Identification of platination sites on human serum transferrin using (13)C and (15)N NMR spectroscopy.

Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl(2)] or cis-[PtCl(2)(NH(3))(2)] have been investigated in solution via observation of [(1)H,(15)N] NMR resonances of the Pt complexes, [(1)H,(13)C] resonances of the eCH(3) groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days.

Synthesis, structural characterization, and antitumor properties of a novel class of large-ring platinum(II) chelate complexes incorporating the cis-1,4-diaminocyclohexane ligand in a unique locked boat conformation.

The first two analogs 5a,b of a new class of neutral large-ring square-planar Pt(II) chelate complexes of the generic structure [Pt(cis-1,4-dach)X2] were synthesized via a refined technique, structurally characterized by NMR (1H, 13C, 195Pt), FAB mass spectrometry, and X-ray crystallography, and evaluated for antitumor activity in vitro and in vivo in sensitive and Pt-resistant murine leukemia cell systems. An X-ray crystal structure analysis confirmed that [Pt(cis-1,4-dach)malonate] 5b is monomeric and that the cis-1,4-diaminocyclohexane (dach) ligand is incorporated in a unique and previously unknown locked boat conformation. Complex 5b crystallized as colorless rectangular plates in the orthorhombic space group Pcmn with Z = 4 and the lattice parameters a = 6.239(1) A, b = 9.965(2) A, and c = 18.437(4) A. Important structural parameters are Pt-O = 2.024(5) A, Pt-N = 2.021(6) A, N-Pt-N = 100 degrees, and N-Pt-O = 85 degrees; R = 0.0515, Rw = 0.0635. Antitumor results in murine tumor models show that the parent molecule 5a (X2 = 2 Cl) (a) is more dose potent than cisplatin against the leukemias and solid tumors examined, (b) possesses significant activity against cisplatin-resistant leukemias, but exhibits partial cross-resistance with cisplatin, and (c) may possess a spectrum of activity different from that of cisplatin. Antitumor test results in vitro indicate that (a) 5a is at least equivalent to cisplatin in dose potency and effectiveness in the leukemia cell systems studied except in the [Pt(1,2-dach)Cl2]-resistant L1210 cell line, (b) the cisplatin-resistant leukemia cell systems exhibit partial cross-resistance to 5a, (c) 5a possesses either comparable or greater cytotoxicity than the reference complexes, CI-973 (3) and bis(platinum) complex 4, and (d) 5a is more effective (approximately 18-fold) than [Pt(1R,2R-dach)Cl2] 2 in inhibiting growth in the Pt(1,2-dach)-resistant L1210 cell line, suggesting that [Pt(cis-1,4-dach)Cl2] is either not recognized as or is not acting as a "typical" Pt(dach) complex. The encouraging antitumor activity of 5a, coupled with a 10-fold higher aqueous solubility compared to [Pt(1R,2R-dach)-Cl2] 2 warrants the following future studies: synthesis of selected analogs, elucidating the nature of Pt-DNA binding sites, the mechanism of action, and the mechanistic basis for the lack of cross-resistance of [Pt(cis-1,4-dach)Cl2] against the [Pt(1,2-dach)Cl2]-resistant L1210 cell line.

In vitro analysis of the interaction between sucralfate and ketoconazole.

