Locoregional cancer treatment with magnetic drug targeting

Locoregional Cancer Treatment with Magnetic Drug Targeting Christoph Alexiou,2 Wolfgang Arnold, Roswitha J. Klein, Fritz G. Parak, Peter Hulin, Christian Bergemann, Wolfgang Erhardt, Stefan Wagenpfeil, and Andreas S. Lu¨bbe Department of Otorhinolaryngology, Head and Neck Surgery C. A., W. A., P. H., Department of Experimental Oncology and Therapeutic Research R. J. K., W. E., and Institute for Medical Statistics and Epidemiology S. W., Klinikum rechts der Isar, Technical University of Munich, 81675 Munich; PhysicsDepartment E 17, Technical University of Munich, 81675 Munich F. G. P.; Chemicell, 10777 Berlin C. B.; and CecilienKlinik, 33175 Bad Lippspringe A. S. L., Germany ABSTRACT The specific delivery of chemotherapeutic agents to their desired targets with a minimum of systemic side effects is an important, ongoing challenge of chemotherapy. One approach, developed in the past to address this problem, is the i.v. injection of magnetic particles ferrofluids (FFs) bound to anticancer agents that are then concentrated in the desired area (e.g., the tumor) by an external magnetic field. In the present study, we treated squamous cell carcinoma in rabbits with FFs bound to mitoxantrone (FFMTX) that was concentrated with a magnetic field. Experimental VX2 squamous cell carcinoma was implanted in the median portion of the hind limb of New Zealand White rabbits (n 5 26). When the tumor had reached a volume of ;3500 mm3 , FFMTX was injected intraarterially (i.a.; femoral artery) or i.v. (ear vein), whereas an external magnetic field was focused on the tumor. FFMTX i.a. application with the external magnetic field resulted in a significant (P < 0.05), complete, and permanent remission of the squamous cell carcinoma compared with the control group (no treatment) and the i.v. FFMTX group, with no signs of toxicity. The intratumoral accumulation of FFs was visualized both histologically and by magnetic resonance imaging. Thus, our data show that i.a. application of FFMTX is successful in treating experimental squamous cell carcinoma. This “magnetic drug targeting” offers a unique opportunity to treat malignant tumors locoregionally without systemic toxicity. Furthermore, it may be possible to use these magnetic particles as a “carrier system” for a variety of anticancer agents, e.g., radionuclides, cancerspecific antibodies, and genes.

[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 Luăbbe Department of Otorhinolaryngology, Head and Neck Surgery [C A., W A., P H.], Department of Experimental Oncology and Therapeutic Research [R J K., W E.], and Institute for Medical Statistics and Epidemiology [S W.], Klinikum rechts der Isar, Technical University of Munich, 81675 Munich; Physics-Department E 17, Technical University 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 targets with a minimum of systemic side effects is an important, ongoing challenge of chemotherapy One approach, developed in the past to address this problem, is the i.v injection of magnetic particles [ferrofluids (FFs)] bound to anticancer agents that are then concentrated in the desired area (e.g., the tumor) by an external magnetic field In the present study, we treated squamous cell carcinoma in rabbits with FFs bound to mitoxantrone (FF-MTX) that was concentrated with a magnetic field Experimental VX-2 squamous cell carcinoma was implanted in the median portion of the hind limb of New Zealand White rabbits (n ‫ ؍‬26) When the tumor had reached a volume of ϳ3500 mm3, FF-MTX was injected intraarterially (i.a.; femoral artery) or i.v (ear vein), whereas an external magnetic field was focused on the tumor FF-MTX i.a application with the external magnetic field resulted in a significant (P < 0.05), complete, and permanent remission of the squamous cell carcinoma compared with the control group (no treatment) and the i.v FF-MTX group, with no signs of toxicity The intratumoral accumulation of FFs was visualized both histologically and by magnetic resonance imaging Thus, our data show that i.a application of FF-MTX is successful in treating experimental squamous cell carcinoma This “magnetic drug targeting” offers a unique opportunity to treat malignant tumors locoregionally without systemic toxicity Furthermore, it may be possible to use these magnetic particles as a “carrier system” for a variety of anticancer agents, e.g., radionuclides, cancer-specific antibodies, and genes the first Phase I clinical trial using this approach in patients with advanced, unsuccessfully treated cancers or sarcomas This “magnetic drug targeting” approach was well tolerated Targeting and prolonged retention of the FF complex at the target site reduces its