B. Silvestrini

Morphological, histochemical and biochemical studies on germ cell mitochondria of normal rats

SummaryMorphological changes in rat germ cell mitochondria are described. In diplotene and secondary spermatocytes and in the spermatids of the Golgi, cap and acrosomal phases, the mitochondria take on a rounded appearance with the inner space containing the matrix flattened against the outer membrane and the intracristal spaces considerably swollen (“condensed” mitochondria).Functional studies on “condensed” mitochondria isolated from the germ cells of normal rats have been performed. The following parameters have been evaluated: ADP/O ratio, respiratory control ratio (RCR) and ADP affinity. The ADP/O values found in the presence of various substrates are in agreement with the theoretical figures. The RCR is remarkably high. Moreover, the ADP affinity of these mitochondria is very high, as demonstrated by the low values of the “apparent Km”. These biochemical findings, which demonstrate a high oxidative capacity coupled with a marked phosphorylation, suggest that the “condensed” appearance of germ cell mitochondria is the expression of an active functional state.

Effects of lonidamine alone or combined with hyperthermia in some experimental cell and tumour systems

Lonidamine or 1-[(2, 4-dichlorophenyl) methyl]-1H-indazole-3-carboxylic acid, studied in a battery of in vitro and in vivo tests currently used for the screening of anti-tumour agents affecting cell division, has been shown to have a narrow spectrum of anti-tumour activity. The significance of this finding is discussed in the light of previous investigations suggesting that lonidamine affects mitochondrial function and not cell replication. Hyperthermia has been shown to sensitize tumour cells to lonidamine. This observation indicates that in combination with hyperthermia lonidamine has some potential for the treatment of cancer, moreover, it suggests that hyperthermia might reproduce a metabolic condition occurring in some stages of the disease. The blood levels corresponding to the anti-tumour action of lonidamine in animals are in the range of those detected in patients treated with the drug.

Researches on the topical activity of benzydamine

Abstract Benzydamine displays a clear-cut anti-inflammatory effect on foot edema induced by carrageenin in rats, both after oral administration and local application on the skin. In both cases it accumulates in the inflamed tissue in concentrations ranging from 5 to 20 μg/g. In the perfused rabbits ear, benzydamine (5 μg/ml) inhibits vascular changes produced by histamine and acetycholine. It is therefore assumed that benzydamine acts in particular on the vascular component of the inflammatory process. The local tolerance to the drug was studied in acute and chronic trials carried out in mice, rats and rabbits. The results obtained suggest that clinical trials aimed at evaluating the efficacy of a topical use of benzydamine in therapy would be useful.

Morphological and biochemical modifications of rat germ cell mitochondria induced by new antispermatogenic compounds: Studies in vivo and in vitro☆

Abstract The morphological changes induced in rat germ cell mitochondria by chlorobenzyl-1 H -indazol-3-carboxylic acid (AF 1312/TS) and lonidamine (AF 1980), two antispermatogenic compounds, are described. Twenty-four hours after treatment, numerous dumbbell-shaped mitochondria appear in the Golgi, cap, and acrosomic spermatids, whereas after 48 hrs severe signs of mitochondrial degeneration are visible in the maturation phase spermatids. Biochemical studies performed on isolated germ cells and their mitochondria, harvested from the testes of normal and treated rats, are also described. In the normal rats, the cells and mitochondria were incubated with the antispermatogenic compounds. Aerobic and anaerobic glycolysis was evaluated on isolated germ cells, whereas the following parameters were evaluated in the mitochondria: ADP O ratio, respiratory control ratio (RCR), and ADP affinity. The results obtained demonstrate that the antispermatogenic agents induce, both in vivo and in vitro , similar changes in energy metabolism; respiration, RCR, and ADP affinity are significantly reduced, while aerobic glycolysis is increased.

Antispermatogenic activity of 1-p-chlorobenzyl-1H-indazol-3-carboxylic acid (AF 1312TS) in rats: I. Trials of single and short-term administrations with study of pharmacologic and toxicologic effects

Abstract A new antispermtogenic agent is described. Following single or short-term administrations, 1- p -chlorobenzyl-1 H -indazol-3-carboxylic acid, or AF 1312 TS , produced in rats a long-lasting inhibition of the spermatogenic process. In adult rats the secondary sex organs were not affected, while in young rats some weight decrease of the prostate, seminal vesicles and levator ani was observed. The interstitial tissue of the testes as well as thymus, adrenals and hypophysis were not affected. AF 1312 TS proved to be devoid of the most common pharmacological activities.

