Locoregional Cancer Treatment with Magnetic Drug Targeting · Locoregional Cancer Treatment with Magnetic Drug Targeting1 Christoph Alexiou,2 Wolfgang Arnold, Roswitha J ... release

[CANCER RESEARCH 60, 6641–6648, December 1, 2000]

Locoregional Cancer Treatment with Magnetic Drug Targeting1

Christoph Alexiou,2 Wolfgang Arnold, Roswitha J. Klein, Fritz G. Parak, Peter Hulin, Christian Bergemann,Wolfgang Erhardt, Stefan Wagenpfeil, and Andreas S. LubbeDepartment of Otorhinolaryngology, Head and Neck Surgery [C. A., W. A., P. H.], Department of Experimental Oncology and Therapeutic Research [R. J. K., W. E.], andInstitute for Medical Statistics and Epidemiology [S. W.], Klinikum rechts der Isar, Technical University of Munich, 81675 Munich; Physics-Department E 17, TechnicalUniversity of Munich, 81675 Munich [F. G. P.]; Chemicell, 10777 Berlin [C. B.]; and Cecilien-Klinik, 33175 Bad Lippspringe [A. S. L.], Germany

ABSTRACT

The specific delivery of chemotherapeutic agents to their desired tar-gets with a minimum of systemic side effects is an important, ongoingchallenge of chemotherapy. One approach, developed in the past to ad-dress this problem, is the i.v. injection of magnetic particles [ferrofluids(FFs)] bound to anticancer agents that are then concentrated in thedesired area (e.g.,the tumor) by an external magnetic field. In the presentstudy, we treated squamous cell carcinoma in rabbits with FFs bound tomitoxantrone (FF-MTX) that was concentrated with a magnetic field.Experimental VX-2 squamous cell carcinoma was implanted in the me-dian portion of the hind limb of New Zealand White rabbits (n 5 26).When the tumor had reached a volume of;3500 mm3, FF-MTX wasinjected intraarterially (i.a.; femoral artery) or i.v. (ear vein), whereas anexternal magnetic field was focused on the tumor. FF-MTX i.a. applica-tion with the external magnetic field resulted in a significant (P< 0.05),complete, and permanent remission of the squamous cell carcinoma com-pared with the control group (no treatment) and the i.v. FF-MTX group,with no signs of toxicity. The intratumoral accumulation of FFs wasvisualized both histologically and by magnetic resonance imaging. Thus,our data show that i.a. application of FF-MTX is successful in treatingexperimental squamous cell carcinoma. This “magnetic drug targeting”offers a unique opportunity to treat malignant tumors locoregionallywithout systemic toxicity. Furthermore, it may be possible to use thesemagnetic particles as a “carrier system” for a variety of anticancer agents,e.g.,radionuclides, cancer-specific antibodies, and genes.

INTRODUCTION

The difference between the success or failure of chemotherapy de-pends not only on the drug itself but also on how it is delivered to itstarget. Because of the relatively nonspecific action of chemotherapeuticagents, there is almost always some toxicity to normal tissue even underoptimal conditions. Therefore, it is of great importance to be able toselectively target the antineoplastic agent to its tumor target as preciselyas possible, to reduce the resulting systemic toxic side effects fromgeneralized systemic distribution and to be able to use a much smallerdose, which would further lead to a reduction of toxicity. In the past,chemotherapy targeted by magnetic fields using magnetic albumin mi-crospheres has shown encouraging results (1, 2). In 1996, Lubbeet al.(3)used a new FF,3 described in detail below, for experiments in whichtumor-bearing experimental animals (nude mice and rats) were injectedi.v. with a FF complex (magnetic drug) that was directed into the tumorusing a magnetic field (permanent magnet; magnetic field strength, 0.5–0.8 Tesla). The FF complex was well tolerated by the animals, and tumorremission was achieved. As a second step, Lubbeet al.(4) also conducted

the first Phase I clinical trial using this approach in patients with ad-vanced, unsuccessfully treated cancers or sarcomas. This “magnetic drugtargeting” approach was well tolerated.

Targeting and prolonged retention of the FF complex at the targetsite reduces its reticuloendothelial system (RES) clearance and facil-itates extravascular uptake. To optimize intratumoral magnetic parti-cle concentration, several features need to be considered: (a) theparticles should be of a size that allows sufficient attraction bythe magnetic field and their introduction into the tumor or into thevascular system surrounding the tumor; (b) the magnetic fields shouldbe of sufficient strength to be able to attract the magnetic nanopar-ticles into the desired area; (c) the FF complex should deliver andrelease a sufficient amount of anticancer agent; and (d) the method ofinjection should have good access to the tumor vasculature and shouldavoid clearance by the reticuloendothelial system (“first pass effect”).