reticuloendothelial system (RES) clearance and facilitates extravascular uptake To optimize intratumoral magnetic particle concentration, several features need to be considered: (a) the particles should be of a size that allows sufficient attraction by the magnetic field and their introduction into the tumor or into the vascular system surrounding the tumor; (b) the magnetic fields should be of sufficient strength to be able to attract the magnetic nanoparticles into the desired area; (c) the FF complex should deliver and release a sufficient amount of anticancer agent; and (d) the method of injection should have good access to the tumor vasculature and should avoid clearance by the reticuloendothelial system (“first pass effect”) The purpose of the present study was to compare different application methods (i.v., i.a.) of magnetic drug targeting for the treatment of experimental VX-2 squamous cell carcinoma Because FFs are visible histologically and by imaging techniques such as MRI, we also wished to demonstrate the morphological intratumoral distribution of these magnetic nanoparticles in conjunction with an external magnetic field focused on the tumor region MATERIALS AND METHODS INTRODUCTION MTX The chemotherapeutic agent used in the experiments, MTX-HCl, (Novantron; Lederle, Wolfratshausen, Germany) is a synthetic anthracendion that inhibits DNA and RNA synthesis by intercalating in DNA molecules, which causes strand breaks Actively dividing cells are the most sensitive, but MTX tends to be non-cell-cycle specific and also inhibits G2-M progression (5) MTX has been used systemically for breast carcinoma, non-Hodgkin’s lymphoma, 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 body surface area) used for the experiments were calculated according the instructions of Kirk and Bistner‘s handbook of veterinarian procedures and emergency treatment (14) Magnetic Nanoparticles (FFs) The FFs used in the experiments were obtained from Chemicell (Berlin, Germany; German patent application no 19624426.9) and consisted of a colloidal dispersion formed by wet chemical methods from iron oxides and hydroxides to produce special multidomain particles (Table 1) The particles were surrounded by starch polymers for stabilization under various physiological conditions and to allow chemoabsorptive binding MTX has cationic characteristics and combines (amine groups of MTX-HCl with phosphate groups of the starch derivates) at a pH of 7.4 (Fig 1) The FF-MTX contained 6.5 mg of MTX per 10 ml Because the drug bond is reversible (ionic binding), desorption of the bound drug was dependent on the physiological environment (pH, osmolality, temperature) and Received 4/21/00; accepted 10/3/00 could be varied by changing the blood electrolyte concentration according to The costs of publication of this article were defrayed in part by the payment of page the specific need In experiments, desorption of MTX took place within 60 charges This article must therefore be hereby marked advertisement in accordance with 18 U.S.C Section 1734 solely to indicate this fact (Fig 2), which ensured that the drug could act freely once localized to the Supported by the Margarete Ammon Foundation, Munich, and grants from the tumor by the magnetic field Pyrogenicity and sterility tests were performed by Technical University of Munich, Germany the Pharmacy Department of the Virchow Medical School (Humboldt-UniverTo whom requests for reprints should be addressed, at Department of Otorhinolarsitaăt, Berlin, Germany) according to good manufacturing practice guidelines yngology, Head and Neck Surgery, Klinikum rechts der Isar, Technical University of Munich, Ismaningerstrasse 22, 81675 Munich, Germany Phone: 49-89-4140-2370; Fax: The characteristics of the FF-MTX are depicted in Table (see also Figs and 49-89-4140-4853; E-mail: C.Alexiou@lrz.tu-muenchen.de 2) The abbreviations used are: FF, ferrofluid; i.a., intraarterial/intraarterially; MR, VX-2 Squamous Cell Carcinoma The VX-2 squamous cell carcinoma magnetic resonance; MRI, MR imaging/image; MTX, mitoxantrone; MTX-FF, FF bound was obtained from the Deutsches Krebsforschungszentrum (Heidelberg, Gerto MTX; MTX-HCl, MTX hydrochloride; MTC, magnetic-targeted carrier 6641 The difference between the success or failure of chemotherapy depends not only on the drug itself but also on how it is delivered to its target Because of the relatively nonspecific action of chemotherapeutic agents, there is almost always some toxicity to normal tissue even under optimal conditions Therefore, it is of great importance to be able to selectively target the antineoplastic agent to its tumor target as precisely as possible, to reduce the resulting systemic toxic side effects from generalized systemic