Plasma Kinetics and Biodistribution of a Lipid Emulsion ... · Plasma Kinetics and Biodistribution of a Lipid Emulsion Resembling Low-Density Lipoprotein in Patients with Acute Leukemia

(CANCER RESEARCH 54. 4660-4666, September 1. 1994]

Plasma Kinetics and Biodistribution of a Lipid Emulsion Resembling Low-Density

Lipoprotein in Patients with Acute Leukemia

Raul C. Maranhao,1 Bernardo Garicochea, Edson L. Silva, Pedro Dorlhiac-Llacer, Silvia M. S. Cadena,

Iris J. C. Coelho, JosÃ©Claudio Meneghetti, Fulvio J. C. Fileggi, and Dalton A. F. Chamone

The Heart Institute 1/nCor) anil Department of Hemalologv ami Hemotherapv IHentocentro) of the Sao Paulo University Medical School Hospital, and Faculty of PharmaceuticalSciences, University of SÃ¤oPaulo. SÃ¤oPaulo, Brazil

ABSTRACT

Low-density lipoprotein (LDL) could be used as a carrier of chemo-

therapeutic agents to neoplastic cells that overexpress LDL receptorsn-I.DI.i, but LDL is difficult to obtain and handle. Recently, it was

observed that a protein-free emulsion resembling the lipid portion of LDL

(LDE) behave like native LDL when injected into the bloodstream. In thisstudy, the evidence that LDE is taken up by rLDL was expanded bycomparing LDL and LDE plasma decay curves in rabbits and by competition experiments with lymphocytes. To verify whether LDE could beremoved from the plasma by neoplastic cells with increased rLDL, LDElabeled with l4Ccholesteryl ester was injected into 14 patients with acute

myeloid leukemia (AMD and into 7 with acute lymphocytic leukemia(ALL). In AML rLDL expression is increased but in ALL it is normal.LDE plasma fractional clearance rate (FCR, in h ' i was calculated from

the remaining radioactivity measured in plasma samples collected during24 h following injection. LDE ECR was 3-fold greater in AML than inALL patients 0.192 Â±0.210 (SD) and 0.066 Â±0.033 IT1, respectively,

P < 0.035. When LDE injection was repeated in 9 AML patients inhematological remission, LDE FCR diminished 66% compared to thepretreatment values (from 0.192 Â±0.210 to 0.065 Â±0.038 h~', P < 0.02), so

that it could be estimated that nearly 66% of the emulsion was taken upby AML cells and only 34% by the normal tissues. As expected, LDE FCRwas unchanged in 4 patients with ALL in hematological remission(0.069 Â±0.044 h~*). Gamma camera images obtained 6 h after the injectionof '"'"'Tr-UilH'k'dLDE into one patient with ALL showed biodistribution

similar to that of LDL. In one AML patient LDE was comparatively moreconcentrated over the areas corresponding to the bone marrow infiltratedby AML cells. Our results indicate that LDE FCR is increased in a diseaseknown to contain malignant cells that overexpress rLDL, suggesting thatLDE is taken up by malignant cells with increased rLDL.

INTRODUCTION

LDL2 carries most of the plasma cholesterol in humans. LDL is a

quasispherical particle composed of a core of cholesteryl esters surrounded by a monolayer of phospholipids and free cholesterol. Apo-lipoprotein B-100, the protein part of LDL, is the ligand for binding

of the lipoprotein to specific receptors (B, E receptors, rLDL) on thecell plasma membrane. After binding to the receptors, LDL is internalized and degraded in lysosomes (1) and its cholesterol is used inseveral cell processes, like membrane synthesis.

In the rapidly proliferating malignant cells, the increased need ofcholesterol for new membrane synthesis may result in overexpressionof the rLDL (2), which allows greater uptake of the lipoprotein by theneoplastic cells. Several investigators showed the uptake of LDL-drugcomplexes by malignant cells (3-7), leading to the possibility of using

Received 10/23/93; accepted 6/28/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely lo indicate this fact.

