Richard D. Olson

Time-related increases in cardiac concentrations of doxorubicinol could interact with doxorubicin to depress myocardial contractile function.

1 The present study evaluated the time‐dependency of acute anthracycline cardiotoxicity by varying the duration of exposure of rabbit isolated atria to doxorubicin and determing changes (1) in contraction and relaxation and (2) in atrial concentrations of doxorubicin and its C‐13 hydroxy metabolite, doxorubicinol. 2 Following addition of doxorubicin (175 μm) to atria, contractility (dF/dt), muscle stiffness (resting force, RF) and relaxation (90% relaxation time, 90% RT) were monitored for a 3.5 h period. 3 Doxorubicin (175 μm) progressively diminished mechanical function (decreased dF/dt, increased RF and prolonged 90% RT) over 3 h. Doxorubicinol (1.8 μm), however, failed to produce time‐related cardiac dysfunction; it depressed contractile function and increased muscle stiffness during the first 30 min without causing additional cardiac dysfunction during the remaining 3 h of observation. Doxorubicinol had no effect on 90% RT. 4 During treatment with doxorubicin, atria contained considerably more doxorubicin than doxorubicinol (ratio of doxorubicin to doxorubicinol ranged from 778 to 74 at 0.5 and 3 h, respectively). Elevations of doxorubicin and doxorubicinol in atria paralleled the degree of dysfunction of both contraction and relaxation; increases in muscle stiffness, however, were more closely associated with increases of doxorubicinol than doxorubicin. 5 To probe the relation between cardiac doxorubicinol and myocardial dysfunction further, without confounding effects of cardiac doxorubicin, concentration‐response experiments with doxorubicinol (0.9–7.2 μm) were conducted. 6 Plots of doxorubicinol concentrations in atria vs contractility indicated that the cardiac concentration of doxorubicinol, at which contractility is reduced by 50%, is five fold lower in doxorubicin‐treated than in doxorubicinol‐treated preparations. Thus, doxorubicin and doxorubicinol appear to interact to depress contractile function. 7 Cardiac concentrations of both doxorubicin and doxorubicinol, as observed in these studies, were found to stimulate markedly Ca2+ release from isolated SR vesicles, but 3 μm doxorubicinol promoted a 15 fold greater release rate than 3 μm doxorubicin. 8 Our observations coupled with the previously reported finding that doxorubicinol inhibits Ca2+ loading of SR, suggests that doxorubicinol accumulation in heart contributes to the time‐dependent component of doxorubicin cardiotoxicity, through a mechanism that could involve perturbations of Ca2+ homeostasis.

Randomized, controlled trial of phytoestrogen in the prophylactic treatment of menstrual migraine

Approximately 30% of women afflicted with migraine have menstrually associated attacks. These migraines are often refractory to treatment. Evidence suggests estrogen and progestin fluctuations may influence menstrual migraine. Phytoestrogens have demonstrated estrogenic effects in some tissues, but are without stimulation of the endometrium, suggesting decreased risk with long-term use. This study was undertaken to assess the efficacy of a phytoestrogen combination in the prophylactic treatment of menstrual migraine. Forty-nine patients were randomized to receive either placebo, or a daily combination of 60 mg soy isoflavones, 100 mg dong quai, and 50 mg black cohosh, with each component standardized to its primary alkaloid. Patients received study medication for 24 weeks. Average frequency of menstrually associated migraine attacks during weeks 9-24 was reduced from 10.3 +/- 2.4 (mean +/- s.e.m.) in placebo treated patients to 4.7 +/- 1.8 (P < 0.01) in patients treated with the phytoestrogen preparation.

Reduction of 13-deoxydoxorubicin and daunorubicinol anthraquinones by human carbonyl reductase.