distribution and to be able to use a much smaller dose, which would further lead to a reduction of toxicity In the past, chemotherapy targeted by magnetic fields using magnetic albumin microspheres has shown encouraging results (1, 2) In 1996, Luăbbe et al (3) used a new FF,3 described in detail below, for experiments in which tumor-bearing experimental animals (nude mice and rats) were injected i.v with a FF complex (magnetic drug) that was directed into the tumor using a magnetic field (permanent magnet; magnetic field strength, 0.5– 0.8 Tesla) The FF complex was well tolerated by the animals, and tumor remission was achieved As a second step, Luăbbe et al (4) also conducted Downloaded from cancerres.aacrjournals.org on October 17, 2018 © 2000 American Association for Cancer Research MAGNETIC DRUG TARGETING Table Characteristics of FFs Composition pH Particle size Magnetites Iron content Stabilizer Number of particles Odor Color Aqueous dispersion of starch polymer-coated magnetic nanoparticles 7.4 100 nm (hydrodynamic diameter) 50 mg/ml 30 mg/ml 25 mg/ml, starch polymer ϳ1010/ml Neutral Black, not translucent in daylight Fig Structural formula of MTX bound to magnetic nanoparticle performed when the tumors had reached a volume of approximately 3500 mm3 For application of the chemotherapy, the animals were anesthetized with an i.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 after separation of the femoral vein and the saphenous nerve ϳ2 cm distal to the inguinal furrow The FF-MTX and the MTX alone were administered by perfusor over a period of 10 To prevent thrombosis, prophylaxis consisting of heparin sodium (heparin/natrium/25,000 IU Ratiopharm; Ratiopharm, Ulm, Germany) was given preoperatively, once immediately postoperatively, and twice daily for days postoperatively (200 IU per kg of body weight, s.c.) Magnetic Field An electromagnet with a magnetic flux density of a maximum 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 a specially adapted pole shoe that was placed in contact with the surface of the tumor On the tip of the pole shoe, the gradient (Fig 3, yellow arrows) has its maximum Fig demonstrates the dependence of the magnetic flux density on the distance to the pole shoe A magnetic flux density of 1.7 Tesla was estimated in the region of the tumor surface and at 10 mm below the tip of the pole shoe, 1.0 Tesla (Fig 3) The magnetic field was focused on the tumor during FF infusion and for 60 in total (Fig 3) Experimental Protocols The 26 animals were divided into six groups, depending on the type of treatment, as shown in Table Group received an i.a infusion of FF-MTX with the magnetic field at a dose equivalent to 20 and 50% of the systemic dose MTX (group 1a and group 1b, respectively) Group received an i.a infusion of MTX alone without the magnetic field at doses equivalent to 20, 50, 75, and 100% of the systemic dose Group received an i.a infusion of FF alone with the magnetic field at equivalent doses compared Fig Desorption of MTX measured by UV-visible-spectroscopy at a wavelength of 648 nm, depending on time many) and originates as a papillomatous reaction to Shope virus infection in Fig Dependence of the magnetic flux density on the distance to pole shoe with the wild rabbits (15) The tumor was preserved through many generations of electromagnet serially transplanted animals and was established as a tumor cell line in our laboratories Its histology and growth characteristics have been extensively Table Experimental protocol described (16, 17) Briefly, after implantation into soft tissue, the tumor ChemotheraExternal enlarges rapidly with increased vascularity in its periphery The animals soon peutic magnetic a b c (within 2–3 weeks) develop central tumor necrosis, locoregional lymph node Group n Application compound Dose fieldd metastases, and hematogenous metastases (e.g., into the lungs) 1a FF-MTX 20% i.a Yes Animals The experimental animals were female New Zealand White rab1b FF-MTX 50% i.a Yes e bits (2000 –2500 g body weight, 12–15 weeks old; Charles River, Sulzfeld, 20%, 50%, 75%, and 100% i.a No MTX f equivalent amounts compared i.a Yes FFs Germany) that were housed individually in a room with an artificial 12/12 h with groups 1a and 1b light/dark cycle (exposed to light from 0700 to 1900 h) The rabbits were fed 4a FF-MTX 20% i.v Yes hard rabbit chow pellets (Altromin, Lage, Germany), carrots, dry bread, and 4b FF-MTX 50% i.v Yes tap water FF-MTX 20% and 50% i.a No Control Control Control group Surgical Intervention Fragments of viable VX-2 tissue, mm in size, a were taken from the tumor periphery in donor animals These fragments were n, number of the tumor bearing animals b Percentage of the regular systemic mitoxantrone