IL-t2 alone. Methods: We implanted poorly immunogenic murine colon cancer cells (MCA26) subcapsularly into the liver to form a solitary metastasis. At a size of 5x5 ram, it was then injected with the gene therapy vectors. We constructed two recombinant edenoviruses expressing the extracellular domain of 4-1BBL under the control of a constitutive viral promoter (RSV); either as a monomer (ADV.mono4-1BBLs), or as a trimer using an isoleucine zipper (ADV.tri-4-1BBLs). Results: I ntretumoral injection of 1 x10^11 viral particles of each 4-1BBL expressing viruses resulted in >70% tumor volume reduction vs. control adenovirus (P<O.01). The combination of ADV.lL-12 plus ADV.tri-4-1BBLs (closed squares) resulted in tumor eradication and in an increased long-term survival compared to ADV.IL-12 alone (open circles) or ADV.IL-12 plus ADV.mono4-1BBLs (crosses) (Fig.). Conclusion: Gene transfer of 1L-12 and trimedzed 4-1BBLs into liver metastases of colon cancer may prove to be an effective treatment that is readily translatable into the clinic.

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................... X

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D a y s P o s t T u m o r I m p l a n t a t i o n

51

Inhibition of the Ras/MAPK Pathway Upregulates the Human Coxsackie and Adenoviras Receptor (CAR) and Increases the Uptake of Adenoviruses in Cancer Cells. Mario Anders, Rong Xiang Ding, Allan Balroain, Frank McCormick, W. Michael Korn, Comprehensive Cancer Ctr, UCSF, San Francisco, CA

Background/Objective: The recently identified human Coxsackie and Adenovirus receptor (CAR) represents the primary cellular site of virus attachment during infection. As genetically modified adenoviruces are currently explored as anti-cancer agents, information about the molecular mechanisms regulating CAR expression is needed. In this study we characterized the expression of CAR in colorectal cancer cell lines and in a mouse skin cancer progression model in relation to the Ras/MAPK pathway. Methods: The human co(orectal cancer cell lines HCT116, SW480, and HT29 were used for this study. CAR expression was analyzed by FACS, Western blotting, Northern Blotting, and real-time PCR. A non-replicating, GFP-expressing adenovirus was used to measure virus uptake and gene delivery. Replication and cytopathic effect of viruses (e.g. Ad5 and Onyx015) were examined by Plaque- and CPE assays, respec- tively. The expression of CAR in tissue sections and cell lines of a mouse skin cancer progression model was determined by immunohistochemistry and Western Blotting. Results: The expression of CAR decreases significantly from high levels in mouse skin papillomas to very tow levels in spindle cell carcinomas, whereas the activity ol the MAPK pathway is increased due to amplification of mutant Ras. This downregulation of CAR is reflected in an impaired virus entry into the highly malignant cell lines. The inhibition of the Ras/MAPK pathway by the MEK inhibitor U0126 leads to a significant upregulation of CAR in all examined cell lines, resulting in a significant increase of uptake, replication and cytopathic effect of adenoviruses. Conclusions: This study demonstrates for the first time that signaling via the Ras/MAPK pathway regulates CAR expression, and affects thereby adenovirus entry into cells. We also show that the inhibition of the Ras/MAPK pathway provides a way to upregulate CAR expression in cancer cells, which may improve the efficacy of adenovirus based cancer therapy.

52

Gene Transfer Of Mutated Sodium-Ion Channel Burst Gastric Cancer In Vivo. Macayoshi Horimoto, Yutaka Sasaki, Takashi Toyama, Takayuki Yakusbijin, Kenya lyoda, Masatsugu Hod, Norio Hayashi, Osaka Univ Graduate Sch of Medicine, Suita Japan

