injection of encapsulated cells producing an ifosfamide-activating cytochrome p450 for targeted...

337

Injection of Encapsulated Cells Producingan Ifosfamide-Activating CytochromeP450 for Targeted Chemotherapy toPancreatic Tumors

PETRA MÜLLER,a,c RALF JESNOWSKI,a PETER KARLE,d REGINA RENZ,d

ROBERT SALLER,c HARTMUT STEIN,a KATRIN PÜSCHEL,a,c KERSTINVON ROMBS,c HORST NIZZE,b STEFAN LIEBE,a THOMAS WAGNER,e

WALTER H. GÜNZBURG,d BRIAN SALMONS,c AND MATTHIAS LÖHRa,f

aDepartment of Medicine and bDepartment of Pathology, University of Rostock, GermanycBavarian Nordic Research Institute, Munich, GermanydInstitute of Virology, University of Veterinary Sciences, Vienna, AustriaeDepartment of Medicine, Medical School of Lübeck University, Germany

ABSTRACT: The prognosis of pancreatic cancer is poor, and current medicaltreatment is mostly ineffective. The aim of this study was to design a new treat-ment modality in an animal model system. We describe here a novel treatmentstrategy employing a mouse model system for pancreatic carcinoma. Embryo-nal kidney epithelial cells were genetically modified to express the cytochromeP450 subenzyme 2B1 under the control of a cytomegalovirus (CMV) immedi-ate early promoter. This CYP2B1 gene converts ifosfamide to its active cyto-toxic compounds, phosphoramide mustard, which alkylates DNA, andacrolein, which alkylates proteins. The cells were then encapsulated in a cellu-lose sulphate formulation and implanted into preestablished tumors derivedfrom a human pancreatic tumor cell line. Intraperitoneal administration oflow-dose ifosfamide to tumor bearing mice that received the encapsulated cellsresults in partial or even complete tumor ablation. Such an in situ chemother-apy strategy utilizing genetically modified cells in an immunoprotected envi-ronment may prove useful for solid tumor therapy in man.

INTRODUCTION

Although chemotherapy is used in the treatment of solid tumors, the efficacy ofthis approach is compromised by a number of factors. These include the accessibilityof the tumor, the degree of drug sensitivity of the tumor and the local and systemictoxicity of the chemotherapeutic agent. Furthermore, tumor cells may become resis-tant to the toxic effects of chemotherapeutic agents. The treatment of pancreatic can-cer is particularly problematic since these tumors are aggressive. Further, pancreaticadenocarcinomas have a poor prognosis and at the time of diagnosis, the tumor is

fAddress for correspondence, reprints and requests: Priv.-Doz. Dr. med. Matthias Löhr, M.D.,Division of Gastroenterology, Department of Medicine, University of Rostock, E. Heyde-mannstr. 6, D-18057 Rostock, Germany. Phone, +49 (381) 494-7497/-7349; fax, +49 (381) 494-7482; e-mail, [email protected]

338 ANNALS NEW YORK ACADEMY OF SCIENCES

generally in an advanced stage no longer suitable for resection.1,2 Even though re-cent advances have been made, for example, using agents such as 5-fluorouracil, cis-platin and gemcitabine,3 the systemic toxicity of these agents at the concentrationsrequired had resulted in only limited success. Local delivery of chemotherapeuticagents would circumvent these problems, but this kind of approach can also beproblematic.

The compounds cyclophosphamide and ifosfamide are used for chemotherapy ofa number of tumors.4,5 These prodrugs are activated in the liver by cytochrome P450to cytotoxic metabolites, i.e., phosphoramide mustard, which alkylates DNA, andacrolein, a protein alkylating agent.6 The active metabolites are released into the cir-culation and are distributed throughout the body, resulting in toxic effects. The abil-ity to relocate the activation step to the vicinity of the tumor should alleviate theundesirable systemic toxicity while at the same time increasing the therapeutic indexof the prodrug. In order to achieve this targeting of the antitumor effect, cells genet-ically modified to express the liver-specific cytochrome P450 2B1 gene have beenencapsulated in cellulose sulphate. The encapsulated cells have been implanted inpreformed tumors derived from a human pancreatic cell line.7,8 The cellulose sul-phate capsules confine the genetically modified cells to the vicinity of the tumor andare expected to protect these cells from the host immune system.

