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Clinical Cancer Research Vol. 6, 2626-2634, July 2000
© 2000 American Association for Cancer Research

Advances in Brief

BBR 3464: A Novel Triplatinum Complex, Exhibiting a Preclinical Profile of Antitumor Efficacy Different from Cisplatin1

Carla Manzotti2, Graziella Pratesi, Ernesto Menta, Roberto Di Domenico, Ennio Cavalletti, Heinz H. Fiebig, Lloyd R. Kelland, Nicholas Farrell, Donatella Polizzi, Rosanna Supino, Gabriella Pezzoni and Franco Zunino

Novuspharma S.p.A., 20052 Monza, Milan, Italy [C. M., E. M., R. D. D., E. C., G. Pe.]; Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy [G. Pr., D. P., R. S., F. Z.]; Tumor Biology Center at the University of Freiburg and Oncotest Institute, Freiburg, Germany D79106 [H. H. F.]; Institute of Cancer Research, Belmont, Sutton, Surrey SM2 5NG, United Kingdom [L. R. K.]; and Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284-2006 [N. F.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 In Vivo Studies
 Results
 Discussion
 REFERENCES
 
Multinuclear platinum complexes represent a new class of anticancer agents, distinct in terms of DNA binding features and the profile of antitumor activity from their mononuclear counterparts, in particular cisplatin. Among complexes of this class, BBR 3464, a trinuclear platinum compound has been selected for preclinical development. In the present study, we describe the preclinical evaluation of BBR 3464 in a series of human tumor cell lines and tumor xenografts, with special emphasis on tumor types known to be resistant to cisplatin. In a panel of seven human tumor cell lines naturally resistant to cisplatin (three ovarian and four melanomas), BBR 3464 was extremely potent with IC50 values at least 20-fold lower than cisplatin. Against eight human tumor xenografts including four tumors refractory to cisplatin, BBR 3464 was confirmed to be very active with a tumor weight inhibition >80% in seven of them. The efficacy of BBR 3464 against cisplatin-resistant tumors was consistent with the ability of the drug to completely overcome resistance in three cell systems characterized by acquired resistance to cisplatin. Moreover, BBR 3464 caused a more prolonged effect than cisplatin, which was reflected by higher specific growth delay values. This prolonged effect is likely to be related to a more persistent perturbation of the cell cycle induced by BBR 3464 than by cisplatin, as shown in one ovarian tumor cell line. Finally, the profile of sensitivity to BBR 3464 within the 60-cell-lines screening panel of the National Cancer Institute, NIH (Bethesda, MD) differed from those of established drugs, thus supporting the hypothesis of a distinct mechanism of cytotoxic activity of BBR 3464. The novel trinuclear platinum complex, in light of its innovative antitumor activity profile, has the potential to become a useful clinical agent for the treatment of unresponsive tumors.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 In Vivo Studies
 Results
 Discussion
 REFERENCES
 
Cisplatin is one of the most active antitumor drugs and is clinically efficacious against a wide range of tumors. Many other mononuclear platinum compounds have been developed as candidates for clinical use (1) . Carboplatin, which is currently the most widely used cisplatin analogue, shows molecular and cellular mechanisms similar to those of cisplatin, and its clinical interest is essentially related to a reduced toxicity profile (2) . 1,2-diaminocyclohexane-platinum complexes were originally developed in an attempt to overcome cisplatin resistance on the basis of their activity in L1210 murine leukemia, but, in other preclinical systems, their superiority over cisplatin is questionable (3) . Among such compounds, only oxaliplatin is currently under clinical investigation and has demonstrated activity against metastatic colon cancer (4) . With the exception of oxaliplatin, clinical trials on mononuclear platinum analogues in general have failed to demonstrate an improved pharmacological profile and a broader spectrum of activity compared with the parent drug cisplatin (5) .