The purpose of the present study was to compare different appli-cation methods (i.v., i.a.) of magnetic drug targeting for the treatmentof experimental VX-2 squamous cell carcinoma. Because FFs arevisible histologically and by imaging techniques such as MRI, we alsowished to demonstrate the morphological intratumoral distribution ofthese magnetic nanoparticles in conjunction with an external magneticfield focused on the tumor region.

MATERIALS AND METHODS

MTX. The chemotherapeutic agent used in the experiments, MTX-HCl,(Novantron; Lederle, Wolfratshausen, Germany) is a synthetic anthracendionthat inhibits DNA and RNA synthesis by intercalating in DNA molecules,which causes strand breaks. Actively dividing cells are the most sensitive, butMTX tends to be non-cell-cycle specific and also inhibits G2-M progression(5). MTX has been used systemically for breast carcinoma, non-Hodgkin’slymphoma, and solid tumors (6–8) and has also been applied locoregionally(9–13). The body surface area and the dose of MTX (10 mg/m2 of bodysurface area) used for the experiments were calculated according the instruc-tions of Kirk and Bistner‘s handbook of veterinarian procedures and emer-gency treatment (14).

Magnetic Nanoparticles (FFs).The FFs used in the experiments wereobtained from Chemicell (Berlin, Germany; German patent application no.19624426.9) and consisted of a colloidal dispersion formed by wet chemicalmethods from iron oxides and hydroxides to produce special multidomainparticles (Table 1). The particles were surrounded by starch polymers forstabilization under various physiological conditions and to allow chemoab-sorptive binding. MTX has cationic characteristics and combines (aminegroups of MTX-HCl with phosphate groups of the starch derivates) at a pH of7.4 (Fig. 1). The FF-MTX contained 6.5 mg of MTX per 10 ml. Because thedrug bond is reversible (ionic binding), desorption of the bound drug wasdependent on the physiological environment (pH, osmolality, temperature) andcould be varied by changing the blood electrolyte concentration according tothe specific need. In experiments, desorption of MTX took place within 60 min(Fig. 2), which ensured that the drug could act freely once localized to thetumor by the magnetic field. Pyrogenicity and sterility tests were performed bythe Pharmacy Department of the Virchow Medical School (Humboldt-Univer-sitat, Berlin, Germany) according to good manufacturing practice guidelines.The characteristics of the FF-MTX are depicted in Table 1 (see also Figs. 1 and2).

VX-2 Squamous Cell Carcinoma.The VX-2 squamous cell carcinomawas obtained from the Deutsches Krebsforschungszentrum (Heidelberg, Ger-

Received 4/21/00; accepted 10/3/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Margarete Ammon Foundation, Munich, and grants from theTechnical University of Munich, Germany.

2 To whom requests for reprints should be addressed, at Department of Otorhinolar-yngology, Head and Neck Surgery, Klinikum rechts der Isar, Technical University ofMunich, Ismaningerstrasse 22, 81675 Munich, Germany. Phone: 49-89-4140-2370; Fax:49-89-4140-4853; E-mail: [email protected].

3 The abbreviations used are: FF, ferrofluid; i.a., intraarterial/intraarterially; MR,magnetic resonance; MRI, MR imaging/image; MTX, mitoxantrone; MTX-FF, FF boundto MTX; MTX-HCl, MTX hydrochloride; MTC, magnetic-targeted carrier.

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many) and originates as a papillomatous reaction to Shope virus infection inwild rabbits (15). The tumor was preserved through many generations ofserially transplanted animals and was established as a tumor cell line in ourlaboratories. Its histology and growth characteristics have been extensivelydescribed (16, 17). Briefly, after implantation into soft tissue, the tumorenlarges rapidly with increased vascularity in its periphery. The animals soon(within 2–3 weeks) develop central tumor necrosis, locoregional lymph nodemetastases, and hematogenous metastases (e.g.,into the lungs).

Animals. The experimental animals were female New Zealand White rab-bits (2000–2500 g body weight, 12–15 weeks old; Charles River, Sulzfeld,Germany) that were housed individually in a room with an artificial 12/12 hlight/dark cycle (exposed to light from 0700 to 1900 h). The rabbits were fedhard rabbit chow pellets (Altromin, Lage, Germany), carrots, dry bread, andtap water.

