Pharmacology and Antitumour Effects of Intraportal Pirarubicin on Experimental Liver Metastases
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CORE Metadata, citation and similar papers at core.ac.uk Provided by PubMed Central '." Macmillan Press 1993 Br. J. Cancer (1993), 68, 277-281277 281 Ltd., Pharmacology and antitumour effects of intraportal pirarubicin on experimental liver metastases L.H. Ramirez', J.-N. Munck', C. BognelP, Z. Zhao', P. Ardouin3, M.-F. Poupon4, A. Gouyettes & P. Rougier' 'Departement de Medecine; 2Departement d'Anatomopathologie; 3Service d'Experimentation Animale; 5Laboratoire de Pharmacologie Clinique U 140 INSERM and URA 147 CNRS, Institut Gustave-Roussy, 94805 Villejuif, France and 4URA 620 CNRS Institut Curie, 75005 Paris, France. Summary Early liver metastases have a predominant portal blood supply. Intraportal (i.port.) vein admini- stration of cytotoxics could theoretically achieve enhanced drug concentrations in tumour cells and be effective as adjuvant therapy after resection of colorectal carcinoma. Pirarubicin (which has a higher hepatic extraction than doxorubicin) was investigated on liver metastases of the VX2 rabbit tumour, which were of less than 2 mm in diameter 7 days after cells injection into the portal vein. To evaluate antitumour activity, 24 rabbits were randomised into three groups 7 days after implantation: (a) control, (b) i.v. pirarubicin, (c) i.port. pirarubicin at doses of 2 mg kg-' in both groups. Portal infusions led to no hematological or hepatic toxicity. Pharmacokinetic parameters showed a significantly reduced systemic exposure after i.port. administration. Fourteen days after treatment, livers and lungs were analysed. The mean number (± s.d.) of tumour foci was (a) 8.62 (± 5.4), (b) 4.62 (± 3.2), (c) 2.25 (± 1.4) (P<0.05 a vs c). The mean tumour area was (a) 6.31 (± 6.1), (b) 1.31 (± 2.2), (c) 0.43 (± 0.4cm2) (P<0.05 a vs c) and the percentage (95% C.I.) of rabbits with lung metastasis was: (a) 87.5% (47-99%), (b) 75% (35-97%), (c) 12.5% (3-52%) (P<0.02 b vs c). Intraportal pirarubicin seems to be well tolerated and more efficient than i.v. administration, particularly in preventing extrahepatic dissemination. Residual cancer cells are responsible for treatment failures in pre-clinical and clinical studies (Miller & Schmidt, 1987). after curative resection for colorectal cancer and the liver is This compound has a faster cellular uptake than that of the most frequent site of relapse (Willett et al., 1984). doxorubicin (Munck et al., 1985), as it is much more Systemic post-operative adjuvant therapy based on lipophilic. Indeed, the apparent partition coefficient (Papp) fluoropyrimidines can reduce the recurrence rates and in- between octanol and phosphate buffer at pH 7 is 35.8 (log crease overall survival, as recently observed in Dukes C stage P: 1.55) for pirarubicin, and 0.26 (log P: - 0.59) for doxo- colon cancer (Moertel et al., 1992). With new active drugs rubicin (unpublished data). Furthermore, pirarubicin has and a more efficient targeting of tumour tissues, further demonstrated its superiority over doxorubicin in an experi- improvements could be obtained. mental study with intraarterial hepatic (i.a.h.) administration Colon cancer cells disseminate through the mesenteric which appeared essentially due to a greater tumour drug veins and portal system (Fisher & Turnbull, 1955), and the uptake (Munck et al., 1993). Subsequent clinical studies have newly growing liver metastases receive their blood supply further demonstrated a high degree of activity, even against from the portal branches, until they develop a main arterial colorectal hepatic metastases (Munck et al., 1990). This vascularisation (Conway et al., 1983; Ackerman, 1986). Fac- prompted our investigation of the putative benefit of in- tors such as intraoperative manipulation of the tumour, traportal infusion of adjuvant pirarubicin in the experimental perioperative impaired immunity and stress, have been VX2 tumour in the rabbit. reported to facilitate the dissemination and seeding of malig- The present study, on a model of early experimental liver nant cells. Regional post-operative adjuvant therapy via the metastases, compares intraportal (i.port.) vs intravenous (i.v.) portal vein could be an effective means of preventing the adjuvant infusion of pirarubicin, taking into account both development of liver metastases, by delivering higher local pharmacokinetic parameters and the effects of this cytotoxic drug concentrations to tumour cells at the onset of metastatic on hepatic and extrahepatic tumoural growth and dissemina- invasion. Several clinical trials have been designed to test this tion. hypothesis (Taylor et al., 1985; Gray et al., 1987; Ryan et al., 1988; Wolmark et al., 1990; Metzger et al., 1990; Wereldsma et al., 1990; Beart et al., 1990; Fielding et al., 1992). Most of them used 5-FU alone, or in association with mitomycin C in Materials and methods different schedules. Current results are controversial and remain inconclusive. Some trials have shown a reduction in Animals and