Paola Della Torre

The Histopathology of Kidney Changes in Rats and Monkeys Following Intravenous Administration of Massive Doses of FCE 26184, Human Basic Fibroblast Growth Factor:

Intravenous administration of human basic fibroblast growth factor up to 100 μg/kg/day to Sprague-Dawley rats caused changes in the kidneys that included enlargement, vacuolation, and karyomegaly of podocytes in glomeruli, dilatation and cast formation in tubules, thickening of the media in the lobular arteries, and hyperplasia of the epithelium of the papilla and collecting ducts. In cynomolgus monkeys there was hyperplasia of the parietal epithelium of Bowmans capsule in the glomeruli, tubular dilatation, and minimal arteriopathy. These changes were only seen at 100 μg/kg/day. The development and eventual recovery over time were investigated in a sequence of sacrifices. In monkeys the first changes were seen after 7 days of treatment, but in rats only after 16 days. In both species the changes had partially resolved after 30 days of recovery and were considered to return to normal after 60 days without treatment. The morphological changes were accompanied by functional alterations that included proteinuria and raised blood urea. Changes that occurred in other tissues including bone, red blood cells, adrenals, ovaries, liver, gall bladder, spleen, mesenteric lymph nodes, thymus, aorta, salivary glands, and injection site are not described in this paper.

Investigations of the effects of the antimalarial drug dihydroartemisinin (DHA) using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX).

Artemisinin derivatives are effective and safe drugs for treating malaria, but they are not recommended during the first trimester of pregnancy because of resorptions and abnormalities observed in animal reproduction studies. Previous studies in rats showed that artemisinin embryotoxicity derives from the depletion of primitive red blood cells (RBCs) over a narrow critical time window (gestation Days 9-14). In order to further investigate the susceptibility of primitive RBCs to artemisinins and to establish whether this susceptibility is species-specific or inherent to the compound, we studied dihydroartemisinin (DHA), both a drug in its own right and the main metabolite of current artemisinin derivatives in use, in the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). This model readily allows investigation and monitoring of primitive and definitive RBCs. Effects on frog larvae exposed to DHA for 48 h during early embryonic development, starting from 24 h post fertilization, were similar to those on rat embryos in terms of reduction in the number of primitive RBCs (clonally produced within the ventral blood island). In contrast, RBCs of older larvae (stage 47, produced at the definitive sites of hematopoiesis) were affected minimally and subsequently recovered. Compared to rat embryos, the frog larvae had no areas of necrosis but they shared similar heart defects. The mitochondrion appeared to be the main subcellular target, similar to observations in Plasmodium. These results implicate artemisinin-induced embryotoxicity through perturbation of metabolically active RBCs; whereas this mode of action does not appear to be species-specific, the stages of susceptibility varied between different species. The window of susceptibility and duration of exposure must be considered to evaluate the clinical relevance of these findings.

Long-Lasting Effect of Dexrazoxane Against Anthracycline Cardiotoxicity in Rats

The long-lasting protective effect of dexrazoxane (ADR-529) against doxorubicin- and epirubicin-induced cardiotoxicity was evaluated in the multiple-dose 35-wk rat model. Groups of 36 male Sprague-Dawley rats were given ADR-529 30 min before administration of cardiotoxic doses of doxorubicin (1 mg/kg/wk) or epirubicin (1.13 mg/kg/wk). The compounds were intravenously injected once weekly for 7 consecutive wk at ADR-529: anthracycline ratios ranging from 5:1 to 20:1. These ratios covered the entire chemotherapeutic range in humans and allowed studying the chronic progressive cardiomyopathy in our rat model. Animals were observed for up to 35 wk to follow the time course of the well-characterized cardiomyopathy, which was evaluated through the well-established qualitative/quantitative morphological grading. It was clearly demonstrated in this rat model that ADR-529, at the ratios administered, provided ample cardioprotection for a duration of 35 wk, which corresponds to 25 yr of equivalent human time.

