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. Trovafloxacin carried /ml) for four hours or more. Other tissues like the liver (16.5 80% mortality without DNR pretreatment but, upon the Ìg /g) and the spleen (14.6 Ìg /g) also retained high tissue addition of DNR, the mortality dropped to nearly 10%. The concentrations of trovafloxacin. The antibiotic concentration most likely explanation for trovafloxacin efficacy may be in its of trovafloxacin was high in all the tissues tested. chemistry. It is a trifluoroquinolone and quinolones with two or more fluorine attachments like sparfloxacin were shown to have Histopathological examination of the lung. Figure 1 shows, in antitumor activity in the murine leukemia P388 model. low power magnification, the normal control lung tissue Difloxacin, a difluoroquinolone, also was shown to potentiate obtained from healthy mice. The blood vessels contained DNR activity in multidrug-resistant leukemia cells (P388/ADR) normal red blood cells and the lung parenchyma was clear (9,10) and human neuroblastoma cells (17). Some quinolones showing normal aeration of the alveoli and no vascular or can inhibit cancer cell growth in vitro. Ofloxacin and its levo alveolar changes (Class O). isomer, levofloxacin, for example inhibited the transitional cell Figure 2 shows, in high power magnification, the normal carcinoma cell lines by inhibiting topoisomerase activity (7). lung with open alveolar spaces and normal blood vessels Similar activity was also reported with ciprofloxacin (18,19). (Class O). We observed that trovafloxacin inhibited P388 leukemia Figures 3, 4 and 5 show the lung of tumor-bearing mice cells and HL-60/AR cells in vitro, but its in vivo activity was treated with ciprofloxacin. In Figure 3 (low power), multiple unknown and, therefore, this experiment was conducted in extensive intravascular and parenchymal tumor metastatic the (tumor-bearing) mouse model with pneumonia induced deposits are visualized (Class 2+).Medium magnification by Klebsiella pneumoniae. In this model, we observed that (Figure 4) shows the majority of metastatic tumors to be the survival rate was nearly 80% with trovafloxacin and 40% inside blood vessels (Class 3+) with areas of necrosis and with ciprofloxacin (7). We could not explain this discrepancy adjacent parenchymal hemorrhage (shown in Figure 5). on the basis of their antimicrobial efficacy because both Figures 6, 7 and 8 show the lung tissue in trovafloxacin- quinolones had very low MIC (0.5 to 1.0 mcg/ml). In fact, treated mice. Lower magnification (Figure 6) shows very both quinolones cured Klebsiella pneumoniae infection in scanty tumor cells in the alveolar and intravascular spaces the mouse (without tumor) 100% of the time. Clearly, the (Class 1+). Most of the lung tissue shows normal aeration failure of ciprofloxacin was somehow related to its effect on with clear alveoli (Class O+). In medium power (Figure 7), the underlying tumor tissue. In clinical studies, ciprofloxacin we see few tumor cells here and there in the parenchyma monotherapy was shown to have a very high failure rate and (Class O+ to 1+) and under higher power (Figure 8) we mortality in the treatment of infections associated in febrile see few blood vessels with very scanty tumor cells mixed granulocytopenic cancer patients (20). with red blood corpuscles, but no masses of intravascular Trovafloxacin (and DNR)-treated animals had longer tumor are seen (Class 1+), like in ciprofloxacin-treated survival and least amount of metastatic deposits in the solid mice (Class 3+). organs. Further, they also exhibited a minimal amount of tumor cells in the tissues. In most instances, at least four or five Discussion high power fields had to be examined in the trovafloxacin- treated animals to detect minute quantities of tumor cells. This Fluoroquinolones are frequently recommended for was in contrast to ciprofloxacin-treated lung tissues that prophylaxis and treatment of bacterial infections in febrile showed massive tumor deposits readily detectable in the tissue neutropenic patients receiving cancer chemotherapy. and in the vasculature. Ciprofloxacin has little or no antimitotic Quinolones represent one of the first classes of activity on the P388 leukemia cell line and therefore antimicrobials to act by inhibiting bacterial DNA gyrase demonstrated higher mortality in our Klebsiella pneumoniae- potentiated by the introduction of fluorine substituent infected tumor-bearing mice despite its excellent antimicrobial found to be cytotoxic to Chinese hamster ovary cells (16). efficacy. However, this is not the case with ciprofloxacin on all The P388 leukemia cells used in the study were found to tumors. For example, ciprofloxacin in vitro inhibited other be unaffected both in vitro and in vivo by many quinolones cancer cell lines such as human transitional cell carcinoma of now in use such as norfloxacin, ofloxacin, ciprofloxacin and the bladder (HTB9) (18,21). Like ciprofloxacin, other others. They neither induce topo II-mediated cleavage in quinolones like ofloxacin and levofloxacin, which are effective vitro nor show antitumor activity in vivo (16). However, against carcinoma of the bladder (HTB9) cell lines, are also quinolones with two or more halogens and a cyclopropyl ineffective against P388 leukemia cells (7,8,19,22). ring attached were observed to increase the life-span of However, trovafloxacin, that has neither a halogen at C-8 tumor-bearing mice by more than 20%. nor a cyclopropyl moity at N-1 in its chemical structure, had