dose (10 mg/m2) placed in a special medium [RPMI 1640, 2.0 g/liter NaHCO3, and L-glutamin c i.a was in femoral artery; i.v was in ear vein (Seromed); Biochrom, Berlin, Germany] and were immediately implanted d Focused on the tumor e under sterile conditions into the hind limb of anesthetized recipient rabbits MTX, chemotherapy (MTX) alone f (n ϭ 26) in the supply area of the femoral artery The experiments were FFs, FFs alone 6642 Downloaded from cancerres.aacrjournals.org on October 17, 2018 © 2000 American Association for Cancer Research MAGNETIC DRUG TARGETING Fig Group 1a: effect of i.a application of FF-MTX [20% of the regular systemic dose (F)] on relative tumor volume after magnetic drug targeting compared with control group [group (‚), control (no treatment)] Symbols, the median tumor volume; bars, the maximum and minimum values; metastases, onset of metastases; treatment, the day of treatment (singular treatment) with groups 1a and 1b Group received an i.v infusion of FF-MTX with the magnetic field at doses equivalent to 20 and 50% of the systemic dose (group 4a and group 4b, respectively) Group received an i.a infusion of FF-MTX without the magnetic field at doses equivalent to 20 and 50% of the systemic dose, and group was the control group without treatment (Table 2) After treatment, the tumor was measured every 3rd day by the same observer (R.K.) with a caliper ruler (measurement scale, 0.1 mm) for a period of months Blood Samples Blood samples were drawn by venipuncture every week and centrifuged at 2000 ϫ g within h Measurements of clinical chemistry parameters (iron, alanine aminotransferase, aspartate aminotransferase, ␥glutamyl transferase, alkaline phosphatase, and lactate dehydrogenase; Hitachi 747 analyzer; Roche Diagnostics, Mannheim, Germany) as well as the blood count parameters (total and differential blood counts; Sysmex SE-9000 analyzer; Sysmex GmbH, Norderstedt, Germany) were performed immediately after sampling Histological Evaluation, MRI Immediately after i.a infusion of 50% FF-MTX into the femoral artery, and after application of the magnetic field for a duration of 60 min, one animal was killed and the tumor was removed and fixed in 3.7% formalin Five-␮m thick paraffin sections of the tumor were cut and stained with H&E After the 3-month observation period, the remaining animals were killed; and the tumor, liver, kidneys, spleen, lungs, brain, and inguinal lymph nodes were removed and examined histologically Six h after 50% FF-MTX application with an external magnetic field, a MRI was performed on four tumor-bearing animals Imaging was done with a 1.5-Tesla clinical MR scanner (ACS-NT; Philips, Best, the Netherlands) A fat-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 formula for an elliptical mass (1/6 ␲ a2b, where a ϭ width on the horizontal axis and b ϭ length on the vertical axis) We considered change of volumes as percentages of tumor volumes (100%) found at day (day of treatment) Statistical analysis for relative tumor volumes was performed using the one sample Welch t test (with a conservatively fixed value of 100% for the control group) and a Welch t test for two independent samples For blood parameters (absolute values), we applied the t test for two independent samples The resulting two-sided Ps were considered significant if Յ0.05 The result was considered significant at P ϭ 0.01 or 0.05 and highly significant if Ͻ0.01 The Ps 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 The animals of group 1a (Fig 4, F), treated i.a with 20% FF-MTX, had a 50% reduction in volume after 3–12 days (mean, days) and complete tumor remission between the 15th and 36th day (mean, 26 days) after treatment This reduction in tumor volume was significant by the 6th day (P ϭ 0.047; P Ͻ ␣) and highly significant by the 15th day (P Ͻ 0.001; P Ͻ ␣) 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– 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 ϭ 0.001; P Ͻ ␣; Figs and 5) In group (i.a MTX alone, no magnetic field), lower dosages (20 and 50% of the systemic dose) did not result in tumor remission (Fig 6, ᭜), and enlarged, palpable inguinal lymph nodes were found after 48 days At higher doses (75 and 100%), complete remission of tumor occurred 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 a progressive increase in tumor volume (Fig 7, Œ) with palpable, enlarged inguinal lymph nodes (metastases) after 45 days Fig Group 1b: effect of i.a application of FF-MTX (50% of the regular systemic dose, f) on relative tumor volume after magnetic drug targeting compared with control group [group (‚), control (no treatment)] Symbols, the median tumor volume; bars, the maximum and minimum values; metastases, onset of metastases; treatment, the