METHODS

Cloning

The cDNA of CYP2B19 was cut out from the plasmid pSW1 using XhoI/XbaIand ligated into the XhoI/XbaI cut nonviral eucaryotic expression vector pcDNA3(Invitrogen). The final construct pc3/2B1 carries the CYP2B1 cDNA under the con-trol of the cytomegalovirus (CMV) immediate early promoter10 (FIG. 1). The yieldedDNA was proved by digest as well as sequencing.

Cell Culture

Human 293 cells used in this study were bought at ATCC (Rockville, MD). Theywere grown in Dulbecco’s modified Eagle’s medium with glutamax (DMEM(glutamax), Gibco/BRL), supplemented with 10% fetal calf serum (FCS). Every 3–4 days the cells were trypsinized and the medium was changed.

Lipofection

For transfection prepared lipofectamine (Gibco/BRL) was used. Before the dayof transfection, 3 × 106 kidney cells were seeded into 100-mm dishes. On the day oftransfection, 4 µg pc3/2B1 were prepared according to the instructions of the pro-ducer and added to the cell layer. After 6 hr, 1 ml DMEM (glutamax) with 10% FCSwas added. The next day the cells were trypsinized, diluted and put in selection. Af-ter a fortnight the resistant clones were isolated and further investigated.

339MÜLLER et al.: ENCAPSULATED CYP2B1 CELLS

Resorufin Assay and Ifosfamide Measurements

The expression of biologically active CYP2B1 in the transfectants was deter-mined using a modified biochemical assay, which is specific for the cytochromeP450 isoforms 1A1 and 2B1.11 The day before measurement different amounts ofcells (5 × 104, 2 × 105, and 2 × 106) were seeded into a 3-cm dish. The next day thecells were washed with phosphate-buffered saline (PBS) and overlayed with 500 µlserum-free medium containing 15 µM 7-pentoxyresorufin (Sigma) and 10 µM dicu-marol (Sigma). After 30 min incubation at 37°C, 375 µl of the supernatant wasmixed with 125 µl 0.1 mM sodium acetate, pH 4.5 containing 75 Fishman units ofβ-glucuronidase/600 Roy units of arylsulfatase (Boehringer). The solution was in-

FIGURE 1. Cloning of the CYP2B1 expression vector. The cDNA for CYP2B1 wascut out from the vector pSW1 and cloned into the eucaryotic expression vector pcDNA3.The gene is now driven by the constitutivly active cytomegalo virus promoter (CMV).


cubated for 3 hr at 37°C, and the reaction stopped by adding 1 ml pure methanol(Sigma). Precipitated proteins were pelleted at 3000 rpm, and the amount of pro-duced resorufin was measured with a fluorometer at 530 nm excitation and 590 nmemission.10

Ifosfamide was measured by flame ionization as described before.12,13 In brief,ifosfamide was extracted into dichloromethane using the oxazaphosphorine deriva-tive trofosfamide as the internal standard. The organic layer was evaporated, and theresidue was redissolved in ethyl acetate, an aliquot of which was injected onto afused silica capillary column under isothermal conditions using N2 as the carrier gas.Blood was drawn at the times indicated. For the comparison of tissue and serum lev-els, blood was drawn at 30 min, and the tumor was removed from the animal at thesame time and put immediately into liquid nitrogen.

Transwell Assay

To test and quantify the bystander effect, a transwell system was used (Falcon).One (1) × 105 pancreatic carcinoma cells PaCa-44 (ATCC)14 were seeded into a fil-ter insert (Falcon) of a 3-cm dish (6-well). Into the lower compartment of the dish1 × 105 wild-type or CYP2B1-expressing 293 cells were seeded. The next day ifos-famide was added resulting in concentrations from 0 up to 2 mM. After one weekthe transwell filters were removed, and the remaining carcinoma cells and theCYP2B1-expressing or wild-type cells were counted.