Carbonyl reductase (CR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of several carbonyls. Anthracyclines used to treat cancer are reduced by CR at the C13 carbonyl and the resulting metabolites are implicated in the cardiotoxicity associated with anthracycline therapy. CR also is believed to have a role in detoxifying quinones, raising the question whether CR catalyzes reduction of anthracycline quinones. Steadystate kinetic studies were done with several anthraquinone-containing compounds, including 13-deoxydoxorubicin and daunorubicinol, which lack the C13 carbonyl, thus unmasking the anthraquinone for study. kcat and kcat/Km values for 13-deoxydoxorubicin and daunorubicinol were nearly identical, indicating that that the efficiency of quinone reduction was unaffected by the differences at the C13 position. kcat and kcat/Km values were much smaller for the analogs than for the parent compounds, suggesting that the C13 carbonyl is preferred as a substrate over the quinone. CR also reduced structurally related quinone molecules with less favorable catalytic efficiency. Modeling studies with doxorubicin and carbonyl reductase revealed that methionine 234 sterically hinder the rings adjacent to the quinone, thus accounting for the lower catalytic efficiency. Reduction of the anthraquinones may further define the role of CR in anthracycline metabolism and may influence anthracycline cytotoxic and cardiotoxic mechanisms.

Prevention by dexrazoxane of down‐regulation of ryanodine receptor gene expression in anthracycline cardiomyopathy in the rat

Anthracyclines can cause cumulative dose‐related cardiotoxicity characterized by changes in Ca2+ metabolism, including dysfunction of the sacroplasmic reticulum (SR) and decreased expression of Ca2+‐handling proteins, such as the ryanodine receptor (RyR2). In this study, we examined the effect of dexrazoxane (ICRF‐187), an iron chelator which prevents anthracycline cardiotoxicity, on RyR2 gene expression in rats treated chronically with daunorubicin. Daunorubicin (2.5 mg kg−1 i.v. weekly for 6 weeks) produced cardiotoxicity as demonstrated by histopathologic changes. The ryanodine receptor/glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA ratio was decreased by 38±3% (P<0.02) compared to values in control rats. Dexrazoxane pre‐treatment (50 mg kg−1; 1 h prior to each daunorubicin injection) prevented the decrease in RyR2/GAPDH mRNA ratio and histopathologic lesions in daunorubicin‐treated rats. This is the first report that a protective agent such as dexrazoxane can ameliorate the decreased expression of a specific gene involved in anthracycline‐induced cardiotoxicity.

Aging alters the force-frequency relationship and toxicity of oxidative stress in rabbit heart

Adult (6 months) and senescent (greater than 5 years) rabbit atria were studied under conditions known to increase cytoplasmic calcium (increased frequency of contraction and oxidative stress). At a contraction frequency of 1/sec, cardiac relaxation (90% relaxation time) was similar in senescent and adult atria but at a frequency of 2 or 3/sec, relaxation was significantly slower in senescent preparations (P less than 0.05). Additional experiments indicated that H2O2 (500 microM), a powerful oxidant, increased resting force and decreased developed force (DF) much more rapidly in senescent than adult atria; the maximum decrease in DF, however, was less in senescent preparations (adult = 81 +/- 6% and senescent = 42 +/- 27% of pre-H2O2 values; P less than 0.05). Age-related differences in effects of H2O2 did not result simply from a decreased ability of senescent hearts to detoxify an oxidative stress by the glutathione pathway. Both basal glutathione (GSH) concentrations and the H2O2-mediated decreases in GSH were similar in adult and senescent ventricular preparations, as were activities of glutathione peroxidase and glutathione reductase. These observations suggest that interventions known to increase cytoplasmic calcium can amplify age-related impairments of cardiac relaxation through mechanisms that may be independent of the glutathione pathway.

Species comparison of adenosine A1 receptors in isolated mammalian atrial and ventricular myocardium.

Various species have been used as models to study the effects of adenosine (ADO) on atrial and ventricular myocardium, but few direct tissue comparisons between species have been made. This study further characterizes adenosine A(1) receptor binding, adenylate cyclase activity and direct and indirect A(1) receptor-mediated functional activity in atrial and ventricular tissue from Sprague-Dawley rats and Hartley guinea pigs. Rat right atria (RA) were found to be significantly more sensitive to cyclopentyladenosine (CPA), while guinea pig left atria (LA) were more sensitive to CPA. After the addition of isoproterenol (ISO), the reduction of CPA response in rat RA was significantly greater than in guinea pig; however, after ISO treatment, the guinea pig LA was more sensitive to CPA than the rat. Adenylate cyclase inhibition by CPA was significantly greater in atria and ventricles obtained from guinea pig than rat. In competition binding experiments, guinea pig RA had significantly more high affinity sites than rat, but the K(i)s were not significantly different. There were no significant differences between guinea pig LA and rat LA. Guinea pig ventricular tissue had fewer high affinity sites than rat without any differences in their K(i) values. In antagonist saturation experiments, the density and affinity of A(1) receptors in guinea pig cardiac membranes were significantly greater than in rat. Our results indicate definite species differences as well as tissue differences between rat and guinea pig. These differences must be considered when interpreting studies using rat and guinea pig tissue as models for cardiac function.

