Modulation of action potential duration by inhibition of the transient outward current in sheep cardiac Purkinje fibers

In sheep cardiac Purkinje fibers concentration-dependent inhibition of transient outward current (i_to) by 4-aminopyridine (4-AP, 3-1000 mol/l) was recorded with the two-microelectrode voltage-clamp technique, and correlated effects on action potential duration measured at — 70 mV (APD-70) were investigatigated.Half-maximal inhibition of i_to-amplitude occurred at 15 mol/l 4-AP. The drug exhibited no major effect on voltage-dependent control of inactivation but reduced the maximally available i_to-current. At different activation frequencies (0.05 Hz, 0.25 Hz, 1 Hz) an equal amount of i_to-current, measured as percentage of the respective control, was inhibited by 4-AP. The APD-70 was on the average increased by 4-AP (3–500 mol/l) in a concentration-dependont manner up to 151 % of control. The drug-induced prolongation, measured as percentage of the respective control, was independent of stimulation frequency (0.05 Hz, 0.25 Hz, 1 Hz). Prolongation of APD-70 was on the average more pronounced for short action potentials (APD-70<150 ms: 169 % of reference) than for longer ones (APD-70 150–300 ms: prolongation to 117 % of reference; 500 mol/l 4-AP; 0.25 Hz stimulation rate). Few long control signals (APD-70 >300 ms) were shortened by 4-AP. These results indicate that inhibition of i_to-current by appropriate drugs will result in a reduction of inhomogeneity of action potential duration.     

The success of limb-salvage surgery in the adolescent patient with osteogenic sarcoma

Malignant bone tumors in the adolescent population are rare but serious problems that are both life- and limb-threatening. Most of these tumors originate in the extremities, hip girdle, or pelvic girdle and require complete surgical resection for adequate therapy. The greatest majority of these tumors are diagnosed as osteogenic sarcomas. In the past, limb ablation was the only effective therapeutic option available to surgical oncologists in adolescent osteosarcoma patients. However, today, after two decades of advances in chemotherapy protocols and reconstructive surgical techniques, limb-salvage surgery has become an accepted treatment standard. Because skeletal immaturity and future bone growth is generally not a major reconstructive consideration in adolescents, 90% of the patients in this age group are today treated with limb-sparing surgery. The most significant question regarding the successful use of limb-salvage surgery is whether it adversely affects long-term outcome compared with standard amputations. The principal studies, both single- and multi-institutional, that compared the risk of local tumor recurrence and overall disease-free survival rate of the two types of procedures, demonstrated no significant difference in disease-free survival rates between the two groups. Similarly, multivariant analyses have shown no survival benefit for choice of surgical procedure in osteosarcoma patients. As a result, limb-sparing surgery for osteosarcoma patients has now been firmly established as a safe, effective, and successful oncology procedure compared with limb ablation.     

Protein tyrosine kinase activity in 350 T1/T2, N0/N1 breast cancer. Preliminary results

Protein tyrosine kinases (PTKs) are a family of enzymes sharing a highly conserved catalytic domain which phosphorylates substrate proteins on tyrosine residues. PTKs play a major role in the transduction of the mitogenic signal and are involved in the control of cell proliferation, differentiation, and transformation processes. PTKs can be subdivided into two major types: membrane associated PTKs consisting essentially of growth factor receptors (receptor tyrosine kinases or RTKs) and cytosolic PTKs involved in the intracellular transduction of mitogenic and differentiation signals. From January 1988 to January 1992, PTK activity was assayed in cytosolic fractions prepared from 350 T1-T2, N0-N1 M0, breast carcinomas. Enzymatic activity was measured using phosphate transfer from [³²P]-ATP to poly-Glu-Tyr as an artificial substrate. According to our previously reported pilot study, we chose a cut-off value of 12 pmol³²P incorporated min^–1 mg^–1 protein, corresponding to the median value. We found positive PTK levels ( 12 pmol/min/mg) to be correlated with a loss of differentiation according to Scarff-Bloom grade (p < 0.001), negative PR (p = 0.03) and ER status (p = 0.04). With a median follow-up of 30 months (0–82), patients with a positive PTK level presented a smaller 3-year disease free survival than in the PTK negative group of patients (p = 0.07). In Cox multivariate analysis including pT, pN, Scarff-Bloom grade, PR and ER, PTK activity does not emerge as a significant prognostic factor.     

A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography

Methods were developed for the efficient routine degradation and fractionation of ethylated and methylated DNA. Alkylated DNA was hydrolyzed by a neutral thermal method to yield 3- and 7- alkylpurines and O2-alkylcytosines. The partially apurinic DNA was separated from the bases by precipitation in 0.1 N HCl. Portions of the DNA precipitate were further hydrolyzed either by 0.1 N HCl to yield purine bases, or by enzymes to yield nucleosides and phosphotriesters. The chemical and enzymic digests were fractionated by a combination of high pressure liquid chromatography systems to yield quantitative estimates of the following products from methylated or ethylated DNA: 1-, 3-, and 7-alkyladenines, O2-alkylcytosines, 3-, O6-, and 7- alkylguanines and O2-, 3-, and O4-alkylthymines. N6-Alkyladenines, 1-alkylguanines and N2-alkylguanines were not detected and the 3- alkylcytosines were detected but not quantified. Phosphotriesters were estimated from the amounts of recovered alkyl phosphotriesters of thymidylyl (3'-5') thymidine. Using these methods, it was possible to account for 98, 81, 98, and 92% of the DNA bound alkyl groups obtained from DNA reacted with [14C]methyl methanesulfonate, [3H]ethyl methanesulfonate, N-[3H]-methyl-N-nitrosourea, and N-[14C]ethyl-N-nitrosourea, respectively. The methods described provide reproducible and quantitative methods of analysis for all the known methylated or ethylated products in a single DNA sample.     