Surgical Intervention. Fragments of viable VX-2 tissue, 1 mm in size,were taken from the tumor periphery in donor animals. These fragments wereplaced in a special medium [RPMI 1640, 2.0 g/liter NaHCO3, andL-glutamin(Seromed); Biochrom, Berlin, Germany] and were immediately implantedunder sterile conditions into the hind limb of anesthetized recipient rabbits(n 5 26) in the supply area of the femoral artery. The experiments were

performed when the tumors had reached a volume of approximately 3500mm3.

For application of the chemotherapy, the animals were anesthetized with ani.m. injection of ketamine [35 mg/kg body weight (Narketan 10; Chassot, Bern,Switzerland)] and xylazine [5 mg/kg body weight (Xylapan; Chassot, Bern,Switzerland)], the femoral artery was cannulized and an indwelling catheter[Venflon (0.8 mm); Ohmeda Co., Helsingburg, Sweden] was placed afterseparation of the femoral vein and the saphenous nerve;2 cm distal to theinguinal furrow. The FF-MTX and the MTX alone were administered byperfusor over a period of 10 min. To prevent thrombosis, prophylaxis consist-ing of heparin sodium (heparin/natrium/25,000 IU Ratiopharm; Ratiopharm,Ulm, Germany) was given preoperatively, once immediately postoperatively,and twice daily for 5 days postoperatively (200 IU per kg of body weight, s.c.).

Magnetic Field. An electromagnet with a magnetic flux density of amaximum of 1.7 Tesla was used to produce an inhomogeneous magnetic field.The magnetic flux density was focused onto the region of the tumor with aspecially adapted pole shoe that was placed in contact with the surface of thetumor. On the tip of the pole shoe, the gradient (Fig. 3,yellow arrows) has itsmaximum. Fig. 3 demonstrates the dependence of the magnetic flux density onthe distance to the pole shoe. A magnetic flux density of 1.7 Tesla wasestimated in the region of the tumor surface and at 10 mm below the tip of thepole shoe, 1.0 Tesla (Fig. 3). The magnetic field was focused on the tumorduring FF infusion and for 60 min in total (Fig. 3).

Experimental Protocols. The 26 animals were divided into six groups,depending on the type of treatment, as shown in Table 2. Group 1 received ani.a. infusion of FF-MTX with the magnetic field at a dose equivalent to 20 and50% of the systemic dose MTX (group 1a and group 1b, respectively). Group2 received an i.a. infusion of MTX alone without the magnetic field at dosesequivalent to 20, 50, 75, and 100% of the systemic dose. Group 3 received ani.a. infusion of FF alone with the magnetic field at equivalent doses comparedFig. 1. Structural formula of MTX bound to magnetic nanoparticle.

Fig. 2. Desorption of MTX measured by UV-visible-spectroscopy at a wavelength of648 nm, depending on time.

Fig. 3. Dependence of the magnetic flux density on the distance to pole shoe with theelectromagnet.

Table 1 Characteristics of FFs

Composition Aqueous dispersion of starch polymer-coated magneticnanoparticles

pH 7.4Particle size 100 nm (hydrodynamic diameter)Magnetites 50 mg/mlIron content 30 mg/mlStabilizer 25 mg/ml, starch polymerNumber of particles ;1010/mlOdor NeutralColor Black, not translucent in daylight

Table 2 Experimental protocol

Group na

Chemothera-peutic

compound Doseb Applicationc

Externalmagnetic

fieldd

1a 5 FF-MTX 20% i.a. Yes1b 5 FF-MTX 50% i.a. Yes2 4 MTXe 20%, 50%, 75%, and 100% i.a. No3 2 FFsf equivalent amounts compared

with groups 1a and 1bi.a. Yes

4a 3 FF-MTX 20% i.v. Yes4b 3 FF-MTX 50% i.v. Yes5 2 FF-MTX 20% and 50% i.a. No6 2 Control Control Control groupa n, number of the tumor bearing animals.b Percentage of the regular systemic mitoxantrone dose (10 mg/m2).c i.a. was in femoral artery; i.v. was in ear vein.d Focused on the tumor.e MTX, chemotherapy (MTX) alone.f FFs, FFs alone.

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MAGNETIC DRUG TARGETING



with groups 1a and 1b. Group 4 received an i.v. infusion of FF-MTX with themagnetic field at doses equivalent to 20 and 50% of the systemic dose (group4a and group 4b, respectively). Group 5 received an i.a. infusion of FF-MTXwithout the magnetic field at doses equivalent to 20 and 50% of the systemicdose, and group 6 was the control group without treatment (Table 2).