Reasons for the failure to expand the clinical spectrum of the second generation analogues are essentially attributable to the high homology shared by all of these potential cisplatin substitutes. Multinuclear platinum compounds represent innovative structures designed and investigated with the aim of discovering new platinum compounds that are structurally dissimilar to cisplatin and possibly characterized by innovative mechanisms of DNA interaction (6, 7, 8) . In particular, the dinuclear platinum complexes, containing two reactive platinum centers firmly linked by a variable diamine length chain, were originally designed to form different types of DNA adducts, such as "long-distance" intra- and interstrand cross-links, which are not available to conventional mononuclear platinum complexes (9) . To improve the DNA binding ability of such complexes, the trinuclear platinum complexes have been synthesized by incorporating a third platinum center within the alkanediamine backbone of the dinuclear complexes. Among them, BBR 3464 was selected for preclinical development. BBR 3464 is best described as two monofunctional [trans-PtCl(NH3)2] platinum units bridged by a platinum tetra-amine unit [trans-Pt(NH3)2(NH2(CH2)6NH2)2]2+ that contributes to DNA binding only through electrostatic and H-bonding interactions (Fig. 1)Citation . The overall 4+ charge (neutralized by four nitrate counter anions), the presence of at least two platinum coordination units, and the consequences of such DNA binding (10) represent a remarkable departure from the cisplatin structural paradigm. The results of antitumor activity evaluation on a panel of cisplatin-resistant human tumor xenografts indicated an impressive efficacy of BBR 3464, particularly in p53 mutant tumors (11) . The in vivo results, which indicated a different spectrum of activity for the trinuclear compared with the mononuclear platinum complex, were supported by a cellular pharmacology study showing the ability of BBR 3464 to overcome cisplatin resistance and different mechanisms of DNA interaction for the two drugs (12) . The findings strongly support the validity of the hypothesis that "nonclassical" platinum structures could generate innovative platinum-based agent of potential clinical relevance.



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  		Fig. 1. Chemical structure of BBR 3464.

 
The aim of the present study was to further investigate the efficacy profile of BBR 3464 in selected preclinical tumor systems, with particular attention to human tumor types known to be relatively resistant to chemotherapy treatment (e.g., melanomas, and gastric and lung carcinomas). To better support the peculiar features of the preclinical efficacy profile, the study design included an antitumor activity evaluation performed in three independent laboratories that use different experimental models and different treatment schedules. The novel compound exhibited a striking lack of cross-resistance in all of the tested tumors with acquired or natural resistance to cisplatin. Taken together, the studies indicate that BBR 3464 is the lead compound of a novel series of platinum-containing DNA-interacting agents.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
In Vivo Studies
 Results
 Discussion
 REFERENCES
 
Drugs
BBR 3464 was synthesized by Boehringer Mannheim Italia, Chemical Department (Monza, Italy), now Novuspharma S.p.A. (Monza, Italy). For in vitro and in vivo studies, BBR 3464 was dissolved in saline (0.9% NaCl) immediately before use. Cisplatin was used as the clinical formulation (Platinex; Bristol Myers Squibb) and diluted in saline.

In Vitro Studies
Cytotoxicity Studies.
The cytotoxic potency of BBR 3464 and cisplatin was assessed on a panel of three human ovarian carcinoma and four human melanoma cell lines. The ovarian carcinoma cell lines OVCAR-5, SW626, and SKOV-3 were obtained from the American Type Culture Collection; the melanoma clones (Me665) originated from a human metastasis (13) , and M14 and JR8 melanoma cell lines were obtained from Dr. G. Zupi (Istituto Regina Elena, Rome). All of the cell lines were maintained as monolayer cultures in RPMI + 10% FCS. The antiproliferative effect of the drugs was assessed by the growth inhibition assay after a 1-h exposure. Cells in the logarithmic phase of growth were seeded in duplicate into six-well plates. Twenty-four h after seeding, drugs were added to the medium, and cells were incubated for 1 h. Cells were harvested 72 h after the beginning of the exposure and counted with a cell counter (Coulter Electonics Ltd, Luton Beds, England).