271 in vivo 19: 269-276 (2005)

Figure 1. Low power magnification of the lung tissue obtained from healthy mice.

Figure 2. High power magnification of the normal lung with open alveolar spaces and normal blood vessels.

272 Thadepalli et al: Antitumor Activity of Trovafloxacin in an Animal Model

Figure 3. Low power magnification of the lung tissue obtained from tumor-bearing mice treated with ciprofloxacin.

Figure 4. Median magnification of the lung tissue obtained from tumor-bearing mice treated with ciprofloxacin.

273 in vivo 19: 269-276 (2005)

Figure 5. High magnification of the lung tissue obtained from tumor-bearing mice treated with ciprofloxacin.

Figure 6. Low magnification of the lung tissue obtained from tumor-bearing mice treated with trovafloxacin.

274 Thadepalli et al: Antitumor Activity of Trovafloxacin in an Animal Model

Figure 7. Median magnification of the lung tissue obtained from tumor-bearing mice treated with trovafloxacin.

Figure 8. High power magnification of the lung tissue obtained from tumor-bearing mice treated with trovafloxacin.

275 in vivo 19: 269-276 (2005) good antitumor activity in this experiment. A fluorine at C-8 9 Gollapudi S, Thadepalli F and Kim CH: Difloxacin reverses position prevents phototoxicity, 18 substitution of C7 with multidrug resistance in HL-60/AR cells that overexpress the multidrug resistance-related protein (MRP) gene. Oncol Res 7(5): cyclopropyl-fused pyrrolidine enhances antibiotic activity 213-25, 1995. against gram-positive bacteria and, finally, replacement of 10 Gupta S, Thadepalli F and Gollapudi S: Difloxacin reverses naphthyridone at N-1 with the difluorophenyl group endowed multidrug resistance in P388/ADR cells via a mechanism the longest half-life for these quinolones (8 hours for independent of P-glycoprotein and without correcting drug transport trovafloxacin in contrast to 4 hours for ciprofloxacin). or subcellular drug distribution. Inter J Oncol 7: 475-480, 1995. Although all quinolones are lipophilic, trovafloxacin has much 11 Riesbeck K, Anderson J, Gullberg M and Forsgren A: Fluorinated -4 quinolones induce hyper production of IL-2. Proc Natl Acad Sci higher lipophilicity compared to ciprofloxacin and, therefore, USA 86: 2809-2813, 1989. higher intracellular drug concentrations are observed with 12 Zehavi-Wilner T and Shalit I: Enhancement of Il-2 production in trovafloxacin. Our experiment shows the potential therapeutic human lymphocytes by two new quinolone derivatives. Lymphokine efficacy of trovafloxacin as an antimicrobial agent and as an Res 8: 35-46, 1986. adjunct to cancer chemotherapy. 13 Thadepalli H, Reddy U, Chuah S , Hanna N, Rana G and Gollapudi S: Evaluation of trovafloxacin in the treatment of In conclusion, we recommend that quinolones with Klebsiella pneumoniae lung infection in tumor-bearing mice. J antimitotic activity be studied in treating infections associated Antimicrob Chemother 45: 69-75, 2002. with cancer chemotherapy and comparison of the outcome 14 Gollapudi S, Thadepalli F and Kim CH: Difloxacin reverses with patients receiving conventional non-quinolone polyanti- multidrug resistance in HL-60/AR cells that overexpress the microbial chemotherapy be made. Presumptuous as it may multidrug resistance-related protein (MRP) gene. Oncol Res 7(5): 213-25, 1995. seem, there are hopes and a significant need for an antibiotic 15 Brigerty KE and Gootz TD: The chemistry and biological profile of that can exert both antimicrobial and antitumor activity. Our trovafloxacin. J Antimicrob Chemother 39: S1-14, 1997. report may augment research in that direction. 