Electron diffraction based analysis of phase fractions and texture in nanocrystalline thin films, part III: application examples.

In this series of articles, a method is presented that performs (semi)quantitative phase analysis for nanocrystalline transmission electron microscope samples from selected area electron diffraction (SAED) patterns. Volume fractions and degree of fiber texture are determined for the nanocrystalline components. The effect of the amorphous component is minimized by empirical background interpolation. First, the two-dimensional SAED pattern is converted into a one-dimensional distribution similar to X-ray diffraction. Volume fractions of the nanocrystalline components are determined by fitting the spectral components, calculated for the previously identified phases with a priori known structures. These Markers are calculated not only for kinematic conditions, but the Blackwell correction is also applied to take into account dynamic effects for medium thicknesses. Peak shapes and experimental parameters (camera length, etc.) are refined during the fitting iterations. Parameter space is explored with the help of the Downhill-SIMPLEX. The method is implemented in a computer program that runs under the Windows operating system. Part I presented the principles, while part II elaborated current implementation. The present part III demonstrates the usage and efficiency of the computer program by numerous examples. The suggested experimental protocol should be of benefit in experiments aimed at phase analysis using electron diffraction methods.

Influence of various administration routes on the antitumor efficacy ofTT-232, a novel somatostatin analog.

TT-232 a novel tumor-selective somatostatin analog with a five residue ring structure (D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2) was developed by us and published in an earlier work. This synthetic heptapeptide had no effect on growth hormone release, but had a remarkable tyrosine-kinase inhibitory effect and inducted apoptosis. The aim of this study was to compare the therapeutic efficacy of TT-232 used in various long-term administration routes and treatment schedules. The effectiveness of TT-232 was studied on different rodent tumors transplanted to inbred mice from SPF breeding. Intermittent treatment by injections and continuous infusion of TT-232 using a s.c., i.p. or i.v. implanted Alzet type osmotic minipump were compared for therapeutic efficacy. The treatments were started either on the day subsequent to tumor transplantation or after the development of a tumor. On the basis of survival and tumor growth inhibition the infusion of TT-232 for 14 days using an implantable osmotic pump proved to be a much more effective route of treatment in both s.c. and i.v. administration than the intermittent injections applied twice a day for 2 weeks. In the case of S-180 sarcoma the continuous administration of TT-232 for 14 days using s.c. implanted osmotic pump resulted in 60% the i.v. infusion produced 40% long-term (over 80 days) and tumor free survivors. By the continuous administration of TT-232, an 80-100% tumor growth inhibitory effect and a considerable retardation of tumor development could be achieved. Continuous infusion from implanted pumps ensured a constant drug level and resulted in a well-defined, consistent pattern of drug exposure over the full duration of drug administration. In our study the route of infusion has been shown to increase drug efficacy relative to conventional delivery methods.

Studies on the melanin affinity of selegiline (deprenyl) and other amphetamine derivatives.

The main aim of our study was to assess the melanin affinity of selegiline as well as the pharmacologic and pharmacokinetic aspects of its binding. The in vivo melanin binding of [14C] selegiline was studied by the method of whole body autoradiography. Extensive accumulation was observed in the pigmented mouse eye while in the albino animal uptake was low in the corresponding tissues. Our in vitro investigations demonstrated that the amphetamine derivatives tested can be taken up by melanins. Scatchard analysis of selegiline binding to the dopamine melanin (structurally similar to the neuromelanin) and beef eye melanin showed that more than one class of binding sites may be implicated. The total binding capacity of the beef-eye melanin was higher than that of the dopamine melanin. The selegiline inhibition of the [3H] MPP+ (the neurotoxic metabolite of MPTP) binding to dopamine melanin was also investigated. In the studied concentration range, the binding of [3H] MPP+ was depressed to about 70 per cent of the original maximum value. In conclusion, as a result of its melanin affinity, which is demonstrated in this study, selegiline may most probably accumulate in the pigmented nerve cells. The observed melanin affinity may contribute to the application of this compound for the treatment of Parkinson's disease or may play a role in its protective effect against MPTP neurotoxicity.

In vitro metabolic study of EGYT-3615 using rat liver microsomes.

In vitro metabolism of EGYT-3615, a prospective new antidepressant was studied by using rat liver microsomes. The metabolite (M) found in earlier in vivo studies was unanimously found to be the product of a microsomal enzymatic process. The Michaelis-Menten constant of the reaction was calculated.

The fate of drotaverine-acephyllinate in rat and man. II. Human pharmacokinetics of drotaverine-14C-acephyllinate.

Pharmacokinetics of Drotaverine-Acephyllinate, Chinoin was investigated in seven male volunteers using 14C labelled drug. Drotaverine-Acephyllinate was administered at a 100 mg single oral dose. Measurements of total radioactivity showed that the drug was absorbed completely and was eliminated by renal and biliary routes. Within 72 hours 39.9 +/- 9.9% and 47.1 +/- 4.9% of the dose were recovered in the urine and faeces respectively. Experimental results were interpreted on the basis of a complex linear compartment model. The structural identifiability of the model was proved by computer analysis, and the pharmacokinetic parameters were determined.