Encapsulation

Capsules were produced as previously described.10,15,16 Briefly, 1 × 107 cells,transfected with CYP2B1, were suspended in 1 ml PBS (pH 7) containing 2–5% cel-lulose sulphate and 5% FCS (Gibco/BRL). The suspension was allowed to drop free-ly into a precipitation bath containing 3% polydiallyldimethyl ammonium in PBS.The capsules were washed twice with normal medium (DMEM) and then either cul-tivated in cell culture dishes or, if empty, stored in PBS at 4°C. To assess possibletoxicity of the capsule material, empty capsules were injected orthotopically into themouse pancreas, as described,17,18 both in nude and immunocompetent Balb/c mice(Charles River, Germany).

Cell Lines, Animals and Experimental Protocol

The human pancreatic adenocarcinoma-derived cell line PaCa-44 (ATCC), wasused to establish tumors in nude mice.7,8 PaCa-44 cells were grown in DMEM me-dium containing 10% FCS and supplemented with penicillin and streptomycin (Gib-co/BRL). Proliferating cells were used to establish tumors in the nude mouse byinjecting 1 × 106 cells subcutaneously in the flanks of nude mice (CD-1 nu/nu;Charles River, Germany). The resultant tumors were allowed to grow for 7–10 daysuntil they reached a size of 1 cm3. The mice were divided into a number of groupson the basis of the treatment that they received (FIG. 5): (1) controls with no treat-ment and no injection; controls with injection of transfected cells (2) with and (3)without encapsulation but without ifosfamide treatment, and three treatment groupsencompassing (4) no injection of cells, (5) injection of naked cells and (6) injectionof encapsulated cells. One (1) × 106 cells were suspended in 100 µl DMEM, filled


into a 1-ml standard syringe. The capsules were delivered through a 21G needle di-rectly into the tumor. In a second series, the capsules were implanted 1 cm distant tothe preestablished tumors by subcutaneous injection. Approximately 40 capsuleswere delivered per injection. Animals were treated intraperitoneally every third dayfor 2 weeks with 100 mg/kg body weight ifosfamide (Holoxan®, Asta Medica, Ger-many). At the same time, sodium 2-mercaptoethanesulphonate (MESNA/Uromitex-an®, Asta Medica) was administered intravenously at the same dosage via the tailvein. Tumor tissue was harvested from anesthetized animals after three weeks. Thetherapeutic effect was defined as a complete response (CR), i.e., total disappearanceof the tumor or a partial response (PR), indicating that more than 50% of the initialtumor mass had been eliminated.19

RESULTS

Characterization of Cytochrome P450 2B1 Expression in 293 Cells

Human 293 cells carrying an expression vector, in which the CYP2B1 cDNA isplaced under the transcriptional control of the CMV immediate early promoter(FIG. 1), were analyzed for expression of functional CYP2B1. The specific 7-pen-toxy-resorufin dealkylating activity of CYP2B1 yields the fluorescent product re-sorufin, which can be detected by excitation at 530 nm and emission at 590 nm.

293 cells containing the gene for CYP2B1 produced measurable amounts offlouroscenic resorufin. The resorufin-dependent flourescence correlated also withthe amount of cells seeded in the assay. This demonstrates that at least the majority,if not all cells, containing the stable integrated vector pc3/2B1 express the CYP2B1gene (FIG. 2).

FIGURE 2. Correlation of resorufin activity with cell count. ! indicates CYP2B1-transfected cells; " indicates controls.


Filter Assay

The activated drug phosphoramide mustard has a small molecular mass and canpass through membranes. This causes a bystander effect that is not dependent on adirect cell cell contact.20 To show whether the released drug can also affect coculti-vated 293 cells that do not express CYP2B1, a transwell system was used. In this sys-tem the cells share the same media, but are seperated by a membrane with 0.45-µmpores.

