Kinetics of the reaction of cis-platinum compounds with DNA in vitro.

The kinetics of the reaction of a series of cis-platinum(II) compounds with DNA in vitro has been studied using their ability to disturb the secondary structure of the macromolecule. The complexation modifies the stacking of the base pairs and causes an inhibition of the intercalation of ethidium bromide which is correlated with the number of platinum atoms bound per nucleotide. The compounds fall into three groups which react in a few minutes, in a few hours or in several days. The inhibition of the complexation by chloride and carboxylato ions indicates that the interaction occurs through hydrolysed species and that hydrolysis is the rate limiting step. In addition the results indicate that the carboxylato entities are able to react with DNA in vitro without enzymatic activation and that there is no correlation between the antitumoral activity of these compounds against L1210 Leukemia cells and their in vitro reactivity towards DNA.

Pharmacological and preclinical toxicological studies of 1,2-diaminocyclohexane(isocitrato)platinum(II).

The antitumor activity of a new highly water-soluble platinum derivative, (1,2-diaminocyclohexane)(isocitrato)platinum(II) (NSC 350602; PHIC), was studied in L1210 leukemia cells inoculated into mice. PHIC was found to be active for i.p. graft-i.p. treatment, i.p. graft-i.v. treatment, and i.v. graft-p.o. treatment. A significant activity was observed on early and advanced L1210 leukemia even when the treatment was delayed 6 days after the graft. A comparison between the activities of PHIC, cisplatin (NSC 119875), and (4-carboxyphthalato)(1,2-diaminocyclohexane)-platinum(II) (NSC 271674; DACCP) for i.p. graft-i.p. treatment indicated that the highest activity was observed for divided doses rather than single dose in the case of PHIC and DACCP and not for cisplatin. Under these conditions, PHIC gave larger treated versus control survival time values or a greater number of surviving animals than did cisplatin and DACCP. No cross-resistance between PHIC and cisplatin could be detected in L1210 leukemia cells resistant to cisplatin. Mutagenicity studies on Salmonella typhimurium revealed that PHIC is far less mutagenic than cisplatin on TA100 and TA98 strains. Other pharmacological parameters, such as growth inhibition rate of cultured L1210 cells, penetration, and DNA binding in L1210 cells inoculated in mice, were compared for PHIC and cisplatin together with their in vitro rates of hydrolysis and platinum:DNA adducts. No nephrotoxicity was detected with PHIC at the maximum nonlethal dose level in mice in contrast to results with cisplatin. A preclinical study was conducted in baboons at 100, 150, and 200 mg/kg. No nephrotoxicity could be detected at a dose of 100 mg/kg without prehydration for six courses at 3-week intervals. At 200 mg/kg, an increase of blood creatinine was controlled by prehydration. Gastrointestinal toxicity was mild during the three regimens. Phase I clinical trials are under way.

Induction of polyploid nuclei in the plasmodium of Physarum polycephalum by platinum antitumor compounds.

The intranuclear mitosis of the plasmodial nuclei of myxomycetes permits the observation of defects in chromosomal repartition which would probably be lethal in other eukaryotic cells with open mitosis. We found that antitumoral platinum-amine compounds perturbed late mitotic events and induced the formation of giant nuclei which were polyploid in plasmodia of Physarum polycephalum. Using 26 platinum-amine complexes, we have shown that all antitumoral compounds induced the formation of polyploid nuclei for drug concentrations at least three times lower than the amount necessary to block the overall plasmodial growth, whereas platinum compounds without antitumor activity did not behave this way. DNA replication appeared to be quantitatively normal during formation of giant nuclei by antitumoral compounds. These observations suggest that platinum-amine compounds exert their antitumor activity by interfering with mitosis rather than by a gross inhibition of DNA synthesis.

Platinum-amine compounds: importance of the labile and inert ligands for their pharmacological activities toward L1210 leukemia cells.

A series of Pt-amine compounds was assayed for their ability to inhibit the growth of cultured L1210 leukemia cells [median inhibitory dose (lD50)], their toxicity in mice [highest nonlethal dose in healthy mice (LD0)], their antitumor activity against leukemia L1210 cells grafted intraperitoneally into mice [mean survival of treated leukemic mice:mean survival of untreated leukemic mice (T/C)], and the ability to hydrolyze their labile ligands in vitro [hydrolysis half-time (t1/2)]. All Pt compounds exhibiting antitumor activity had a pair of labile ligands in the cis geometry with different charges, and the leaving groups had a wide range of hydrolysis rates. Among the compounds that showed antitumor activity, ID50 depended more on the inert ligands than on the labile ligands and was correlated with T/C. A relationship between LD0 and t1/2 was verified in the series of cis-Pt(II) compounds with the exception of the oxalate derivatives. Pt(II) and Pt(IV) compounds exhibited similar ID50, LD0, and T/C.

Whole-body autoradiographic study of the distribution of 195mPt in healthy and tumor-bearing mice treated with labeled cisplatin.

