Constancy in Integrated Cisplatin Plasma Concentrations Among Pediatric Patients

The authors report on the variability in the integrated quantity of free (unbound) plasma cisplatin (area under curve of plasma concentration versus time, AUC). The AUC was measured in 19 patients receiving cisplatin doses proportional to body surface areas (BSA), 30mg/m2 over 1 hour. The relative standard deviation (RSD, population standard deviation divided by mean value) for the maximum free plasma cisplatin concentration (Cmax, μM) was 0.338; for the half-life (t½, minute), 0.210; and for the AUC (μM minute), 0.320. Thus, BSA-based dosing gave significant variability in the AUC. We attempted to use (weight)a(height)b, seeking values of a and b that gave the smallest RSD in AUC, but only minimal improvement could be obtained by deviating from the BSA formula (a = b = 0.5). However, dosing proportional to (weight)d(Cmax)f (with d ≈ 3/4 and f ≈ –1) reduced the RSD in AUC from ~1/3 to ~1/10. Dosing proportional to (weight)m(Cmax)n(t½)p (with m≈ 0.7, n ≈ –1, and p ≈ –½) reduced it further, to ~1/32. In contrast, using (weight)d(Cmax)f(age)g gave no improvement over (weight)d(Cmax)f. The authors conclude that the inconsistency in AUC can be reduced 10-fold with dosing proportional to the weight and the drug pharmacokinetic parameters [(weight0.7) (Cmax t½ 0.5)]

Oxygen Measurement via Phosphorescence: Reaction of Sodium Dithionite with Dissolved Oxygen

A homemade instrument for the measurement of oxygen concentration in aqueous solutions measures the decay rate of the phosphorescence of a Pd-porphyrin complex (phosphor) dissolved in the solution, which is flashed every 0.1 s with 630 nm light. The concentration of O 2 is a linear function of the decay rate. The instrument is used to study the reaction of dithionite (S 2O 4 2-) with O 2 at 25°C and 37°C. It is found that the ratio of dithionite to oxygen consumed in the reaction is 1.2 ± 0.2 at 25°C and 1.7 ± 0.1 at 37°C, suggesting a temperature-dependent stoichiometry. At both temperatures, the initial rate of O 2 consumption, -d[O 2]/dt, is found to be 1/2 order in S 2O 4 2- and first order in O 2. This finding is consistent with a previously proposed mechanism: S 2O 4 2- ↔ 2SO 2 - comes to a rapid equilibrium, and SO 2 - reacts with O 2 in the rate-determining step

Quantitative Measure of Cytotoxicity of Anticancer Drugs and Other Agents

Many anticancer drugs act on cancer cells to promote apoptosis, which includes impairment of cellular respiration (mitochondrial O 2 consumption). Other agents also inhibit cellular respiration, sometimes irreversibly. To investigate the sensitivity of cancer cells to cytotoxins, including anticancer drugs, we compare the profiles of cellular O 2 consumption in the absence and presence of these agents. Oxygen measurements are made at 37 °C, using glucose as a substrate, with [O 2] obtained from the phosphorescence decay rate of a palladium phosphor. The rate of respiration k is defined as -d[O 2]/dt in a sealed container. Different toxins produce different profiles of impaired respiration, implying different mechanisms for the drug-induced mitochondrial dysfunction. The decrease in the average value of k over a fixed time period, I, is proposed as a characteristic value to assess mitochondrial injury. The value of I depends on the nature of the toxin, its concentration, and the exposure time as well as on the cell type. Results for several cell types and 10 cytotoxins are presented here

Quantitative Measure of Cytotoxicity of Anticancer Drugs and Other Agents.

Many anticancer drugs act on cancer cells to promote apoptosis, which includes impairment of cellular respiration (mitochondrial O(2) consumption). Other agents also inhibit cellular respiration, sometimes irreversibly. To investigate the sensitivity of cancer cells to cytotoxins, including anticancer drugs, we compare the profiles of cellular O(2) consumption in the absence and presence of these agents. Oxygen measurements are made at 37 degrees C, using glucose as a substrate, with [O(2)] obtained from the phosphorescence decay rate of a palladium phosphor. The rate of respiration k is defined as -d[O(2)]/dt in a sealed container. Different toxins produce different profiles of impaired respiration, implying different mechanisms for the drug-induced mitochondrial dysfunction. The decrease in the average value of k over a fixed time period, I, is proposed as a characteristic value to assess mitochondrial injury. The value of I depends on the nature of the toxin, its concentration, and the exposure time as well as on the cell type. Results for several cell types and 10 cytotoxins are presented here

