in vivo 19: 269-276 (2005) Antitumor Activity of Trovafloxacin in an Animal Model HARAGOPAL THADEPALLI1, FRANK SALEM1, SEE KEAN CHUAH1 and SASTRY GOLLAPUDI1,2 1Charles R. Drew University, School of Medicine, Los Angeles, CA, 90059; 2University of California, Irvine, CA 92697, U.S.A. Abstract. Quinolone antibiotics come close to being ideal showed antitumor activity and it was found that these chemotherapeutic agents in that they are administered orally, are quinolones had two halogens at C-6, C-8 and N-1 cyclopropyl concentrated in cells and tissue, are readily available, relatively (not methyl or ethyl) subtituents on their chemical structures. safe and exhibit increased activity against both bacteria and Previously, we have reported that certain quinolones possess tumor cells both in vitro and in vivo. Our objective was to chemosensitizing activity and they reverse multidrug resistance evaluate the in vivo activity of trovafloxacin and ciprofloxacin in cancer cells (9,10). Furthermore, quinolones appear to have against murine leukemic cells in neutropenic mice with lung immunopotentiating properties (11, 12). Clearly these infection due to Klebsiella pneumoniae. The results showed that properties of quinolones are relevant to their therapeutic both trovafloxacin and ciprofloxacin were effective in clearing efficacy against infections associated with cancer. lung infection. However, trovafloxacin, but not ciprofloxacin, Trovafloxacin is a trifluoroquinolone that is effective against was effective in preventing metastasis of leukemia cells to the various aerobic and anaerobic gram-positive and gram-negative lungs and other tissue and in prolonging the survival of mice. organisms (see ref in 13). We recently reported that trovafloxacin was as effective as ciprofloxacin in clearing An antimicrobial agent that is active against both gram-negative Klebsiella pneumoniae lung infection in mice (13). However, in and gram-positive aerobic and anaerobic bacteria can be used tumor-bearing mice, trovafloxacin was found to be more as a single antibiotic for prophylaxis as well as treatment of effective in clearing the K. pneumoniae infection. This could not bacterial superinfections in cancer patients receiving be explained by the antimicrobial activity of these quinolones chemotherapy. Obviously, if such an agent is also effective in because the organism was equally susceptible to both preventing the growth of tumor cells, it is a double bonus. trovafloxacin and ciprofloxacin. In vitro evaluation of these Fluoroquinolones are inhibitors of prokaryotic DNA gyrase, a quinolones showed that trovafloxacin (but not ciprofloxacin) DNA topoisomerase. The quinolone antimicrobials share was very effective in inhibiting the growth of P388 murine structural features with flavonoid anticancer agents (1-4). leukemia cells (14). The purpose of this study was to examine Several quinolone derivatives were observed to inhibit the effect trovafloxacin and ciprofloxacin might have on the eukaryotic cell growth in vitro (5-8). Yamashita et al. (7) resolution of invasive cancer disease in the above animal model. evaluated several quinolones for their direct antitumor activity in the murine leukemia P388 model. They showed that many Materials and Methods antimicrobial quinolones at low concentrations do not induce mammalian topoisomerase II-mediated cleavage of DNA and Animals. Six to 8 week-old male DBA/2 mice were used. do not exhibit antitumor activity. However, 12 quinolones Tumor cells. P388 murine leukemia cells were maintained in vitro as a suspension culture in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 Ìg/ml penicillin and 50 Ìg/ml streptomycin. Correspondence to: Haragopal Thadepalli, MD, Professor of Medicine and Pathology, Division of Geriatrics/Gerontology, Tumor induction and therapy. One million P388 murine leukemia Department of Internal Medicine, Charles Drew University of cells were injected into the peritoneal cavity of the mice. The mice Medicine and Science, 1731 East 120th Street, Los Angeles, CA were treated with daunorubicin (DNR 10 Ìg/kg/dose, once a day) 90059, U.S.A. Tel: (323) 563-4822, Fax: (323) 563-9393, e-mail: for 3 days. DNR was administered intraperitoneally. Chemotherapy [email protected] was initiated 24 hours after the introduction of tumor cells. ∫ey Words: Trovafloxacin, antitumor activity, quinolone antibiotics, Antimicrobial chemotherapy. Therapy was initiated on the fourth ciprofloxacin. day. The mice were treated with alatrovafloxacin (CP-116, 517-27), 0258-851X/2005 $2.00+.40 269 in vivo 19: 269-276 (2005) Table I. Survival of tumor-bearing mice treated with trovaflocacin, Table II. Survival of tumor-bearing mice treated with trovaflocacin, ciprofloxacin, or Daunorubicin (DNR) (First trial). ciprofloxacin, or Daunorubicin (DNR) (Second trial). Tumor-bearing Survivors/ Survival Number of Tumor-bearing Survivors/ Survival Number of mice treated with total rate survivors mice treated with total rate survivors number (%) with sterile