JPS-1-094 Author Proof Amphotericin B/Emulsion Admixture ... Final.pdf · Amphotericin B/Emulsion Admixture Interactions:A ... Mixing FungizoneTM with a fat emulsion used for nutritional

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AAmphotericin B/Emulsion Admixture Interactions:An Approach Concerning the Reduction ofAmphotericin B Toxicity

E. SOCRATES T. EGITO,1,2 IVONETE B. ARAUJO,1 BOLIVAR P.G.L. DAMASCENO,1 JAMES C. PRICE2

1Programa de Pos-graduacao em Ciencias Farmaceuticas (PPCF), Universidade Federal do Rio Grande do Norte (UFRN),Rua Praia de Areia Branca, 8948–Natal-RN, 59094-450, Brazil

2Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, Georgia

Received 24 October 2001; revised 5 March 2002; accepted 24 April 2002

Published online ? ? ? ? in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.10223

ABSTRACT: Mixing FungizoneTM with a fat emulsion used for nutritional purpose(IntralipidTM or LipofundinTM) was reported to decrease Amphotericin B (AmB) toxicityin clinical use. In an effort to understand the reason for this phenomenon, spectral andmorphological analyses were done for the FungizoneTM and FungizoneTM/LipofundinTM

admixture (FLmix). The absorption spectra analyses showed that not only FungizoneTM

but also FLmix presented spectra that were concentration dependent. Moreover, thespectra of FLmix remained stable until the concentration of 5� 10ÿ7 M, and only at5� 10ÿ8 M did they become similar in shape to the FungizoneTM spectra. Morphologicalstudies revealed that even though emulsion droplets with or without FungizoneTM pre-sented the same particle size, the former was less electron dense compared withLipofundinTM alone. These results suggest a kind of association between FungizoneTM

and LipofundinTM that remains over the whole range of concentrations. This hypothesiswas confirmed by in vitro studies in which FLmix presented an important selectivityagainst human and fungal cells compared with FungizoneTM. These findings suggestthat parenteral emulsions should be able to reduce the AmB toxicity probably bychanging the AmB self-association state by binding it with emulsion droplets.ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1–13, 2002

Keywords: amphotericin B; red blood cells; Candida tropicalis; absorption spectra;parenteral emulsion; transmission electron microscopy (TEM); toxicity

INTRODUCTION

Acquired immune deficiency syndrome, cancer,and organ transplants are the three principal rea-sons for the increase in the number of immuno-compromised patients who easily become hosts offungal infection.1 Although discovered more than50 years ago, Amphotericin B (AmB) remains the

drug of choice for the treatment of life-threaten-ing fungal infections, primarily because of itsbroad spectrum of activity.2 However, the outstan-ding efficacy of its commercially available form,FungizoneTM, in which the drug is solubilized bysodium deoxycholate, is tempered by sometimessevere and frequent adverse reactions.3

To reduce its toxicity, several studies have sug-gested formulating AmB, which is lipophilic, inlipid carriers such as small unilamellar liposomes(AmBisomeTM), colloidal dispersion (ABCD,AmphocilTM, or AmphotecTM), and lipid complex(ABLC or AbelcetTM).4 All of these lipid-basedAmB preparations are reported to have better

JPS-1-094

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 00, XXXXX 2002 1

Correspondence to: Dr. E. S. T. Egito ((at UniversidadeFederal do Rio Grande do Norte) Telephone: 84-9431-8816;Fax: 84-219-2836; E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 91, 1–13 (2002)ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association

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Atherapeutic indices than FungizoneTM, but theiruse is limited by their high cost and difficultpreparation.5–7

An alternative delivery system, based on thelipid emulsions used for parenteral nutrition inclinical practice (IntralipidTM or LipofundinTM),has been shown to reduce the AmB toxicity withlittle compromise in efficacy.8–16

In vitro studies revealed that although Fungi-zoneTM/IntralipidTM admixtures (FImix) were atleast as active as FungizoneTM against Candida,no hemolytic activity was observed against redblood cells at AmB concentrations up to 80 mg/L.In contrast, FungizoneTM was hemolytic at anAmB concentration of 0.1 mg/L. Moreover, con-cerning potassium ion (Kþ) leakage, the FImixwas reported to be half as toxic to red blood cells asFungizoneTM.15 In contrast, a recent study thatalso examined the relative toxicity of these for-mulations as measured by Kþ release from redblood cells during incubation at 378C,16 showedthat FImix seems to be slightly more toxic thanFungizoneTM. However, Joly et al.12 found thatFImix presented a reduced toxicity against renaltubular cells compared with FungizoneTM.

In vivo studies showed that although the toxi-city of FungizoneTM was 1.7- to 2.5-fold lower inthe presence of IntralipidTM, the antifungalactivity of FungizoneTM was not reduced.12 There-fore, it was concluded that FImix was more effec-tive than FungizoneTM when both drugs weregiven at the maximum tolerated dose. Indeed,IntralipidTM improved the therapeutic index ofAmB by allowing larger doses of FungizoneTM tobe infused12 rather than by altering its antifungaleffect of a given dose or by targeting the infectedsite.

As found for the in vitro studies, there areconflicting results from clinical studies. At leastthree reports have shown that FImix was respon-sible for a reduction in fever and chills in neu-tropenic patients,9,17,18 whereas its effectivenessin the treatment of systemic candidiasis wasmaintained.10 In a pharmacokinetic study, Hei-nemann et al.14 found an important decrease inboth maximum concentration of AmB in serum(Cmax) value and area under the concentration-time curve value when FImix was used. In addi-tion, a recent study with critically ill patientsshowed that FImix compared with FungizoneTM

was not only effective in eradicating Candidainfections but also better tolerated in that thefrequency rate and severity of immediate sideeffects (hypotension, chills, fever, anaphylaxis,

renal impairment) were diminished.13 Contraryto these authors, two other clinical trials revealedno significant advantage with respect to safetyand tolerance in the administration of FImixcompared with FungizoneTM.19,20

The reasons for poor or good therapeutic effi-cacy of AmB in FImix are unclear, and there is alack of understanding regarding how this mixtureworks when administered to patients with estab-lished infections. The goal of this report was to usespectral and morphological analysis of the Fungi-zoneTM/emulsion admixture (FLmix) and to cor-relate the results with a study of the effects ofFungizoneTM and FLmix on toxicity to red bloodcell (RBC) membranes and effectiveness againstCandida tropicalis. The effects of concentrationand temperature on the spectra shape were eva-luated. In addition, in vitro effects of FungizoneTM

and FLmix on the membranes of RBCs and C.tropicalis were studied. Afterward, a correlationwith the spectra and morphological studies wasmade.