Cardioprotection by dexrazoxane in rats treated with doxorubicin and paclitaxel

Purpose: Results of several clinical studies suggest that the combination of doxorubicin (DOX) and paclitaxel (PTX) is highly active against solid tumors. Both drugs are known to cause adverse cardiac effects, cardiomyopathy in the case of DOX and acute changes in cardiac rhythm in the case of PTX. It has been suggested that the addition of dexrazoxane (DZR) to this regimen may reduce the risk of cardiotoxicity. A model of chronic cardiomyopathy in the rat was used to determine whether DZR was tolerated and cardioprotective in a DOX+PTX combination. Methods: Male rats were treated once weekly for 7 weeks with one of the following vehicle and/or drug sequences: Group A, M/6 sodium lactate/saline/Cremophor EL (CEL); Group B, lactate/DOX/CEL; Group C, DZR/DOX/CEL; Group D, lactate/DOX/PTX; and Group E, DZR/DOX/PTX. DZR and DOX or their respective vehicles were given i.v. whilst PTX or CEL were given i.p. DZR, DOX and PTX were administered at 16u2009mg/kg, 0.8u2009mg/kg and 2.4u2009mg/kg, respectively, doses which caused minimal noncardiac toxicities. The hearts were examined histologically 5 weeks following the last treatment. Results: There were no deaths and no signs of overt toxicity during the 12 weeks of study. There was a significant decrease (Pu2009<u20090.01) in white blood cell count in rats treated with DZR+DOX, DOX+PTX or DZR+ DOX+PTX but not in those given DOX alone. Liver and kidney weights were increased in rats given DOX (Pu2009<u20090.05) but not in those given DZR+DOX. PTX had no effect on the DOX-induced liver and kidney changes and did not interfere with the protective effect of DZR on the kidney. The severity and extent of cardiomyopathy expressed as the mean total score (MTS) for each treatment group, was similar for DOX and DOX+PTX (4.6 and 4.2, respectively). DZR provided significant cardioprotection (Pu2009<u20090.01) when added to either DOX (MTS 2.0) or to DOX+PTXu2009(MTS 2.1). Conclusions: The results suggest that PTX does not exacerbate the chronic cardiomyopathy caused by DOX and when added to the DOX+PTX combination, DZR retains its protective activity against DOX-induced cardiotoxicity without increasing noncardiac toxicity.

Protection against doxorubicin-induced cardiotoxicity in weanling rats by dexrazoxane.

Purpose: Dexrazoxane (DZR) protects against anthracycline-induced cardiotoxicity in several laboratory animal species and in patients with breast cancer. Encouraging results have also been obtained in a limited number of pediatric oncology patients. We conducted studies to determine the safety and cardioprotective activity of DZR in the doxorubicin (DOX)-treated weanling rat simulating the rapidly growing immature child. Methods: Male weanling rats and young adult rats, 20␣days old and 7 weeks old, respectively, were given 1u2009mg/kg DOX i.v., either alone or with 20u2009mg/kg DZR, once weekly for 7 weeks. Rats were sacrificed at weeks 8, 12 or 26 following blood collection for hematology and serum chemistry. Hearts were weighed and examined histologically. Results: DOX, either alone or with DZR, inhibited growth, and body weight remained below that of controls throughout the 26 weeks of study. There were no biologically significant hematologic changes in either the DOX- or DZRu2009+u2009DOX-treated young rats. DOX caused a slight increase in liver and kidney weights relative to body weight and a slight increase in serum cholesterol and triglycerides in the young rats. These effects were ameliorated or delayed by DZR. DOX, either alone or with DZR, caused a marked atrophy of the testes in the young rats which had recovered by week 26. In the mature rats, DOX caused a significant decrease in the WBC 1 week after the last treatment, and the WBC was significantly lower in the rats given DZRu2009+u2009DOX compared to those given DOX alone. There were marked increases in liver and kidney weight, serum cholesterol and triglycerides in the mature rats given DOX alone but not in those given DZRu2009+u2009DOX. There was also a marked testicular atrophy in the mature rats given either DOX or DZRu2009+u2009DOX but, unlike that observed in the young rats, this had not returned to normal by week 26. DOX-induced cardiotoxicity was less severe in the younger rats than in the mature rats but in both age groups, the lesion progressed rapidly until week 12, 5 weeks after the last dose, and remained relatively stable or progressed slightly thereafter. DZR provided significant cardioprotection in both age groups at all time points examined. Moreover, in both age groups, the severity of the cardiomyopathy in the DZR-treated rats was somewhat less at week 26 than it was at week 12. Conclusions: The results indicate that the pharmacologic effects of DZR, including its ability to protect against cardiotoxicity, are similar in immature and adult male animals treated with DOX.