The cross-resistance profile of BBR 3464 and cisplatin was determined at the Institute of Cancer Research, Sutton, England, on a panel of human ovarian carcinoma cell lines, including three pairs of parental and cisplatin-resistant sublines, 41M and 41McisR, CH1 and CH1cisR, and A2780 and A2780cisR (14 , 15) . Cells were seeded in quadruplicate into 96-well microtiter plates, and, after allowing an attachment overnight, the drugs were added. Drug exposure lasted for 96 h, and cell survival was determined by the SRB3 protein assay.

The cytotoxicity profile of BBR 3464 was also investigated at NCI, NIH (Bethesda, MD) against the 60-cell-lines panel according to standard conditions as described previously (16) . Briefly, after 48 h of drug exposure, the SRB assay was used to assess cell viability or growth.

The IC50 (defined as the drug concentration causing a 50% reduction of cell growth over that of the untreated control) was assessed from the dose-response curves. The resistant index was calculated as the ratio of IC50 in the cisplatin-resistant cell line:IC50 in the parental cell line.

Cell Cycle Analysis.
Cell cycle perturbation induced by the drugs was investigated in the A2780 human ovarian carcinoma cell line. Cells (5 x 104/ml) were seeded in 25-cm2 tissue culture flasks. Twenty-four h after seeding, cells were treated for 1 h with the drugs at equitoxic concentrations (IC80; 5 µg/ml and 1 µg/ml for cisplatin and BBR 3464, respectively). After 24, 48, and 72 h in drug-free medium, the cell cycle distribution was investigated by flow cytometric analysis, as already described (17) . Briefly, cells were trypsinized, washed, fixed, and stored at -20°C. Cells were then rehydrated and stained with propidium iodide solution for 30 min. Fluorescence intensity was determined by a FACScan flow cytometer equipped with an argon laser (Becton Dickinson, Mountain View, Ca).


   In Vivo Studies
Top
 ABSTRACT
 Introduction
 Materials and Methods
 In Vivo Studies
Results
 Discussion
 REFERENCES
 
Animals and Tumor Lines.
In vivo studies were performed at three different institutions: INT and BMIt in Italy and the Oncotest Institute of Freiburg in Germany. Eight-ten-week-old nude athymic mice of two different strains were used; CD-1-nuBR (Charles River Laboratory, Calco, Italy) by INT and BMIt, and NMRI nu/nu by Oncotest Institute. They were maintained according to the European guidelines, and the Ethics Committees for Animal Experimentation of the different Institutions approved the experimental protocols. Most tumor lines originated and were established at the INT (POVD/DX) and Oncotest Institute (GFX 214, LXFA 526, LXFS 650 and OVXF 899). NCI kindly supplied the N592 and A2780 tumor lines to INT, and Dr. Ishitsuka (Hoffman-La Roche, Kamakura, Japan) kindly supplied the MKN45 tumor line to BMIt. Tumor lines were maintained in nude mice by serial s.c. passages of tumor fragments. The human origin was routinely confirmed by lactate dehydrogenase isoenzyme analysis.

Assessment of Chemosensitivity.
Adult mice were s.c. transplanted on one or both flanks with tumor fragments. TW was calculated biweekly from caliper-derived diameter measurement, according to the formula: TW (mg) = tumor volume (mm3) = a2 x b/2, where a and b are the shortest and the longest diameter, respectively. Mice bearing comparably sized tumors (approximately 50–100 mg) were randomized into treatment and control groups (5–6 mice with bilateral tumors or 7–8 mice with a single tumor). Both of the drugs were given i.v. in a volume of 10 ml/kg of body weight, according to different treatment schedules depending on the investigating laboratory. Because the tumor line may affect drug toxicity, the maximal tolerated doses, defined on the basis of lethal toxicity in each experiment, ranged from 0.3 to 0.45 and 4 to 6 mg/kg per injection for BBR 3464 and cisplatin, respectively.