MATERIALS AND METHODS

Materials

FungizoneTM and LipofundinTM (MCT/LCT 20%)were obtained from Bristol-Myers Squibb (SaoPaulo, SP, Brazil) and B. Braun (Sao Goncalo,RJ, Brazil), respectively. Sodium deoxycholate,and water for injection were from Sigma (St.Louis, MO).

Methods

AmB Formulations

FungizoneTM solution at the concentration of 5�10ÿ3 M (5 mg/mL) was obtained by dissolving thecomponents of a vial with 10 mL of water for in-jection. Each vial of FungizoneTM contains 50 mgof AmB, approximately 41 mg of sodium deox-ycholate, and appropriate phosphate buffer tomaintain a pH 7.4. Control vials were prepared,containing equivalent amounts of each of theabove ingredients except AmB, to yield doses ofsodium deoxycholate in the controls equivalent tothe FungizoneTM. FLmix was achieved by mixing(Vortex Genie 2, model G-560; Scientific Indus-tries, Inc., Bohemia, NY) 10 mL of LipofundinTM

with 10 mL of FungizoneTM solution for 15 min at258C. This mixture was used within 2 h after itspreparation.

2 EGITO ET AL.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 00, XXXXX 2002

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ASpectral Studies

To determine absorption spectra (Olis-Cary 118Spectrophotometer; Olis, Inc., Bogart, GA), dis-persions of FungizoneTM and FLmix were pre-pared in phosphate buffer solution, pH 7.4, atvarious AmB concentrations. FLmix was recordedagainst a blank consisting of LipofundinTM inphosphate buffer solution, in the reference beam.The optical path of the quartz cuvettes used was0.1, 1, and 10 cm for the concentrations of 5�10ÿ5 M (50 mg/L), 5� 10ÿ6 M (5 mg/L), and5� 10ÿ7 M (0.5 mg/L) and 5� 10ÿ8 M (0.05 mg/L),respectively. These paths were chosen to obtainspectra with absorbance values less than 1.2.Their molar extinction coefficients (e) were calcu-lated using the Beer-Lambert equation. Allspectra were recorded at 258C or 378C.

Quantitation of AmB Species

AmB in aqueous media is present as a mixtureof various species in equilibrium, monomers,and soluble and insoluble aggregates.21,22 Theconcentration of each species depends on thetotal AmB concentration,22 on the method ofpreparing the solutions,22 and on the AmBpharmaceutical delivery system.23 Typically, itis assumed that the spectral bands indicat-ing the existence of monomers and aggregatesspecies of AmB are located around 409 nm, and327 nm, respectively.23 Previous studies show-ed that the selectivity against mammalian andfungal cells also depends on the aggregationstate of AmB.22,24–26 Only self-associated AmBwas shown to increase Kþ permeability ofcholesterol-containing egg phosphatidyl cholinevesicles, whereas both monomer and aggregatespecies modify Kþ permeability in ergosterol-containing liposomes.26 Therefore, the equili-brium between monomers and aggregates seemsto have a key role in drug activity. Consequently,the structural study and quantitation of theAmB species constitute an essential step inunderstanding the molecular mechanism of thisantifungal agent concerning both selectivity andcytotoxicity.21

To estimate the quantities of each species inaqueous media as a function of the AmB carrier,we propose the following algorithm to process theabsorption data. The absorption spectrum of AmB,which results from the spectroscopic contribu-tions of aggregates (b) and monomers (d), dependson the concentration of the forms. Therefore,

absorbances can be expressed according to theBeer-Lambert formulation as:

Al ¼ ebl1� ½b� � lþ edl2

� ½d� � l ð1Þ

Where l1¼wavelength for aggregates formed(usually around 327 nm); l2¼wavelength formonomers formed (usually around 409 nm);l¼ cuvette path length; and e¼molar extinctioncoefficients.

Assuming that AmB in aqueous media corre-sponds to a mixture of these two spectroscopicspecies, the total AmB concentration ([T]) was ex-pressed as the sum of b and d concentrations (eq. 2):

½T� ¼ ½b� þ ½d� ð2Þ

Molar extinction coefficients (e) for each spec-troscopic species were deduced from their typicalabsorption spectra at l1 and l2 nm for eachconcentration studied. The mean number of AmBmonomeric units in each aggregated form isunknown and probably corresponds to a wide sizedistribution. Consequently, the e values wereestimated considering monomeric AmB. There-fore, the concentration of b and d was expressed interms of monomeric AmB and calculated accord-ing to the expressions:

½b� ¼ Al1

l ebl1þ edl2

� � ð3Þ

d½ � ¼ T½ � ÿ b½ � ð4Þ

Mean Particle Size Studies

The mean particle size of LipofundinTM andFLmix at two AmB concentrations was estimatedby photon correlation spectroscopy using aNICOMP Particle Sizing Systems, SubmicronParticle Sizer, Autodilute, model 370A (SantaBarbara, CA).

Morphological Analysis

Transmission electron microscope (TEM) exam-ination of LipofundinTM and FLmix at two AmBconcentrations was performed using a TEMJEOL, model JEM100cx II (Electron MicroscopeLtd., Tokyo, Japan) after negative staining withosmium tetroxide solution at 2%(w/v).

Preparation of RBC Suspension

One healthy adult female donor provided allnormal human RBCs for the in vitro experiments;

AMPHOTERICIN B AND EMULSION ADMIXTURE 3


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Athis was done to minimize sources of variability.Five milliliters of venous blood was collectedin sterile EDTA [1 mg/mL, ethylenediaminete-traacetate at 10%(w/v); Labtest, Lagoa Santa, MG,Brazil] syringes and promptly centrifuged (Refri-gerated Centrifuge, ALC, model PK121R, Milan,Italy) in tubes at 1100g for 10 min at 48C. Plasmawas aspirated with precautions, and the exposedbuffy coat was removed and discarded. The RBCswere washed three times by centrifugation(1100g for 5 min at 48C) and suspension in fivevolumes of normal saline [NaCl at 0.9% %(w/v);B. Braun]. They were then resuspended in 4 mLof saline, counted in a NeubauerTM chamber, andresuspended again until desired concentration(5� 107 cells/mL). They were used within 24 h ofcollection.