After treatment, the tumor was measured every 3rd day by the sameobserver (R.K.) with a caliper ruler (measurement scale, 0.1 mm) for a periodof 3 months.

Blood Samples.Blood samples were drawn by venipuncture every weekand centrifuged at 20003 g within 2 h. Measurements of clinical chemistryparameters (iron, alanine aminotransferase, aspartate aminotransferase,g-glutamyl transferase, alkaline phosphatase, and lactate dehydrogenase; Hitachi747 analyzer; Roche Diagnostics, Mannheim, Germany) as well as the bloodcount parameters (total and differential blood counts; Sysmex SE-9000 ana-lyzer; Sysmex GmbH, Norderstedt, Germany) were performed immediatelyafter sampling.

Histological Evaluation, MRI. Immediately after i.a. infusion of 50%FF-MTX into the femoral artery, and after application of the magnetic field fora duration of 60 min, one animal was killed and the tumor was removed andfixed in 3.7% formalin. Five-mm thick paraffin sections of the tumor were cutand stained with H&E. After the 3-month observation period, the remaininganimals were killed; and the tumor, liver, kidneys, spleen, lungs, brain, andinguinal lymph nodes were removed and examined histologically.

Six h after 50% FF-MTX application with an external magnetic field, a MRIwas performed on four tumor-bearing animals. Imaging was done with a1.5-Tesla clinical MR scanner (ACS-NT; Philips, Best, the Netherlands). Afat-suppressed, T1-weighted turbo-spin echo sequence was used for imaging(TR 535; TE 20; echotrain length, 5).

Statistical Analysis. The tumor volume was calculated using the formulafor an elliptical mass (1/6p a2b, wherea 5 width on the horizontal axis andb 5 length on the vertical axis). We considered change of volumes aspercentages of tumor volumes (100%) found at day 0 (day of treatment).Statistical analysis for relative tumor volumes was performed using the onesample Welcht test (with a conservatively fixed value of 100% for the controlgroup) and a Welcht test for two independent samples. For blood parameters(absolute values), we applied thet test for two independent samples. Theresulting two-sidedPs were considered significant if#0.05. The result wasconsidered significant atP 5 0.01 or 0.05 and highly significant if,0.01. ThePs were calculated using the Statistical Package for Social Sciences (SPSS)version 9.0 and Microsoft EXCEL version 97.

RESULTS

Tumor Volume. In the control group without treatment (Group 6,‚, Figs. 4–10) the tumor volume increased to 14.723 mm3 (median

value) at 12 days, and palpable metastases appeared after 30 days. Theanimals of group 1a (Fig. 4,F), treated i.a. with 20% FF-MTX, hada 50% reduction in volume after 3–12 days (mean, 6 days) andcomplete tumor remission between the 15th and 36th day (mean, 26days) after treatment. This reduction in tumor volume was significantby the 6th day (P5 0.047;P , a) and highly significant by the 15thday (P, 0.001;P , a). The animals of group 1b (50% FF-MTX; Fig.5,f) had a decrease in tumor volume similar to that of group 1a (Fig.5,f), with a 50% decrease in volume after 3–6 days (mean, 4.2 days)and complete tumor remission after 12–57 days (mean, 21.8 days).The decrease in tumor volume was highly significant by the 6th day(P 5 0.001;P , a; Figs. 4 and 5).

In group 2 (i.a. MTX alone, no magnetic field), lower dosages (20and 50% of the systemic dose) did not result in tumor remission (Fig.6, r), and enlarged, palpable inguinal lymph nodes were found after48 days. At higher doses (75 and 100%), complete remission of tumoroccurred at the 36th (75%) and 33rd day (100%; Fig. 6,f).

The two group-3 animals (i.a. FF alone with the magnetic field,amount of FFs alone equivalent to groups 1a and 1b) demonstrated aprogressive increase in tumor volume (Fig. 7,Œ) with palpable,enlarged inguinal lymph nodes (metastases) after 45 days.

Fig. 4. Group 1a: effect of i.a. application of FF-MTX [20% of the regular systemicdose (F)] on relative tumor volume after magnetic drug targeting compared with controlgroup [group 6 (‚), control (no treatment)].Symbols, the median tumor volume;bars, themaximum and minimum values;metastases, onset of metastases;treatment, the day oftreatment (singular treatment).