195mPt-labeled cisplatin was administered iv and ip to control mice and to mice bearing Sarcoma 180. The delivered dose was 8 mg/kg and the injected activity was 80 muCi/kg. Whole-body autoradiography was performed at various times after treatment. For all animals, concentration of radioactive material was observed in the kidneys as early as 5 minutes after treatment. Most of the delivered radioactivity was present in the urine-bladder for the first 4 hours after the injection. Autoradiography also showed concentration of 195mPt in the bile ducts. These results suggest that a lower but significant part of the delivered radioactivity was also eliminated in the bile, which may explain in part the presence of the isotope in the gut lumen. In addition, 195mPt-equivalents were observed in the kidney parenchyma, the cartilages, the teeth, the skin, and the hair. Only trace amounts of 195mPt were present in the hypophysis and the testes. The distribution of radioisotope in healthy and tumor-bearing animals was comparable. Measurable uptake occurred in the Sarcoma 180 tumor.

cis-Pt(NH3)2Cl2 and trans-Pt(NH3)2Cl2 inhibit DNA synthesis in cultured L1210 leukemia cells.

A comparison of the inhibition of DNA synthesis by the two geometrical bidentate isomers cis- and trans-Pt(NH3)2Cl2 and by the monodentate [Pt(dien)Cl]Cl in a model used for screening potential antitumor compounds, the L1210 leukemia cells, is presented. The efficacy of penetration after a 2 hours Pt treatment is in the order trans (8) greater than cis (1) approximately dien (0.7). DNA replication is reduced to 50% of the control when 1.8 X 10(-4), 2.4 X 10(-4) and 80 X 10(-4) Pt atoms were bound per nucleotide for cis, trans and dien derivatives, respectively. If we admit that DNA is the pharmacological target of Pt antitumor compounds, these results suggest that a quantitative inhibition of DNA synthesis is certainly not correlated with antitumor activity.

Structures of the adducts formed between [Pt(dien)Cl]Cl and DNA in vitro.

The products of the reaction between [Pt(dien)Cl]Cl and salmon sperm DNA have been purified and their structures determined. [Pt(dien)Cl]Cl binds at the N7 position of guanine for levels of fixation below 0.1 platinum per DNA base. Above this level of binding, [Pt(dien)Cl]Cl also reacts at the N7 position of adenine. 1,7-[Pt(dien)]2Ade was observed when more than 0.3 platinum per base were bound to the DNA. Platination at the N7 position of guanosine, unlike alkylation, stabilized the glycosyl linkage and did not lead to fission of the imidazole ring at high pH.

Immunochemical studies of DNA modified by cis-dichlorodiammineplatinum(II) in vivo and in vitro.

Two rabbits were immunized with native DNA modified in vitro by cis-dichlorodiammineplatinum(II) (cis-Pt). The interactions between the antiserum and several natural and synthetic nucleic acids were studied primarily by radioimmunoassays. Native DNA's modified by platinum compounds with labile groups in the cis position are recognized by the antiserum. No cross-reaction is found with native DNA's substituted by other platinum compounds [trans-dichlorodiammineplatinum(II), cis-diamminotetrachloroplatinum(IV), and chlorodiethylenetriamminoplatinum(II)] and with natural and synthetic modified polyribonucleotides. cis-Pt-modified oligodeoxyribonucleotides and enzymatically hydrolyzed cis-Pt-modified native DNA do not bind to the antiserum. cis-Pt-modified double-stranded synthetic polydeoxyribonucleotides are either hardly recognized or not recognized at all. The antiserum recognized a modified double-stranded helix in DNA which is not formed in several modified synthetic DNA's and RNA's. At least two different antigenic determinants are present in cis-Pt-modified DNA at a ratio of cis-Pt to bases equal to 0.05. Finally, the antiserum does not react with in vivo cis-Pt-modified DNA.

Kinetics of cisplatin in an anuric patient undergoing hemofiltration dialysis.

After in vitro tests, a tracer dose of cisplatin was administered to an anuric cancer patient. Plasma total platinum levels were monitored as a function of time, as was filterable platinum elimination by hemofiltration dialysis. Based on the derived pharmacokinetic parameters, the patient was later given 50-mg doses of cisplatin, once as a single agent, then four times in combination with doxorubicin, VM-26, and cyclophosphamide. Plasma levels could be fitted by a sum of three exponentials after the infusion was stopped. The mean corresponding half-lives are 30 minutes, 6 hours, and 5 days. Depending on the rate of infusion, plasma peaks ranged between 1.1 micrograms/ml (5.7 X 10(-6) M) and 2.27 micrograms/ml (1.16 X 10(-5) M). During the hemofiltration process immediately following the infusion, the elimination rate was first-order, with a mean half-life of 60 minutes. The mean amount eliminated during the first hemofiltration session was 8% of the dose, decreasing to about 3% on Days 2 and 3. A partial tumor response was observed after two treatment courses, evidenced by the relief of abdominal pain and bowel obstruction episodes and a 50% decrease in peritoneal metastases.

Viscosity, nicking, thermal and alkaline denaturation studies on three classes of DNA-platinum complex.