Preparation Inocula of C. tropicalis

A strain of C. tropicalis isolated from a patient’surine culture was maintained on Sabouraud-Dextrose-Chloramphenicol agar (SDC; MicroMed,Sao Paulo, SP, Brazil) at room temperature andpassaged monthly. Before experiments, an inocu-lum from the culture was transferred to an SDCagar scope, and incubated at 378C for 16–18 h.The fungal cells were then washed three timeswith normal saline, resuspended, counted in thecentral reticule of a NeubauerTM chamber, andresuspended again to obtain the desired concen-tration (5� 107 cfu/mL).

Toxicity of AmB Formulations

Four milliliters of RBCs (5� 107cells/mL) wereincubated for 1 h at 378C with the vehicle controlor with different concentrations (50, 5, 0.5, and0.05 mg/L) and formulation of AmB, eitherFungizoneTM and FLmix. The RBCs were thencentrifuged for 5 min at 1100g and washed threetimes with normal saline. The pellet of RBC waslysed by 4 mL of distilled water, stirred, andcentrifuged (1100g for 5 min) to remove mem-branes. Kþ content of the supernatant was deter-mined using a Flame Photometer 7000 (Tecnow,Sao Paulo, SP, Brazil) calibrated with Kþ refer-ence at 5 mEq/L; hemoglobin was estimated fromits absorption at 540 nm recorded with a ColemanSpectrophotometer (model SP395U; Sao Paulo,SP, Brazil). The total potassium and hemoglobincontent was estimated from the control RBCtubes. Release was calculated as the differencebetween control and treated cells and was

expressed as a percentage of the total hemoglobinor potassium content. At least three differentexperiments were performed with each formula-tion and each experimental point was performedin triplicate.

Efficacy of AmB Formulations

Two-milliliter samples of fungal suspension con-taining 5� 107 cfu/mL were incubated for 1 h at378C with FungizoneTM and FLmix at AmBconcentrations of 50, 5, 0.5, and 0.05 mg/L. Cellswere centrifuged for 10 min at 2200g, washedthree times in normal saline, and 2 mL of purifiedwater was added to the pellet of fungal cells. Analiquot of this pellet was lysed for 5 min at 1008Cand centrifuged to remove membranes, and freepotassium was measured. The Kþ leakage wascalculated similarly to the RBCs. For the cellviability evaluation, 100-mL aliquots of appropri-ate dilution of the fungal pellet were seeded, induplicate, onto agar plates and incubated at 378C.The number of colony-forming units was countedat 24 and 48 h and expressed as a percentage ofthose obtained from a control inoculum incubatedwithout AmB. Three different experiments wereperformed with each formulation and each experi-mental point was performed in duplicate.

Statistical Analysis

All potassium and hemoglobin release, and cfuviability data were expressed as the mean�SE.Statistical analysis was performed using analysisof variance test and significance was defined asp< 0.05.

RESULTS

Spectral Studies

The absorption spectra of FungizoneTM (Fig. 1)and FLmix (Fig. 2), at 258C are concentrationdependent. At low concentration, spectra weresimilar to those obtained in methanol or otherpolar organic solvents, exhibiting maxima at364, 383.5, and 407.5 nm and a shoulder around347 nm. They are traditionally assigned to mono-meric AmB.26 For increasing FungizoneTM con-centrations, a new band appeared around 326 nm,and two other ones at 383 and 418 nm. The in-crease of amplitude of the band at 326 nm,assigned to AmB self-associated species,26 occur-red at the expense of those of monomeric AmB. In

4 EGITO ET AL.


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Athe FLmix spectra, these bands were slightly blueshifted (4 nm). It should be noted that 5� 10ÿ8 Mspectra are given to illustrate the described ten-dency but should not be considered on a quanti-tative basis in the low wavelength region (theybecome negative) because of the weakness of thesignals (maximum absorbance at 407 nm: 0.05)and the corresponding uncertainty. Beside thesmall wavelength differences between Fungizo-neTM and FLmix, the concentration dependencywas also different: the formation of self-associatedspecies (monitored by the band around 326 nm)only occurred above 5� 10ÿ7 M whereas it startedto occur above 5� 10ÿ8 M for FLmix.

It was also noticed that the characteristics ofthe spectra remained unchanged at 12 h.

At 378C, similar changes on concentration wereobserved. For FLmix, the band characteristic ofself-association was blue-shifted compared withthe corresponding band at 258C (319.5 nm).Quantitative changes are described below.

Consequently, although variation in tempera-ture did not provoke any important change on theFungizoneTM spectra, the AmB–emulsion com-plex presented a quite different behavior with a128C temperature increase. In this case, moremonomeric forms are present, probably releasedfrom the AmB–emulsion complex, because of theincrease of thermodynamic energy on the water/oil interface.

Therefore, it can be concluded from theseresults that a kind of association between AmBand LipofundinTM remains over the whole rangeof concentrations.

Quantitation of AmB Species

The evaluation of concentration of aggregates [b]and monomers [d] is summarized in Figures 4and 5Q2. At 258C (Fig. 4), FungizoneTM presentedthe profile mentioned in the literature.21,22 At lowconcentration, 5� 10ÿ8 M, 100% of the AmB wasin the monomeric form; as the concentrationincreased, aggregates started to appear and theb/d50%, where sub-exists 50% of aggregates and50% of monomers, was retrieved at 1.5� 10ÿ6 M.At high concentrations, 5� 10ÿ5 M, almost all thespecies found were aggregates (4.6� 10ÿ5 M,92.7%), and the existence of a small quantity ofmonomer (0.4� 10ÿ5 M, 7.3%) was the result of

Figure 1. Concentration-induced changes in theFungizoneTM spectra. The spectra were obtained at258C, in phosphate buffer solution, pH 7.4, at fourAmB concentrations. The optical path of the quartzcuvettes used was 0.1, 1, and 10 cm for the concentra-tions of 5� 10ÿ5 M (50 mg/L), 5� 10ÿ6 M (5 mg/L), and5� 10ÿ7 M (0.5 mg/L) and 5� 10ÿ8 M (0.05 mg/L),respectivelyQ1.

Figure 2. Concentration-induced changes in theFungizoneTM/LipofundimTM admixture spectra at 258C.

Figure 3. Concentration-induced changes in theFungizoneTM/LipofundimTM admixture spectra at 378C.