Quantification of the voltage-response relationship between punctate and field electrical stimulation and the function of isolated rat left atria and papillary muscles

The effects of varying the electrical stimulation voltage on the noradrenergic tone and systolic and diastolic cardiac function of isolated rat cardiac preparations is unknown. If a wide range of voltages substantially alters the basal noradrerengic tone of isolated cardiac preparations, highly variable responses may complicate the interpretation of the cardiac effects of interventions, such as drugs or toxins. This study was designed to determine whether field or punctate electrode stimulation altered systolic and diastolic cardiac function of isolated rat atrial and ventricular muscle preparations through a beta-adrenergic receptor-mediated mechanism. Isolated left atria and right ventricular papillary muscles from adult Sprague-Dawley rats were stimulated to contract isometrically in Krebs bicarbonate buffer (30 degrees C, pH 7.4, 0.5 Hz). Cardiac muscle function was assessed by determining cardiac contractility (dF/dt), relaxation (80% relaxation time), and muscle stiffness (changes in resting force). Muscles were electrically stimulated to contract using either punctate or field electrodes. Basal values of cardiac function were determined at threshold voltages, and voltage was increased in stepwise fashion, over a wide range, to obtain a voltage response-relation to cardiac function. In left atrial preparations, both punctate and field electrical stimulation caused a 200% increase in cardiac contractility. The greatest changes in contractility occurred at near threshold voltages (less than 5 volts for punctate and 19 volts for field stimulation). The voltage-dependent increases in cardiac contractility were attenuated or abolished by pretreatment with atenolol (10 micromol/L; selective beta 1-antagonist) or reserpine (5 mg/kg i.p., 24 hr before euthanasia). In contrast to left atrial preparations, neither field or punctate electrical stimulation had any effect on the cardiac contractility of papillary muscle preparations. Neither field or punctate electrical stimulation had any effect on cardiac relaxation (80% relaxation time) or muscle stiffness (changes in resting force) of left atrial or papillary muscle preparations. This study demonstrates that punctate and field electrical stimulation can significantly increase cardiac contractility of rat left atrial preparations at near threshold voltages by increasing noradrenergic tone. Thus, control of the voltage range may be required to reduce variation in cardiac function of isolated rat left atrial preparations.

Pharmacokinetic calculator program for generation of initial parameter estimates from a three-compartment infusion model

A polyexponential curve-stripping program, KIN, is described for use on the HP-41CV programmable calculator. The program may be used in the analysis of plasma-concentration-time curves for a three-compartment intravenous bolus or infusion model with linear elimination processes. The coefficients and hybrid rate constants of the exponential function are then used to compute pharmacokinetic parameters (volume of the central compartment, intercompartmental rate transfer constants), which may be used as initial estimates of model parameters in non-linear regression curve-fitting procedures.

PIII-12Prevention of chronic anthracycline cardiotoxicity in the Fischer 344 rat and effects on iron metabolism

Anthracyclines, such as doxorubicin and daunorubicin, can cause chronic, cumulative dose‐related cardiotoxicity. This can be prevented by dexrazoxane, a putative iron chelator. Disorders of iron metabolism, including altered IRP1‐IRE binding may be an important mechanism of anthracycline cardiotoxicity. This study was designed to examine the role of IRP1‐IRE binding in a chronic model of daunorubicin cardiotoxicity and whether dexrazoxane could prevent such changes in IRP1‐IRE binding due to daunorubicin.

Inhibition of forskolin‐stimulated adenylyl cyclase activity by PD 81, 723

Clinical Pharmacology & Therapeutics (1996) 59, 175–175; doi: 10.1038/sj.clpt.1996.200