In healthy volunteers, the bioavailability of ketoconazole is significantly decreased during simultaneous administration with sucralfate. In an effort to address this problem, we examined the interaction between sucralfate and ketoconazole in aqueous solutions and in simulated gastric fluid (SGF) at various initial pHs (1, 2, 3, and 6) in the presence or absence of glutamic acid hydrochloride (GA). Samples from each solution were taken 30 min and 2 h after the addition of ketoconazole to evaluate the solubility of ketoconazole over the usual time period of maximal absorption of ketoconazole in humans. The addition of GA to SGF leads to an increase in solution acidity, while the pHs of SGF at a pH of 1, 2, or 3 are markedly increased by the addition of sucralfate. There is a net decrease in acidity from initial pHs for the pH 1, 2, and 3 solutions when GA and sucralfate are combined. The concentration of ketoconazole in SGF at pHs of 1, 2, 3, 4, and 6 was evaluated in order to assess the pH-dependent solubility properties of the drug in the absence of other interacting species. Regardless of the initial pH, combinations of GA plus ketoconazole showed high concentrations of ketoconazole (approximately 100%) in solution. In contrast, significant decreases in the concentration of soluble ketoconazole were observed when sucralfate was mixed with ketoconazole, and, in some cases, soluble ketoconazole was not detectable. The addition of GA to a mixture of sucralfate and ketoconazole leads to a significant increase in the concentration of solubilized ketoconazole. Nonetheless, important sucralfate-ketoconazole interactions are still observed. After 2 h, approximately 35% of the maximal ketoconazole concentration remained in solution. Comparison of the ketoconazole concentrations at different pHs with the predicted concentrations of the three protonation species of ketoconazole [H2(ketoconazole)(2+), H(ketoconazole)(+), or ketoconazole] showed no correlation. Therefore, the decrease in ketoconazole solubility is not simply a reflection of pH perturbation associated with the dissolution of sucralfate. The observed data are most consistent with a model that has H2(ketoconazole)(2+) or H(ketoconazole)(+) forming an electrostatic interaction with the sucralfate polyanion. The findings of this study suggest that the coadministration of sucralfate with other azole antifungal agents should be investigated.

Comparative nephrotoxicity of a novel platinum compound, cisplatin, and carboplatin in male Wistar rats.

The nephrotoxicity of three platinum-containing antitumor agents was compared at doses that approximate the LD10 (cisplatin) or the LD50 (CI-973, carboplatin) doses. Male Wistar rats were administered single iv doses of 45 mg/kg CI-973, 6.5 mg/kg cisplatin, or 65 mg/kg carboplatin and observed for 4 days. Cisplatin treatment increased blood urea nitrogen (4x), creatinine (3x), glucose, and fractional electrolyte excretions, and decreased creatinine clearance by Day 4. These parameters were not significantly altered in CI-973- and carboplatin-treated animals. Cisplatin increased urinary excretion of LDH (six-fold), GGT (twofold), and NAG (twofold); CI-973 and carboplatin increased GGT excretion (approximately twofold). Cisplatin induced the following functional changes as a consequence of direct nephrotoxicity: decreases in GFR (84%), ERPF (97%), ERBF (96%), and ERTS (95%), and increases in FF (fivefold). Functional changes, attributed to prerenal effects of CI-973, included a decrease in ERPF (35%) and an increase in FF (48%). No changes were seen following carboplatin treatment. All cisplatin-treated rats had proximal tubular necrosis in the outer stripe of the outer medulla, extending multifocally into inner cortical medullary rays. No renal lesions were detected by light or electron microscopy in the control or CI-973- or carboplatin-treated rats. Cisplatin produced marked nephrotoxicity as determined by biochemical, functional, and histopathologic endpoints. CI-973 and carboplatin were significantly less nephrotoxic than cisplatin.

Anticancer activity in murine and human tumor cell lines of bis(platinum) complexes incorporating straight-chain aliphatic diamine linker groups.