1To whom requests for reprints should be addressed at Instituto do Coracao HC-FMUSP. Av. Dr. Eneas de Carvalho Aguiar, 44, 1Â°Andar. Sao Paulo, SP, 05403, Brazil.

2 The abbreviations used are: LDL, low-density lipoprotein; HDL, high-density li

poprotein; VLDL, very-low-density lipoprotein; rLDL, LDL receptor; LDE, protein-freelipid emulsion resembling LDL; FCR, fractional clearance rate; ALL. acute lymphocyticleukemia; AML, acute myeloid leukemia.

LDL as drug carrier in cancer chemotherapy (3, 8-10). This would

increase the antineoplastic effects while reducing the toxic effectsupon normal cells. However, isolation from plasma and handling ofnative LDL are difficult; therefore its use in cancer treatment mayremain confined to experimental trials (11).

Recently, Maranhao el al. demonstrated that LDE, a protein-free

emulsion with composition resembling the lipid portion of LDL,behaves similarly to LDL when injected into rats (12) and humansubjects (13). In the present study, the evidence that LDE is taken upby rLDL was further strengthened in studies with rabbits and humanleukocytes. LDE labeled with 14Ccholesteryl oleate was then injected

i.v. in two groups of patients: group 1, with AML; and group 2, withALL. AML cells overexpress rLDL, whereas rLDL activity in ALL issimilar to normal cells (2, 14) and the plasma decay curves of the LDElabel were determined. The effects of chemotherapy on the LDEclearance were also examined. The images of the biodistribution ofLDE labeled with "Tc were obtained in one ALL and in one AML

patient. Taken together, our results indicate that LDE removal fromplasma is increased in a disease known to contain malignant cells thatoverexpress rLDL, suggesting that LDE is taken up by malignant cellswith increased rLDL.

MATERIALS AND METHODS

Study Subjects. Fourteen patients with AML and 7 with ALL were studiedat the Infirmary of the Department of Hematology and Hemotherapy of the SÃ¤oPaulo University Medical School Hospital. The experimental protocol wasapproved by the Ethics Committee of the Sao Paulo University Medical Schooland an informed consent was obtained from each participant. Individualcharacteristics and clinical data are shown in Table 1. Classification of theacute leukemias was according to the French-American-British criteria (15).

When they were first studied, the patients were newly diagnosed. All patientsexhibited bone marrow hypercellularity, and chemotherapy had not yet beeninitiated. In 9 patients with AML the study was repeated after they were treatedwith induction chemotherapy and achieved complete hematological remission(less than 5% of blasts in the bone marrow biopsy and normal blood cellcount). Treatment was performed with daunorubicin (50 mg/m2 during 3 days)and 1-ÃŸ-D-arabinofuranosylcytosine (100 mg/m2 during 7 days). Among the

remaining 5 patients, complete hematological remission was not obtained in 3patients, and 2 died during induction therapy. The study was also repeated afterremission in 4 of the patients with ALL submitted to treatment according to thefollowing protocol: vincristine, 1.4 mg/m2 i.V., at days 1, 8, 15, 22, 28, and 36of treatment; daunorubicin, 50 mg/m2 i.v., at days 1, 2, 3, and 15; predntsone,60 mg/m2 p.o. during the first 28 days of treatment; L-asparaginase, 6000units/m2 i.v., from the 17th to the 28th day of treatment; methotrexate, 12 mg

i.V., on the 8th day of treatment. Among the remaining three ALL patients,complete remission was not obtained in two and one of them died during theinduction chemotherapy. Both groups of AML and ALL patients had alreadystopped treatment for at least 10 days when the LDE clearance study wasrepeated. One ALL and one AML patient were given "VmTc LDE for acqui

sition of biodistribution images. The two patients were newly diagnosed andhad not been treated. In those the LDE clearance study was not performed.

Determination of Cholesterol and TriglycÃ©rides. The total plasma cholesterol and triglycÃ©rides(TG) of the subjects were determined after a 12-h fastwith the aid of enzymatic kits (CHOD-PAP, Merck, and Abbott, respectively).

