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Background: HIPEC is a new treatment modality for abdominal cancers that combines cytoreductive surgery with Hyperthermic, Intraoperative Peritoneal Chemotherapy, followed by systemic chemotherapy. A significant survival benefit has been shown for HIPEC compared with systemic therapy alone. However, it is not clear what is the contribution of i.p. drug delivery and what influence the mild hyperthermia has on the uptake of cisplatin in abdominal tumors., Materials and Methods: We used a peritoneal perfusion system in rats to compare the pharmacokinetics and pharmacodynamics of cisplatin, after normothermic (37 degrees C/90 minutes) and hyperthermic (40 degrees C/90 minutes) intra-peritoneal perfusion, with an i.p. bolus injection., Results: Hyperthermic perfusion with 15 micrograms/ml (in 200 ml) cisplatin gave equivalent plasma drug levels to a maximum tolerated dose (MTD) i.p. bolus injection of 4 mg/kg (36 micrograms/ml in 20 ml). The drug concentration in small (1-5 mm) intra-peritoneal tumors was also comparable for both these treatments, and for normothermic perfusion., Conclusion: Mild hyperthermic perfusion with cisplatin (40 degrees C/90 minutes) did not improve drug uptake in small intra-peritoneal tumors, relative to normothermic perfusion or i.p. bolus injection.

We have designed and used an in vitro bone marrow cell culturing system for investigating pharmacodynamic interactions between platinum anti-cancer drugs and taxanes. With this system, in which the bone marrow progenitor cell CFU-GM is proliferating and differentiating into granulocytes and monocytes, we could show a strong antagonistic cytotoxicity of the combination carboplatin and Taxotere, in three different schedules, and of the combination cisplatin and Taxol, in two out of the three schedules tested. Modulation of intracellular platinum drug accumulation in granulocytes and monocytes does not seem to be a plausible explanation for the observed antagonism. In vitro co-incubation of granulocytes/monocytes with the combination carboplatin and Taxotere did not reveal an effect of Taxotere on intracellular platinum accumulation. Although Taxol reduced intracellular cisplatin levels by 12%, this effect was not significantly different from the co-incubation of cisplatin with Cremophor EL, the solvent for paclitaxel in Taxol. The toxicity data obtained in this study seem to be in accordance with recent clinical trials where combination therapies with platinum drugs and taxanes resulted in marked reductions in myelosuppression in patients. Therefore, these types of assays could be useful as to the assessment of bone marrow toxicities of clinically important drug combinations.

We have studied the formation and repair of cisplatin-DNA adducts in wild-type mouse leukemia L1210/0 cells and in the sublines L1210/2 and L1210/5, which differ in cisplatin sensitivity. In a colony-formation assay these sublines were 9- and 22-fold more resistant compared to L1210/0, respectively. Cisplatin-induced DNA modification was studied at the cellular level by immunocytochemistry with antiserum NKI-A59 raised against cisplatin-treated DNA. Levels of nuclear staining immediately after a 1-h treatment were similar to those seen after a 24-h post-incubation in drug-free medium. Clear differences in DNA platination were found between the cell lines: immediately after exposure, L1210/2 and L1210/5 showed only 32 and 14%, respectively, of the nuclear staining observed in L1210/0, and 48 and 13% after 24 h. In these experiments, adduct-specific nuclear staining was quantified as the area under the adduct versus concentration curves (AUC). The formation and repair in these cell lines of the bifunctional adducts cis-Pt(NH3)2d(pGpG) (Pt-GG), cis-Pt(NH3)2d(pApG) (Pt-AG) and cis-Pt(NH3)2(dGMP)2 (G-Pt-G) were studied with an enzyme-linked immunosorbent assay (ELISA). No relation between repair and resistance was observed. The results suggest that differences in induced DNA platination levels, rather than in repair, are responsible--at least in part--for the differences in cisplatin resistance. A mechanism such as an increased tolerance of the resistant cells to plantinum-DNA damage may also be involved.