Inhibition of potassium outward currents and pacemaker current in sheep cardiac Purkinje fibres by the verapamil derivative YS 035

Summary The electrophysiologic mode of action and potency of the verapamil derivative YS 035 (N,N-bis-(3,4-dimethoxyphenethyl)-N-methyl amine) were investigated in sheep cardiac Purkinje fibres. Action potential duration measured at a repolarization level of –60 mV (APD-60) and membrane currents recorded with the two-microelectrode voltage-clamp technique were evaluated. At 10 mol/l YS 035 APD-60 was increased to about 115% of reference. Prolongation measured as percentage of the respective control exhibited on the average no dependence on stimulation frequency (0.17–2 Hz). At 100 mol/l membrane became depolarized to about –50 mV and action potentials could no longer be elicited. Further study was focussed on effects on outward currents, mostly activated at a frequency of 0.05 Hz. Transient outward current (i_to) was completely blocked at 100 mol/l and half-maximal inhibition occurred at about 14 mol/l. Inwardly rectifying potassium current (i_K1) was reduced to 47% of reference at 100 mol/l. An initially activating outward current at positive membrane potentials (i_inst) was reduced to 73% at 100 mol/l. Time-dependent (delayed) outward current (i_K) was on the average not affected up to 100 ol/l. Besides inhibition of repolarizing outward currents YS 035 completely blocked pacemaker current (i_f) at 100 mol/l and half-maximal reduction was achieved at 5 mol/l. YS 035 (1–100 mol/l) did not clearly affect time constants of activation at selected test potentials (I_K: +35 mV; i_f: –90 mV) or inactivation (i_to: 0 mV). Voltage-dependent control mechanisms of currents (it_to, i_f) were not influenced by YS 035 but the amount of available current was reduced.In conclusion, the verapamil derivative YS 035 inhibited pacemaker current and potassium outward currents which correlated to a prolongation of cardiac action po tentials. Electrophysiological actions of the compound favour it to be tested in vivo as an antiarrhythmic drug candidate. Send offprint requests to U. Borchard at the above address     

Near-fatal cerebellar swelling caused by acute multifocal cerebellar necrosis.

We report a case of acute near-fatal cerebellar swelling, which was accompanied by multifocal cerebellar necrosis. Acute, near-fatal cerebellar swelling is a rare problem thought to be of parainfectious aetiology. Initiation by multifocal cerebellar necrosis has not been reported so far with this disorder.     

Plasma and cerebrospinal fluid population pharmacokinetics of temozolomide in malignant glioma patients.

PURPOSE: Scarce information is available on the brain penetration of temozolomide (TMZ), although this novel methylating agent is mainly used for the treatment of malignant brain tumors. The purpose was to assess TMZ pharmacokinetics in plasma and cerebrospinal fluid (CSF) along with its inter-individual variability, to characterize covariates and to explore relationships between systemic or cerebral drug exposure and clinical outcomes. EXPERIMENTAL DESIGN: TMZ levels were measured by high-performance liquid chromatography in plasma and CSF samples from 35 patients with newly diagnosed or recurrent malignant gliomas. The population pharmacokinetic analysis was performed with nonlinear mixed-effect modeling software. Drug exposure, defined by the area under the concentration-time curve (AUC) in plasma and CSF, was estimated for each patient and correlated with toxicity, survival, and progression-free survival. RESULTS: A three-compartment model with first-order absorption and transfer rates between plasma and CSF described the data appropriately. Oral clearance was 10 liter/h; volume of distribution (V(D)), 30.3 liters; absorption constant rate, 5.8 h(-1); elimination half-time, 2.1 h; transfer rate from plasma to CSF (K(plasma-->CSF)), 7.2 x 10(-4)h(-1) and the backwards rate, 0.76 h(-1). Body surface area significantly influenced both clearance and V(D), and clearance was sex dependent. The AUC(CSF) corresponded to 20% of the AUC(plasma). A trend toward an increased K(plasma-->CSF) of 15% was observed in case of concomitant radiochemotherapy. No significant correlations between AUC in plasma or CSF and toxicity, survival, or progression-free survival were apparent after deduction of dose-effect. CONCLUSIONS: This is the first human pharmacokinetic study on TMZ to quantify CSF penetration. The AUC(CSF)/AUC(plasma) ratio was 20%. Systemic or cerebral exposures are not better predictors than the cumulative dose alone for both efficacy and safety.