HDL cholesterol was determined with the same method, after precipitation of

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LIPID EMULSION RESEMBLING LDL IN ACUTE LEUKEMIA PATIENTS

Table 1 Subject characteristics, hematological and lipid data, and plasma clearance of the injected ^Ccholesteryl oleate of IDE

SubjectsAML1234567891(111121314Mean

Â±SD/*'ALL16171819202122Mean

Â±SDAge

(yr)/sex28/F15/F36/M50/M19/F58/F21/M44/M38/M44/F16/M29/F49/M34/M12/M35/F28/F55/F41/F15/M20/MClass(FAB)"MlMlMlMlM2M2M2M2M3M3M3M4MSMSLILILIL2L2L2L2WBC*Xl<r312.06.866.017.827.211.77.742.6138.052.122.937.510.41.976.621.619.615.7108.024.316.2%LCBM956558958068839893736078559090827160926755Cholesterol

(mg/dl)Total8810312172106118961241831601261098581112

+300.0501125142118190129135140140

Â±24LDL41527432537042671121017562504863

Â±230.032978916511269849685

Â±16HDL192419102918232534282120131721

Â±60.42692225203328211724

Â±5TG

(mg/dl)139136140150120150155159236155150135112129148

Â±290.5846124131165224160150136156

Â±34FCR'(h'1)0.8120.4380.1350.1510.0650.1350.3380.1340.0460.0490.0630.1170.1360.0860.193

Â±0.2100.3090.0270.0890.0910.0970.0410.0890.0260.066

Â±0.033" FAB, French-American-British classification; % LC BM, % of leukemic cells found in bone marrow; TG, triglycÃ©rides.* WBC in the peripheral blood.' Calculated from the disappearance curves of the 14Ccholesteryl oleate moiety of LDE, as described in "Materials and Methods."d Comparison with ALL pretreatment values.

LDL and VLDL with MgCl2 and phosphotungstic acid. VLDL concentration glycerols. LDE was used in the experiments described below within 1 weekwas calculated assuming the relation

VLDL = TG/5

(16). LDL was calculated using the Friedewald equation where LDL = totalcholesterol - (VLDL + HDL) (17).

Preparation of LDE. Egg phosphatidylcholinewas purchasedfrom LipidProducts (Surrey, United Kingdom); triolein, cholesteryl oleate, and cholesterol were from Nu-Chek Prep (Elysian, NJ), and radioactive lipids wereacquired from Amersham (United Kingdom). Lipids were >98% pure asdetermined by thin layer chromatography. The LDE used in this study wasprepared from lipid mixtures composed of 40 mg of phosphatidylcholine, 20mg of cholesteryl oleate, l mg of triolein, and 0.5 mg of unesterified cholesterol. Emulsification of lipids and purification of the emulsions were doneaccording to the procedure described by Ginsburg et al. (18). Wheneverpossible, all the materials and instruments used in the preparation of LDE wereput into oven at 180Â°Cfor 4 h and autoclaved. Lipids were dissolved inchloroform:methanol (2:1) and dispensed into vials. 14Ccholesteryloleylester

was added to the vials. The mixtures were dried under a nitrogen streamfollowed by overnight vacuum desiccation at 4Â°Cto remove residual solvents.The dried lipids were resuspended in 10 ml of 0.1 MKC1-0.01MTris-HCl, pH8.0. The suspension was sonicated using a Branson model 450 cell disrupter,with a 125-W output in the "continuous" operating mode, for 180 min underN2 atmosphere. Temperature was kept above 52Â°C,the melting point of

cholesteryl oleate, as monitored by a thermocouple inserted in the vials duringthis procedure. The emulsified lipid suspension was then transferred to cleantubes for ultracentrifugation at 195,000 X g (30 min) in a TH 641 rotor of aSorvall OTD-Combi ultracentrifuge at 4Â°C.The top 10% of the solution,