In the presence of ifosfamide the pancreatic carcinoma cells on top of the filterinsert of the transwell system showed growth inhibition when cocultured withCYP2B1-expressing cells, but not when sharing the media with 293 wild-type cells.At a concentration of 0.5 mM ifosfamide the cell number was reduced by more than50% after one week. The presence of ifosfamide alone without activating cellscaused no cell growth inhibition. Thus, ifosfamide can be activated by CYP2B1, bereleased from the activating cell and freely diffuse in the surrounding media.

Tolerance of Empty Capsules

In order to determine the toxic potential of the capsules per se, empty capsuleswere injected orthotopically into the pancreas of nude and immunocompetent mice.A slight foreign-body reaction with macrophages and a few granulocytes could bedetected in both animal models; however, no excessive scaring or proliferation ofconnective tissue was detected (FIG. 3). No signs of pancreatitis could be observed.

FIGURE 3. Empty cellulose sulphate capsules injected orthotopically into the nudemouse pancreas. Note the sparse inflammatory reaction surrounding the capsule. Bar equals100 µm (×25).


A

B

FIGURE 4. Confocal laser microscopy of encapsulated cells in a ‘Life&Dead’ assay.Yellow/green signal indicates living cells. (A) Capsules cultured for 4 weeks after encapsu-lation. Bar equals 100 µm. (B) Capsules prepared from xenotransplanted tumors after threeweeks of in vivo treatment with ifosfamide.


Encapsulation of CYP2B1-Expressing Cells

Cells were encapsulated as described. Typically, 1–2 × 105 cells are contained byone capsule of 500 µm diameter. Enzyme activity was determined with the resorufinassay to be in the range of 0.2 pmol/L/capsule, which would be equivalent to 1 × 105

capsules since 1 × 106 cells present an enzyme activity of 2 pmol/L. After 4 weeksof continuous cultivation, viability was measured by Life&Dead assay and laser con-focal microscopy. At that time, about two thirds of the cells appeared to be viable(FIG. 4).

In order to determine the therapeutic potential of CYP450-expressing cells im-planted in the vicinity of a preformed pancreatic tumor, in combination with ifosfa-mide administration, the cells were first encapsulated in cellulose sulphate15 toensure that they would be confined to the tumor. The cellulose sulphate capsules car-rying the CYP2B1-expressing cells had a diameter of around 400 µm, so that theycould easily be injected through a 21G needle (inner diameter 0.6 mm) withoutdamage.

Evalulation in a Pancreatic Tumor Model System

The encapsulated cells were injected into a preformed tumor grown in nude miceand derived from the human pancreatic-derived PaCa-44 cells, an established animalmodel for pancreatic cancer.7,8 When injected subcutaneously into nude mice, thehuman pancreatic cancer derived cell line PaCa 44 forms tumors. Such preformedtumors were allowed to reach a size of about 1 cm3. Some of the tumors were theninjected with either 1 × 106 CYP2B1-expressing cells or about 40 capsules carryingCYP2B1 cells. The mice were then given ifosfamide systemically. Tumor growth

FIGURE 5. Effect of ifosfamide treatment in pancreatic tumors implanted withCYP2B1-producing, encapsulated cells and controls.


A

B

FIGURE 6. Gross appearance of a pancreatic tumor xenotransplanted to nude micewithout treatment (A,C) and at the same time after 3 weeks of ifosfamide treatment and in-jection of CYP2B1-expressing, encapsulated cells (B,D). Direct injection of capsules (A,B)and subcutaneous implantation of capsules 1 cm apart from the tumor (C,D).


was slightly reduced in mice that did not recieve the CYP2B1 cells (either encapsu-lated or not), presumably due to conversion of the ifosfamide in the livers of thesemice to the toxic metabolites. However, tumor reduction was most evident in micethat received encapsulated CYP2B1-expressing cells by injection into the tumor and

C

D

FIGURE 6. Continued.