Antitumor activity was assessed by two end points: (a) relative TWI (TWI%) in treated (T) versus control (C) mice, according to the formula:

where the relative TW was calculated for each single tumor by dividing its TW at a given day by that at the beginning of the treatment; and (b) SGD, calculated as:

where T and C represent the mean time (in days) to quadruple initial weight for treated and control tumors, respectively.

Death of mice was ascribed to drug toxicity either when they died before the first untreated control or when they presented a small tumor and spleen and/or liver size reduction at necropsy.

Statistical comparison of TW in cisplatin versus BBR 3464-treated tumors was assessed by the Student’s t test for unpaired observations (two-tailed) or the Mann-Whitney-Wilcoxon U test.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 In Vivo Studies
 Results
Discussion
 REFERENCES
 
Cytotoxicity Studies.
The pattern of cytotoxic activity of BBR 3464 was assessed in a panel of human tumor cell lines, including ovarian carcinomas and melanomas, that were selected for their intrinsic resistance to cisplatin (Table 1)Citation . The IC50 values obtained after 1 h of exposure indicated a substantial increase in cytotoxic potency of BBR 3464 compared with that of cisplatin (up to 800-fold in ME665/2/60). The IC50 values of BBR 3464 ranged from 0.04 to 1.6 µM, whereas those of cisplatin ranged from 9 to 42 µM. The effect of a 96-h exposure was also evaluated in the SKOV-3 cell line. It is noteworthy that IC50 values after 1 and 96 h of exposure markedly differed (about 20-fold) for cisplatin, but only slightly (2-fold) for BBR 3464.


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  		Table 1 Cellular response to BBR 3464 and cisplatin in human tumor cell lines after 1 h of drug exposure

 
The ability of BBR 3464 to overcome cisplatin-acquired resistance was evaluated in three pairs of human ovarian carcinoma cell lines in which resistance is probably mediated by different mechanisms (14 , 15) . After a 96-h exposure, BBR 3464 was much more potent than cisplatin against all of the tested cell lines. As clearly indicated by the resistance index values for each pair of cell lines (Fig. 2)Citation , BBR 3464 was able to completely overcome cisplatin resistance in the tested cell systems.



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  		Fig. 2. Resistance index (ratio between IC50 value in the resistant subline and in the parental cell line) in three pairs of ovarian carcinoma cell lines (A2780 and A2780cisR; 41M and 41McisR; CH1 and CH1cisR). Continuous exposure for 96 h to BBR 3464 ({blacksquare}) or cisplatin ({square}).

 
BBR 3464 cytotoxicity was evaluated in the 60-cell-lines screening system of the NCI. As described by the mean graph profile (Fig. 3)Citation , BBR 3464 was more potent than cisplatin in the inhibition of cell growth (mean Log10 GI50, -7.29 and -5.18, respectively) and did not show any subpanel selectivity (Fig. 3A)Citation . Indeed, the pattern of cytotoxicity was markedly different from that of cisplatin (Fig. 3B)Citation . Moreover, from the mean graph a further analysis was performed by NCI using a specific algorithm: the COMPARE assay, which evaluates the similarity of the mean graphs (18) . The comparison of the mean graph indicated no similarity for BBR 3464 and other drugs. This suggests that BBR 3464 does not share a mechanism of action with any established drug, including cisplatin and other platinum complexes.



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  		Fig. 3. Chemosensitivity profile to BBR 3464 (A) or cisplatin (B) of the 60-cell-lines panel of the NCI. Drug exposure was continuous for 48 h. GI50, drug concentrations causing a 50% growth inhibition.