Fig. 5. Group 1b: effect of i.a. application of FF-MTX (50% of the regular systemicdose,f) on relative tumor volume after magnetic drug targeting compared with controlgroup [group 6 (‚), control (no treatment)].Symbols, the median tumor volume;bars, themaximum and minimum values;metastases, onset of metastases;treatment, the day oftreatment (singular treatment).

Fig. 6. Group 2: effect of i.a. application of MTX—20 and 50% (r) and 75 and 100%(f) of the regular systemic dose—on relative tumor volume compared with control group[group 6 (‚), control (no treatment)].Symbols, the median tumor volume;bars, themaximum and minimum values;metastases, onset of metastases;alopecia, onset ofalopecia;treatment, the day of treatment (singular treatment).

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The six animals of group 4 (i.v. injection via the ear vein of 20%and 50% FF-MTX with magnetic field) showed a slight tumor remis-sion, but the reduction of volume was not statistically significant incomparison to the control group (Ps: group 4a 0.48- 0.70, group 4b0.26- 0.96 (P. a; Fig. 8,E; Fig. 9, □).

The two animals of group 5 (i.a. FF-MTX 20 and 50%, without amagnetic field) showed a discontinuation of tumor growth and noevidence of metastases, but no remission of the tumor was seen (Fig.10, FF-MTX; 20%,r; FF-MTX 50%,f). At the time of treatment,,5% of the animals showed a small necrotic fraction in the area of thetumor area (Fig. 10).

Local and Systemic Effects.Similar to the description in theliterature (18), the general condition of the control group animals(limited to two animals for ethical reasons) worsened during theobservation period, and the animals developed pneumonia, whichexplains the increase of leukocytes as seen in Fig. 11.

All of the animals in the groups treated with FF and a magneticfield developed a slight gray discoloration of the skin covering thetumor. In addition, scattered, dark injected vessels were seen in thetumor region. The gray discoloration, caused by the strong magnetic

field strength which attracted the FFs throughout the whole tumor tothis layer (not shown as a figure), was completely reversible andlasted for approximately 48 h.

None of the animals of group 1 had any evident side effects such asalopecia, ulcers, or muscular atrophy; and their general condition(weight, food intake, excrement, urine, activity, fur condition) re-mained normal during the whole 3-month observation period com-pared with the physiological data of healthy animals (breeder’s state-ment by Charles River, Sulzfeld, Germany). No significant changes inserum iron or leukocyte values were seen in this group (Fig. 11a).

The urine of one animal in group 2 (50% MTX) showed blue-greendiscoloration, and this animal developed mild alopecia in the region ofthe digits after 48 days. Both animals with low-dose MTX (20 and50%) had a decrease in leukocyte values, but this was not statisticallysignificant (P5 0.29). Both of the group-2 animals with high-dose(75 and 100%) MTX had temporary blue-green urine discoloration, aswell as a unilateral alopecia (palmar region of the digits to the kneejoint) of the limb in which the tumor was implanted developing after33 days. This hairless area developed cutaneous inflammation andulceration, followed by mild alopecia of the ipsilateral fore limbs andhead. The musculature of the treated limb became atrophic, and the

Fig. 7. Group 3: effect of i.a. application of FFs with magnetic field (Œ).‚, control (notreatment).Symbols, the median relative tumor volume;bars, the maximum and minimumvalues;metastases, onset of metastases;treatment, the day of treatment (singular treat-ment).

Fig. 8. Group 4a: effect of i.v. application of FF-MTX 20% with magnetic field (E).‚, control (no treatment).Symbols, the median relative tumor volume;bars, the maximumand minimum values;metastases, onset of metastases;treatment, the day of treatment(singular treatment).

Fig. 9. Group 4b: effect of i.v. application of FF-MTX 50% with magnetic field (M).‚, control (no treatment).Symbols, the median relative tumor volume;bars, the maximumand minimum values;metastases, onset of metastases;treatment, the day of treatment(singular treatment).

Fig. 10. Group 5: effect of i.a. application of FF-MTX 20% (r) and 50% (f) withoutmagnetic field.‚, control (no treatment).Symbols, the median relative tumor volume;bars, the maximum and minimum values;metastases, onset of metastases;treatment, theday of treatment (singular treatment).