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Athe thermodynamic equilibrium between bothspecies. However, the profile of aggregates andmonomers for FLmix was quite distinct. At lowconcentration, 80% was monomeric and 20% wasin the form of aggregates (3.95� 10ÿ8 M and1.05� 10ÿ8 M, respectively) and the b/d50% wasdetected at 7� 10ÿ7 M. Also, it was observedthat the variation of monomers and aggregates infunction of the log of the concentration was quitelinear.

At 378C, FungizoneTM maintained the sameprofile as that at 278C (Fig. 5). The only significantvariation concerned its b/d50% which was slightlydislocated to the right, at 2.1� 10ÿ6 M. Never-theless, the FLmix profile presented considerablechanges; not only did its b/d50% shift to the right

(at 9� 10ÿ7 M) but also the aggregation structuredisappeared at low concentration. Probably, the128C increase in the temperature was able torelease the AmB from the oil/water interface be-cause of the high level of thermodynamic energythere, caused by the surfactants.

Particle Size and Morphological Evaluation

No significant changes in the LipofundinTM emul-sion particle size were observed when the AmBwas incorporated to the system (Table 1). BothAmB concentrations studied, 2.5� 10ÿ3 M and5� 10ÿ5 M, showed a droplet size around 280�70 nm, which was similar to the system withoutAmB. Only after 12 h of preparation did FLmix at5� 10ÿ5 M reveal some variation on its distribu-tion profile of droplets (Fig. 6), and its particlesize, analyzed in volume by weight, changed from299.6� 81.5 to 347.8� 131.8 nm.

The morphological analysis of FungizoneTM

(Fig. 7) and LipofundinTM with (Fig. 8) or without(Fig. 9) AmB had different aspects. Indeed, thefree AmB–FLmix was less electron-dense thanAmB-loaded FLmix. Nevertheless, no changes inparticle size, which was confirmed to be around250 nm, were observed (Fig. 10A and B). However,FungizoneTM micelles presented a particle sizethat were five times lower, around 50 nm, andwere high electron-dense because of the unsatu-rated carbons of the AmB.

In Vitro Evaluation of FungizoneTM and FLmix

Toxicity against Human RBCs

Potassium and hemoglobin leakage after the in-cubation of 2� 108 RBCs (1 h at 378C) with increas-ing concentrations of FungizoneTM and FLmix areshown in Figures 11 and 12, respectively.

No significant potassium leakage was observ-ed below 0.1 mg/L with either FungizoneTM orFLmix. At higher concentrations, 0.5 and 5 mg/L,the permeability of the cell membrane, as re-flected by potassium release, was higher withFungizoneTM (46.73� 11.33% and 98.96� 0.26%,respectively) than with FLmix (9.61� 5.99%and 40.01� 7.74%, respectively), and only at thehighest concentration, 50 mg/L, were the releaseprofiles similar, 99.35� 0.16% and 96.13� 0.36%,respectively. At AmB concentrations of 5 and0.5 mg/L, the potassium release induced byFungizoneTM was significantly different from thatwith FLmix ( p< 0.0001).

Figure 4. Distribution of aggregated [b] and mono-meric [d] species as a function of the concentration at258C. The percentages of both species were calculatedfrom the absorption data at around 326 and 407.5 nmaccording to eqs. 3 and 4, respectively, described inMaterial and Methods.

Figure 5. Distribution of aggregated [b] and mono-meric [d] species as a function of the concentration at378C.

6 EGITO ET AL.


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Table 1. Mean Particle Sizes of LipofundinTM and FungizoneTM/LipofundinTM Admixture (FLmix) Immediately and after 12 h of Preparationa

Sample

Immediately after Preparation

Number Weighted Intensity Weighted Volume Weighted

MeanDiameter SD CV

MeanDiameter SD CV

MeanDiameter SD CV

Blank 1 243.2 68.6 0.282 286.4 80.8 0.282 310.1 87.4 0.282FLmix at 2.5� 10ÿ3 235.1 70.3 0.299 283.3 84.7 0.299 309.7 92.6 0.299Blank 2 238.3 66.3 0.278 279.3 77.7 0.278 301.4 83.8 0.278FLmix at 5� 10ÿ5 239.0 65.0 0.272 278.3 75.7 0.272 299.6 81.5 0.272

After 12 h

Blank 1 239.5 64.2 0.268 277.6 74.4 0.268 298.3 79.9 0.268FLmix at 2.5� 10ÿ3 241.5 64.5 0.267 279.4 74.6 0.267 300.0 80.1 0.267Blank 2 245.1 67.6 0.276 286.5 79.1 0.276 309.1 85.3 0.276FLmix at 5� 10ÿ5 220.5 83.6 0.379 302.0 114.5 0.379 347.8 131.8 0.379

CV, coefficient of variation; SD, Standard deviation.aBlank 1 and Blank 2 samples were prepared from LipofundimTM diluted 1:2 and 1:5 in phosphate buffer solution, pH 7.4, respectively.

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A(5.36� 9.28%) and at 5 mg/L reached 96.99�2.53%. When FLmix was used at 5 and 50 mg/L,the toxicity against RBCs, as reflected by hemo-globin leakage, was significantly reduced com-pared with FungizoneTM ( p< 0.0001).

Activity against C. tropicalis

Potassium leakage after the incubation of 108 cfu(1 h at 378C) with increasing concentrations ofFungizoneTM and FLmix is shown in Figure 12.Whatever the system tested, a significant potas-sium release was observed at the lowest con-centration (0.05 mg/L) and reached 100% at the

5 mg/L level. However, FungizoneTM at 0.05 mg/Linduced a significantly larger potassium release(88.96� 3.49%), compared with the FLmix (60.59�21.36%) ( p< 0.0001).

The antifungal activity of FungizoneTM andFLmix is shown in Figure 13. Determination ofthe viability of C. tropicalis cells (ability to formcolonies) showed that whereas FungizoneTM wasable to kill all seeded fungal cells from a con-centration of 5 mg/L (0.36� 0.62 cfu%), FLmixwas unable to do that. Indeed, after treatmentof C. tropicalis cells with FLmix at the highestconcentration studied, 50 mg/L, a viability of11.89� 4.65 cfu% was retrieved. Nevertheless,

Figure 8. TEM of FungizoneTM/LipofundimTM ad-mixture showing the incorporation of FungizoneTM onthe emulsion droplet, in the oil/water interface. Thesample was stained with osmium tetroxide 2% andanalyzed at 58,000� magnification. Bar, 170 nm.