containing particles that float at background density of approximately 1.006g/ml, was removed by aspiration with a needle. The remaining solution wasadjusted to a background density of 1.22 g/ml by adding solid KBr. A secondultracentrifugation step was then performed at 195,000 X g (120 min) at 4Â°C.The top 20-30% of the sample was collected by aspiration, after attaining theroom temperature and dialyzed overnight in Tris-HCl buffer to remove KBrcontained in the solution. This LDE fraction was sterilized through passage in0.2-fim filter and analyzed for lipid composition by standard laboratory methods (16, 19, 20). LDE prepared as described had approximately 64% phos-pholipids, 33% cholesteryl ester, 1% unesterified cholesterol, and 2% triacyl-

after it was prepared.Preparation of HDL and LDL. HDL and LDL were obtainedfrom the

blood of healthy normolipidemic donors by sequential ultracentrifugation (21).In Vitro Acquisition of Apolipoproteins by LDE. Two ml of '"C-LDE,

prepared as described above, were incubated with 30 mg of HDL, during 2 hat 37Â°C.After incubation, the density was adjusted to 1.063g/ml with KBr andthe mixture was submitted to ultracentrifugation at 195,000 X g at 4Â°Cfor

24 h. The emulsion containing apolipoproteins was recovered at the top of thetube and dialyzed against 3 changes of 2 liters of 0.01 M Tris-HCl buffer,containing 0.15 MNaCl and 1 mMEDTA. The resulting artificial lipoprotein(14C-LDE-apo)was submitted to 10% sodium dodecyl sulfate-polyacrylamide

gel electrophoresis (22) to analyze the apolipoprotein content and used in thecell binding studies.

Competition between 14C-LDE-apoand LDL. Mononuclearleukocyteswere isolated from the blood of healthy normolipidemic subjects by centrifu-gation on a Ficoll-Hypaque gradient. The cells were incubated during 24 h inRPMI 1640 containing 100 units/ml of penicillin, 5 fig/ml of gentamicin, andsupplemented with 10%of lipoprotein-deficientserum. This procedure inducesthe maximal expression of rLDL (2). After this period, the cells were harvestedand the viability was evaluated by trypan blue exclusion. Viable cells (IO6)were incubated with 10 /xg of 14C-LDE-apoand increasing concentrations ofLDL for 4 h at 37Â°Cin 1 ml of RPMI. After incubation, the cells were washedthree times with RPMI at 4Â°Cand dissolved in 0.2 MNaOH for 2 h at 37Â°C.

An aliquot was removed for protein determination (23) and the incorporatedradioactivity was determined in the liquid scintillation counter.

Preparation of Tc-LDE and "Tc-LDL. The procedureof labelingLDE with "Te for imaging of its biodistribution in rabbits and patients hadbeen developed and tested previously in rabbits.3The technique was based on

the procedure for obtaining labeled native LDL described by Lees et al. (24).LDE was labeled with Wnltcusing sodium dithionite as a reducing agent. The"Tc-LDE complex was purified by gel filtration and sterilized by passingthrough a 0.2-/im Millipore filter. WmTc-LDE(370 MBq) was injected i.v. into

subjects within 30 min of radiolabeling. As tested by thin-layer chromatography, 90% of the radiation were found at the LDE phospholipids. LDL was also

3 R. C. Maranhao et al., unpublished results.

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labeled with WmTc according to the technique of Lees el al. (24) for injection

in rabbits.Clearance Studies in Rabbits. Two groups of male New Zealand rabbits

weighing 2-3 kg were given injections 111 MBq Tc-LDE or ""Tc-LDL

via one ear vein and blood samples were collected from the other ear vein atpreestablished intervals during 24 h. Plasma was separated by a 15-min

centrifugation (3.000 X g) and radioactivity was determined in a gamma

counter.Clearance Studies in Patients. The participants were fasting at the com

mencement of the clearance studies, at approximately 9 a.m., but they wereallowed two standard meals during the study, at approximately 12:30 p.m. andat approximately 7 p.m. They received injections of 37 kBq l4C-cholesteryl

ester, roughly 5-6 mg of emulsion total lipids, at a volume of 500 fi\,

administered i.v. in a bolus. Plasma samples were collected during 24 h, inintervals of 5 min and 1, 2, 4, 6, 8, 12, and 24 h. Aliquots (1.5 ml) of bloodplasma were extracted with chlorofornrmethanol (2:1, v/v) (25), and thesolvent phase was transferred into counting vials and dried under a nitrogenstream. Radioactivity was measured in a scintillation solution (PPO:dimethyl-POPOP:Triton X-100:toluene, 5g:0.5g:333ml:667 ml) using a Beckman LS-