The biological activity of a series of dinuclear bis(platinum) complexes of formula [(cis-PtX2-(NH3)]2(NH2(CH2)nNH2)] (X = Cl, n = 4-9, compounds 6-11; X2 = malonate, n = 5 or 6, compounds 12 and 13) is described in selected murine leukemia, murine solid tumor, and human tumor cell lines and in murine leukemia cell lines rendered resistant to cisplatin (cis-[Pt(NH3)2Cl2]). The bis(platinum) compounds showed greater activity in vitro against murine tumor cell lines resistant to either cisplatin or DACH ([Pt(DACH)Cl2]). The resistance factor is dependent on chain length of the diamine, and the structural feature of a dinuclear complex is of general use in reducing cross-resistance with cisplatin. In vivo [(cis-PtCl2(NH3)]2(NH2(CH2)5NH2)] (7) showed a % T/C of 204 against murine L1210 leukemia resistant to cisplatin compared to a % T/C of 104 for cisplatin itself at optimal doses. The complex [(Pt(mal)(NH3)]2(NH2(CH2)6NH2)] (13) was highly active in the colon 26 tumor line with 3/10 tumor-free survivors (dose of 186 mg/kg, ip D1,5,9); however, 13 was subject to substantial cross-resistance in the cisplatin resistant L1210 leukemia (% T/C 139 versus % T/C of 223 in the sensitive line). In four selected human tumor lines in vitro, compounds 6-11 were uniformly more potent than cisplatin. In the corresponding xenografts, compound 7 showed greater activity in the HCT-8 (coloadenocarcinoma) and H23 (nonsmall cell lung), but diminished potency in AH125 and H520 (both nonsmall cell lung) lines in comparison to cisplatin. Retention of activity against cisplatin-resistant cell lines and a different spectrum of activity compared to cisplatin in some human tumor cell lines suggest that this class of complexes is mechanistically different from mononuclear complexes and worthy of further development toward clinical trials.

Inorganic concepts relevant to metal binding, activity, and toxicity in a biological system.

Selected physical and inorganic concepts and factors which might be important in assessing and/or understanding the fate and disposition of metal compounds in a biological environment are reviewed. The stereochemistry (geometry), thermodynamic stability, redox properties, and intrinsic reactivity/lability are properties of metal compounds which can have a major influence on metal-target binding and on the eliciting of activity/toxicity. Concepts and factors are illustrated with appropriate examples where possible. Efforts to correlate the toxicity of metal compounds with a suitable indicator of toxicity should be expanded to include other parameters or combinations of parameters.

In vivo and in vitro binding of platinum to metallothionein.

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammineplatinum(II]. Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl2 significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl2 pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.

Pharmacokinetics of Ip cisplatin in refractory ovarian carcinoma.

Four patients with small residual ovarian carcinoma following treatment with cisplatin, doxorubicin, and cyclophosphamide have subsequently received 57 courses of ip cisplatin. Cisplatin (120-270 mg in 2 L of Ringer's lactate) was administered via Tenckhoff catheter, with a dwell time of 15-20 mins. Courses were given weekly for 12 weeks, with response documented by laparoscopy or laparotomy prior to and following the trial. With a dwell time of 20 mins, 75% +/- 5% (mean +/- SD) of platinum was recovered. With 120 mg of cisplatin and a dwell time of 20 mins, total plasma platinum peaked at 1.23 +/- 0.42 microgram/ml and by 8 hrs decreased to 0.67 +/- 0.12 microgram/ml. Filterable (non-protein-bound) platinum peaked at 0.73 +/- 0.21 microgram/ml and by 8 hrs fell to 0.03 microgram/ml. Excretion rate paralleled the filterable plasma curve, peaking at 40 mins; 30% +/- 7% of absorbed drug was recovered in urine within 24 hrs. Renal clearance of filterable platinum was 106 +/- 20 ml/min. Creatinine clearance was 76 +/- 7 ml/min. Three responses, one complete and two partial, were noted. Zero to two episodes of vomiting occurred in each course. One patient had a creatinine clearance decrease to 40 ml/min, one had two episodes of thrombocytopenia, and one had mild abdominal pain with a cisplatin dose of greater than or equal to 210 mg. No neurotoxicity, catheter infection, or peritonitis was encountered.

Investigation of correlations between chemical parameters of metal ions and acute toxicity in mice and Drosophila.

We are studying correlations between physicochemical properties associated with metal ions and observed toxicity. In order to test correlations, we obtained, under uniform conditions, LD50 values for acute toxicity in mice for 24 metal ions. The new data show a better correlation between LD50 and Pearson and Mawby's softness parameter sigma p, defined using chemical concepts of hard and soft acids and bases, than had been obtained by others. From a wide range of physicochemical parameters, the electrode potential can give almost as good a correlation as sigma p. Better correlations might exist for parameters more relevant to biological systems.

Comparison of the iv and ip routes of administration of cisplatin in dogs.