day of treatment (singular treatment) Fig Group 2: effect of i.a application of MTX—20 and 50% (᭜) and 75 and 100% (f) of the regular systemic dose— on relative tumor volume compared with control group [group (‚), control (no treatment)] Symbols, the median tumor volume; bars, the maximum and minimum values; metastases, onset of metastases; alopecia, onset of alopecia; treatment, the day of treatment (singular treatment) 6643 Downloaded from cancerres.aacrjournals.org on October 17, 2018 © 2000 American Association for Cancer Research MAGNETIC DRUG TARGETING Fig Group 3: effect of i.a application of FFs with magnetic field (Œ) ‚, control (no treatment) Symbols, the median relative tumor volume; bars, the maximum and minimum values; metastases, onset of metastases; treatment, the day of treatment (singular treatment) Fig 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 maximum and minimum values; metastases, onset of metastases; treatment, the day of treatment (singular treatment) The six animals of group (i.v injection via the ear vein of 20% and 50% FF-MTX with magnetic field) showed a slight tumor remission, but the reduction of volume was not statistically significant in comparison to the control group (Ps: group 4a 0.48- 0.70, group 4b 0.26- 0.96 (P Ͼ ␣; Fig 8, E; Fig 9, □) The two animals of group (i.a FF-MTX 20 and 50%, without a magnetic field) showed a discontinuation of tumor growth and no evidence of metastases, but no remission of the tumor was seen (Fig 10, FF-MTX; 20%, ᭜; FF-MTX 50%, f) At the time of treatment, Ͻ5% of the animals showed a small necrotic fraction in the area of the tumor area (Fig 10) Local and Systemic Effects Similar to the description in the literature (18), the general condition of the control group animals (limited to two animals for ethical reasons) worsened during the observation period, and the animals developed pneumonia, which explains the increase of leukocytes as seen in Fig 11 All of the animals in the groups treated with FF and a magnetic field developed a slight gray discoloration of the skin covering the tumor In addition, scattered, dark injected vessels were seen in the tumor region The gray discoloration, caused by the strong magnetic field strength which attracted the FFs throughout the whole tumor to this layer (not shown as a figure), was completely reversible and lasted for approximately 48 h None of the animals of group had any evident side effects such as alopecia, ulcers, or muscular atrophy; and their general condition (weight, food intake, excrement, urine, activity, fur condition) remained normal during the whole 3-month observation period compared with the physiological data of healthy animals (breeder’s statement by Charles River, Sulzfeld, Germany) No significant changes in serum iron or leukocyte values were seen in this group (Fig 11a) The urine of one animal in group (50% MTX) showed blue-green discoloration, and this animal developed mild alopecia in the region of the digits after 48 days Both animals with low-dose MTX (20 and 50%) had a decrease in leukocyte values, but this was not statistically significant (P ϭ 0.29) Both of the group-2 animals with high-dose (75 and 100%) MTX had temporary blue-green urine discoloration, as well as a unilateral alopecia (palmar region of the digits to the knee joint) of the limb in which the tumor was implanted developing after 33 days This hairless area developed cutaneous inflammation and ulceration, followed by mild alopecia of the ipsilateral fore limbs and head The musculature of the treated limb became atrophic, and the Fig Group 4b: effect of i.v application of FF-MTX 50% with magnetic field (Ⅺ) ‚, control (no treatment) Symbols, the median relative tumor volume; bars, the maximum and 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% (᭜) and 50% (f) without magnetic field ‚, control (no treatment) Symbols, the median relative tumor volume; bars, the maximum and minimum values; metastases, onset of metastases; treatment, the day of treatment (singular treatment) 6644 Downloaded from cancerres.aacrjournals.org on October 17, 2018 © 2000 American Association for Cancer Research MAGNETIC DRUG TARGETING regional lymph nodes or in any other organs Some FF particles were found in the spleen of the animals, but none were evident in the liver, lungs, or brain or in the implantation site and surrounding musculature and skin No other macroscopic or histological pathological changes were found in any of the investigated organs In group 2, the VX-2 tumors of the two low-dose animals were 8.644 mm3 (50% MTX) and 2.497 mm3 (20% MTX) in size, with a large area of central necrosis and viable tumor at the periphery The two animals with high-dose MTX (75 and 100%) had