residence times in the circulation (6,12).The rapid release of the radiolabel in human tumor homogenateindicated the potential of esterases to serve as a trigger torelease DEX in tumors after passive accumulation of DEX-PNPs via the enhanced permeability and retention (EPR) effect.CONCLUSIONStudies with nanoparticles containing amphipathic estersshould be conducted with caution in rodents. Our studies clearlydemonstrate that their esterase-rich plasma will alter biodistri-bution. This limitation may be overcome, to some extent, byusing a carboxylesterase-deficient mousemodel. In humans, it isspeculated that PEGylated dexamethasone palmitate deliveredin a solid lipid nanoparticle will not be de-esterified in plasma,but rapid de-esterification to dexamethasone will occur in thetumor after accumulation via the EPR effect. The releaseddexamethasone can then act to reduce interstitial hydrostaticpressure resulting in the enhancement of the uptake ofsystemically administered chemotherapeutic agents.ACKNOWLEDGMENTSThe authors are grateful for financial support from theMarkey Cancer Center Experimental Therapeutics Program(ML), the Buck-Kentucky Lung Cancer Research Chair(JJR), the Benedict Cassen Postdoctoral Fellowship fromthe Education and Research Foundation for the Society ofNuclear Medicine (XL), the Lyman T. Johnson PostdoctoralFellowship (DT), and grant DGE-0653710 from the NSFIGERT Program (MDH).REFERENCES1. J. Rinehart, L. Keville, J. Neiddhart, L. Wong, L. DiNunno, P.Kinney, M. Aberle, L. Tadlock, and G. Cloud. Hematopoieticprotection by dexamethasone or granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients treated with carboplatinand ifosfamide. Am. J. Clin. Oncol. 26:448–458 (2003).2. J. J. Rinehart, and L. R. Keville. Reduction in carboplatinhematopoietic toxicity in tumor bearing mice: comparativemechanisms and effects of interleukin-1 beta and corticosteroids.Cancer Biother. Radiopharm. 12:101–109 (1997).3. H. Wang, M. Li, J. J. Rinehart, and R. Zhang. Dexamethasone asa chemoprotectant in cancer chemotherapy: hematoprotectiveeffects and altered pharmacokinetics and tissue distribution ofcarboplatin and gemcitabine. Cancer Chemother Pharmacol.53:459–467 (2004).4. H. Wang, M. Li, J. J. Rinehart, and R. Zhang. 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Nanoparticles Containing Anti-inflammatory Agents as Chemotherapy Adjuvants II: Role of Plasma Esterases in Drug Release

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Nanoparticles Containing Anti-inflammatory Agents as Chemotherapy Adjuvants II: Role of Plasma Esterases in Drug Release

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