BACKGROUND & AIM: Cancer gene therapy must target cancer cells in order to be effective. The promoter element of tumor-expressing molecules can be used to induce specific expression of the transfected genes in the tumor. However, tumor-specific promoters such as CEA induce less gene expression than do virus promoters such as the SV4O- and CMV-promoters. Therefore, we sought a gene whose expression could induce tumor cell death regardless of the transcriptional efficiency of the promoters. Mammalian degenerin (MDEG) is a member of the novel amilodde-sensitive sodium-ion channel family, and its site-directed active mutant (MDEG-G430F) induces massive Na* influx into cells, leading to cell ballooning and cell bursting. We attempted a novel therapeutic approach for gastric cancers by transferring MDEG-G430F into cancer cells using tumor-specific promoters. METHODS:MDEG-G43OF cDNA in which Glycine430 was replaced by Phenylalanine430, was prepared using the QuickChange Site-Directed Mutagenesis kit. MKN45-P which was used as the CEA-producing cell line,and lx10 ~ cells were injected into the peritoneal cavities of each female BALB/c nude mouse. The inoculated mice were divided into three groups(Experimental group, Mock group, and Control group). Intrepedtoneal injection of Fusogenic-liposomes containing plasmids was started on day 3 and repeated once a week for each group.Serum CEA levels were measured each week and the mortality rate was monitored. RESULTS: In carcinoembryonic antigen (CEA)-producing gastric cancer cells, MDEG-G43OF produced similar levels of cell death when using a CEA- promoter as when using a potent nonspecific promoter such as the CMV-promoter. In an in vivo study, fusogenic-liposome complexes containing MDEG-G43OF driven by the CEA-pro- meter were intraperitoneally injected into CEA-producing gastric cancer cells in a mouse peritoneal dissemination model. Although all 15 of the control mice were dead 50 days after inoculation, 13 of the 15 mice treated with MDEG-G430F survived. CONCLUSION: These results indicate that transferring mutated sodium-ion channel into cancer tissues using tumor- specific promoters can achieve striking and selective cancer cell death irrespective of the

transcriptional efficiency of the promoters used in vivo, and suggest that this approach is a promising new strategy for cancer gene therapy.

53

High Efficiency Gene Transfer to Pancreatic Cancer Cells Using Epidermal Growth Factor Receptor and Integrin Targeted Adenoviral Vectors John G. Wesseling, Univ of Amsterdam, Amsterdam Netherlands; Masato Yamamoto, Univ of Alabama at Birmingham, Birmingham, AL; Piter J. Bosma, Univ of Amsterdam, Amsterdam Netherlands; Victor Krasnykh, Jerry L. Blackwell, Salwyn M. Vickers, Igor Dmitriev, David T. Curiel, Univ of Alabama at Birmingham, Birmingham, AL

Background: Pancreatic cancers are highly aggressive and mostly diagnosed late in its clinical stage. As available therapies show limited success, new therapeutic approaches have been sought, i.e. adenoviral (Ad) suicide gene therapy. However, poor efficiency of adenoviral gene transfer in pancreatic cancer cells due to coxsackie-adenovirus receptor (CAR) deficiency has hampered the application of adenovirus-based suicide gene therapy in this field. In this study, to overcome the poor gene transfer by CAR independent adenoviral infection, we apply epidermal growth factor receptor (EGFR) mediated targeting with sCAR-EGF complex as well as integrin mediated targeting using the adenovirus vector with integrin binding motif (RGD) in the knob region (RGD-Ad). Methods: Two primary (p6.3 and p10.5) and four established (BxPC-3, Capan-f, Hs766T and MIAPaCa-2) pancreatic carcinoma cells were used for the analysis. The expression of CAR, EGFR, a v ~ integrin and ~v~ integrin was analyzed by flowcytometry. The gene transfer with Ad/sCAR-EGF was compared with Ad/sCAR-6His (non specific) and parental Ad at various multiplicity of infection (MOI). The gene transfer with RGD-Ad was compared with regular Ad at different MOIs and the effect of blocking with unmodified knob protein was also analyzed. Results: The flowcytometry revealed all 6 pancre- atic carcinoma cells were CAR negative but EGFR positive. Also, they were all positive for either ~/33 or ~,85 integrin. When sCAR-EGF was combined with the Ad vector, 1.5-5 fold enhancement of gene transfer was observed on EGFR positive pancreatic cancer cells in comparison with sCAR-6His. When RGD-Ad was compared with regular Ad vector, RGD-Ad showed 100-500 fold higher gene transfer in pancreatic cancer cells. The existence of the regular Ad knob during infection did not affect the gene transfer with RGD-Ad, while it did inhibit the gene transfer with the regular Ad vector. Conclusion: Both sCAR-EGF and RGD- Ad could increase the gene transfer dramatically. These methods to overcome low Ad infectivity due to CAR deficiency may help the development of really feasible gene therapy for pancre- atic cancers.