no viable tumor at the implantation site None of the other investigated organs in the animals of group (liver, kidneys, spleen, lungs, or brain) had any pathological changes The tumors of both animals of group measured 13.324 mm3 and 17.649 mm3, respectively, with a large area of central necrosis and viable tumor at the periphery No FF particles were found within the tumor or in the surrounding musculature and skin Some FFs were found in the spleen Metastases were found in the inguinal lymph nodes and liver of both animals None of the other investigated organs (kidneys, spleen, lungs, brain) had any pathological changes MRI Fig 16, a and b, 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 h after treatment The tumor is situated at the medial portion of the hind limb (dotted circle), and the concentration of FF is seen by extinction of signal Fig 16a (i.a FF-MTX) shows definite extinction of signal and Fig 16b (i.v FF-MTX) only a very discrete signal extinction The area marked f is at the head of the femur and appears to be hypodense DISCUSSION 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 (no treatment) MTX, MTX alone; FF, FFs (alone) in correspondence to FF-MTX 20% and FF-MTX 50%; i.a., i.a in femoral artery; i.v., i.v in ear vein; percentage (%), the amount of the regular systemic MTX dose a, Ⅺ, control; f, i.a FFs alone in correspondence with groups 1a and 1b with magnetic field; p, i.v FF-MTX 20% with magnetic field; o, i.v FF-MTX 50% with magnetic field b, Ⅺ, control; s, i.a FF-MTX 20% with magnetic field; 2, i.a FF-MTX 50% with magnetic field; z, i.a MTX 20% and 50%; `, i.a MTX 75% and 100% circumference was noticeably smaller (by cm) at the end of the 3-month observation period There was no marked difference in the severity of the side effects between the two animals, except for the fact that the animal with the higher MTX dose (100%) developed the changes several days sooner Group-2 animals with 50, 75, and 100% MTX steadily lost weight after an initial lag-phase and were underweight at the end of the observation period (mean value, 1800 mg below the lower reference values according to the breeder’s statement; Charles River) These animals became leucocytopenic (Յ2.95 ϫ 103/ ␮l) in the early phase (highly significant drop; P ϭ 0.004; Fig 11b), but recovered slightly in the middle and late periods None of the animals of group or showed any significant changes in serum iron (not shown in figures) or leukocyte counts (group 3, Fig 11a; groups 4a and 4b, Fig 11a) during the observation period when compared with initial values Histological Findings Fig 12 shows a whole-mount cross-section of the tumor that was excised just after treatment Brown-black granules, FF particles distributed throughout the entire tumor A higher magnification of a blood vessel (Fig 13) shows that the intraluminal FF particles were concentrated and deposited on the endothelium nearest to the magnetic field and were separated from the erythrocyte pool, but, as can be seen from Fig 14, FF particles were also found in the tumor interstitium and in the adjacent surrounding tissues as well (Fig 15) After the 3-month observation period, no viable tumor tissue was histologically evident in the animals of group 1, with only fibrosis seen in the tumor implantation site No metastases were found in the Chemotherapy is a balancing act between efficacy and toxicity and a number of strategies have been developed that aim to resolve this dilemma Regional chemotherapy via a regional artery administers a more concentrated dose of the active agent directly into the tumor (19) The advantage of this approach is limited, however, by drain-off via the venous blood, which limits exposure time and reduces the overall efficacy Magnetic drug targeting is a means of holding the chemotherapeutic agent at the desired site of activity, thus increasing efficacy and diminishing systemic toxicity In the present study, the authors found that this approach led to complete tumor remission with reduced doses of 20 and 50% FF-MTX (Figs and 5) The application was well tolerated by the animals, 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 developed metastases and suffered from chemotherapeutic side effects Only when the dose of MTX alone was increased to 75% and 100%, was a tumor remission seen, but this resulted in severe side effects (alopecia, ulcers, and leukocytopenia as seen in Fig 11b) i.v infusion of the FF-MTX complex was also ineffective inasmuch as only a slight tumor remission that was not statistically significant resulted (Figs and 9) The same was true of i.a infused FF-MTX without an external magnetic field, because the 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 the tumor even though