 
Cell Cycle Analysis.
The drug-induced perturbations of cell cycle distribution (G1 and G2 phases), assessed 24, 48, and 72 h after a 1-h exposure to equitoxic concentrations (IC80), are shown in Fig. 4Citation . The increase over time in the G1 phase observed in untreated samples was due to the crowding of cell cultures. Starting at 24 h, BBR 3464 produced a decrease of the cell population in the G1 phase and a parallel increase in the G2 phase. The perturbations in cell cycle distribution were maintained for at least 72 h. In contrast, the cell cycle perturbation induced by equitoxic concentrations of cisplatin had recovered by 48 h (Fig. 4A)Citation . No significant modification of the S phase was induced by either drug (not shown). The extent of cell arrest (or delay) in the G2 phase was concentration-dependent (Fig. 4B)Citation ; thus, the persistence of cell cycle perturbations was most evident at high drug concentrations.



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  		Fig. 4. Cell-cycle perturbation after a 1-h drug treatment in the A2780 human ovarian carcinoma cells. A, percentages of cells in G1 or G2 phases after 24-h ({blacksquare}), 48-h (), and 72-h ({square}) treatment with cisplatin (5 µg/ml) or BBR 3464 (1 µg/ml). B, percentages of cells in G1 phase ({square} and {triangleup}) and in G2 phase ({blacksquare} and {blacktriangleup}) 72 h after 1-h exposure to cisplatin ({triangleup} and {blacktriangleup}) or BBR 3464 ({square} and {blacksquare}).

 
Antitumor Activity Studies.
The antitumor activity profile of BBR 3464 was assessed in a panel of eight human tumor xenografts. The panel included tumor models representative of tumor types generally refractory to chemotherapy (i.e., NSCLC and gastric carcinoma). Ovarian carcinomas and SCLCs were also included because, despite a relative responsiveness to cisplatin, the development of drug resistance is a clinically frequent phenomenon in both tumor types. Previous studies have shown that BBR 3464 is tolerated at doses about 15- to 20-fold lower than those of cisplatin (11) . The finding is confirmed in the present study, in which the maximal tolerated dose of BBR 3464 ranged from 0.3 to 0.45 compared with 4 to 6 mg/kg per injection for cisplatin, depending on treatment schedule and tumor model.

The results of the studies are reported in Table 2Citation . Against the GFX214 gastric carcinoma, two dose levels of BBR 3464 were investigated (0.3 and 0.45 mg/kg) and both were highly active, in contrast to the lack of activity observed for cisplatin (in this tumor model the maximal tolerated dose of cisplatin was 4 mg/kg given i.v. on days 1 and 15; Fig. 5Citation ). The same pattern of efficacy was observed on the other gastric tumor investigated, MKN45, in which BBR 3464 (0.3 mg/kg) induced a TWI% of 71, whereas cisplatin (6 mg/kg) was inactive (TWI%, 28). The triplatinum complex was also more active than cisplatin against the NSCLC LXFA526, in particular in terms of SGD (1–1.4 for the two doses of BBR 3464 and 0–0.4 for cisplatin). In LXFA526 tumor-bearing mice, drug-associated toxicity appeared more marked, with evidence of lethal toxicity at low doses. Both BBR 3464 and cisplatin showed comparable and very high antitumor efficacy against two SCLCs, LXFS650 and N592, with drug doses that induced some lethal toxicity in almost all of the treated groups. The two drugs proved to be highly effective even against the ovarian carcinoma A2780, and the range of active doses was wider for BBR 3464 than for cisplatin. Against the other ovarian carcinoma investigated, OVXF899, cisplatin was completely inactive at a dose level already presenting some lethal toxicity, whereas the trinuclear platinum complex was impressively efficacious even at a subtoxic dose (0.3 mg/kg). Moreover, on this tumor model, BBR 3464 induced partial tumor regression (defined as 10–50% reduction of initial TW), as depicted in Fig. 6ACitation .