100C spectrometer.Imaging Studies in Rabbits. The planar images were acquired in anterior

view 24 h postinjection of 111 MBq WmTc-LDE or ""Tc-LDL. Gamma

camera (Orbiter model; Siemens, Des Plaines, IL) images were obtained witha high resolution collimator and stored in 128 x 128 matrices in a dedicated

computer system for subsequent analysis.Imaging Studies in Subjects. The planar images were obtained in anterior

and posterior views 6 h postinjection of 370 MBq WmTc-LDE. Digitized

images of a view gamma camera (Siemens-Basicam, Des Plaines, IL) equippedwith a general-purpose parallel-hole collimator were stored in 128 X 128

matrices in a dedicated computer system and analyzed later.Estimation of FCR. FCR of the 14C-cholesteryl oleate of the LDE was

calculated according to the method described by Matthews (26) as

FCR=hr +-r\b, b2

where al, al, b\, and b2 were estimated from biexponential curves obtainedfrom the remaining radioactivity found in plasma after injection, fitted by leastsquare procedure, as

y Â»(a,â€¢Â«-*")+(Â«,â€¢*-â€¢")

where v represents the radioactivity plasma decay.Statistics. Differences in LDE FCR and triglycÃ©ridesbetween AML and

ALL groups and before and after chemotherapy were assessed using thenonparamctric Mann-Whitney test (27). Student's i test was used to test for

differences in values of total cholesterol and fractions, between the two groupsand before and after hematological remission (27). Two-tailed P values below0.05 were considered significant. Pearson's product moment correlation coef

ficient was used to assess the strength of association between LDE FCR andLDL-cholesterol and total cholesterol parameters.

RESULTS

LDE Binding to Apolipoproteins. Fig. 1 shows the sodium do-decyl sulfate-polyacrylamide gel electrophoresis gels of the apoli-

poproteins that bound to LDE recovered after the incubation withHDL. Apo E was the predominant apolipoprotein that bound to LDE,albeit residues of other apolipoproteins can also be seen.

Competition between LDE and LDL for Binding to Lymphocytes. Fig. 2 shows that the uptake of 14Ccholesteryl oleate-labeled

LDE by lymphocytes was progressively diminished as increasingLDL mass was added to the incubates. This indicates thatLDE compete with LDL for the same receptor-mediated uptake

mechanism.Comparison of LDE with LDL Plasma Kinetics in Rabbits. Fig.

3 shows that 99mTc-LDE and WmTc-LDL injected into the two groups

of rabbits had similar plasma-decaying curves.

KDa116

* 66

45

â€”29

Fig 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of apolipoproteins associated with LDE (Â£)after incubation with human HDL (left lane) and themolecular weight standards ÃŸ-galactosidase, phosphorylase h, bovine serum albumin,ovalbumin. and carbonic anhydrase (righi lane). kDu. molecular weight in thousands.

100-

a

UOJIU

75-

50-

25-

0 100 200 300 400LDL (ug.mr1)

Fig 2. Effect of unlabeled plasma LDL on the uptake by lymphocytes of LDEassociated with apolipoproteins and labeled with MCcholesteryl ester (l4C-LDE-apo).Cells were incubated with 10 /Â¿gof l4C-LDE-apo and increasing concentrations of LDL,during 4 h at 37Â°C,in 1 ml of RPMI. The experiment was performed in triplicate.

Comparison of LDE with LDL Biodistribution Images Acquired in Rabbits. The biodistribution images acquired from the twogroups of four rabbits that received injections of WmTc-LDE orWmTc-LDL did not differ importantly. The images of two of those

rabbits given injections of the two different preparations (Fig. 4) showthat the uptake of radioactivity by the different organs is alike.