the dose of chemotherapeutic agent was markedly reduced At present, i.a delivery of chemotherapeutic agents is approved and well accepted for treatment of liver metastases (20) and has occasionally been used for other tumor types also (e.g., inoperable head and neck tumors); but it has often necessitated complicated, time-consuming operative procedures, including general anesthesia (21) Experimentally, Swistel et al (18) described encouraging results using i.a chemotherapy for VX-2 squamous cell carcinoma They achieved complete tumor remission after i.a application of 6645 Downloaded from cancerres.aacrjournals.org on October 17, 2018 © 2000 American Association for Cancer Research MAGNETIC DRUG TARGETING Fig 12 Illustration of a cross-section of VX-2 squamous cell carcinoma of the rabbit immediately after magnetic drug targeting Brown-black particles, FFs scattered within the complete tumor Yellow frames, areas that are described in more detail in Figs 13, 14, and 15 Adriamycin in four of six animals, whereas i.v infusion of Adriamycin caused severe toxicity and resulted in complete remission in only two cases A potential complication that could arise with the use of FF com- Fig 14 Section B from Fig 12 Yellow arrows, tumor tissue with interstitial FF concentrations Fig 13 Section A from Fig 12 The vessel supplying the tumor shows an intramural concentration of FFs oriented toward the magnetic field pounds is the fact that, with larger particles, embolization could occur, preventing a sufficient concentration of the chemotherapeutic agent from reaching the tumor On the other hand, if the particles are too small, the external magnetic field might not provide sufficient attraction so that the particles are drawn into the tumor The particles used 6646 Downloaded from cancerres.aacrjournals.org on October 17, 2018 © 2000 American Association for Cancer Research MAGNETIC DRUG TARGETING Fig 15 Section C from Fig 12 Transitional region between musculature and tumor tissue Black condensations, FFs within and outside the tumor in the present study had a size of 100 nm No embolization was seen in the main vascular system of the tumor, and the particles were attracted throughout the entire tumor including its surface (Fig 12) An additional helpful factor is that microvascular permeability in neoplastic tissues is increased (8-fold compared with normal tissue) as is diffusion (33-fold; Ref 22) Our histological findings showing distribution of FF particles throughout the tumor strongly support the concept that high-molecular-weight substances such as chemotherapeutic agents or monoclonal antibodies can be effectively targeted to tumor tissue In addition, the fact that the FF alone with a magnetic field failed to cause tumor remission (Fig 7) indicates that the therapeutic effect resulted from the action of the chemotherapeutic agent itself, rather than intratumoral embolization by the particles The electromagnet used for this study produced a magnetic flux density of a maximum of 1.7 Tesla, which decreased depending on the distance to the pole shoe (Fig 3) The magnetic gradient can be seen as a collection of vectors that point in the direction of increasing values as shown in Fig (yellow arrows) The arrow sizes correspond to the strength of the magnetic gradient Both factors (direction and magnitude) reflect the inhomogeneous character of the magnetic field, which is of key importance for magnetic drug targeting In previous studies, it was suggested that a magnetic field strength of 8000 Gauss (0.8 Tesla) is sufficient to exceed linear blood flow in the intratumoral vasculature and allow 100% localization of magnetic carrier containing 20% magnetite (23) In contrast, Goodwin et al (24) applied MTCs i.a in a swine model, focusing a magnetic field of only 250-1000 Gauss (0.025– 0.1 Tesla; permanent neodymium magnet) to the desired compartments in the liver and lungs The depth of this MTC targeting was –12 cm and the particle size was 0.5–5 ␮m With this model, MTCs with a predefined activity had a concentration of 67% in the liver and 50% in the lung localized by the magnet The magnetic field strength with a maximum of 1.7 Tesla used in the present investigation was the strongest ever applied for magnetic drug targeting We achieved a high concentration of FFs within the tumor after i.a infusion of FFs, which was seen by histological (Figs 12–15) and MRI (Fig 16a) methods The VX-2 squamous cell carcinoma in the present study was superficially exposed and had no migratory motion, as was the case with the liver and lung targets (breathing fluctuations) in the swine model of Goodwin et al (24) In addition these organs lie deeply in the body cavity (8 –12 cm from the body surface), greatly