Derangements of liver tissue bioenergetics in Concanavalin A-induced hepatitis

BACKGROUND: A novel in vitro system was employed to investigate liver tissue respiration (mitochondrial O(2) consumption) in mice treated with concanavalin A (Con A). This study aimed to investigate hepatocyte bioenergetics in this well-studied hepatitis model. METHODS: C57Bl/6 and C57Bl/6 IFN-γ(−/−) mice were injected intravenously with 12 mg ConA/kg. Liver specimens were collected at various timepoints after injection and analyzed for cellular respiration and caspase activation. Serum was analyzed for interferon-gamma (IFN-γ) and aminotransferases. Fluorescence activated cell sorting analysis was used to determine the phenotype of infiltrating cells, and light and electron microscopy were used to monitor morphological changes. Phosphorescence analyzer that measured dissolved O(2) as function of time was used to evaluate respiration. RESULTS: In sealed vials, O(2) concentrations in solutions containing liver specimen and glucose declined linearly with time, confirming zero-order kinetics of hepatocyte respiration. O(2) consumption was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. Enhanced liver respiration (by ≈68%, p<0.02) was noted 3 hr after ConA treatment, and occurred in conjunction with limited cellular infiltrations around the blood vessels. Diminished respiration (by ≈30%, p=0.005) was noted 12 hr after ConA treatment, and occurred in conjunction with deranged mitochondria, areas of necrosis, and prominent infiltrations with immune cells, most significantly, CD3(+)NKT(+) cells. Increases in intracellular caspase activity and serum IFN-γ and aminotransferase levels were noted 3 hr after ConA treatment and progressed with time. The above-noted changes were less pronounced in C57Bl/6 IFN-γ(−/−) mice treated with ConA. CONCLUSIONS: Based on these results, liver tissue bioenergetics is increased 3 hr after ConA exposure. This effect is driven by the pathogenesis of the disease, in which IFN-γ and other cytokines contribute to. Subsequent declines in liver bioenergetics appear to be a result of necrosis and active caspases targeting the mitochondria within hepatocytes

Experimental and Theoretical Studies on the Pharmacodynamics of Cisplatin in Jurkat Cells

For Jurkat cells in culture exposed to cisplatin (1), we measured the number of platinum adducts on DNA and showed that it is proportional to the AUC, the area under the concentration vs time curve, for cisplatin. The number of platinum-DNA adducts is measured immediately following exposure to drug. The AUC is calculated either as the product of the initial cisplatin concentration and the exposure time or as the integral under the concentration vs time curve for the unreacted dichloro species, which decreases exponentially. We also show that the number of adducts correlates with decreases in respiration, with the amount of DNA fragmentation, and with cell viability, all measured 24 h after exposure to the drug. To study the reactions of cisplatin at concentrations approaching clinical relevance (65 microM), we use two-dimensional [1H15N]HSQC NMR and the 15N-labeled form of the drug, cis-Pt(15NH3)2Cl2, 1. In the absence of cells, 1 reacts with components of the growth medium and also transforms slowly (k(h) = 0.205 h-1 at 37 degrees C) into the chloro-aquo species, cis-[Pt(15NH3)2Cl(H2O)]+ (2), which at the pH of the medium (pH 7.15), is mainly in the deprotonated chloro-hydroxy form, cis-Pt(15NH3)2Cl(OH) (4). The concentration of 2 (4), as measured by HSQC NMR, decreases due to reaction with components of the medium. In the presence of 5 million or more cells, the concentration of 1 decreases with time, but the NMR signal for 2 (4) is not seen because it is rapidly removed from solution by the cells, keeping its concentration very low. These experiments confirm that the species preferentially removed from the medium by cells is 2 (4) and not 1. Our findings are discussed in the context of a kinetic model for platination of nuclear DNA by cisplatin, which includes aquation of cisplatin outside the cell, passage of 2 (4) through the cell membrane, reaction of reactive platinum species (RPS) in the cytosol with thiols, formation of adducts between RPS and accessible sites on genomic DNA, and removal of platinum from DNA by repair. Some of the rate constants involved are measured, but others can only be estimated. Calculations with this model show that little of the platinum reacts with intracellular thiols before reaching the nuclear DNA, indicating that binding to thiols is not important in cisplatin resistance. The model also predicts the circumstances under which the amount of platination of nuclear DNA is proportional to AUC