Plasma Lipids and LDE Kinetics before Chemotherapy. Table1 shows the data for AML and ALL patients studied before chemotherapy. Plasma LDL-cholesterol was lower in the AML patientscompared to the ALL patients (P < 0.04), but HDL-cholesterol and

4662




triglycÃ©rideplasma concentration did not differ between the twogroups. The cholesteryl ester FCR was approximately 3-fold greater in

the AML patients than in the ALL patients 0.193 Â±0.210 (SD) and0.666 Â±0.033 h~', respectively; P < 0.04 (Table 1; Fig. 5). It is

noteworthy that the individual FCR values varied widely in AML, butnot in ALL. A negative correlation was found between the cholesterylester FCR and the plasma concentration of total (r = 0.33, P = 0.04)and LDL cholesterol (f = 0.44, P = 0.02) in the AML but not in ALL

group. In both, the cholesteryl esters FCR did not correlate with theplasma levels of HDL cholesterol and triglycÃ©ridesor the bloodleukocyte count.

Plasma Lipids and LDE Kinetics in Hematological Remission.In hematological remission (Table 2), the plasma concentration oftotal and LDL cholesterol of AML patients rose 38 and 54%, respec-

100-

â€¢r.75-ufÃÃâ€¢250H--oa

8 12 16Time (h)

20 24

Fig 5. Comparison of the curves of removal from plasma of LDE labeled withl4CcholesteryI ester in 14 AML ( ) and 7 ALL ( ) patients determined prior to

chemotherapy. Points represent mean Â±SEM (bars).

100-1

ofO

â€¢9 50HT3n)OC. 25-

O 4 8 12 16 20 24Time (h)

Fig 3. Comparison of the plasma decaying curves of ""Te LDE ( ) and Wl"Tc

LDL ( ) injected into rabbits. Each point represents mean Â±SEM (bars) of 4experiments.

Fig 4. Comparison of the biodistribution of """Te LDL (a) and Tc LDE (b) in the

rabbit. Images were acquired 24 h after injection of the labeled preparations. /, liver; 2,adrenals: 3, kidneys; 4, intestine.

lively, compared to the pretreatment levels (P < 0.0025 for bothparameters). The correlation between total and LDL cholesterolplasma concentration and FCR of the emulsion cholesteryl ester wasno longer observed. Remarkably, the LDE removal from plasmadecreased approximately 66% compared with the pretreatment results(from 0.193 Â±0.0210 to 0.065 Â±0.038 h'1, P < 0.02; Fig. 6). In

contrast, the LDE FCR did not change significantly in the ALLpatients studied after remission, as well as their plasma levels of totaland LDL cholesterol. HDL cholesterol levels remained unchanged inboth groups after remission (Table 2).

Biodistribution of LDE. Fig. 7 shows the gamma camera imagesof the biodistribution of ''''"'Tc-LDE in one ALL and one AML

nontreated patient, obtained 6 h postinjection. In the ALL subject, theliver was the predominant uptake site. LDE uptake by the liver wasmarkedly lower in the AML patient (Fig. le) compared with the ALLpatient (Fig. la). On the other hand, 1)9nTc activity was pronouncedly

higher in the AML patient over the areas corresponding to the pelvicbones (Fig. If), femur (Fig. 7#), and skull (Fig. Ih), the sites presumably containing bone marrow invaded by the malignant cells.

DISCUSSION

The major goal in cancer chemotherapy is to selectively kill thetumor cells without damaging normal tissues and organs. Therefore,the achievement of vehicles capable of delivering drugs to targetedcells has long been pursued. In the last decade, several authorssuggested the possibility of using LDL to shuttle antitumor drugs toneoplastic cells, based on the increased expression of rLDL in certaintypes of cancer. Although it was successful in experimental trials, thedifficulties in isolating LDL from plasma and handling the preparationare serious drawbacks for its use in routine treatment.