number (%) with sterile of mice cultures of mice cultures per group (% cure ) per group (% cure ) NO DNR/Saline 0/11 0 0(0) NO DNR/Saline 1/20 5 0(0) NO DNR/ Trovafloxacin 1/11 9 1(100) NO DNR/ Trovafloxacin 9/24 38 9 (100) NO DNR/Ciprofloxacin 2/11 18 2(100) DNR/Saline 1/17 6 0 (0) DNR/Saline 1/11 9 0 (0) DNR/Trovafloxacin 14/20 70 13(93) DNR/Trovafloxacin 10/11 91 10 (100) DNR/Ciprofloxacin 5/19 26 3( 60) DNR/Ciprofloxacin 2/11 18 1 (50) No DNR/Trovafloxacin/rGCSF 10/25 40 2 (20) No DNR/Trovafloxacin/rGCSF 6/12 50 5 (83) DNR/Trovafloxacin/rGCSF 12/17 71 12(100) DNR/Trovafloxacin/rGCSF 10/12 83 10 (100) DNR/Ciprofloxacin/rGCSF 7/17 41 7 (100) a prodrug of trovafloxacin (Pfizer Inc., Groton, CT, USA) or Autopsy. At autopsy, the tumor was found metastatic to the alatrovafloxacin plus GCSF. It was compared with another liver, spleen and mostly the lungs. In control animals, gross quinolone ciprofloxacin alone and ciprofloxacin with the rGCSF. sections of the solid organs showed metastasis. The lung was Trovafloxacin, ciprofloxacin and GCSF were given intraperitoneally leathery hard and grossly consolidated with minimal air in three times a day at 4-hour intervals. The dosage of alatrovafloxacin and ciprofloxacin were 30 mg/kg/dose and 20 controls as well as animals treated with DNR with or mg/kg/dose, respectively. The prodrug alatrovafloxacin is readily without ciprofloxacin, as well as trovafloxacin alone without soluble in water at neutral pH and 30 mg of this prodrug is DNR. On the other hand, the majority of DNR-pretreated equivalent to 20 mg of the active trovafloxacin compound (15). The animals that received trovafloxacin therapy had no grossly dosage of GCSF was 10 Ìg/kg/dose. Mice that were treated without detectable metastatic deposits in the solid organs and the any antibiotic (only saline) were used as controls. lungs were spongy and aerated. In trial I (Table I), the survival rate without any antibiotic Quantitative analysis. Antibiotic therapy was given for 7 days. One to 2 days after the distribution of antibiotics, all mice were sacrificed by (saline injection) was 0% and it did not improve on DNR cervical dislocation. At autopsy, lungs were removed aseptically and therapy. On ciprofloxacin therapy, the survival rate was 18% transferred to 10% formalin. They were later embedded in paraffin without DNR and it was 18% with the addition of DNR. On blocks, sectioned and stained with hematoxylin/eosin and examined the other hand, on trovafloxacin the survival rate jumped for the spread of tumor cells in the lungs. Infiltration of malignant from 9% to 91%, a ten-fold increased survival when DNR cells into the lung tissue was classified as 0 to 3+ arbitrarily. No was added. Upon addition of rGCSF, the survival rate was cells=0, infiltration into bronchus or blood vessel 3+, readily 83%, i.e., rGCSF therapy had no additional benefit. detectable in every field 2+, occasional malignant cells seen after searching more than four high power fields=1+. No malignant cells In trial II (Table II), this result was reproducible on seen after searching four high powerfields=0+. repeat experiment. Briefly, the survival rate was 5% to 6% on saline with or without DNR and 9% to 26% on Results ciprofloxacin with or without DNR. The survival rate on trovafloxacin was 38% without DNR, Survival. Each experiment was done twice to verify the which improved to 70% when DNR therapy was added. reproducibility of results. Each experiment was done with or Addition of rGCSF and DNR therapy to ciprofloxacin had without the addition of daunorubicin (DNR) and with or minimal effect with a cure rate of 41% (26% with DNR alone), without the addition of rGCSF. The cumulative mortality whereas trovafloxacin plus DNR plus rGCSF had a 71% cure without any therapy was 50% on day 10. After the inoculation rate. of tumor cells, there was a cumulative mortality of 50% In summary, there was 28% survival without any quinolone. without any therapy on day 10 and it was 12 days with DNR The survival rate on ciprofloxacin plus DNR was 23%, which and ciprofloxacin therapy without the addition of DNR. The improved to 41% on addition of rGCSF. On the other hand, 50% cumulative mortality on trovafloxacin without DNR alatrovafloxacin therapy had a survival rate of 77% to 80% occurred by the 12th day, whereas on DNR + trovafloxacin and it was unmodified (75% survival) when rGCSF was the survival was about 80% on day 15, which was not added. Thus, trovafloxacin exhibited superior efficacy to influenced by the addition of GCSF. ciprofloxacin in protecting these animals from cancer. 270 Thadepalli et al: Antitumor Activity of Trovafloxacin in an Animal Model Tissue concentration of trovafloxacin. Following 20 mg/kg of In our study, ciprofloxacin exhibited low antitumor activity intraperitoneal injection, the trovafloxacin level in the lung in the P388 leukemia animal model with nearly 80% mortality (17.5 Ìg/g) was more than three times the serum level (5.16 with or without the addition of DNR.