54

Improved Gene Transfer To Esophageal Adenocarcinoma And Squamous Carcinoma Cells Using Targeted Adenovirus Vectors. Willem A. Marsman, Chriatianne J. Buskens, John G. Wesseling, Acad Medical Ctr, Amsterdam Netherlands; Hidde J. Haisma, Univ of Groningen, Gruningen Netherlands; David T. Curiei, Univ of Alabama, Birmingham, AL; Jacques J. Bergman, Jan Jb Lanschot, Piter J. Bosma, Aced Medical Ctr, Amsterdam Netherlands

BACKGROUND: The incidence of esophageal adenocarcinoma is rising and conventional therapies remain inadequate. Adenoviral (Ad) gene therapy is a promising alternative. An important limitaton, however, is the low expression in many malignancies of the Coxsackie- Adenovirus receptor (CAR), involved in adenoviral entry. A retargeted edenovirus, that can enter independently of CAR expression, should improve the infection efficiency in many malignancies. AIM: To investigate different targeting strategies for adenovirel gene-therapy in esophageal carcinomas. METHODS: Genetic retargeting was performed by the insertion of Arg-Gly-Asp (RGD) in the fiber knob (AdRGD), which a[tows CAR independent ce[I entry via integrins. Also bispecific antibodies were used, directed to the adenovirus knob on one side and on the other side to the Epithelial cell adhesion molecule (EpCAM) or the epidermal growth factor receptor (EGFR). These proteins are often upregulated in malignancies. Infections were performed in two esophageal squamous carcinoma cell lines (TE-1 and TE-2) and two esophageal adenocarcinoma cell lines (JROECL33 and OACM1.4C). Two cell lines known to be efficiently infected by all three modified Ad-vectors were used as positive controls. All recombinant Ad-vectors contained the luciferese gene behind a CMV promoter. The cell lines were incubated with non-targeted adenovirus and the three targeted vectors at an m.o.i, of 1 and 1 O, in duplicate. 48 hours after infection the cells were homogenized and luciferase activity was measured. RESULTS: Targeting towards EGFR or EpCAM using bispecific antibodies did not increase the infection efficiency in either squamous carcinoma cell line. In both esophageal adenocarcinoma cell lines a 2 to 3 fold increase was seen, compared to the non-targeted vector. In contrast, targeting towards the integrins using AdRGD resulted in a 50 to 500 fold increase of luciferase expression in the squamous- and adenocarcinoma esophageal cells. CONCLUSION: These data show that AdRGD infects adenocaminoma and squamous esopha- geal carcinoma much more efficiently than non-targeted adenovirus. Thus, an adenovirus targeted to the integrins seems a more promising vector for gene therapy in esophageal carcinoma. Similar experiments in an ex vivo explant system of biopsies of normal and cancerous tissue from the esophagus are ongoing to investigate these targeting methods in a more physiologic setting.