Figure 9. TEM of LipofundimTM emulsion showing areduction on the droplet electron density. The samplewas stained with osmium tetroxide 2% and analyzed at58,000� magnification. Bar, 170 nm.

Figure 10. TEM of FungizoneTM/LipofundimTM

admixture (A) LipofundimTM emulsion (B) stained withosmium tetroxide 2% and analyzed at 19,000� magni-fication. Bar, 500 nm.

Figure 11. In vitro release of potassium from humanRBCs (&, &) and C. tropicalis(*, *) induced byFungizoneTM (*, &) and FungizoneTM/LipofundimTM

admixture [FLmix (*, &)]. Each point on the figure isthe mean (�SD) of three determinations. *Significantdifference between the two forms for RBCs ( p< 0.0001).

8 EGITO ET AL.


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Athe difference of activity between both prepara-tions was significantly different only at 0.5 mg/L,when FungizoneTM was more effective thanFLmix ( p< 0.0001).

DISCUSSION

The use of electronic absorption analysis has beenpreviously reported as a powerful tool to followthe interaction of AmB with a lipidic carrier.23,27

In fact, the strong absorbance that AmB exhibitsbetween 300–450 nm, because of the seven

conjugated double bonds existing on its apolardomain (Fig. 14), is heavily influenced by con-formational changes provoked by its self-associa-tion in water media or by its interaction withother compounds such as drug carriers.

The physicochemical properties of AmB, main-ly its amphiphilic and zwitterionic nature, attrib-uted respectively to the apolar and polar sides ofthe lactone ring and to the presence of ionizablecarboxyl and amine groups (Fig. 14), as well as itsasymmetrical distribution of hydrophobic andhydrophilic groups, make the drug extremelyinsoluble in aqueous solvents and in many organicsolvents where it generates aggregates beyond athreshold concentration. In fact, beneath 10ÿ6 M,which is the critical aggregation concentration(CAC), AmB can be present in aqueous media as asoluble monomer, and its absorption spectrum ischaracterized by a peak at 409 nm. However,above the CAC, AmB molecules are able to self-associate to form oligomers and then aggregatesof oligomers, which are characterized in absorp-tion spectra by a broad intense single band at340 nm.28–30

It has been reported that the AmB maypresent various patterns of activity againstergosterol-containing fungal cells with respectto the toxicity against cholesterol-containingmammalian cells, depending on its molecularpresentation.22,24,26,31,32 Indeed, monomers weremore efficient than aggregates of oligomersat inducing the permeability of a cholesterol-containing membrane to potassium.33 Self-asso-ciated AmB was shown to trigger permeabi-lity changes in RBC membranes and to inducecytotoxic events.22 Moreover, the toxic chemo-therapeutic effects in mice were previouslydemonstrated to be correlated to the aggregationstate of AmB.25

Binding of the monomeric or oligomeric formsof an amphiphilic drug can lead to an active formeither at once or after reorganization in micel-les within the lipid bilayer, but only when the

Figure 12. In vitro release of hemoglobin fromhuman RBCs induced by FungizoneTM (&) and Fungi-zoneTM/LipofundimTM admixture [FLmix (&)]. Eachpoint on the figure is the mean (�SD) of three deter-minations. *Significant difference between the twoformulations ( p< 0.0001).

Figure 13. In vitro antifungal activity of Fungizo-neTM (&) and FungizoneTM/LipofundimTM admixture[FLmix (&)] on C. tropicalis (mean�SD of threedeterminations run in duplicate). *Significant differ-ence between the two formulations ( p< 0.0001). Figure 14. AmB structure.



Author Proof

Aconcentration exceeds the critical micellar con-centration in the membrane.34,35 Consequently,any factor modifying the equilibrium between thedifferent species of AmB in aqueous media maychange its overall activity.

In the present study, the spectral evaluation ofFungizoneTM and FLmix clearly demonstrate thatthe behavior of AmB molecules was perturbedfrom one system to another. In fact, the spectra ofAmB in the FungizoneTM (Fig. 1) presented aconcentration dependency that was reported byother groups.23,28,36,37 At higher concentrations,the spectra presented an intense band at 326 nmand two small ones at 383 and 418 nm. However,as the concentration decreased, the predominantband disappeared and a new one, at 407.5 nm,was observed when it reached 5� 10ÿ7 M. Thefact that the aggregation state band was shifted toa slightly lower wavelength, 326 nm, for theFungizoneTM system compared with the classicalAmB self-associated band location, which is at340 nm, indicates that the AmB molecules arecomplexed with the sodium deoxycholate. How-ever, because the deoxycholate concentration inFungizoneTM diluted to 5� 10ÿ7 M AmB is verylow and below its critical micellar concentration(2.8� 10ÿ3 M),38 we believe that the detergentmolecules dissociate from the AmB–micelle com-plex and dissolve in water. Therefore, at highdilutions, FungizoneTM behaved like an aqueoussolution of AmB.39

For FLmix, the results indicate that the AmB–emulsion complex generated is quite different(Fig. 2). Firstly, except at very low AmB concen-tration (5� 10ÿ8 M), the variation of the absorp-tion spectrum was great, and its behavior wassimilar to FungizoneTM at the same AmB con-centration. The self-associated AmB band (at324 nm in this preparation) was maintained athigh dilutions (5� 10ÿ7 M), whereas the propor-tion of the monomeric form remained low (Fig. 5).This suggests that the AmB molecules werestrongly associated with the emulsion droplet.Indeed, it was observed that despite the highdilution of FLmix at AmB concentrations of5� 10ÿ7 M and 5� 10ÿ8 M, the emulsion systemremained stable and presented a milky appear-ance. Thus, we believe that, when FungizoneTM

was mixed with LipofundinTM, the AmB–micellecomplex existing in the former dissociates, per-mitted the detergent to go to the external aqueousphase and the AmB molecules to be incorporat-ed into the oil/water phospholipid monolayerinterface that composes the emulsion system.