MALE FACTOR Inhibition of human sperm respiration by 4-hydroperoxycyclophosphamide and protection by mesna and WR-1065

Objective: To determine the effect of 4-hydroperoxycyclophosphamide (4OOH-CP) on the respiration of human sperm, and investigate the protective properties of mesna and WR-1065. Setting: SUNY Upstate Medical University, Syracuse, NY. Patient(s): Men (n ¼ 12) visited the Andrology Department for fertility evaluation. Intervention(s): None. Main Outcome Measure(s): Sperm respiration. Result(s): Immediate decline in the rate of respiration was observed when 4OOH-CP was added to washed sperm or semen. The inhibition was concentration dependent. The respiration was less affected when 4OOH-CP was added to semen, suggesting the presence of protective factors in the seminal plasma. Excess of mesna or WR-1065 ameliorated the effect of 4OOH-CP. Mesna was the more potent of the two compounds. 4OOH-CP also inhibited the respiration of mitochondria from beef heart. Conclusion(s): These findings emphasize the adverse effects of alkylating agents on sperm function. The results also provide a framework for thiol drug administration with high-dose alkylating agents to protect male fertility. The protective capacity of seminal plasma deserves further testing. (Fertil Steril Ò 2009;91:173-8. Ó2009 by American Society for Reproductive Medicine.

Inhibition of Cellular Respiration by Doxorubicin

Doxorubicin executes apoptosis, a process known to produce leakage of cytochrome c and opening of the mitochondrial permeability transition pores. To define the loss of mitochondrial function by apoptosis, we monitored cellular respiration during continuous exposure to doxorubicin. A phosphorescence analyzer capable of stable measurements over at least 5 h was used to measure [O(2)]. In solutions containing glucose and cells, [O(2)] declined linearly with time, showing that the kinetics of oxygen consumption was zero order. Complete inhibition of oxygen consumption by cyanide indicated that oxidations occurred in the respiratory chain. A decline in the rate of respiration was evident in Jurkat and HL-60 cells exposed to doxorubicin. The decline was abrupt, occurring after about 2 h of incubation. The inhibition was concentration-dependent and was completely blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Respiration in resistant HL-60/MX2 cells, characterized by an altered topoisomerase II activity, was not inhibited by doxorubicin. A decline in cellular ATP was measured in Jurkat cells after 2-4 h of incubation with 20 microM doxorubicin, paralleling the decline in respiration rate. Thus, cells incubated with doxorubicin exhibit caspase-mediated inhibition of oxidative phosphorylation

Dactinomycin Impairs Cellular Respiration and Reduces Accompanying ATP Formation

The effect of dactinomycin on cellular respiration and accompanying ATP formation was investigated in Jurkat and HL-60 cells. Cellular mitochondrial oxygen consumption (measured by a homemade phosphorescence analyzer) and ATP content (measured by the luciferin-luciferase bioluminescence system) were determined as functions of time t during continuous exposure to the drug. The rate of respiration, k, was the negative of the slope of [O2] versus t. Oxygen consumption and ATP content were diminished by cyanide, confirming that both processes involved oxidations in the mitochondrial respiratory chain. In the presence of dactinomycin, k decreased gradually with t, the decrease being more pronounced at higher drug concentrations. Cellular ATP remained constant for 5 h in untreated cells, but in the presence of 20 microM dactinomycin it decreased gradually (to one-tenth the value at 5 h for untreated cells). The drug-induced inhibition of respiration and decrease in ATP were blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethyl ketone (zVAD-fmk). A rapid but temporary decrease in cellular ATP observed on the addition of zVAD-fmk was shown to be due to DMSO (added with zVAD-fmk). The effect of dactinomycin on respiration differed from that of doxorubicin. Plots of [O2] versus t were curved for dactinomycin so that k decreased gradually with t. The corresponding plots for doxorubicin were well fit by two straight lines; so k was constant for approximately 150 min, at which time k decreased, remaining constant at a lower level thereafter. The results for cells treated with mixtures of the two drugs indicated that the drugs acted synergistically. These results show the onset and severity of mitochondrial dysfunction in cells undergoing apoptosis induced by dactinomycin