Biodistribution and antitumour efficacy of long-circulating N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates in nude mice

The aim of this study was to evaluate the influence of the molecular weight (mol. wt) of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (DOX) conjugates (P-DOX) on biodistribution and therapeutic efficacy in nu/nu mice bearing human ovarian carcinoma OVCAR-3 xenografts. Copolymerisation of HPMA, a polymerisable derivative of DOX (N-methacryloylglycylphenylalanylleucylglycyl doxorubicin) and a newly designed crosslinking agent, N(2),N(5)-bis(N-methacryloylglycylphenylalanyl-leucylglycyl)ornithine methyl ester monomers resulted in novel, high mol. wt, branched, water-soluble P-DOX containing lysosomally degradable oligopeptide sequences as crosslinks and side-chains terminated in DOX. Four conjugates with mol. wt of 22, 160, 895 and 1230 kDa were prepared. The results indicated that the half-life in blood and the elimination rate from the tumour were up to 28 times longer and 25 times slower, respectively, for P-DOX (mol. wt=1230 kDa) than for free DOX. Treatment with P-DOX (mol. wt > or = 160 kDa) inhibited tumour growth more efficiently than that of 22 kDa P-DOX or free DOX (P<0.02) at a 2.2 mg/kg DOX equivalent dose. In conclusion, the administration of long circulating P-DOX resulted in enhanced tumour accumulation with a concomitant increase in therapeutic efficacy.     

Switching to a low-fat diet attenuates the intensified doxorubicin cardiotoxicity associated with high-fat feeding

Purpose

A high-fat diet has been shown to exacerbate the cardiotoxicity associated with the chemotherapy drug doxorubicin (DOX); however, it is unknown whether switching from a high-fat diet to a low-fat diet can attenuate the intensified DOX cardiotoxicity. The purpose of this study was to investigate the effects of a low-fat diet on DOX-induced cardiotoxicity in rats previously fed a high-fat diet.

Methods

Male rats were randomly assigned to consume a Western diet or a low-fat diet for 6 weeks. Western diet-fed rats were then further randomized to switch to the low-fat diet (WD-LF) or continue with the Western diet (WD). One week later, WD-LF and WD received 1 mg/kg DOX per day for 10 consecutive days and continued with their diets (WD-LF + DOX, WD + DOX). LF was further randomized to receive 1 mg/kg DOX per day for 10 consecutive days (LF + DOX) or saline injections as a control (LF + SAL). Four weeks following the first injection, cardiac function was analyzed, and left ventricles were analyzed for cardiotoxicity indices.

Results

When compared to LF + SAL and LF + DOX, WD + DOX exhibited an enhanced cardiotoxicity as evidenced by reduced septal wall thickness, fractional shortening, and sarco-endoplasmic reticulum Ca²⁺ ATPase expression as well as increased left ventricular cavity dimensions, lipid peroxidation, and β-myosin heavy-chain expression. This exacerbated cardiotoxicity was not observed in WD-LF + DOX.

Conclusions

Switching to a low-fat diet 1 week prior to, during, and following DOX treatment attenuated the exacerbated cardiotoxicity observed in the previously Western diet-fed rats.     

The influence of the time interval between monoHER and doxorubicin administration on the protection against doxorubicin-induced cardiotoxicity in mice

Purpose: Despite its well-known cardiotoxicity, the anthracyclin doxorubicin (DOX) continues to be an effective and widely used chemotherapeutic agent. DOX-induced cardiac damage presumably results from the formation of free radicals by DOX. Reactive oxygen species particularly affect the cardiac myocytes because these cells seem to have a relatively poor antioxidant defense system. The semisynthetic flavonoid monohydroxyethylrutoside (monoHER) showed cardioprotection against DOX-induced cardiotoxicity through its radical scavenging and iron chelating properties. Because of the relatively short final half-life of monoHER (about 30 min), it is expected that the time interval between monoHER and DOX might be of influence on the cardioprotective effect of monoHER. Therefore, the aim of the present study was to investigate this possible effect. Methods: Six groups of 6 BALB/c mice were treated with saline, DOX alone or DOX (4 mg/kg i.v.) preceded by monoHER (500 mg/kg i.p.) with an interval of 10, 30, 60 or 120 min. After a 6-week treatment period and additional observation for 2 weeks, the mice were sacrificed. Their cardiac tissues were processed for light microscopy, after which cardiomyocyte damage was evaluated according to Billingham (in Cancer Treat Rep 62(6):865–872, 1978). Microscopic evaluation revealed that treatment with DOX alone induced significant cardiac damage in comparison to the saline control group (P<0.001). Results: The number of damaged cardiomyocytes was 9.6-fold (95% CI 4.4–21.0) higher in mice treated with DOX alone than that in animals of the control group. The ratio of aberrant cardiomyocytes in mice treated with DOX preceded by monoHER and those in mice treated with saline ranged from 1.6 to 2.8 (mean 2.2, 95% CI 1.2–4.1, P=0.019). The mean protective effect by adding monoHER before DOX led to a significant 4.4-fold reduction (P<0.001, 95% CI 2.3–8.2) of abnormal cardiomyocytes. This protective effect did not depend on the time interval between monoHER and DOX administration (P=0.345). Conclusion: The results indicate that in an outpatient clinical setting monoHER may be administered shortly before DOX.This work was supported in part by grant VU-97-1525 from the Koningin Wilhelmina Foundation, Amsterdam, The Netherlands.     