Cisplatin at a dose of 3 mg/kg was administered to dogs either iv or ip. Cisplatin concentrations in serum, urine, and tissues were measured with a radioisotope tracer method employing 195mPt cisplatin. Systemic toxicity was monitored by serial BUN, creatinine, and wbc and platelet counts. The mode of administration did not affect systemic toxicity since the changes in renal and bone marrow functions were identical in the two groups. Serum cisplatin levels following iv administration peaked at 13.5 micrograms/ml at 5 minutes and were biphasic with rapid initial decline and a prolonged elimination phase. In contrast, levels following ip administration increased rapidly to 1.5 micrograms/ml at 4 hours and then decreased with the iv levels. The amount of drug recovered in the urine was similar regardless of method of administration, with approximately 50% of the injected dose excreted by Day 4. The drug levels within the tissues on Days 4 and 8 were similar, with the exception of the tissues lining the peritoneal cavity. On Day 4 the tissues lining the peritoneal cavity had 2.5-8 times higher levels of drug after ip administration, and this difference was statistically significant (P less than 0.01). Local toxic effects encountered with ip administration consisted of bloody ascites on Day 4 (four of none dogs) and filmy adhesions on Day 8 (one of four dogs). It is concluded that ip cisplatin chemotherapy may increase the therapeutic index for small tumors which are confined to the peritoneal cavity.

The metabolism of cis-dichlorodiammineplatinum (II): distribution, clearance, and toxicity.

The distribution and toxicity of 195mPt-labeled cis-platinum were studied in two groups of dogs. Serum and tissue levels, renal and marrow toxicities, and urinary excretion were compared in dogs given cis-platinum, 3 mg/kg, intravenously with a group treated by intraperitoneal administration. Peak serum levels occurred immediately after intravenous administration and 8 hours after intraperitoneal instillation, at which time the two were identical and declined at similar rates. Mean levels of cis-platinum measured in peritoneal tissues on day 4 were higher in the intraperitoneal group. Blood urea nitrogen, creatinine, and white blood cell and platelet counts were similar in both groups. Bloody ascites and adhesion formation occurred in 37.5% of dogs treated by intraperitoneal administration. The intraperitoneal route of chemotherapy administration results in higher drug levels in target peritoneal tissues without a corresponding increase in systemic toxicity.

Therapeutic synergism of hyperthermia-cis-platinum in a mouse tumor model.

A small-animal model was developed as a guide to whole-body hyperthermia in cancer patiens. Anesthetized DBA/2 mice were secured to a platform, and their hindlimbs were immersed in a 42.3 degrees C water bath for 30-60 minutes. Hindlimb hyperthermia reulted in steady-state rectal and femoral bone marrow and muscle temperatures of 42 degrees C and upper extremity muscle and esophagus temperatures of 41 degrees C. With this hyper thermia technique, the mouse spleen colony assay could be used to quantitate the lethality of hyperthermia and/or cis-dichloro-diammineplatinum(II) (cis-platinum) on clonogenic bone marrow and leukemia cells. Hyperthermia prior to cis-platinum administration increased cis-platinum inhibition of leukemia colony formation as much as 2 logs; however, antileukemia synergism ws greatest when cis-platinum administration immediately preceded hyperthermia and no evidence existd of synergism against normal bone marrow colonies. Correlative in vivo drug uptake studies showed a marked increase in leukemia cell uptake of 195mPt-cis-platinum at elevated temperatures, which suggested a potential mechanism for the apparent antileukemia synergism of cis-platinum and heat.

Mutagenicity, cytotoxicity, and DNA binding of platinum(II)-chloroammines in Chinese hamster ovary cells.