Viscosity, nicking, thermal denaturation and alkaline denaturation studies were used to investigate perturbations induced in the DNA secondary structure after complexation with platinum compounds. Three types of DNA-platinum complex, representative of the different modes of platinum binding, have been studied. Cis-Pt(NH3)2Cl2, which forms a cis-bidentate complex with DNA, strongly decreased the viscosity and, according to thermal and alkaline denaturations, destabilized the macromolecule. On the contrary, trans-Pt(NH3)2Cl2, a trans-bidentate complex, stabilized the DNA secondary structure and decreased the viscosity but much less than did cis-Pt(NH3)2Cl2. [Pt(dien)Cl]Cl, a monodentate complex, had no effect on the viscosity; however, addition of this compound stabilized DNA up to 0.01 platinum bound per nucleotide while further Pt binding destabilized the macromolecule. Cis- and trans-Pt(NH3)2Cl2 renatured thermally denatured DNA, which has been interpreted as evidence for the presence of interstrand crosslinks. [Pt(dien)Cl]Cl, on the other hand, did not renature DNA. If the renaturation observed for the bidentate compounds is due only to the presence of interstrand crosslinks, then one interstrand crosslink is found when 400-1000 molecules of the platinum isomer are bound per T7 DNA molecule. Electron microscopy results show that the three types of DNA-platinum complex do not nick DNA up to 0.01 bound platinum per nucleotide.

A circular dichroism study of DNA.platinum complexes. Differentiation between monofunctional, cis-bidentate and trans-bidentate platinum fixation on a series of DNAs.

The circular dichroism (CD) spectra of a series of DNA . platinum complexes are presented. The following platinum compounds, [Pt(dien)Cl]Cl, cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, trans-Pt-(NH3)2Cl2, K[Pt(NH3)Cl3] and K2[PtCl4] were complexed with the DNA extracted from bacteria Micrococcus lysodeikticus (72% dG + dC), Escherichia coli (50% dG + dC), Clostridium perfringens (32% dG + dC) and salmon sperm (41% dG + dC). Strong differences were found between the different DNA . Pt complexes. Three types of spectra clearly demonstrate the different platinum binding modes on DNA. In the first type, the platinum compound, i.e. [Pt(dien)Cl]Cl, is fixed to DNA with only one bond (monofunctional complex formation) and no significant change of the CD positive band of DNA is found. The main feature of the second type is a continuous intensity decrease of the positive band as observed for trans-Pt(NH3)2Cl2 (trans-bidentate complex formation). The third type concerns the cis-bidentate platinum fixation obtained with cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, K[Pt(NH3)Cl3] and K2[PtCl4]. The CD spectra are in this case characterized by an increase in the positive Cotton effect which is dG + dC-dependent up to an rb value around 0.10 (where rb = number of platinum atoms bound per nucleotide), followed by a decrease until DNA saturation with platinum is reached. A linear decrease in the amplitude of the negative band is detected in all the complexes except in the case of the monofunctional DNA . Pt complexes. For the cis-bidentate and trans-bidentate platinum fixation, a continuous bathochromic shift occurs.

Relative efficiencies of a series of square-planar plantinum(II) compounds on Salmonella mutagenesis.

Twelve Pt(II) compounds have been tested for mutagenicity on Salmonella typhimurium (strain TA 100). Very high mutagenic activities were found for the cis derivatives. A correlation is suggested between these results and a formerly described model of chemical reactivity towards DNA, according to which cis derivatives from intra-strand chelates with guanine. A smaller activity was found with monodentate complexes with DNA.

Differentiation between chelated and non-chelated DNA-Pt complexes by atomic absorption spectrophotometry.

As a continuation of our study of the properties of the cis-trans platinum series we have investigated the DNA-Pt complexes of [Pt(dien)Cl]Cl, cis-Pt (en)Cl2, cis-Pt (NH3)2Cl2 and K2[PtCl4] by atomic absorption spectrophotometry. The DNA-Pt complexes correspond to the saturation of the N7-(Guanine) sites. It has been found that the chelate complexes obtained with cis-Pt(en)Cl2, cis-Pt(NH3)2Cl1 and K2[PtCl4] show the same absorbance. The [Pt(dien)Cl]Cl and trans-Pt (NH3)2Cl2 which are bound to the N7 (tuanine) site only, show an absorbance greater than the chelate complexes by a factor of two. In addition, it has been possible in the case of the trans-Pt (NH3)CCl1 complex to follow the fixation of platinum to DNA by atomic absorption spectrophotometry. The result is similar to the ionic chlorine liberation procedure reported previously.

X-ray photoelectron spectroscopy of DNA-Pt complexes. Evidence of O6 (Gua)-N7 (Gua) chelation of DNA with cis- dichlorodiamine platinum(II).

The binding energies of nitrogen, oxygen, phosphorus, chlorine and Pt in several DNA - Pt (II) complexes are reported and discussed. The nitrogen band of DNA is slightly shifted upon complexation with Pt. Oxygen binding energies in the complexes studied clearly show that cis-Pt(NH3)2Cl2 forms a specific chelate N7(Gua) - O6 (Gua) with DNA as opposed to trans-Pt(NH3)2Cl2 and the other Pt compounds which react only with the N7(Gua) site of DNA.