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circumference was noticeably smaller (by 3 cm) at the end of the3-month observation period. There was no marked difference in theseverity of the side effects between the two animals, except for thefact that the animal with the higher MTX dose (100%) developed thechanges several days sooner. Group-2 animals with 50, 75, and 100%MTX steadily lost weight after an initial lag-phase and were under-weight at the end of the observation period (mean value, 1800 mgbelow the lower reference values according to the breeder’s statement;Charles River). These animals became leucocytopenic (#2.953 103/ml) in the early phase (highly significant drop;P 5 0.004; Fig. 11b),but recovered slightly in the middle and late periods.

None of the animals of group 3 or 4 showed any significant changesin serum iron (not shown in figures) or leukocyte counts (group 3, Fig.11a; groups 4a and 4b, Fig. 11a) during the observation period whencompared with initial values.

Histological Findings. Fig. 12 shows a whole-mount cross-sectionof the tumor that was excised just after treatment.Brown-black granules,FF particles distributed throughout the entire tumor. A higher magnifi-cation of a blood vessel (Fig. 13) shows that the intraluminal FF particleswere concentrated and deposited on the endothelium nearest to themagnetic field and were separated from the erythrocyte pool, but, as canbe seen from Fig. 14, FF particles were also found in the tumor intersti-tium and in the adjacent surrounding tissues as well (Fig. 15).

After the 3-month observation period, no viable tumor tissue washistologically evident in the animals of group 1, with only fibrosisseen in the tumor implantation site. No metastases were found in the

regional lymph nodes or in any other organs. Some FF particles werefound in the spleen of the animals, but none were evident in the liver,lungs, or brain or in the implantation site and surrounding musculatureand skin. No other macroscopic or histological pathological changeswere found in any of the investigated organs.

In group 2, the VX-2 tumors of the two low-dose animals were 8.644mm3 (50% MTX) and 2.497 mm3 (20% MTX) in size, with a large areaof central necrosis and viable tumor at the periphery. The two animalswith high-dose MTX (75 and 100%) had no viable tumor at the implan-tation site. None of the other investigated organs in the animals of group2 (liver, kidneys, spleen, lungs, or brain) had any pathological changes.

The tumors of both animals of group 3 measured 13.324 mm3 and17.649 mm3, respectively, with a large area of central necrosis andviable tumor at the periphery. No FF particles were found within thetumor or in the surrounding musculature and skin. Some FFs werefound in the spleen. Metastases were found in the inguinal lymphnodes and liver of both animals. None of the other investigated organs(kidneys, spleen, lungs, brain) had any pathological changes.

MRI. Fig. 16,a andb, show the left hind limb (implantation site)of two rabbits that received 50% FF-MTX i.a. and i.v., respectively.The MRI was made 6 h after treatment. The tumor is situated at themedial portion of the hind limb (dotted circle), and the concentrationof FF is seen by extinction of signal. Fig. 16a(i.a. FF-MTX) showsdefinite extinction of signal and Fig. 16b (i.v. FF-MTX) only a verydiscrete signal extinction. The area markedf is at the head of thefemur and appears to be hypodense.

DISCUSSION

Chemotherapy is a balancing act between efficacy and toxicity and anumber of strategies have been developed that aim to resolve this di-lemma. Regional chemotherapy via a regional artery administers a moreconcentrated dose of the active agent directly into the tumor (19). Theadvantage of this approach is limited, however, by drain-off via thevenous blood, which limits exposure time and reduces the overall effi-cacy. Magnetic drug targeting is a means of holding the chemotherapeuticagent at the desired site of activity, thus increasing efficacy and dimin-ishing systemic toxicity. In the present study, the authors found that thisapproach led to complete tumor remission with reduced doses of 20 and50% FF-MTX (Figs. 4 and 5). The application was well tolerated by theanimals, and no signs of toxicity were detected. On the contrary, i.a.infusion of the same doses, 20 and 50%, of MTX alone (group 2, Fig. 6)resulted in no reduction of tumor volume, and the animals developedmetastases and suffered from chemotherapeutic side effects. Only whenthe dose of MTX alone was increased to 75% and 100%, was a tumorremission seen, but this resulted in severe side effects (alopecia, ulcers,and leukocytopenia as seen in Fig. 11b). i.v. infusion of the FF-MTXcomplex was also ineffective inasmuch as only a slight tumor remissionthat was not statistically significant resulted (Figs. 8 and 9). The same wastrue of i.a. infused FF-MTX without an external magnetic field, becausethe tumor remained at the same size, without remission (Fig. 10). Thus,the combination of i.a. infusion with a magnetic field was safe, effective,and well tolerated by the animals and was very effective in treating thetumor even though the dose of chemotherapeutic agent was markedlyreduced.