subsequent treatment with ifosfamide, with 4 of the mice showing complete tumorregression (FIG. 5). Even though the ifosfamide dosage was identical in all the treat-ed mice, animals treated with encapsulated cells also appeared healthier comparedto those injected directly with CYP2B1 cells, which at best showed only a partial re-sponse (FIG. 6). A similar significant tumor reduction could also be observed if theencapsulated CYP2B1-expressing cells were implanted 1 cm apart from the pre-established tumor (FIG. 6). Upon histology, capsules appeared to be intact with via-ble cells surrounding the remaining tumor cells (FIG. 7). In capsules prepared fromtumors removed after three weeks of treatment, a substantial number of cells re-mained viable after intermediate culture (FIG. 4B) and showed resorufin activity inthe range of 0.16 pmol/capsule (data not shown). The serum levels of ifosfamide fol-lowing a single injection of one dose (100 mg/kg BW) was comparable to results ob-tained earlier in the nude mose system (FIG. 8). Tissue levels reached about 50% ofserum levels 30 min after injection (FIG. 8).

DISCUSSION

Ifosfamide is an approved chemotherapeutic agent that has been widely used inoncology for the treatment of solid tumors, including pancreatic cancer,21,22 formany years. Nevertheless, the necessary local concentrations of the toxic metabo-lites of ifosfamide, i.e., phosphoramide mustard and acrolein, are only achieved atthe expense of high systemic concentrations, since the liver is the normal site of con-

FIGURE 7. CYP2B1-expressing cells in capsules three weeks after injection into pre-established human pancreatic tumors. Note the living capsules in the intact cells and tumorcells surrounding. Magnification ×40.


version. High systemic concentrations lead to severe effects on nontarget organs.12

The local delivery of either ifosfamide or the active metabolites may circumvent thisproblem. However, ifosfamide is only poorly activated if at all outside of the liver,and direct delivery of the activated metabolites after in vitro production is hamperedby the short half-life (∼45 min) of these compounds.23

FIGURE 8. Ifosfamide levels in mice bearing pancreatic tumors injected withCYP2B1-expressing encapsulated cells. (A) Serum kinetics after a single intraperitonealdose of ifosfamide (100 mg/kg BW). (B) Serum and tissue levels. Mean ± 1 SD.


In the approach described here, cells that have been genetically modified to pro-duce CYP2B1, an enzyme that converts ifosfamide to cytotoxic metabolites, havebeen enclosed in capsules implanted in the vicinity of the tumor. The cells are thusphysically targeted to, and confined around, the tumor. Previously we have shownthat encapsulated cells injected directly into the tumor are able to activate ifosfa-mide, thus killing the cells.10 More importantly, we demonstrate a bystander effect,not only in cell culture but also in mice, since in this study the encapsulated cyto-chrome P450-expressing cells were injected in subcutaneously, 1 cm away from thetumor, yet there was still a measurable therapeutic effect after ifosfamideadministration.

The implantation of encapsulated cells as described here should allow higherlocal concentrations of the active metabolite to be maintained without systemic tox-icity. Further, such an approach should be useful, since such encapsulated cells areprotected from the host immune response.

This novel approach to targeting of chemotherapy was tested in human pancreatictumors that had been preformed in nude mice. Treatment of mice receiving the en-capsulated cells with ifosfamide resulted in a complete tumor disappearance in about20% of the animals and a significant reduction in the tumor burden in the remaining80%.10 Treatment of these mice was much more successful than control mice receiv-ing either nonencapsulated CYP2B1-expressing cells or mice that did not receivecells at all.

As demonstrated by the injection of capsules with 293 cells in the unaffected pan-creas of both nude and immunocompetent mice, no tissue reaction or pancreatitiscould be observed seven days after injection. In the case of the pancreas, this is animportant issue, since the organ is very sensitive to manipulation and ischemia. In-jection of adenovirus for gene therapy into the pancreatic duct, for instance, causedpancreatitis. Thus, the local application of such capsules seems feasible.