16 Suto MJ, Domagala JM, Ronald GE, Mailloux GB and Cohen MA: Fluoroquinolones: relationships between structural variations, References mammalian cell cytotoxicity, and antimicrobial activity. J Med Chem 35: 4745-4750, 1992. 17 Norris, MD, Madafiglio J, Gilbert J, Marshall GM and Haber M: 1 Cheng CC: Common structural pattern among many biologically Reversal of multidrug resistance-associated protein-mediated drug active compounds of natural and synthetic origin. Med Hypothesis resistance in cultured human neuroblastoma cells by the quinolone 20: 157-172, 1986. antibiotic difloxacin. Med Pediat Oncol 36: 177-180, 2001. 2 Kuo SC, Lee HJ, Juang JP, Lin YT, Wu TS, Lednicer D, Paul KD, 18 Ebisuno S, Inagaki T, Kohjimoto Y and Ohkawa T: The cytotoxic Lin CM, Hamel E and Lee KH: Synthesis and cytotoxicity of effect of fleroxacin and ciprofloxacin on transitional cell carcinoma 1,6,7,8-substituted 2-(4' substituted phenyl)-4-quinolones and in vitro. Cancer 80: 2263-2267, 1997. related compounds: identification of antimitotic agents interacting 19 Kamat AM, DeHaven JI and Lamm DL: Quinolone antibiotics: a with tubulin. J Med Chem 36: 1146-1156, 1993. potential adjunct to intravesical chemotherapy for bladder cancer. 3 Xia Y, Yang Z, Morris-Natschke S and Lee KH: Recent advances Urology 54(1): 56-61, 1999. in the discovery and development of quinolones and analogs as 20 Meunier F, Zinner SH, Gaya H, Calandra T, Viscoli C, Klastersky antitumor agents. Curr Med Chem 6: 179-194, 1999. J and Glauser M: (EORTC Committee). Prospective randomized 4 Domagala JM: Structure-activity and structure-side effect evaluation of ciprofloxacin versus piperacillin plus amikacin for relationships for the quinolone antibacterials. J Antimicrob empiric antibiotic therapy of febrile granulocytopenic cancer Chemother 33: 685-706, 1994. patients with lymphomas and solid tumors. Antimicrob Agents 5 Ghoneum M, Gollapudi S, Gill G and Thadepalli H: Effect of Chemother 35: 873-878, 1991 quinolone derivatives on tumor cell growth in vitro. Cancer P388, 1991. 21 Aranha O, Wood DP Jr and Sarkar FH: Ciprofloxacin mediated 6 Gollapudi S, Vayuvegula B, Gupta S, Fox M and Thadepalli H: cell growth inhibition, S/G2-M cell cycle arrest, and apoptosis in a Aryl-fluoroquinolone derivatives A-56619 (difloxacin) and A-56620 human transitional cell carcinoma of the bladder cell line. Clin inhibit mitogen-induced human mononuclear cell proliferation. Cancer Res 6: 891-900, 2000. Antimicrob Agents Chemother 390-394, 1996. 22 Xia Y, Yang Z, Morris-Natschke S and Lee KH: Recent advances 7 Yamakuchi M, Nakata M, Kawahara K, Kitajima I and Maruyama in the discovery and development of quinolones and analogs as I: New quinolones, ofloxacin and levofloxacin inhibit telomerase antitumor agents. Curr Med Chem 6: 179-194, 1999. activity in transitional cell carcinoma cell lines. Cancer Lett 119(2): 213-219, 1997. 8 Yamashita Y, Ashizawa T, Morimoto M, Hosomi J and Nakano H: Antitumor quinolones with mammalian topoisomerase II mediated Received November 23, 2004 DNA cleavage activity. Cancer Res 52: 2818-2822, 1992. Accepted December 20, 2004

276