Cytotoxic effects of cis-diamminedichloroplatinum-(II) (cis-DDP) are thought to be mediated by binding to DNA. Studies on binding of cis-DDP to cellular DNA rely heavily on the availability of specific antibodies. We therefore raised and characterized four rabbit antisera: one against cis-DDP-modified DNA (antiserum NKI-A59) and three others against the cis-DDP-modified (di)nucleotides cis-Pt(NH3)2d(pApG) (NKI-A68), cis-Pt(NH3)2d(GMP)2 (NKI-A10), and Pt(NH3)3dGMP (NKI-A39). Reactivities to platinum compounds were determined in an enzyme-linked immunosorbent assay (ELISA) and in a quantitative immunocytochemical assay. In the ELISA, NKI-A59 showed a high affinity for DNA heavily substituted with either cis-DDP or CBDCA [cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)]; amounts of platinum per well giving 50% inhibition (IA50) were as low as 15 and 76 fmol, respectively. NKI-A59 also showed affinity to cis-DDP-modified poly[d(G-C)].poly[d(G-C)], poly(dC), and poly(dG). No affinity was found for trans-DDP [trans-diamminedichloro-platinum(II)]-modified DNA, enzymatically digested cis-DDP-DNA, or cis-DDP-DNA, or cis-DDP-modified poly(dA).poly(dT), oligo(dA)15.oligo(dT)15, oligo(dG)21, oligo(dG)42, or oligo(dAAAG)10. The efficiency of binding to cis-DDP-DNA decreased with decreasing DNA modification levels. Although other cis-DDP-DNA- and cis-DDP-(di)nucleotide-specific antisera have been identified, NKI-A59 is the first antiserum described that is suitable for the in situ detection of cis-DDP-DNA adducts at clinically relevant platinum levels. Adduct-specific immunostaining signals in cultured RIF-1 cells or rat liver paralleled platinum-DNA binding as measured by atomic absorption spectroscopy. The antisera NKI-A68, NKI-A10, and NKI-A39 showed high affinity for their corresponding haptens and varying affinity for non-hapten cis-DDP-DNA adducts. Their affinity for digested cis-DDP-modified DNA was up to 30 times that for intact cis-DDP-DNA. Neither NKI-A68 nor NKI-A10 resulted in specific immunocytochemical staining of cis-DDP-DNA adducts. We conclude that NKI-A68, NKI-A10, and NKI-A39 are suitable for platinum-DNA adduct analysis of digested DNA in ELISA and that NKI-A59 is suitable for platinum-DNA adduct detection at the single-cell level using immunocytochemical methods.

The relationship between cell killing and the binding of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) to DNA was studied in six mammalian cell lines. Two of the human cell lines (COV413B) were of the same origin, comprising one sensitive to cis-DDP and the other with induced resistance to the drug. The four other lines, two rodent (RIF-1, Chinese hamster ovary) and two human (A2780, A1847), were unrelated. The cell lines differed in their sensitivity to cis-DDP, as tested in a clonogenic assay. cis-DDP-DNA binding was determined by quantitative immunocytochemistry using an antiserum against cis-DDP-modified DNA. The resistance factors relative to RIF-1, calculated from full survival curves for cis-DDP, were 3.8 +/- 0.4 for Chinese hamster ovary cells and 8.8 +/- 0.7 for both A2780 and A1847 lines. Using quantitative immunocytochemistry, the levels of the adduct-specific nuclear staining density compared with RIF-1 cells were 4.8 +/- 0.2 for Chinese hamster ovary cells, 9.1 +/- 0.2 for A2780, and 10.0 +/- 0.1 for A1847 cells, i.e., in good agreement with the resistance factors. In studies with the COV413B cells and their cis-DDP-resistant counterpart COV413B-PtR, immunologically detected adduct levels again correlated closely with resistance factors (correlation coefficient = 0.97). The kinetics of cis-DDP-DNA adduct formation and loss was investigated in RIF-1, A2780, and A1847 cells by the immunocytochemistry technique. Adduct levels after a 1-h incubation with approximately equitoxic doses of cis-DDP increased by 18 to 32% (average, 27%) between 0 and 6.5 h after treatment and then declined. Adduct half-lives in this latter phase did not correlate with the sensitivities of the cells for cis-DDP. These results indicate that the initial level of cis-DDP-DNA binding measured by quantitative immunocytochemistry may be a reasonable predictor of sensitivity to this chemotherapeutic drug.