55

Epidermal Growth Factor Enemas Are Effective in The Treatment Of Leg-sided Ulcerative Colitis Atul Sinha, Jeremy Md Nightingale, Kevin P. West, Univ Hospitals of Leicester, Leicester United Kingdom; Jorge Berlanga-Acosta, CIGB, Havana Cuba; Raymond J. Playford, Imperial Coil Sch of Medicine, London United Kingdom

Background: Epidermal Growth Factor (EGF) is a 53 amino acid molecule produced by the salivary glands that stimuletea intestinal mucosal cell proliferation. Studies have suggested a role for EGF in wound repair and ulcer healing. Aim: To examine whether EGF enemas are effective in the treatment of active left-sided ulcerative colitis. Methods: A randomized double

A - 1 1

blind controlled clinical trial was performed. Outpatients with active left-sided ulcerative colitis were either started on mesalazine 1.2 gm/day or had the dose increased by an equivalent amount. They were taught to self-administer enemas which contained either EGF (5 mcg EGF in 100 ml) or an inert carrier (control) once a day for 14 days. They were reviewed at 14 and 28 days. Results: Eleven patients received EGF and 12 placebo. Both groups had similar baseline characteristics. Eight patients in the EGF group and nine in the p~acebo group were on mesalazine before recruitment to the study. Two patients from the placebo group developed worsening colitis and were withdrawn. After 2 weeks of enema treatment, there were significant improvements in the EGF treated group in symptom score (score 0 or 1 for the absence or presence of liquid stools, nocturnal diarrhoea and visible blood in stools) from median 3 to 1 (p=O.O02), diarrhoea from median 5 to 2 motions/24 hours (p<O.05), sigmoidoscopic score (Baron et al Br Med J 1:89-92, 1964) from median 3 to 1 (p = 0.004) and histological score (Richards eta/ Br Med J 1:160-165, 1960) from median 3 to 1 (p=O.O02). These parameters were significantly better in the EGF group than in the placebo group at 2 and 4 weeks (p<O.O05 for all). No significant change in these parameters was seen in the placebo group. Nine (82%) patients in the EGF group as compared to one (8%) in the placebo group achieved remission after 2 weeks (p = 0.005). Eight of these 9 maintained their remission up to the end of the study. At 4 weeks, nine patients in the EGF group and three in the placebo group were in remission (p<O.O01). Conclusions: EGF enema is an effective treatment for left-sided ulcerative colitis. A dose increase of 1.2 gm mecalazine had little effect in inducing remission in these patients.

56

Idiosyncratic Adverse Reactions to 6-Meroaptopudne (6-MP) and Azathiopdne (AZA) Can Be Averted by Switching to Thioguanine(6-TG) in Patients with IBD. Maria C. Dubinsky, Edward Feldman, Maria T. Abreu, Cedars-Sinai Medical Ctr, Los Angeles, CA; Ernest G. Seidman, Ste-Justine Hosp, Montreal Canada; Darren Baroni, Tripler AMC, Honolulu, HI; Asher Kornbluth, Mount Sinai Medical Ctr, New York, NY; Stephan R. Targan, Eric A. Vasiliauskas, Cedars-Sinai Medical Ctr, Los Angeles, CA

Background: Approximately 15% of IBD patients receiving 6-MP/AZA develop idiosyncratic adverse drug reactions necessitating early discontinuation of traditional thiopurines. These reactions, which include pancreatitis, abdominal pain, fever, arthralgias, royalgias, dermatitis, nausea, fatigue, typically reoccur upon rechallenge. Our recent pilot study suggested that 6- TG, a closely related thiopurine, was efficacious and well tolerated in a subgroup of IBD patients resistant to 6-MP. Aim: To determine if 6-MP/AZA induced idiosyncratic adverse reactions can be avoided by swifcbing to 6-TG therapy. Methods: This cohort of IBD patents was compiled from the experience of a group of gastroenterologists who used 6-TG as an alternate thiopurine in patients intolerant to 6-MP/AZA. Idiosyncratic reactions must have been documented within 1 month of 6-MP/AZA initiation. Results: 6-TG was initiated in 17 IBD patients at a median [range] dose of 20 [10-40] mg/day. Median age was 43 [9-63] years. An idiosyncratic reaction reoccurred in only 1/17 patients (see table) at the same 2 week time point as had been previously experienced with 6-MP. Conclusions: The results of this observational study suggest that 6-TG is well tolerated and can be used as an alternate thiopurine in patients intolerant to traditional 6-MP/AZA. Based on these results, combined with the previously suggested efficacy data, further evaluation of 6-TG is warranted in IBD.