The cytotoxicity and mutagenicity at the six platinum(II)chloroammines have been investigated in Chinese hamster ovary cells. For these compounds, the observed slopes of the mutation-induction curves (mutants/10(6) cells/microM) were: cis-Pt(NH3)2Cl2 (31.5), K[Pt(NH3)Cl3] (2.78), [Pt(NH3)3Cl]Cl (0.11), K2[PtCl4] (0.12), trans-Pt(NH3)2Cl2 (0.013), and [Pt(NH3)4]Cl2 (0.0). The relative cytotoxicity of these compounds follows the same order and is of similar magnitude. The observed relative mutagenicities of these compounds paralleled their reported potencies in the Ames assay and their relative antitumor activities. Results indicate that Chinese hamster ovary cells are useful in quantifying low mutagenic activity of chemicals such as platinum compounds. Studies with 195mPt-labeled cis- and trans-Pt(NH3)2Cl2 showed that during treatment both compounds enter the cell and bind to the DNA with comparable efficiency. Hence, the relative mutagenicities of cis- and trans-Pt(NH3)2Cl2 are not a consequence of different initial levels of DNA binding.

Kinetic analysis of the in vitro binding of radioactive cis- and trans-dichlorodiammineplatinum(II) to DNA.

The binding of cis(c)- and trans(t)-Pt(NH3)2Cl2 to DNA at platinum/DNA-nucleotide ratios (Ri) of 0.1 or less has been studied by means of radioactive 195mPt-labeled compounds. Kinetic data are consistent with the following scheme: (Formula: see text). At 25 degrees C and pH 5-6 in 5 mM NaClO4, the values for the rate constants in the above scheme for the c-isomer are k2 = 2.2 X 10(-5) sec -1, k7 = 0.32 (sec M)-1, and k8 = 143 (sec M)-1; for the t-isomer the values are k2 less than 0.5 X 10(-5) sec-1 and k7 = 0.95 (sec M)-1. Platinum-DNA adducts do not undergo detectable exchange for 3 days at 37 degrees C, indicating the absence of a dynamic equilibrium. For both isomers the rate of binding is the same for single- and double-stranded DNA. The conclusions derived from Ag+ and H+ titration studies are consistent with binding at guanine N(7) for Ri less than 0.1. The reaction rate is competitively inhibited by various salts and buffers and is suppressed by raising the pH (50% inhibition of initial rates at pH 7.3). At 37 degrees C and pH 7 in 0.15 M NaCl, 6-8% of both the c- and t-isomers bind to DNA in 24 h, suggesting that both compounds should bind to DNA under biological conditions.

Binding of cis- and trans-dichlorodiammineplatinum(II) to the nucleosome core.

The binding of the antitumor drug cis-dichlorodiammineplatinum(II) and its inactive trans isomer with the nucleosome core particle has been investigated. Kinetic studies show that platinum binding increases with incubation time, from a few bound platinum atoms per nucleosome core in the first 0.5 hr to 40-50 after 40 hr. There is no crosslinking or dissociation of nucleosome cores upon platinum binding, as revealed by sedimentation velocity measurements. Electrophoresis through 0.1% sodium dodecyl sulfate/18% polyacrylamide gels after platinum binding reveals striking differences in the DNA and protein band patterns for the two isomers. cis-Dichlorodiammineplatinum(II) binds first to the DNA, retarding and spreading its migration in the gel. A comparison study with the 146-base-pair nucleosome core DNA showed the binding to be little affected by the presence of the histone octamer. The trans complex, on the other hand, produces DNA-histone and histone-histone crosslinks that only appear for the cis isomer after long incubation times. The protein-protein crosslinks were reversed by soaking the gel in cyanide solution to form [Pt(CN)(4)](-2). Subsequent two-dimensional gel electrophoresis revealed that trans-dichlorodiammineplatinum(II) forms specific crosslinks between histone protein pairs H3 and H2a and H2b and H4 in the nucleosome core. The occurrence of DNA-protein crosslinks was demonstrated by treating the platinum/nucleosome core reaction mixtures with a protease or with nucleases prior to electrophoresis and observing changes in the gel patterns. Platinum was located in the gels through autoradiography using (195m)Pt-labeled complexes. This work clearly demonstrates the greater propensity of trans-dichlorodiammineplatinum(II) to form histone-histone and histone-DNA crosslinks compared with the antitumor active cis isomer, which binds first to the DNA and only forms crosslinks to the histones when the nucleosome core is heavily loaded with platinum.