The formation and stability of interaction products between the anti-cancer drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA were studied in buccal epithelial and urinary cells from ten cancer patients who received cis-DDP-based therapy. Buccal cells were collected 1 h before and 1-2 h after i.v. infusions with cis-DDP. The interaction products were visualized in an immunocytochemical peroxidase assay, using an antiserum against cis-DDP-modified calf thymus DNA. The nuclear staining density was measured by microdensitometry. Nuclear staining densities in buccal cells after infusions of greater than or equal to 20 mg/m2 cis-DDP were always higher than pretreatment values. Repeated sampling from individual patients treated for 2-5 consecutive days with daily doses of 20-70 mg/m2 cis-DDP indicated that cis-DDP-DNA binding in buccal cells increased in proportion to the cumulative total dose of cis-DDP. The variation in dose-density response between patients was 17%. Apparent adduct loss in buccal cells from four patients, as measured 8-17 days after the last infusion, amounted to 67-86%. Platinum-induced DNA modifications could also be detected in buccal cells from two cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)-treated patients. In vitro experiments with human buccal cells and lymphocytes indicated linear relationships between DNA modification and either cis-DDP concentration or incubation time. Nuclear staining densities in pretreatment buccal cells from ten cancer patients treated in vitro with 33 microM cis-DDP for 1 h revealed that interpatient variation in in vitro DNA modification by cis-DDP was low. No quantitative correlation was found between in situ and in vitro DNA modification.

The in situ binding of the anticancer drug cis-diamminedichloroplatinum(II) (cisDDP) to DNA was studied in the rat dorsal root spinal ganglion (DRG), using an antiserum against cisDDP-modified calf thymus DNA in a quantitative immunocytochemical assay. Rats received a dose of cisDDP (1 mg/kg), two times a week, up to a cumulative dose of 15 mg/kg (group I) or 34 mg/kg (group II). Rats of group III were given a single dose of 15 mg/kg. Rats were killed 48 hr (groups I and II) or 6 hr (group III) after the last injection. In groups I and II cisDDP-induced neurological damage was assessed by measuring both motor and sensory nerve conduction velocities (MNCV and SNCV). Whereas the MNCV was not influenced by the treatment with cisDDP, the SNCV decreased significantly. The level of cis-DDP-DNA binding in DRG satellite cells equalled that in liver cells, but binding could not be shown in DRG neuron nuclei. The level of cisDDP-DNA binding in spinal cord and brain was very low. The neuroprotecting peptide ORG.2766, an ACTH4-9 analog, was given sc (10 micrograms/rat) four times a week concomitantly with cisDDP to some rats of groups I and II. ORG.2766 prevented the decrease of the SNCV, but did not change the extent of cisDDP-DNA binding in satellite or liver cells. It is concluded that the amelioration of cisDDP toxicity by ORG.2766 is not directly related to the cisDDP-DNA binding in satellite cells.

Calf thymus DNA was modified in vitro by cis-diamminedichloroplatinum(II) (cisDDP), complexed with methylated bovine serum albumin and used to immunize rabbits. The anti-cisDDP-DNA antiserum obtained was applied in a double peroxidase-antiperoxidase staining procedure to localize cisDDP-DNA and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA)-DNA interaction products in cryostat tissue sections of mice and rats. Rats received cisDDP (0-10 mg/kg) and were killed after 24 h. Mice received cisDDP (0-15 mg/kg) or CBDCA (200 mg/kg), and were killed after 2 h-162 days. For each time-dose combination two mice or one rat were used; agents were given i.p. Specific nuclear staining was observed in all tissues examined from cisDDP- or CBDCA-treated animals. No significant nuclear staining could be observed in tissue sections from control rats and mice. The extent of staining after cisDDP was dose and time dependent. The lowest dose of cisDDP after which specific nuclear staining could be detected varied from tissue to tissue [e.g., 0.1 mg/kg, pancreas (mouse); 0.5 mg/kg, liver, kidney (mouse, rat)]. The longest time interval after a single dose of 6 mg/kg cisDDP in which adducts could be visualized also depended on the tissue and varied between 9 days (spleen, testis) and 162 days (kidney). The staining intensity in liver and kidney, measured microdensitometrically, decreased relatively fast in the first days after treatment, but much slower thereafter. In the kidney, cisDDP-induced DNA modification showed regional variation: inner cortex greater than outer cortex greater than medulla (rat) and cortex greater than medulla (mouse). In the mouse kidney, a small subpopulation of tubular cells in close association with the renal corpuscles showed a remarkably high staining intensity after both cisDDP and CBDCA administration. Tissues that showed clear cisDDP-induced histological alterations (kidney, pancreas, testis, and duodenum) also showed moderate to high levels of cisDDP-DNA interaction products. A correlation between cell damage (measured histologically) and cisDDP-DNA binding within one tissue type was demonstrated in the rat inner renal cortex, the murine renal cortex, and in duodenal epithelial cells of both mice and rats.