The fate of drotaverine-acephyllinate in rat and man. I. Absorption, distribution and excretion in the rat.

Two different labelled forms were used for the pharmacokinetic investigations: the carbon 1 in the isoquinoline ring (Drotaverine-14C-Acephyllinate) and the carboxyl group of theophylline-7-acetic acid (Drotaverine-Acephylline-14C-ate). Drotaverine-14C-Acephyllinate was rapidly absorbed from duodenal and ileal segments. Biliary excretion was substantial after oral administration and radioactivity was excreted mostly in the feces. Absorption of Drotavenine-Acephylline-14-C-ate from the gastrointestinal tract was very poor and radioactivity was therefore excreted for the most part in the feces. The results of the study were confirmed by whole body autoradiography.

The fate of p-bromo-methylamphetamine (V-111) in the body.

The fate of p-bromo-methylamphetamine (V-111) in the body was studied by means of its radioactive labelled forms in mouse and rat experiments. It was found with the whole body autoradiographic method and liquid-scintillation measurements that the compound is rapidly absorbed by whatever routes of administration and it is rapidly taken up by the tissues from the blood stream. In the central nervous system, it reaches higher concentration than methyl-amphetamine and it leaves the central nervous system more slowly. We have shown with differential centrifugation that V-111 is bound much more avidly to the mitochondrial and microsomal fractions of rat brain than methyl-amphetamine and o-bromo-methyl-amphetamine (V-104). The intensity of binding is proportional to the lipid solubility of the compounds. V-111 and its metabolites are excreted mainly in the urine, and they can be found in small amounts also in the stool. In the case of V-111-3-14C a small amount of 14CO2 appeared in the expired air, too, which is a consequence of the disintegration of the molecule. It has been shown by the radiochromatographic and gas chromatographic, mass-spectrometric analysis of the metabolites that V-111 is excreted partly in unchanged form, nevertheless, the N-demethylated and subsequent products, viz. p-bromo-phenyl-acetone, p-bromo-phenylpropanol, p-bromo-benzoic acid and p-bromo-hyppuric acid are also excreted in the urine. The main metabolic pathway of amphetamine and of its methyl-derivative in rat is p-hydroxylation, which does not take place in the case of p-halogenated V-111. Thus the secondary metabolic pathway (demethylation, oxidative desamination) becomes the main metabolic route of V-111 in this species. The vigorous demethylation of V-111 was proved both in vivo and in vitro. In the rat, demethylating activity increases during prolonged treatment. The latter fact has to be taken into consideration when interpreting the pharmacological tolerance that develops during chronic administration of the compound.

Qualitative and quantitative determination of drotaverine metabolites in rat bile.

After oral and intravenous administration of drotaverin-14C its metabolites were determined in rat bile. Three major metabolites were identified by tlc. All the metabolites appeared in conjugated form. No unchanged drotaverine was detectable in the bile, except after treatment with doses much in excess of the therapeutic range. The ratio of major metabolites to unchanged product was determined by two-dimensional densitometry using a Telechrom Video Densitometer.

The intestinal absorption and excretion of 14C drotaverin in rats.

The absorption and biliary excretion of drotaverin/a papaverine analogue/were studied in rats. In vivo loop technique was used. The absorption of the compounds from the jejunum was found to be rapid and the activity injected into the duodenal sac to disappear only when the common bile duct was ligated. An appreciable activity was detected in the bile collected by cannulation following both i.v. (67%) or per os (31%) administration. Considerable activity was excreted with the bile even 24 hours after drug administration. A partial entero-hepatic cycle for the drotaverin metabolites is suggested.

Long-term fate of [14C]nicotine in the mouse: retention in the bronchi, melanin-containing tissues and urinary bladder wall.

N-methyl-14C and 2'-14C-labelled nicotine were used for whole-body autoradiographic distribution studies on C57BL- and NMRI-mice. Radioactivity was retained in the melanin-containing tissues, in the bronchial walls, and in the urinary bladder wall, up to 1 month after administration. The activity levels in the bronchi decreased faster if [2'(14)C] nicotine was used. Quantitative measurements of the retention of the 2 14C-labelled nicotine preparations confirmed the autoradiographic findings. It is proposed that nicotine is N-demthylated in the bronchial mucosa, the off-coming methyl group being incorporated into the cell constituents of the mucosa. Thin-layer chromatographic studies showed that no nicotine was present in the lungs after 24 h. In melanin, however, only unmetabolized nicotine was found from 4 h on. Some reactive nicotine metabolites may be responsible for the retention in the urinary bladder wall. Also in the full-term fetuses radioactivity accumulated in the pigmented eyes and in the respiratory tract. The accumulation and long-term retention of nicotine in the melanin-containing structures might accelerate the development of drug-induced or senile changes in these tissues. The retention in the urinary bladder wall persisted even after rinsing. This may indicate an accumulatory mechanism worth considering in the pathogenesis of urinary bladder cancer.