HPMA copolymer–anticancer drug–OV-TL16 antibody conjugates. II. Processing in epithelial ovarian carcinoma cells in vitro

The binding, internalization, subcellular trafficking and in vitro cytotoxicity of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–anti-cancer drug–OV-TL16 antibody (Ab) conjugates in the ovarian carcinoma OVCAR-3 cell line have been investigated. Adriamycin (ADR) and meso chlorin e₆ mono(N-2-aminoethylamide) (Mce₆) photosensitizer were used as anti-cancer drugs. Targeted (Ab-containing) conjugates were compared with non-targeted HPMA copolymer–drug conjugates and with free drugs. Targeted conjugates were taken up rapidly by cells and detected within lysosomes by confocal fluorescence microscopy. The ADR attached to polymer chains via a degradable GFLG spacer was released from the conjugate, diffused via the lysosomal membrane into the cytoplasm and ultimately accumulated in the cell nuclei. In contrast, conjugates containing ADR bound via the GG spacer accumulated in the lysosomes, but no fluorescence could be detected in the cell nuclei. Binding the drugs to a non-targeted HPMA copolymer decreased their cytotoxicity in vitro. The IC₅₀ dose increased from 2 μM for free ADR to 150 μM for P(GFLG)–ADR (P is the HPMA copolymer backbone) and from 0.34 μM for free Mce₆ (with light) to 290 μM for P–(GG)–Mce₆. However, attachment of OV-TL16 Abs rendered HPMA copolymer–drug conjugates biorecognizable by OVCAR-3 cells and markedly increased their cytotoxicity. The IC₅₀ doses were 4.4 and 0.38 μM for the targeted conjugates P(GFLG)–ADR–Ab and P(GG)–Mce₆–Ab (with light), respectively. Biorecognition was shown to be specific by inhibition experiments with free Ab. The findings indicate the potential of these conjugates as effective agents in the treatment of ovarian cancer. Int. J. Cancer 75:600–608, 1998. © 1998 Wiley-Liss, Inc.     

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro

During recent years N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been developed as targetable drug carriers. These soluble synthetic polymers are internalized by cells by pinocytosis and they can be tailor-made to include peptidyl side-chains degradable intracellularly by specific lysosomal enzymes. Thus they provide the opportunity fo achieve controlled intracellular delivery of anticancer agents. The anthracycline antibiotic daunomycin, and protein synthesis inhibitor puromycin, were bound to HPMA copolymers via several different peptide side-chains, including Gly-Gly, Gly-Phe-Leu-Gly and Gly-Phe-Phe-Leu. Incubation of polymer-drug conjugates with isolated lysosomal enzymes (either a mixture of rat liver lysosomal enzymes or purified thiol-dependent lysosomal proteinases, cathepsins L and B) showed that significant release of drug occurred over 20 h, more than 20% of daunomycin and more than 80% of puromycin being liberated. To test their pharmacological activity conjugates were incubated with either the mouse leukaemia L1210, or the human lymphoblastoid leukaemia CCRF in vitro. The conjugates tested were all less effective than free daunomycin, but they showed differential toxicity against L1210 depending on the aminoacid sequence of their drug-polymer linkage. Inclusion of fucosylamine-terminating side-chains into the HPMA copolymer structure increased the affinity of conjugates for the L1210 cell membrane and resulted in increased toxicity. In contrast HPMA-daunomycin conjugates with or without fucosylamine affected CCRF cells equally, but this cell line was more sensitive than the mouse leukaemia to both free and polymer-bound daunomycin. Incubation of L1210 cells in polymer-bound daunomycin for 72 h, followed by plating cells out in low density in drug-free medium, showed that a concentration of polymer-bound drug (184 micrograms ml-1) could be selected to achieve a cytotoxic effect.     