Evaluation of 4'-Iodo-4'=Deoxydoxorubicin-Induced Cardiotoxicity in Two Experimental Rat Models*

In the present study, 1 single-dose and 1 multiple-dose models were applied in studying 4′-iodo-4′-deoxydoxorubicin (I-DX) cardiotoxicity. Anthracycline cardiotoxicity has been reproduced in several animals including mice, rats, hamsters, rabbits, dogs, and monkeys. Of these species, the rat can be considered the most suitable species for the study of anthracycline-induced cardiomyopathy. The cardiotoxicity induced by I-DX in male Sprague-Dawley rats was compared to that of doxorubicin (DX), used as standard positive control. Groups of 36–42 rats were given single or repeated doses of the compounds, injected intravenously in a volume of 5.0 ml/kg. Animals were observed for up to 35 wk to follow the progression of the lesions. Cardiomyopathy was evaluated through well-established qualitative/quantitative morphological grading. The new DX derivative proved to be clearly less cardiotoxic than DX with both treatment schedules. Although both models can be considered useful for evaluating and comparing the cardiotoxicity of new anthracycline derivatives and mimicking the transvenous endomyocardial biopsies in humans, the chronic test seems to be more suitable for compounds like I-DX, which possess a low cardiotoxic potential and which could go undetected in the single-dose test.

Tolerability and toxicological profile of pixantrone (Pixuvri®) in juvenile mice. Comparative study with doxorubicin.

The tolerability of pixantrone dimaleate (Pixuvri(®)), an aza-anthracenedione for non-Hodgkin lymphoma, was assessed in juvenile mice after intraperitoneal injection. Twenty animals/sex/dose received pixantrone 15 or 27 mg/kg/day on Post-Natal-Days (PND) 10, 13, 17, 20, 35, 39 and 42 in comparison with doxorubicin, 3 mg/kg/day. Animals were sacrificed on PND 42, 73 and 96. All pixantrone animals survived, while doxorubicin induced 52.5% mortality and the surviving animals were sacrificed early due to severe toxicity. Recoverable bone marrow toxicity (pixantrone), and toxicity to thymus and reproductive organs (pixantrone, doxorubicin) were observed without nephro- or hepatotoxicity. Pixantrone was measurable in plasma up to 2h (occasionally 6h) post-dose. At PND 42, mean Cmax and AUC values increased proportionally with dose, without gender difference or accumulation. Pixantrone showed minimal cardiotoxicity in males and negligible in females at PND 96. Doxorubicin induced significant heart weight reduction at PND 42, however early sacrifice impeded further cardiac assessments.

PNU-159548, a novel cytotoxic antitumor agent with a low cardiotoxic potential

Purpose: PNU-159548 (4-demethoxy-3-deamino-3aziridinyl-4-methylsulphonyl-daunorubicin), a derivative of the anticancer idarubicin, has a broad spectrum of antitumoral activity in vitro and in vivo attributable to its DNA intercalating and alkylating properties. The present study was conducted to determine the cardiotoxic activity of PNU-159548 relative to doxorubicin in a chronic rat model sensitive to anthracycline-induced cardiomyopathy. Methods: Young adult male rats were allocated to the following treatment groups: group 1, PNU-159548 vehicle control (colloidal dispersion); group 2, doxorubicin control (saline); groups 3, 4, 5, 6, and 7, PNU-159548 at 0.12, 0.25, 0.50, 0.75, and 1.0 mg/kg, respectively; and group 8, 1.0 mg/ kg doxorubicin. Treatments were administered intravenously once weekly for 4 weeks (first sacrifice time) or for 7 weeks (rats killed at weeks 8, 12, 22, 27, or 35). Body weights, organ weights, serum chemistry, hematology, serum troponin-T, and cardiac histopathology were followed throughout the study. Results: Doxorubicin caused irreversible cardiomyopathy evident at week 4 in some rats and progressing in severity in all rats by week 8. There were also marked myelotoxicity, increased liver and kidney weights, testicular atrophy, and about 20% mortality by week 27 in doxorubicin-treated rats. The deaths were attributed to cardiomyopathy and/ or nephropathy. PNU-159548 caused a dose-dependent myelotoxicity, with the dose of 0.5 mg/kg per week being equimyelotoxic to 1.0 mg/kg per week doxorubicin.PNU-159548 also caused an increase in liver weight that was reversible and a non-reversible testicular atrophy but, unlike doxorubicin, had no effect on kidney weight. At equimyelotoxic doses, the cardiotoxicity caused by PNU-159548, expressed as the mean total score, was less than one-twentieth of that induced by doxorubicin, and much less than that predicted on the basis of its content of idarubicin, which is in turn markedly less cardiotoxic than doxorubicin. Conclusions: The novel cytotoxic antitumor derivative, PNU-159548, is significantly less cardiotoxic than doxorubicin at equimyelosuppressive doses. The combination of intercalating and alkylating activities within the same molecule without the cardiotoxic side effects of anthracyclines makes PNU-159548 an excellent candidate for clinical development in oncology.