complicating focusing of the magnetic flux density onto the tumor area Two approaches to overcome this problem are possible: (a) the use of larger particles, as previously suggested by Luăbbe and Bergemann (25); or (b) the use of a stronger magnetic field The particles (FF-MTX) used in the present study were 100 nm in size (hydrodynamic diameter) and have shown good 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 to external magnetic field The MR images were taken h later still showing stable concentration of FFs within the area of interest 6647 Downloaded from cancerres.aacrjournals.org on October 17, 2018 © 2000 American Association for Cancer Research MAGNETIC DRUG TARGETING therapeutic results in smaller animals (mouse, rat) as well (3, 4) The strong magnetic field was very efficacious in combination with these particles, but additional experiments (which we have already begun) should be performed using marked FFs to clarify the optimal magnetic field strength and particle size For example, to more effectively treat in deep body cavities (i.e., pancreatic cancer and so forth) rotating magnetic fields could be used to focus the particles to the region of interest It is also important that the tumor has a sufficient blood supply so that the particles have access to the particular area A remarkable feature of using ionically bound pharmaceuticals is that the anticancer agents are able to desorb from the carrier (FF) after a defined time span and the low-molecular-weight substances (e.g., the molecular weight of MTX 517) can then pass through the vascular wall or interstitium into the tumor cells This is important because once the FF-MTX complex has been directed to the tumor by the magnetic field, the drug must dissociate to act freely within the tumor As shown in Fig 2, MTX desorbs from the FF after 30 (half-life), and, therefore, 50% of the drug is free to act on the tumor after 30 Dextran-coated iron oxides have been shown to produce signal loss by MRI and have been used as a contrast medium for the detection of metastatic lymph nodes (negative contrast; Ref 26) We found total signal loss and therefore a very high concentration of FF by MRI after focusing by means of the magnetic field (Fig 16a) Recent studies have shown that i.a application of radioactively labeled magnetic carriers with an external magnetic field resulted in retention of at least 50% in the target site (27) In comparison, after i.v injection, only very slight signal loss was seen, which indicates a very low concentration (Fig 16b) This underscores the advantage of i.a versus i.v infusion in magnetic drug targeting Previous studies by Bacon et al concerning FF with a particle size of 0.5–1.0 ␮m found no acute or chronic toxicity after the i.v infusion of 250 mg of iron/kg of body weight in rats (28), and 1–3 mg of iron/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 not associated with any signs of toxicity Magnetic microspheres loaded with the ␥-emitting radioisotope 90Y have also been successfully used as a form of radionuclide therapy In one study, this compound was maneuvered within the body of a mouse to a s.c lymphoma, resulting in eradication of the tumor (30) Magnetic fluids have also been used for the so-called “magnetic fluid hyperthermia” that has been used to 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Carriers, pp 501–516 New York: Plenum Publishing Corp., 1997 31 Jordan, A., Scholz, R., Wust, P., Fahling, H., Krause, J., Wlodarcyk, W., Sander, B., Vogl, T., and Felix, R Effects of magnetic fluid hyperthermia (MFH) on C3H mammary carcinoma in vivo Int J Hyperthermia, 13: 587– 605, 1997 32 Partridge, M., Phillips, E., Francis, R., and Li, S R Immunomagnetic separation for enrichment and sensitive detection of disseminated tumor cells in patients with head and neck SCC J Pathol., 189: 368 –377, 1999 6648 Downloaded from cancerres.aacrjournals.org on October 17, 2018 © 2000 American Association for Cancer Research Locoregional Cancer Treatment with Magnetic Drug Targeting Christoph Alexiou, Wolfgang Arnold, Roswitha J Klein, et al Cancer Res 2000;60:6641-6648 Updated version Cited articles Citing articles E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/60/23/6641 This article cites 25 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magnetic field; 2,... metastases; treatment, the day of treatment (singular treatment) 6644 Downloaded from cancerres.aacrjournals.org on October 17, 2018 © 2000 American Association for Cancer Research MAGNETIC DRUG TARGETING. .. treatment, the day of treatment (singular treatment) 6643 Downloaded from cancerres.aacrjournals.org on October 17, 2018 © 2000 American Association for Cancer Research MAGNETIC DRUG TARGETING Fig Group