Theoretically, AmB could be located either in theoily core of emulsion droplets or at the interface.However, because AmB is not soluble either insoybean oil or in medium-chain fatty acids,40 weassume that it is located in the phospholipidmonolayer, as already suggested by Washingtonet al.41

This hypothesis was partially confirmed by thevariation found in the FLmix spectra measuredat 378C (Fig. 3). In fact, the behavior of AmBmolecules located at the phospholipid monolayerwas severely perturbed by the temperature in-crease of 128C: the aggregate’s band was redshifted from 324 to 319.5 nm; the intensity ofthe spectrum aggregate’s band at 5� 10ÿ6 M and5� 10ÿ7 M was reduced with a consequent in-crease in the monomeric one; the presence ofmonomeric species was increased to 100% insteadof 80% that was found at 258C (Figs. 4 and 5); andthe b/d50% was strongly altered. All these eventscould have happened because of the increase onthermodynamic energy on the oil/water interfacecaused by the increase in temperature thatprovokes a complete change in the thermody-namic equilibrium of aggregate and monomericspecies, because of the release of the latter fromthe AmB–emulsion droplet complex.

The particle size analysis and the morpholo-gical evaluation once again indicated that aninteraction between AmB and emulsion dropletshad occurred. First, a variation in the distribu-tion profile of droplets charged with AmB wasfound (Fig. 6). Second, a small variation of theparticle size after 12 h was detected (Table 1).Finally, the TEM pictures revealed that notonly FungizoneTM micelles (Fig. 7), which pre-sent a granulometric size fivefold lower thanFungizoneTM/LipofundinTM complex, disappearedfrom the FLmix external phase (Fig. 10), but alsoFLmix droplets were more electron-dense (Fig. 8)than LipofundinTM droplets (Figs. 9 and 10). Thisindicates that FungizoneTM micelles were incor-porated into LipofundinTM.

The in vitro FLmix evaluation permitted usto go forward on the AmB phospholipid mono-layer localization hypothesis. In fact, these stu-dies showed that FLmix presented an importantselectivity against human and fungal cells andwas less toxic than FungizoneTM. When the targetcells were human RBCs, whereas RBC did notpresent hemoglobin leakage over the whole rangeof doses tested, the C. tropicalis viability startedto decrease at 5� 10ÿ7 M (85.32� 10.19% cfu)and became 11.89� 4.65% cfu at 5� 10ÿ5 M. No

10 EGITO ET AL.


Author Proof

Asignificant Kþ leakage, which has often been usedas an index of polyene toxicity,22,42–45 from RBCwas observed below 5� 10ÿ7 M (2.97� 2.11% at5� 10ÿ8 M), but for C. tropicalis, the lowest con-centration studied caused 60.59� 21.36% leak-age, reached at 87.77� 6.81% as early as the5� 10ÿ7 M level. For RBCs, this degree of toxicitywas observed only at 5� 10ÿ5 M. The results forFungizoneTM were in agreement with those pub-lished recently.46 The FLmix presented nearly thesame efficacy against C. tropicalis and a lowertoxicity against RBCs. However, the ensemble ofactivity results of FLmix point out that at aparticular AmB concentration (0.5 mg/L), theactivity toward C. tropicalis is slightly decreased,probably because of the variation of AmB speciesin equilibrium. In fact, some authors have foundthat the association of AmB with lipids reduced itsactivity against fungal cells.47–49 Nevertheless,the high protective effect on RBCs induced byFLmix allows the use of larger doses of Fungizo-neTM, thus improving its therapeutic index.

All the results together suggest that Lipofun-dinTM should be able to reduce the FungizoneTM

toxicity probably by changing the AmB self-association state by binding it with the emulsiondroplet. Indeed, FLmix may be considered as areservoir of the monomeric form of AmB thatreleases only limited amounts of free AmB in theaqueous media. As a consequence, the level of freeAmB might be below its CAC and the drug couldbe in its monomeric form. This form would be ableto bind to the ergosterol of fungal cells but wouldbe inactive against the cholesterol of mammaliancells.

Liposomal and other lipid-based forms of AmBhave been extensively studied, and some of themare already marketed.4 However, the FLmix formmay have some practical advantages in that itdoes not include expensive semi-synthetic lipidsand is even now in use on clinical trials. Never-theless, as shown by our results, the mixture oftwo approved pharmaceutical entities induceslarge physicochemical changes in both of them.In this specific case, FLmix should be consideredas a new delivery system and its use should bemade with caution.

ACKNOWLEDGMENTS

We thank Elayne C. M. T. Egito, for her drawingof the AmB structure, and Glenn Hawes, forediting the manuscript. The authors are indebted

to one of the referees for his constructive remarks.Dr. Egito is grateful for the financial support fromCAPES (Brazilian Department of Education),Brasılia/Brazil. This work was funded in part bythe FMC Foundation.

REFERENCES

1. de Pauw BE, Meunier F. 1999. The challenge ofinvasive fungal infection. Chemotherapy 45:1–14.

2. Hartsel S, Bolard J. 1996. Amphotericin B: New lifefor an old drug. Trends Pharmacol Sci 17:445–449.

3. Gallis HA. 1996. Amphotericin B: A commentary onits role as an antifungal agent and as a comparativeagent in clinical trials. Clin Infect Dis 22:S145–S147.

4. Groll AH, Muller FMC, Piscitelli SC, Walsh TJ.1998. Lipid formulations of amphotericin B: Clin-ical perspectives for the management of invasivefungal infections in children with cancer. KlinPadiatr 210:264–273.

5. Hiemenz JW, Walsh TJ. 1996. Lipid formulations ofamphotericin B: Recent progress and future direc-tions. Clin Infect Dis 22:S133–S144.

6. Joly V, Geoffray C, Reynes J, Goujard C, Mechali D,Maslo C, Raffi F, Yeni P. 1996. Amphotericin B ina lipid emulsion for the treatment of cryptococ-cal meningitis in AIDS patients. J AntimicrobChemother 38:117–126.

7. Wong-Beringer A, Jacobs RA, Guglielmo BJ. 1998.Lipid formulations of amphotericin B: Clinicalefficacy and toxicities. Clin Infect Dis 27:603–618.

8. Kirsh R, Goldstein R, Tarloff J, Parris D, Hook J,Hanna N, Bugelski P, Poste G. 1988. An emulsionformulation of amphotericin B improves the thera-peutic index when treating systemic murine candi-diasis. J Infect Dis 158:1065–1070.

9. Moreau P, Milpied N, Fayette N, Ramee JF,Harousseau JL. 1992. Reduced renal toxicity andimproved clinical tolerance of amphotericin Bmixed with intralipid compared with conventionalamphotericin B in neutropenic patients. J Anti-microb Chemother 30:535–541.