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  		Table 2 Antitumor activity of BBR 3464 and cisplatin after i.v. treatments on s.c. human tumor xenografts

 


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  		Fig. 5. Growth curves of two gastric carcinomas, GFX 214 (A) and MKN45 (B) xenografted into nude mice. •, untreated tumors. Treated tumors: BBR 3464, 0.3 mg/kg ({square}) or 0.45 mg/kg ({blacksquare}); cisplatin, 4 mg/kg ({triangleup}) or 6 mg/kg ({blacktriangleup}). Treatments were administered i.v., on days 1 and 15 (A) and 1, 8, and 15 (B).

 


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  		Fig. 6. Growth curves of ovarian carcinoma OVXF 899 (A) and SCLC N592 (B) xenografted into nude mice. •, untreated tumors. Treated tumors: BBR 3464, 0.3 mg/kg {square}; cisplatin, 4 mg/kg ({triangleup}); cisplatin, 6 mg/kg ({blacktriangleup}). Treatments were administered i.v., on days 1 and 15 (A) and 1, 8, and 15 (B).

 
It is worth mentioning that in tumor models exhibiting responsiveness to both BBR 3464 and cisplatin in terms of TWI, BBR 3464 caused a prolonged tumor growth inhibition even after the end of treatment, a property not shared by cisplatin. The observation was reflected by higher SGD values for BBR 3464 than for cisplatin (Table 2)Citation and by different growth curve profiles (Fig. 6B)Citation . Moreover, the SGD values reported in Table 2Citation were generally higher for BBR 3464 than for cisplatin (range, 1–12 and 0–7.7, respectively) in all of the investigated tumors. To explore the potential of BBR 3464 in overcoming acquired resistance that was not cisplatin-related, its efficacy was investigated on a SCLC (POVD/DX), with acquired resistance to doxorubicin exhibiting a multidrug resistant phenotype. Both BBR 3464 and cisplatin proved to be highly effective, achieving an almost complete TWI. Nevertheless, BBR 3464 activity was significantly higher than that of cisplatin (Table 2)Citation .


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 In Vivo Studies
 Results
 Discussion
REFERENCES
 
The cellular pharmacology studies and antitumor efficacy evaluation reported in the present paper clearly document that the novel trinuclear platinum complex BBR 3464 exhibited a peculiar profile of cytotoxic and antitumor activity different from that of cisplatin. BBR 3464 represents a nonclassical structure compared with cisplatin, of considerable preclinical interest for its ability to form different types of platinum-DNA adducts and for its outstanding activity on a series of human non small cell lung and ovarian carcinomas xenografts (10 , 11) . Despite the large molecular size and its charged nature, BBR 3464 was characterized by a marked increase in cytotoxicity that was reflected in an in vivo potency of 15- to 20-fold that of cisplatin. Moreover, it is noteworthy that BBR 3464 showed comparable cytotoxic activity between 1 and 96 h of exposure, as observed on SKOV-3 cell lines (Table 1)Citation , thus supporting previous observations indicating a fast intracellular accumulation and DNA binding (12) .

The triplatinum complex showed lack of cross-resistance in all of the three sublines of human ovarian carcinoma cell lines with acquired resistance to cisplatin. Several resistance mechanisms such as impaired drug accumulation, increase in DNA repair, glutathione content, or alterations in the DNA mismatch repair system have been identified in resistant cell sublines (14 , 15 , 19 , 20) . The high efficacy of the trinuclear platinum compound against human tumor xenografts with acquired resistance to cisplatin (the ovarian carcinomas A2780/DDP and IGROV/DDP and the SCLC POVD/DDP) has already been reported (11) . Taken together, these results strongly support the ability of the new triplatinum complex to overcome several mechanisms associated with cisplatin-resistance. Moreover, BBR 3464 was extremely active against a human tumor xenograft (POVD/DX) with acquired resistance to doxorubicin, achieving a quite complete TWI (99%).