anaesthesia the number of hepatic recurrences with no benefit for overall Female New Zealand white rabbits weighing 2.7-3.2 kg were survival (Wereldsma et al., 1990). Others have demonstrated used (Elevage Scientifique des Dombes, Romans, France). an increase in overall and disease free survival, although no The rabbits were maintained under standard conditions on a differences were noted in the number of hepatic recurrences laboratory diet and water ad libitum. All procedures were (Wolmark et al., 1990). carried out under general i.v. anaesthesia using ketamine Other antitumoural compounds could be tested for hydrochloride (50 mg kg-'; KetamineO, Parke Davis) and regional therapy, focusing on drugs with a high hepatic xilazine 2% (0.1 ml kg-'; Rompun®, Bayer). All experiments extraction and limited heptatic toxicity. Anthracyclines, are were conducted in accordance with the European Council generally considered inactive against colorectal cancer, but directive 86/609/CEE, and French legislation concerning pirarubicin, a new derivative, has been recently investigated animal welfare. Drugs and chemicals Correspondence: J.-N. Munck, Institut Gustave-Roussy, rue Camille- Desmoulins, 94805 Villejuif Cedex, France. Doxorubicin hydrochloride, daunorubicin, doxorubicinol, Received 26 October 1992; and in revised form 31 March 1993. doxorubicinone, pirarubicinol, and pirarubicin hydrochloride 278 L.H. RAMIREZ et al. were provided by Laboratoire Roger Bellon (Neuilly-sur- counts at 5, 7 and 14 days, which were compared to pre- Seine, France). The chemical structure of doxorubicin and treatment baseline values. pirarubicin is depicted in Figure 1. Solvents used for extrac- tion and high performance liquid chromatography (HPLC) analyses were all of HPLC grade or of highest available Determination ofmaximal tolerated single i.port. doses of purity. pirarubicin The maximal tolerated dose (MTD) in rabbits for the i.v. VX2 tumour inoculation and surgical procedures route had been previously determined at 2 mg kg-' (unpub- lished data). Higher i.v. doses (2.5 mg kg-') were associated The VX2 tumor was kindly provided by Dr G. Orth (U 190 with lethal toxicity. The determination of the MTD for the INSERM, Institut Pasteur, Paris, France) and was main- i.port. route was achieved by escalating the 2 mg kg-' dose tained by serial passages in carrier rabbits. A VX2 tumour by 0.5 mg increments. Pirarubicin was administered at single was removed from one animal, minced in NCTC 109 doses of 2 mg, 2.5 mg, 3 mg, and 3.5 mg to four groups of medium (Eurobio, Paris, France) and filtered through a cot- four rabbits without a tumour graft. ton gauze. The filtrate was adjusted to 3 x I07 cells ml-' with the above medium containing 10% dimethylsulfoxide (DMSO) and 20% foetal calf serum (Gibco, Paris, France) to Determination ofpirarubicin plasma concentration constitute a homogeneous stock that was frozen in liquid Plasma concentrations were determined using reversed-phase nitrogen in 1 ml aliquots and used throughout the study. HPLC. Daunorubicin was added as the internal standard This procedure minimised inter-subject variations in tumour (100 ng ml-'). Half a ml of plasma was extracted on 100 mg growth rate. octadecyl (C18) columns (1 ml Bakerbond spe®, Baker, Phil- Hepatic implantation of the VX2 carcinoma was accomp- lipsburg, NJ) preconditioned with 1 ml of methanol, followed lished through a small median subxyphoid incision. A 1 ml by 1 ml of water. After air drying, elution was accomplished frozen sample of VX2 cells was rapidly thawed and centri- with 1 ml of methanol/dichloromethane (1:1, v:v) following fuged. The cell pellet was adjusted to 0.5 ml (NCTC 109 the addition of 200 flI of DMSO. The volume was then medium), and injected with a 24-gauge catheter into the main reduced to approximately 200 ftl under a nitrogen stream portal vein. before HPLC injection. This procedure allowed a 95% recovery of pirarubicin, internal standard and metabolites. Establishment of a model ofearly hepatic metastases The HPLC system consisted of a C18 column (Nucleosil C, 10 ltm, 3.9 x 300 mm, SFCC, Neuilly-sur-Seine, France), a In order to establish the optimal time for i.port. drug Wisp automatic injector (710B, Waters Associated, Milford, administration, the kinetics of VX2 tumour growth after Ma, USA), a 6000A pump (Waters), and a fluorescence i.port. inoculation were determined. For this purpose three detector (Shoeffel FS 970) set at 251 nm (ex.) and 550 nm groups of four rabbits each were studied, and the animals (em.). The mobile phase consisted of water (adjusted to were sacrificed at 7, 14, and 21 days after VX2 cell injections. pH 2.4 with phosphoric acid) and acetonitrile (68:32, v:v) at Livers were cut into 5 mm slices, and the number of tumours a flow rate of 1.75 ml min-'. Under these conditions, the and their diameters were recorded and cross-sectional areas retention times of doxorubicinol, doxorubicin, pirarubicinol, calculated. Lung slides were screened for the presence or daunorubicin, and pirarubicin were 3.59, 4.48, 5.94, 6.90 and absence of metastases.