10. Caillot D, Casasnovas O, Solary E, Chavanet P,Bonnotte B, Reny G, Entezam F, Lopez J, BonninA, Guy H. 1993. Efficacy and tolerance of anamphotericin B lipid (Intralipid) emulsion in thetreatment of candidaemia in neutropenic patients.J Antimicrob Chemother 31:161–169.

11. Thakur CP. 1994. Comparison of glucose versus fatemulsion in the preparation of amphotericin B foruse in kala-azar. Trans R Soc Trop Med Hyg 88:698–699.

12. Joly V, Farinotti R, Saintjulien L, Cheron M,Carbon C, Yeni P. 1994. In-vitro renal toxicityand in-vivo therapeutic efficacy in experimental



Author Proof

Amurine cryptococcosis of amphotericin B (Fungi-zone) associated with intralipid. Antimicrob AgentsChemother 38:177–183.

13. Sorkine P, Nagar H, Weinbroum A, Setton A,Israitel E, Scarlatt A, Silbiger A, Rudick V, KlugerY, Halpern P. 1996. Administration of amphoter-icin B in lipid emulsion decreases nephrotoxicity:Results of a prospective, randomized, controlledstudy in critically ill patients. Crit Care Med 24:1311–1315.

14. Heinemann V, Kahny B, Jehn U, Muhlbayer D,Debus A, Wachholz K, Bosse D, Kolb HJ, WilmannsW. 1997. Serum pharmacology of amphotericin Bapplied in lipid emulsions. Antimicrob AgentsChemother 41:728–732.

15. Shadkhan Y, Segal E, Bor A, Gov Y, Rubin M,Lichtenberg D. 1997. The use of commerciallyavailable lipid emulsions for the preparation ofamphotericin B-lipid admixtures. J AntimicrobChemother 39:655–658.

16. Jensen GM, Skenes CR, Bunch TH, Weissman CA,Amirghahari N, Satorius A, Moynihan KL, EleyCGS. 1999. Determination of the relative toxicity ofamphotericin B formulations: A red blood cellpotassium release assay. Drug Deliv 6:81–88.

17. Chavanet PY, Garry I, Charlier N, Caillot D,Kisterman JP, Dathis M, Portier H. 1992. Trial ofglucose versus fat emulsion in preparation ofamphotericin for use in HIV infected patients withcandidiasis. Br Med J 305:921–925.

18. Caillot D, Reny G, Solary E, Casasnovas O,Chavanet P, Bonnotte B, Perello L, Dumas M,Entezam F, Guy H. 1994. A Controlled trial of thetolerance of amphotericin-B infused in dextrose orin intralipid in with hematological malignancies.J Antimicrob Chemother 33:603–613.

19. Schoffski P, Freund M, Wunder R, Petersen D,Kohne CH, Hecker H, Schubert U, Ganser A. 1998.Safety and toxicity of amphotericin B in glucose 5%or intralipid 20% in neutropenic patients withpneumonia or fever of unknown origin: randomisedstudy. Br Med J 317:379–384.

20. Nath CE, Shaw PJ, Gunning R, McLachlan AJ,Earl JW. 1999. Amphotericin B in children withmalignant disease: A comparison of the toxicitiesand pharmacokinetics of amphotericin B adminis-tered in dextrose versus lipid emulsion. AntimicrobAgents Chemother 43:1417–1423.

21. Gaboriau F, Cheron M, Leroy L, Bolard J. 1997.Physico-chemical properties of the heat-induced‘superaggregates’ of amphotericin B. Biophys Chem66:1–12.

22. Legrand P, Romero EA, Cohen BE, Bolard J. 1992.Effects of aggregation and solvent on the toxicity ofamphotericin B to human erythrocytes. AntimicrobAgents Chemother 36:2518–2522.

23. Egito EST, Fessi H, Appel M, Barratt G, Legrand P,Bolard J, Devissaguet JP. 1996. A morphological

study of an amphotericin B emulsion-based deliv-ery system. Int J Pharm 145:17–27.

24. Lambing HE, Wolf BD, Hartsel SC. 1993. Tem-perature effects on the aggregation state andactivity of amphotericin B. Biochim Biophys Acta1152:185–188.

25. Barwicz J, Christian S, Gruda I. 1992. Effects of theaggregation state of amphotericin B on its toxicityto mice. Antimicrob Agents Chemother 36:2310–2315.

26. Bolard J, Legrand P, Heitz F, Cybulska B. 1991.One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium. Biochemistry 30:5707–5715.

27. Espuelas MS, Legrand P, Cheron M, Barratt G,Puisieux F, Devissaguet JP, Irache JM. 1998.Interaction of amphotericin B with polymeric col-loids: A spectroscopic study. Colloids Surf B Bio-interfaces 11:141–151.

28. Mazerski J, Bolard J, Borowski E. 1982. Self-association of some polyene macrolide antibioticsin aqueous media. Biochim Biophys Acta 719:11–17.

29. Mazerski J, Grzybowska J, Borowski E. 1990.Influence of net charge on the aggregation andsolubility behavior of amphotericin B and its de-rivatives in aqueous media. Eur Biophys J 18:159–164.

30. Bolard J, Seigneuret M, Boudet G. 1980. Interac-tion between phospholipid-bilayer membranes andthe polyene antibiotic amphotericin B: Lipid stateand cholesterol content dependence. Biochim Bio-phys Acta 599:280–293.

31. Barwicz J, Gruszecki WI, Gruda I. 1993. Sponta-neous organization of amphotericin B in aqueous-medium. J Colloid Interface Sci 158:71–76.

32. Maher P, Singer SJ. 1984. Structural changesin membranes produced by the binding of smallamphipathic molecules. Biochemistry 23:232–240.

33. Hartsel SC, Benz SK, Ayenew W, Bolard J. 1994.Naþ, Kþ and Clÿ selectivity of the permeabilitypathways induced through sterol-containing mem-brane-vesicles by amphotericin B and other polyeneantibiotics. Eur Biophys J 23:125–132.

34. Milhaud J, Hartmann MA, Bolard J. 1989. Interac-tion of the polyene antibiotic amphotericin-B withmodel membranes: Differences between small andlarge unilamellar vesicles. Biochimie 71:49–56.

35. Oneill LJ, Miller JG, Petersen NO. 1986. Evidencefor nystatin micelles in L-cell membranes fromfluorescence photobleaching measurements of dif-fusion. Biochemistry 25:177–181.