Moreover, on the basis of the unique profile of chemosensitivity in the 60 cell lines of the NCI screening system (by COMPARE analysis) and of the different pattern of cellular response in our panel of human tumor cell lines, it is likely that BBR 3464 and cisplatin differ substantially in their mechanisms of action or resistance, or both. The conclusion is consistent with the observation that the mechanism of drug-DNA interaction is at least in part different for the two complexes in an osteosarcoma cell system (12) . The formation of different types of platinum-DNA adducts between the mononuclear and trinuclear platinum complexes is based on drug structural features. It is possible that the types of DNA adducts formed by BBR 3464 are recognized and/or repaired by different cellular processes. Indeed, preliminary evidence supports an involvement of cell cycle checkpoints as a critical determinant of chemosensitivity of yeast mutants to BBR 3464 (21) . A different cellular response is also suggested by the more persistent cell cycle perturbation effects of BBR 3464 than those of cisplatin at equitoxic dose levels. A short-term exposure (1 h) to BBR 3464 caused a marked and persistent accumulation of cells in the G2 phase. Such cytokinetic effects are consistent with persistent tumor growth inhibition observed in tumors responsive to BBR 3464. In fact, the SGD values determined by BBR 3464 were consistently higher than those produced by cisplatin, even in those tumors that showed comparable sensitivity to the two drugs in terms of TWI.

The trinuclear platinum complex exhibited a substantial improvement in antitumor efficacy over cisplatin, in particular in tumors generally refractory to chemotherapy treatment such as gastric and NSCLC carcinomas. The finding is consistent with the results of a previous study performed on a different panel of tumor xenografts including NSCLC, SCLC, and ovary and prostate carcinomas (11) . Among the 18 tested tumors, including 10 tumors highly resistant to cisplatin (TWI < 50%), BBR 3464 exhibited a sustained antitumor activity in 10 (61%) either in terms of TWI% (>80%) or in terms of persistence of response (SGD values). The antitumor activity of BBR 3464 was impressive, if tumors refractory to cisplatin treatment are considered: all such tumors were responsive to the triplatinum complex. The pharmacological interest for the multinuclear complex is further supported by the significant efficacy on human tumor models carrying the mutant p53 gene (11) . The lack of functional p53 after mutation is a frequent alteration in human tumors and may be responsible for a relative resistance to conventional DNA-damaging agents, including cisplatin (22) .

In conclusion, considering the peculiar cytotoxicity profile, the lack of cross-resistance with cisplatin in a variety of in vitro and in vivo systems, and the wide spectrum of activity in human tumor xenografts, BBR 3464 appears to represent a genuinely new platinum compound with promising therapeutic activity. The interest of the novel compound for clinical development is also supported by the toxicological profile, which indicates toxic effects comparable with those of cisplatin in terms of neurotoxicity and myelotoxicity (23) . The acute manifestations of nephrotoxicity were less marked than those of cisplatin and could be prevented by hydration and/or slow infusion. In contrast to cisplatin, BBR 3464 did not induce emetic effects in ferrets. Phase II clinical studies are ongoing.



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  		Fig. 3B. Continued.

 

   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was partially supported by the Associazione Italiana per la Ricerca sul Cancro, Milan, Italy. Back

2 To whom requests for reprints should be addressed at, Novuspharma S.p.A., via G. B. Stucchi 110, 20052 Monza, Milan, Italy. Phone: 39-039-2814306; Fax: 39-039-2814609; E-mail: carla.manzotti{at}novuspharma.com Back

3 The abbreviations used are: SRB, sulforhodamine B; NCI, National Cancer Institute; INT, Istituto Nazionale Tumori of Milan; BMIt, Boehringer Mannheim Research Center in Italy; SCLC, small cell lung carcinoma; NSCLC, non-SCLC; TW, tumor weight; TWI, TW inhibition; SGD, specific growth delay. Back

Received 7/ 9/99; revised 12/ 6/99; accepted 12/20/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 In Vivo Studies
 Results
 Discussion
 REFERENCES
 

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