Protein-free emulsions can mimic the metabolism of plasma li-poproteins in vivo (28-32). MaranhÃ¤oet al. (12) demonstrated in rats

that LDE has a plasma kinetic behavior resembling that of native LDLand that the emulsion is probably taken up by the B,E receptors thatsequestrate LDL into the cell. The structure and physical properties ofprotein-free microemulsions identical to LDE had been well defined

previously (18, 33). LDE is devoid of apo B, but it acquires apo Efrom the circulating lipoproteins after injection into the blood. Because rLDL can also recognize apo E, LDE is thus taken up by thetissues via the LDL receptor-mediated endocytic pathway (12). In

patients with familial hypercholesterolemia LDE plasma clearancewas pronouncedly reduced compared to normal subjects (13). This isalso expected for native LDL, because B,E receptors are defective inthis disease (1).

The assumption that LDE is removed from the plasma by rLDL wasstrengthened in this study by the finding in rabbits that the 9l)mTc-LDE

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Table 2 Leukocyte count, lipids, and plasma clearance of LDE in patients after remission

SubjectsAML1235610111214Mean

Â±SDP*ALL17IX2122Mean

Â±SDPdWBC"

x10"'8.85.47.74.97.36.29.412.35.811.07.14.69.1Total138165142139198136192132155155

Â±240.0020140172141135147

Â±170.6111Cholesterol

(mg/dl)LDL85IOS8688127H124689997

Â±190.001294111927994

Â±130.3728HDL23232422302124262424

Â±30.24642028202523

Â±40.8848TG"(mg/dl14917016(114620512822219(1161170

Â±300.0456130166146155149

Â±15>0.9999FCRCOr')0.0440.0940.0340.0250.0760.1500.0510.0670.0440.065

Â±0.0380.01580.0470.0860.1220.0210.069

Â±0.0440.9273

a WBC in the peripheral blood.

TG, triglycÃ©rides.Calculated from the disappearance curves of the 14Ccholesteryl oleate moiety of LDE as described in "Materials and Methods."

1 Comparison with pretreatment values.

oÃ¼_

.25

.20

.15

.10

.05

-I

AML ALLFig 6. Comparison of the fractional clearance rate (FCR) of LDE radioactive label

before treatment (D) and after achievement of hematological remission (M) in subjectswith AML and with ALL. *P < 0.04, compared with AML pretreatment values. Columns,

mean; bars, SEM.

plasma decay curve was similar to that of 99mTc-LDL. Moreover, the

biodistribution images in rabbits of both preparations showed a closeresemblance, which also suggests that they are removed by the tissuesby the same mechanism. Finally, the competition experiment indicatesthat LDE cellular uptake was in fact by rLDL, since the binding ofLDE to human lymphocytes was progressively dislocated by theaddition to the incubates of increasing LDL mass.

Since LDE was shown to behave like native LDL, we hypothesizedwhether it could also be selectively taken up by malignant cells withup-regulation of the rLDL. Our preliminary results showed that in

AML patients LDE removal from plasma was faster than in healthynormolipidemic subjects (34). The comparison of LDE FCR obtainedin AML with that in ALL patients confirmed our hypothesis. FCR was3 times greater in AML patients than in ALL, indicating that whenAML cells with overexpression of rLDL are present they sequestratemost of the emulsion from the plasma.

Other important observation in our study was the negative correlation between LDE plasma clearance and the concentration of plasmaLDL-cholesterol found in AML patients. It is conceivable that the

greater the expression of rLDL, the greater is the LDL clearance andthe lower are the plasma levels of LDL, as shown in previous studies(34). Thus, the observed correlation suggests that LDE was removedfrom the circulation by the neoplastic cells with enhanced receptors

that also remove the native lipoprotein (35). The lack of correlation inALL and in AML after hematological remission further confirms thisassumption.

We also demonstrated that when AML cells were removed from theblood and bone marrow by chemotherapy the LDE plasma clearancewas pronouncedly reduced, approaching the values of the ALL patients. This confirms that the high clearance rates observed in AMLpatients before treatment were due to the presence of the neoplasticcells which avidly take up the emulsion particles. Since LDE FCR wasreduced by 66% after the hematological remission, it can be estimatedthat approximately 66% of the emulsion injected into the nontreatedpatients was taken up by the AML cells, whereas only the remaining34% was removed by the normal tissues. The lack of effect ofchemotherapy on LDE clearance in the ALL patients agrees withthese statements.