36. Strauss G, Kral F. 1982. Borate complexes ofamphotericin B: Polymeric species and aggregatesin aqueous-solutions. Biopolymers 21:459–470.

37. Ernst C, Grange J, Rinnert H, Dupont G. 1981.Structure of amphotericin B aggregates as revealed

12 EGITO ET AL.


Author Proof

Aby UV and CD spectroscopies. Biopolymers 20:1575–1588.

38. Pramauro E, Pelezetti E. 1996. Surfactants in ana-lytical chemistry: Applications of organized amphi-philic media, Vol. 31. New York: Elsevier.

39. Lamyfreund MT, Schreier S, Peitzsch RM, Reed WF.1991. Characterization and time-dependence of am-photericin B: Deoxycholate aggregation by quasi-elastic light-scattering. J Pharm Sci 80:262–266.

40. Asher IM, Schwartzman G, USASRGQ3. 1972.Amphotericin B In: Florey K, editor. Analyticalprofiles of drug substances, Vol. 6. New York:Academic Press, pp 1–42.

41. Washington C, Taylor SJ, Davis SS. 1988. Thestructure of colloidal formulations of amphotericinB. Int J Pharm 46:25–30.

42. Butler WT, Cotlove E. 1971. Increased permeabilityof human erythrocytes induced by amphotericin B.J Infect Dis 123:341–350.

43. Davis SS, Washington C, West P, Illum L, Liver-sidge G, Sternson L, Kirsh R. 1987. Lipid emulsionsas drug delivery systems. Ann NY Acad Sci 507:75–88.

44. Forster D, Washington C, Davis SS. 1988. Toxicityof solubilized and colloidal amphotericin B formu-lations to human erythrocytes. J Pharm Pharmacol40:325–328.

45. Souza LC, Maranhao RC, Schreier S, Campa A.1993. In-vitro and in-vivo studies of the de-crease of amphotericin B toxicity upon associationwith a triglyceride-rich emulsion. J AntimicrobChemother 32:123–132.

46. Egito EST, Appel M, Fessi H, Barratt G, PuisieuxF, Devissaguet JP. 1996. In-vitro and in-vivoevaluation of a new amphotericin B emulsion-baseddelivery system. J Antimicrob Chemother 38:485–497.

47. Janoff AS, Perkins WR, Saletan SL, Swenson CE.1993. Amphotericin B lipid complex (ABLCTM): Amolecular rationale for the attenuation of ampho-tericin B related toxicities. J Liposome Res 3:451–471.

48. Jullien S, Contrepois A, Sligh JE, Domart Y, YeniP, Brajtburg J, Medoff G, Bolard J. 1989. Study ofthe effects of liposomal amphotericin B on Candidaalbicans, Cryptococcus neoformans, and erythro-cytes by using small unilamellar vesicles preparedfrom saturated phospholipids. Antimicrob AgentsChemother 33:345–349.

49. Clark JM, Whitney RR, Olsen SJ, George RJ,Swerdel MR, Kunselman L, Bonner DP. 1991.Amphotericin-B lipid complex therapy of experi-mental fungal infections in mice. AntimicrobAgents Chemother 35:615–621.

Q1: OK to delete symbols since an explanatory legendappears on the figure?

Q2: Please cite Figure 3 in order in text.

Q3: What is USASRG?



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NEW TO JOURNAL OF PHARMACEUTICAL SCIENCES

Online Submission and Peer ReviewIn order to facilitate online collaboration and expedite the publication process, theJournal of Pharmaceutical Sciences is pleased to now offer web-basedsubmission and peer review. The online submission site offers new functionalitynot offered before, including:

• Instant confirmation of manuscript receipt

• The ability to track a manuscript’s progress through the peer review process

• The ability to submit manuscripts 24/7 from anywhere in the world via webaccess

We invite authors to visit the online submission site athttp://jpharmsci-wiley.manuscriptcentral.com/.

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Softproofing for advanced Adobe Acrobat Users - NOTES toolNOTE: ACROBAT READER FROM THE INTERNET DOES NOT CONTAIN THE NOTES TOOL USED IN THIS PROCEDURE.

Acrobat annotation tools can be very useful for indicating changes to the PDF proof of your article.By using Acrobat annotation tools, a full digital pathway can be maintained for your page proofs.

The NOTES annotation tool can be used with either Adobe Acrobat 3.0x or Adobe Acrobat 4.0.Other annotation tools are also available in Acrobat 4.0, but this instruction sheet will concentrateon how to use the NOTES tool. Acrobat Reader, the free Internet download software from Adobe,DOES NOT contain the NOTES tool. In order to softproof using the NOTES tool you must havethe full software suite Adobe Acrobat Exchange 3.0x or Adobe Acrobat 4.0 installed on your com-puter.

Steps for Softproofing using Adobe Acrobat NOTES tool:

1. Open the PDF page proof of your article using either Adobe Acrobat Exchange 3.0x or AdobeAcrobat 4.0. Proof your article on-screen or print a copy for markup of changes.

2. Go to File/Preferences/Annotations (in Acrobat 4.0) or File/Preferences/Notes (in Acrobat 3.0)and enter your name into the “default user” or “author” field. Also, set the font size at 9 or 10point.

3. When you have decided on the corrections to your article, select the NOTES tool from theAcrobat toolbox and click in the margin next to the text to be changed.

4. Enter your corrections into the NOTES text box window. Be sure to clearly indicate where thecorrection is to be placed and what text it will effect. If necessary to avoid confusion, you canuse your TEXT SELECTION tool to copy the text to be corrected and paste it into the NOTEStext box window. At this point, you can type the corrections directly into the NOTES textbox window. DO NOT correct the text by typing directly on the PDF page.

5. Go through your entire article using the NOTES tool as described in Step 4.

6. When you have completed the corrections to your article, go to File/Export/Annotations (inAcrobat 4.0) or File/Export/Notes (in Acrobat 3.0). Save your NOTES file to a place on yourharddrive where you can easily locate it. Name your NOTES file with the article numberassigned to your article in the original softproofing e-mail message.

7. When closing your article PDF be sure NOT to save changes to original file.

8. To make changes to a NOTES file you have exported, simply re-open the original PDFproof file, go to File/Import/Notes and import the NOTES file you saved. Make changes and re-export NOTES file keeping the same file name.

9. When complete, attach your NOTES file to a reply e-mail message. Be sure to include yourname, the date, and the title of the journal your article will be printed in.

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