F~equency of Idiosyncratic reaclJons on 6-MP/AZA vs 6-TG

IDIOSYNCRATIC 6-MPI/~ 6-TG REACTION (n) (n)

pancreatitis 6 0 acute abdominal pain 3 0 feverlmyalgiaslarth ralgias 4 1 rash 1 0 nausea 1 o fatigue 2 0 TOTAL 17 1

57

Continuous Oral Plus Topical 5-ASA Administration SignificaMly Modifies The Clinical Course Of Ulcerative Colitis Mariateresa Mt Pimpo, Brigida B. Galleth, Giovanni G. Latella, Maria M. Chlaramonte, Gastroenterology Unit, L'Aquila Italy; Renzo R. Caprilli, Gastroenterology Unit, Rome Italy; Giuseppe G. Fried, Gastroenterology Unit, L'Aquila Italy

Backgrouod/Aim A cross-sectional study has shown that concentration of Meealazine (5-ASA) in the large bowel mucoca is inversely correlated to severity of ulcerative colitis ~. This study was carried out with the aim to evaluate whether maintaining a long term high mucosal colonic concentration of 5-ASA could modify the clinical course of moderate to severe disease. To date, the only established way to obtain the highest 5-ASA mucocal concentration in distal cotonic segments is the association of oral plus topical treatment 2. Methods. Eighteen consecutive UC patients in clinical remission which had had at least four moderate to severe relapses in the preceding two years (controlperiod), were assigned to a two years continuous oral plus topical 5-ASA treatment (study period). The previous treatment for all patients consisted of continuous oral 5-ASA and, during recurrences, steroids and/or topical 5-ASA. The treatment of the study period consisted in oral 5-ASA 2.4/4.8 g/day plus topical 5-ASA 12/28 g/week. Humber of recurrences, number of non-programmed visits and endoscopy, courses of steroids and days of hospitalisation were evaluated in the study period and compared with the same data recorded in the control period. The comparison was possible since in our IBD study group all patients are submitted to an identical follow-up procedure that assure a periodical or critical assessment of standardised parameters. A non parametric test for paired data (signed rank test) was used for statistical analysis. Results. Significantly lower number of recurrence (p =0.002) number of non-programmed visits (p=O,O02) and endoscopy (p = 0,016), courses of steroids (p = 0.004) and days of hospitalication (p = 0.03) were observed during the two years of oral plus topical treatment in comparison with the control period. In particular, only two patients had one mild recurrence while the remaining

patients maintained a complete remission during the two years of follow-up. Conclusion. The combined oral plus topical 5-ASA treatment, continuously administered along two years, s~gnificantly modifies the clinical course of the moderate to severe UC. It is speculated that this effect has been obtained by increasing mucosal drug concentration in the study period with respect to control period. • G Fneri, Giacomelh R, Pimpo MT et al Gut 2000, 47: 410- 14 ;G Frieri, MT Pimp& GC Palumbo et al : Aliment Pharmacol Thor 1999; 13:1413-17

58

Molticenter, Randomized Trial Comparing Oral, Topic and Oral Plus Topic Mesalazine in PreveMion of Relapse in Distal Ulcerative Colitis (DUC). Jnaquin Hinojosa, Agueda Abad, Julian Panes, Ignacio Fernandez-Blanco, Carlos M de SOl& Miquel A. Gassull, Spanish Group for the Study of IBD (GETECCU) Spain