At present, i.a. delivery of chemotherapeutic agents is approvedand well accepted for treatment of liver metastases (20) and hasoccasionally been used for other tumor types also (e.g.,inoperablehead and neck tumors); but it has often necessitated complicated,time-consuming operative procedures, including general anesthe-sia (21). Experimentally, Swistelet al. (18) described encouragingresults using i.a. chemotherapy for VX-2 squamous cell carcinoma.They achieved complete tumor remission after i.a. application of

Fig. 11. Values of the WBC before treatment (day 0) and on days 3–15 (early period),18–48 (middle period), and 51–81 (late period) after the respective treatment regimes.Columns, the median values;bars, the maximum and minimum values.‚, control (notreatment).MTX, MTX alone;FF, FFs (alone) in correspondence to FF-MTX 20% andFF-MTX 50%; i.a., i.a. in femoral artery;i.v., i.v. in ear vein;percentage(%), the amount ofthe regular systemic MTX dose.a, M, control;f, i.a. FFs alone in correspondence with groups1a and 1b with magnetic field;p, i.v. FF-MTX 20% with magnetic field;o, i.v. FF-MTX 50%with magnetic field.b, M, control;s, i.a. FF-MTX 20% with magnetic field;2, i.a. FF-MTX50% with magnetic field;z, i.a. MTX 20% and 50%; , i.a. MTX 75% and 100%.

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Adriamycin in four of six animals, whereas i.v. infusion of Adria-mycin caused severe toxicity and resulted in complete remission inonly two cases.

A potential complication that could arise with the use of FF com-

pounds is the fact that, with larger particles, embolization could occur,preventing a sufficient concentration of the chemotherapeutic agentfrom reaching the tumor. On the other hand, if the particles are toosmall, the external magnetic field might not provide sufficient attrac-tion so that the particles are drawn into the tumor. The particles used

Fig. 12. Illustration of a cross-section of VX-2 squa-mous cell carcinoma of the rabbit immediately after mag-netic drug targeting.Brown-black particles, FFs scatteredwithin the complete tumor.Yellow frames, areas that aredescribed in more detail in Figs. 13, 14, and 15.

Fig. 13. SectionA from Fig. 12. The vessel supplying the tumor shows an intramuralconcentration of FFs oriented toward the magnetic field.

Fig. 14. SectionB from Fig. 12. Yellow arrows, tumor tissue with interstitial FFconcentrations.

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in the present study had a size of 100 nm. No embolization was seenin the main vascular system of the tumor, and the particles wereattracted throughout the entire tumor including its surface (Fig. 12).An additional helpful factor is that microvascular permeability inneoplastic tissues is increased (8-fold compared with normal tissue) asis diffusion (33-fold; Ref. 22). Our histological findings showingdistribution of FF particles throughout the tumor strongly support theconcept that high-molecular-weight substances such as chemothera-peutic agents or monoclonal antibodies can be effectively targeted to

tumor tissue. In addition, the fact that the FF alone with a magneticfield failed to cause tumor remission (Fig. 7) indicates that thetherapeutic effect resulted from the action of the chemotherapeuticagent itself, rather than intratumoral embolization by the particles.

The electromagnet used for this study produced a magnetic fluxdensity of a maximum of 1.7 Tesla, which decreased depending on thedistance to the pole shoe (Fig. 3). The magnetic gradient can be seenas a collection of vectors that point in the direction of increasingvalues as shown in Fig. 3 (yellow arrows). The arrow sizes correspondto the strength of the magnetic gradient. Both factors (direction andmagnitude) reflect the inhomogeneous character of the magnetic field,which is of key importance for magnetic drug targeting. In previousstudies, it was suggested that a magnetic field strength of 8000 Gauss(0.8 Tesla) is sufficient to exceed linear blood flow in the intratumoralvasculature and allow 100% localization of magnetic carrier contain-ing 20% magnetite (23). In contrast, Goodwinet al. (24) appliedMTCs i.a. in a swine model, focusing a magnetic field of only250-1000 Gauss (0.025–0.1 Tesla; permanent neodymium magnet) tothe desired compartments in the liver and lungs. The depth of thisMTC targeting was 8–12 cm and the particle size was 0.5–5mm. Withthis model, MTCs with a predefined activity had a concentration of67% in the liver and 50% in the lung localized by the magnet.