Antispermatogenic activity of 1-p-chlorobenzyl-1H-indazol-3-carboxylic acid (AF 1312TS) in rats: II. A study of treatments of duration between 5 and 180 days

Abstract 1-p Chlorobenzyl-1H-indazol-3-carboxylic acid or AF 1312 TS was given to rats in the diet for periods of time ranging from 5–180 days. Body weight, weight and histology of the main internal organs, food consumption, serum levels of the drug and blood composition were determined. The most extensive experiment was performed in young rats treated up to 180 days with a diet containing 0.5% AF 1312 TS . A marked and constant reduction in the weight of the testes with inhibition of spermatogenesis was observed during the entire experiment. In rats treated for 20–80 days a reduction in the weight of the ventral prostate, seminal vesicles and levator ani was also observed with a histological picture of hyposecretion of the above-mentioned glands: These effects were no longer detected following a 180-day treatment. The influence of doses and age on the effects of AF 1312 TS was also studied on the basis of a 30-day treatment period. In young rats, effects on spermatogenesis were not separated from effects on the accessory sex organs even with the lowest doses; in mature rats, instead, the spermatogenic process was specifically inhibited even with the highest doses. AF 1312 TS was without effects in female rats. The toxicological tests gave no evidence of systemic toxicity of the drug.

The effect of the association of Gossypol and Lonidamine on the energy metabolism of Ehrlich ascites tumor cells

The effect of the association of Gossypol and Lonidamine on the energy metabolism of Ehrlich ascites tumor cells has been investigated. The action of the drug on tumor cells was studied by addition of the drugs to cells harvested from Swiss male mice. The results may be summarized as follows: (1) Low concentrations of Gossypol increase the rate of oxygen consumption by uncoupling oxidative phosphorylation. High concentrations result in an inhibition of oxygen consumption with a mechanism that must be regarded as not directly related to the uncoupling activity. (2) Gossypol, at concentrations at which it exerts an uncoupling activity, stimulates mitochondrial ATPase which in turn increases the aerobic and anaerobic rates of lactate production. The decrease of glycolysis at high concentrations of Gossypol does not depend on the inhibition of enzymes of the glycolytic pathway, but must be ascribed to cell death. (3) The association of a low concentration of Gossypol with Lonidamine brings about a further inhibition of oxygen consumption. Moreover, Lonidamine abolishes the stimulation of glycolysis induced by Gossypol and lowers lactate production to values that are quite similar to those found with Lonidamine alone. (4) It may be concluded that the association of Gossypol and Lonidamine results in a very effective decrease of the energy requirements of cancer cells.

Toxicology of benzydamine

Abstract In acute studies, benzydamine produced pharmacologic effects and death at doses far above the human therapeutic levels. Human therapeutic doses are of the order of 0.7-1.0 mg/kg p.o. The LD 50 values (mg/kg) in mice were 33, i.v.; 110, i.p.; 218, s.c.; and 515, p.o.; and in rats, 100 i.p., and 1050 p.o. The general effects of benzydamine were similar in mice, rats, rabbits, and cats, and consisted of muscle relaxation and sedation, while at higher doses, ataxia and convulsions were observed. In dogs the drug produced sedation, but the occurrence of vomiting prevented the study of high dosages. Benzydamine in chronic studies produced lethal effects only in those experimental conditions and doses favorable to the production of acute pharmacodynamic effects. A depression of growth rate and enlargement of the liver were observed in mice and rats medicated with large doses of benzydamine. The latter effect is probably related to enzyme induction. Hematologic parameters, liver function, reproduction, and histologic structure of the major organs were not altered by benzydamine. The observation of helminthic infestation in control dogs, but not in those given benzydamine, suggested an antihelminthic activity of the drug.

A Long-Term Clinical Experience with Lonidamine

Lonidamine (LNA) has been investigated both alone and in combination with different chemotherapeutic agents in various types of tumors at an advanced stage. Acute and long-term treatments were studied. LNA has been shown to be devoid of the most typical side effects of currently used chemotherapeutic agents. It has revealed a characteristic profile of side effects, comprising myalgia, photosensitivity and altered hearing. LNA alone, tested in a limited number of patients, produced a partial remission in one ovary adenocarcinoma and a stabilization in two breast carcinomas and one microcytoma. When used in combination with chemotherapeutic agents, it potentiated them, particularly in brain metastases and lung adenocarcinomas.

The effect of gossypol and Lonidamine on electron transport in Ehrlich ascites tumor mitochondria.

The effect of the association of gossypol and Lonidamine on the electron transport in Ehrlich ascites tumor mitochondria has been investigated by addition of drugs to isolated mitochondria. The results may be summarized as follows. (1) Low concentrations of gossypol increase the rate of oxygen consumption at the level of three energy-conserving sites of the respiratory chain. Higher concentrations result in an inhibition of oxygen consumption at (or near) both energy-conserving sites 1 and 2, while energy-conserving site 3 is unaffected. (2) Gossypol, at concentrations at which it exerts its uncoupling effect, stimulates ATPase activity. Higher concentrations inhibit the enzyme activity. (3) The addition of gossypol to mitochondria respiring on pyruvate plus malate or succinate induces a more oxidized state of NAD+ and cytochrome b, respectively. (4) Gossypol enhances the effect of Lonidamine on oxygen consumption. Lonidamine does not affect state 4 respiration, but in the presence of gossypol, it determines a marked decrease in the rate of oxygen consumption. The inhibition of oxidation of NAD-linked substrates is greater than that of FAD-linked substrates. (5) It may be concluded that gossypol is very effective in potentiating the effect of Lonidamine. Moreover, it may be suggested that the antitumor activity of Lonidamine is enhanced if it is used in combination with other drugs and/or treatments, such as hyperthermia, which modify the energy status of mitochondria.