It is worthwhile to point out that there was a great interindividualvariation in the LDE FCR measured in AML but not in ALL patients.This can be explained by the wide variation of rLDL expression inAML cells, which could range from a 3- to 100-fold increase (2, 14).

It is remarkable that this variation became considerably smaller afterremission, indicating that AML cells had been responsible for suchvariation in FCR. It is also noteworthy that the same trend occurred inAML LDL-cholesterol concentration values, which were much more

dispersed before than after the treatment. Among the nine AMLpatients who were studied twice, in only one (case 10) LDE FCR didnot decrease posttreatment as expected, but otherwise it pronouncedlyincreased. Although this paradoxical behavior cannot be explained, itwas again similar to LDL behavior, since that case was also the onlyone in whom LDL-cholesterol plasma concentration decreased instead

of increasing in posttreatment.LDE removal from the plasma of ALL patients equaled that of

normolipidemic healthy subjects: LDE-FCR was 0.078 Â±0.033 h"1, asobserved in 19 healthy volunteers.1 Moreover, the 99mTc-LDE images

acquired in the ALL patient resembled those obtained from twohealthy subjects.3 Again, LDE behaved like LDL, since the images

obtained from the ALL patient and the two controls are similar tothose obtained with 99mTc-LDL (36), the liver being the main uptakeorgan. In the AML patient the lower hepatic uptake of 99mTc-LDE

associated with enhanced uptake over bone marrow areas was expected considering the overall results described in this study. Shift of

4664



I.IPID EMULSION RESEMBLING LDL IN ACUTE LEUKEMIA PATIENTS

Fig 7. Biodistribution of LDE in one ALL (left) and one AML patient (right). Both had been recently diagnosed, and chemotherapy had not yet been started. In both bone marrowwas hypercellular. The images were acquired 6 h after the injection of WmTc LDE: liver (a, e)'t pelvic bones (b, f); femur (c, g); skull (d, h).

4665




the label from the liver to the bone marrow infiltrated by AML cellswas also shown when WmTc-LDL was injected (37).

The clearcut results obtained in the present study fully confirmedour hypothesis that LDE can be a preparation perfectly suited for useas drug vehicle in routine cancer therapy. It is easy to prepare frommaterials produced by the chemical industry; hydrophobic drugs canbe easily incorporated into it by cosonication with the lipid mixturesor incubation with the formed emulsion, and when stored at 4Â°Cit can

be preserved for a long period.Moreover, the possibility of using LDE as a chemotherapeutic

vehicle may not be limited to AML, since up-regulation of rLDL,

the mechanism that allows targeting of cancer cells by both nativeLDL and LDE, was also observed in several other malignantneoplasias: glioma cells (38); endometrial adenocarcinoma (8);uterine cervical carcinoma (8); gallbladder cancer cells (39); breastcancer (40); myeloproliferative diseases (41); and metastaticprostate carcinoma (42).

ACKNOWLEDGMENTS

The authors arc grateful to Dr. Odaly Tofoletto and Carmen G. Vinagre forhelp with the experiments; Maria C. M. Latrilha, Cristiane J. Furlanetto, andJacques Malesys for expert technical assistance, and Julia T. Fukushima andDr. Rita H. A. Cardoso for the statistical analysis.

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1994;54:4660-4666. Cancer Res Raul C. Maranhão, Bernardo Garicochea, Edson L. Silva, et al. LeukemiaResembling Low-Density Lipoprotein in Patients with Acute Plasma Kinetics and Biodistribution of a Lipid Emulsion

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Plasma Kinetics and Biodistribution of a Lipid Emulsion ... · Plasma Kinetics and Biodistribution of a Lipid Emulsion Resembling Low-Density Lipoprotein in Patients with Acute Leukemia