This novel strategy for the treatment of solid tumors combines gene/cell therapywith chemotherapy. However, since a well characterized CYP2B1-expressing cellclone is encapsulated and delivered to the tumor, no direct gene therapeutic interven-tion is necessary in the patient. This simplifies the treatment and enhances the safetyof the system as compared to other gene therapy approaches for cancer. A prerequi-site for the use of the same cell line in different patients is that the cells are not elim-inated by immune responses. In a number of experiments we have not detected anyobvious immune response in immunocompetent mice to encapsulated cells, in thepancreas (not shown) and in the mammary gland. Our previous study reported theuse of feline kidney cells as the cell line for expression of CYP2B1. Although thesecells expressed good levels of cytochrome P450, resulting in a therapeutic antitumoreffect upon application of ifosfamide, such cells may not be suitable for eventualclinical use. These cells produce endogenous retroviruses, which may be releasedfrom the capsules and result in the establishment of a productive infection. The 293cell line does not appear to produce endogenous retroviruses.24 A further advantageto the use of cell lines of human origin may be more applicable for human clinicaltrials, because they are complement resistant.25

In summary, we have demonstrated the feasibility of a gene therapy approach toa model system of human pancreatic carcinoma. The marked response including asubstantial number of complete remissions led to a phase I clinical protocol.26


ACKNOWLEDGMENTS

We thank Jim Halpert for the cytochrome P450 cDNA, Jörg Pohl (Asta Medica)for helpful discussions about ifosfamide, and Nora Sartori for her help in setting upthe chemotherapy protocol. We would like to express our sincere thanks to GiselaSparmann for her input and support. This work was supported by a Vaestfond grantfrom the Danish government to Bavarian Nordic Research Institute and EC GrantBIO4-CT-0100.

REFERENCES

1. ROSEWICZ, S. & B. WIEDENMANN. 1997. Pancreatic carcinoma. Lancet 349: 485–489.2. KALTHOFF, H., M. LÖHR, C. ROEDER & W. SCHMIEGEL. 1995. Das Pankreaskarzinom:

Zellbiologie, Matrixproteine und Wachstumsregulation. In Erkrankungen des exkre-torischen Pankreas. 2nd edit. G. Adler, U.R. Fölsch, J. Mössner & M.V. Singer, Eds.:385–404. Fischer. Jena.

3. CARMICHAEL, J., U. FINK, R.C.G. RUSSELL et al. 1996. Phase II study of gemcitabinein patients with advanced pancreatic cancer. Br. J. Cancer 73: 101–105.

4. KEIZER, H.J., J. OUWERKERK, K. WELVAART, C.J.H. VAN DER VELDE & F.J. CLETON.1995. Ifosfamide treatment as a 10-day continuous intravenous infusion. J. CancerRes. Clin. Oncol. 121: 297–302.

5. AHLGREN, J.D. 1996. Chemotherapy for pancreatic carcinoma. Cancer 78: 654–663.6. DIRVEN, H.A.A.M., B. VAN OMMEN & P.J. VAN BLAEDEREN. 1996. Glutathione conju-

gation of alkylating cytostatic drugs with a nitrogen mustard group and the role ofglutathione S-transferases. Chem. Res. Toxicol. 9: 351–360.

7. LÖHR, M., B. TRAUTMANN, M. GÖTTLER et al. 1994. Human ductal adenocarcinomasof the pancreas express extracellular matrix proteins. Br. J. Cancer 69: 144–151.

8. LÖHR, M., B. TRAUTMANN, S. PETERS et al. 1996. Expression and function of integrinsin human pancreatic adenocarcinoma. Pancreas 12: 248–259.

9. KEDZIE, K.M., C.A. BALFOUR, G.Y. ESCOBAR et al. 1991. Molecular basis for a func-tionally unique cytochrome P450IIB1 variant. J. Biol. Chem. 266: 22515–22521.

10. LÖHR, M., P. MÜLLER, P. KARLE et al. 1998. Targeted chemotherapy by encapsulat-ing cells engineered to deliver CYP2B1, an ifosfamide activating cytochrome P450gene. Gene Ther. 5: 1070–1078.