A previous study of GETECCU (Spanish Group for Study of IBD) has proved that topical and combined treatment with mesalazine are equally effective and better than oral therapy alone in inducing remission in active Oista[ Ulcerative Colitis (OUC) (Gastroenterology 2000;118:A778-9). However,ther'snt defined guides-lines in the prevention of relapse with mecalazine in the patients with DUC. Aim:Randomized,controlled trial comparing, in a large series of patients, the long-term effectiveness of oral, topic (foam) or combined mesalazine treatment in maintaining clinical and endoscopic remission Jrl patients with mild to moderately active OUC. Method:67 patients in complete remission were randomized to receive mesalazine as rectal foam (1 g/48h, n=27), oral tablets (500 mg tid,n=17)or combined treatment (n = 23). Disease activity was assessed by a score combining clinical and endoscopic parame- ters, ranging from 0 to 6. Complete remission was defined as a score --2 and relapse when it was>2. Treatment was administered for 12 months; relapses rates were assassed each 3 months. Results: All groups were homogeneous at baseline. The global relapse was 15%(10/ 67 patients) at 12 months (8/10 at 6 m). The table shows the relapse rates in each treatment group. All treatmentS were well tolerated and mild side effects were observed only in 7 patients. Condosions:l )Mesalazine is effective in maintaining remission in patients with DUC;2) Oral,topical and combined treatment with mesalazine are equally effective in prevention of relapse

ReJape= reZe=

TOPICAL ORAL COMBINED N Lose R N Lose R N Lose R

3m 27 5 1 17 6m 21 1 2 11 9m 18 5 1 10 t2m 12 1 0 8 ToMI 2 12 4(15%) 1

i)>0.05 R=r~p~

5 t 23 3 1 0 1 19 5 2 2 0 12 3 1 t 0 8 0 0 8 2(t1%) 23 11 4(17%)

59

InpatieM And OutpMieM Costs Of Care For IMlammMoP/Bowel Disease In The Year 2000 Michael 0. Rice, Kristen O. Arsefleau, Stephen J. Bickston, Fabio Cominelli, Univ of Virginia Health System, Charlottesville, VA

BACKGROUND: Inpatient and outpatient direct costs for inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are substantial and have not been estimated in a single US population since 1990. Changes in health care delivery and introduction of costly new drugs may have impacted total direct costs, as well as the distribution of costs among inpatient and outpatient care. The aim of this study was to assess the distribution of inpatient and outpatient costs for IBD patients admitted to a University Hospital (UVA) during fiscal year 2000 (FY2000). METHODS: The Clinical Data Repository (COR) is an administrative database containing clinical, financial, and demographic data on all inpatient and outpatient admissions to UVA since 1993. Adult I BD patients treated at UVA during FY2OO0 were identified by ICD-9 codes. Costs for inpatient and outpatient visit were calculated using cost-charge ratios and aggregated by revenue code. Previously published cost data from 1990 were inflation-adjusted to FY2000 US dollars (Hays etal. J Clin Gastroenterol 1992;14:318-27). Two physicians reviewed the medical records of a random 30% subsample to validate ICD- 9 diagnosis coding. RESULTS: We identified 310 CD and 182 UC patients (90 inpatient cases, 1198 outpatient cases). Our cohort was 85% white and 53% female. ICD-9 coding accuracy was 97%. During FY2000, total annual direct costs were $1,037,360 for CO and $412,147 for UC. The average cost per CD patient was lower in 2000 than in 1990 ($3,346 vs. $8,521 [inflation-adjusted)), but similar for UC ($2,265 vs. $1,934 [inflation-adjusted)). CD total costs were evenly distributed between inpatient and outpatient care (54.8% vs. 45.2%), but UC costs were heavily weighted towards inpatient care (89.6%). CD total direct costs were distributed as follows: drug/pharmacy=44.9%, surgery=10.9%, laboratory=4.8%, radiol- ogy = 3.6%, endoscopy = 3.8%, pathology = 1.1%, clinic = 2.3%, and other (room/board, emergency room, etc)=28.6%. The distribution for UC was: drug/pharmacy=7.3%, sur- gery = 34.1%, laboratory = 4.4%, radiology = 1.5%, endoscopy = 5.0%, pathology = 3.9%, clinic = 1.0%, and other = 42.8%. CONCLUSIONS: The average cost per CD patient in FY2000 was lower than in 1990, but remained constant for UC. Our results suggest that despite the high cost of new biological therapies for CD, the overall direct medical costs per patient have decreased due to a shift in practice away from hospitalizations and surgery.

A-12