The magnetic field strength with a maximum of 1.7 Tesla used inthe present investigation was the strongest ever applied for magneticdrug targeting. We achieved a high concentration of FFs within thetumor after i.a. infusion of FFs, which was seen by histological (Figs.12–15) and MRI (Fig. 16a) methods. The VX-2 squamous cell car-cinoma in the present study was superficially exposed and had nomigratory motion, as was the case with the liver and lung targets(breathing fluctuations) in the swine model of Goodwinet al. (24). Inaddition these organs lie deeply in the body cavity (8–12 cm from thebody surface), greatly complicating focusing of the magnetic fluxdensity onto the tumor area. Two approaches to overcome this prob-lem are possible: (a) the use of larger particles, as previously sug-gested by Lubbe and Bergemann (25); or (b) the use of a strongermagnetic field. The particles (FF-MTX) used in the present studywere 100 nm in size (hydrodynamic diameter) and have shown good

Fig. 15. SectionC from Fig. 12. Transitional region between musculature and tumortissue.Black condensations, FFs within and outside the tumor.

Fig. 16. MRI of tumorous (VX-2 carcinoma)hind limbs of rabbits after i.a. (a) and i.v. (b)application of FFs with 60-min exposure time toexternal magnetic field. The MR images were taken6 h later still showing stable concentration of FFswithin the area of interest.

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therapeutic results in smaller animals (mouse, rat) as well (3, 4). Thestrong magnetic field was very efficacious in combination with theseparticles, but additional experiments (which we have already begun)should be performed using marked FFs to clarify the optimal magneticfield strength and particle size. For example, to more effectively treatin deep body cavities (i.e.,pancreatic cancer and so forth) rotatingmagnetic fields could be used to focus the particles to the region ofinterest. It is also important that the tumor has a sufficient bloodsupply so that the particles have access to the particular area.

A remarkable feature of using ionically bound pharmaceuticals isthat the anticancer agents are able to desorb from the carrier (FF) aftera defined time span and the low-molecular-weight substances (e.g.,the molecular weight of MTX 517) can then pass through the vascularwall or interstitium into the tumor cells. This is important becauseonce the FF-MTX complex has been directed to the tumor by themagnetic field, the drug must dissociate to act freely within the tumor.As shown in Fig. 2, MTX desorbs from the FF after 30 min (half-life),and, therefore, 50% of the drug is free to act on the tumor after 30 min.

Dextran-coated iron oxides have been shown to produce signal lossby MRI and have been used as a contrast medium for the detection ofmetastatic lymph nodes (negative contrast; Ref. 26). We found totalsignal loss and. therefore. a very high concentration of FF by MRIafter focusing by means of the magnetic field (Fig. 16a). Recentstudies have shown that i.a. application of radioactively labeled mag-netic carriers with an external magnetic field resulted in retention ofat least 50% in the target site (27). In comparison, after i.v. injection,only very slight signal loss was seen, which indicates a very lowconcentration (Fig. 16b). This underscores the advantage of i.a.versusi.v. infusion in magnetic drug targeting.

Previous studies by Baconet al. concerning FF with a particle sizeof 0.5–1.0mm found no acute or chronic toxicity after the i.v. infusionof 250 mg of iron/kg of body weight in rats (28), and 1–3 mg ofiron/kg of body weight in humans have been shown to be safe as well(29). This agrees with our findings, inasmuch as FF infusion was notassociated with any signs of toxicity.

Magnetic microspheres loaded with theg-emitting radioisotope90Yhave also been successfully used as a form of radionuclide therapy. In onestudy, this compound was maneuvered within the body of a mouse to as.c. lymphoma, resulting in eradication of the tumor (30). Magnetic fluidshave also been used for the so-called “magnetic fluid hyperthermia” thathas been used to control the local growth of murine mammary carcinoma(31). Additional modification of the magnetic particles so that they couldbind monoclonal antibodies, lectins, peptides, hormones or genes couldmake delivery of these compounds more efficient and also highly spe-cific. Therefore, magnetic particles could make important contributions tomolecular and cell biology (e.g.,in vitro transfection with genes), whichwould result in advances in both basic science and clinical practice (32).

ACKNOWLEDGMENTS

We thank M. Settles, Department of Radiology (Ernst J. Rummeny, Direc-tor) for the magnetic resonance imaging and P. Luppa, Department of ClinicalChemistry, (Dieter Neumeier, Director) for blood analysis.

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2000;60:6641-6648. Cancer Res Christoph Alexiou, Wolfgang Arnold, Roswitha J. Klein, et al. Locoregional Cancer Treatment with Magnetic Drug Targeting

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