11. DONATO, M.T., M.J. GOMEZ-LECHON & J.V. CASTELL. 1993. A microassay for mea-suring cytochrome P450IA1 and P450IIB1 activities in intact human rat hepato-cytes cultured on 96-well plates. Anal. Biochem. 213: 29–33.

12. KUROWSKI, V. & T. WAGNER. 1993. Comparative pharmacokinetics of ifosfamide, 4-hydroxyifosfamide, chloracetaldehyde, and 2- and 3-dechloroethylifosfamide inpatients on fractionated intravenous ifosfamide therapy. Cancer Chemother. Phar-macol. 33: 36–42.

13. KUROWSKI, V. & T. WAGNER. 1997. Urinary excretion of ifosfamide, 4-hydroxyifos-famide, 3- and 2-dechloroethylifosfamide, mesna, and dimesna in patients on frac-tionated intravenous ifosfamide and concomitant mesna therapy. CancerChemother. Pharmacol. 39: 431–439.

14. OLSEN, D., T. NAGAYOSHI, M. FAZIO et al. 1989. Human laminin: cloning andsequence analysis of cDNAs encoding A, B1 and B2 chains, and expression of thecorresponding genes in human skin and cultured cells. Lab. Invest. 60: 772–782.

15. STANGE, J., S. MITZNER, H. DAUTZENBERG et al. 1993. Prolonged biochemical andmorphological stability of encapsulated liver cells—a new method. Biomat. Art.Cells Immob. Biotech. 21: 343–352.

16. SALLER, R.M., J. STANGE, S. MITZNER, U. HEINZMANN, B. SALMONS & W.H. GÜN-ZBURG. 1997. Encapsulated cells producing retroviral vectors for in vivo gene ther-apy. Submitted.


17. REYES, G., A. VILLANUEVA, C. GARCIÁ et al. 1996. Orthotopic xenografts of humanpancreatic carcinomas acquire genetic aberrations during dissemination in nudemice. Cancer Res. 56: 5713–5719.

18. LÖHR, M. 1996. Zell- und Molekularbiologie der Bindegewebsreaktion bei Pankre-atitis und Pankreaskarzinom. Solvay. Hannover.

19. WHO. 1979. WHO Handbook for Reporting Results of Cancer Treatment. WorldHealth Organization (WHO). Geneva.

20. CHEN, L. & D.J. WAXMAN. 1995. Intratumoral activation and enhanced chemothera-peutic effect of oxazaphosphorines following cytochrome P-450 gene transfer:development of a combined chemotherapy/cancer gene therapy strategy. CancerRes. 55: 581–589.

21. CERNY, T., G. MARTINELLI, A. GOLDHIRSCH et al. 1991. Continuous 5-day infusion ofifosfamide with mesna in inoperable pancreatic cancer patients: a phase II study. J.Cancer Res. Clin. Oncol. 117(Suppl. IV): S135–S138.

22. EINHORN, L.H. & P.J. LOEHRER. 1986. Ifosfamide chemotherapy for pancreatic carci-noma. Cancer Chemother. Pharmacol. 18(Suppl. 2): 51–54.

23. ZHENG, J.J., K.K. CHAN & F. MUGGIA. 1994. Preclinical pharmacokinetics and sta-bility of isophosphoramide mustard. Cancer Chemother. Pharmacol. 33: 391–398.

24. PEAR, W.S., G.P. NOLAN, M.L. SCOTT & D. BALTIMORE. 1993. Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA90: 8392–8396.

25. TAKEUCHI, Y., C.D. PORTER, K.M. STRAHAN et al. 1996. Sensitization of cells andretroviruses to human serum by (alpha 1-3) galactosyltransferase. Nature 379: 85–88.

26. LÖHR, M., Z.T. BAGO, H. BERGMEISTER et al. 1999. Cell therapy using microencap-sulated 293 cells transfected a gene construct expressing CYP2B1, an ifosfamideconverting enzyme, instilled intra-arterially in patients with advanced-stage pan-creatic carcinoma. A phase I study. J. Mol. Med. 77: 393–398.

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