HPMA copolymer platinates as novel antitumour agents: in vitro properties,pharmacokinetics and antitumour activity in vivo

The aim of this study was to compare in vitro and in vivo HPMA copolymer platinates with cisplatin in terms of platinum release, toxicity and antitumour activity. N-(2-hydroxypropyl)methacrylamide (HPMA) conjugates containing peptidyl side-chains (Gly–Gly or Gly–Phe–Leu–Gly) terminating in either carboxylate or amino species were prepared. The carboxylate polymeric intermediate was reacted with cisplatin, and the polymeric diamine with potassium tetrachloroplatinate to produce HPMA copolymer platinates of Mw 25 000–31 000 Daltons with a platinum loading of 3–7 wt%. The diglycyl spacer was selected because it is non-biodegradable, whereas the tetrapeptide spacer is known to be cleaved by the lysosomal thiol-dependent proteases. In vitro the HPMA copolymer platinates displayed a range of platinum release rates at pH 7.4 and 5.5; from <5%/24 h in the case of the diamino species which require enzymatic activation, to >80%/24 h in the case of the carboxylate. Cisplatin and the fast releasing carboxylate species displayed IC₅₀ values of 10 μg/ml Pt-equivalent against B16F10 cells in vitro, whereas the slow releasing conjugates were not cytotoxic over the dose range studied. Antitumour activity of HPMA copolymer platinates was first evaluated against L1210 and B16F10 tumours inoculated intraperitoneally (i.p.). When conjugates were administered i.p., the antitumour activity observed against L1210 tumours was within the range seen for free cisplatin (ratio of mean survival of treated animals to mean survival of controls, T/C, 1.20–1.70). Neither cisplatin nor HPMA copolymer platinates were active against intraperitoneal (i.p.) B16F10 tumours when administered i.p. However, when conjugates were administered intravenously (i.v.) to treat subcutaneous (s.c.) B16F10 tumours grown to palpable size, free cisplatin was still not active but the HPMA copolymer platinates bearing carboxylate and diamine platinates showed significant antitumour activity (T/C>1.35). Throughout these studies, the polymer platinates were 5–15-fold less toxic than cisplatin in vivo. After i.v. administration, the blood clearance of HPMA copolymer platinate was considerably slower (t_1/2α∼10 h) than seen for free cisplatin (t_1/2α<5 min). HPMA copolymer platinates (15 mg/kg Pt-equivalent) gave rise to an approximately 60-fold increase in Pt AUC in B16F10 tumour tissue than was achieved after administration of cisplatin at its maximum tolerated dose (MTD) (1 mg/kg).     

The protective activity of ICRF-187 against doxorubicin-induced cardiotoxicity in the rat

Summary The protective activity of the bisdioxopiperazine ICRF-187 against the cardiotoxicity of doxorubicin was evaluated in the rat using both functional and histological assays. Animals that had received a single i. v. dose of doxorubicin (4 mg/kg) alone were compared with those that had been pretreated with a single i. v. injection of saline or ICRF-187 (40 or 60 mg/kg). All rats showed a transient reduction in body weight during the first 3 weeks after drug administration. The greatest reduction (16%) was observed in animals that had received a combination of ICRF-187 (40 or 60 mg/kg) and doxorubicin. Deaths related to cardiotoxicity were observed only in rats that had received doxorubicin alone and in those treated with saline; most of the deaths occurred at between 8 and 13 weeks after drug administration. Sequential assessments of heart function showed a persistent depression of cardiac output in animals that had received doxorubicin, with or without pretreatment with ICRF-187. The reduction in cardiac output observed in rats that had been pretreated with ICRF-187 (40 or 60 mg/kg) amounted to 15% and 30% after 12 and 20 weeks, respectively, indicating that cardioprotection was only partial. Nevertheless, this represented a marked improvement as compared with the 35% reduction in cardiac output measured at 12 weeks in animals that had received doxorubicin but without pretreatment with ICRF-187. Histological examination of animals that had died during the course of the study and had received doxorubicin after pretreatment with saline revealed severe myocardial lesions typical of doxorubicin-induced damage. In contrast, animals that had been pretreated with ICRF-187 and survived for up to 20 weeks after treatment showed a marked amelioration of these lesions. The present findings may be interpreted as a true cardioprotection or a delay in the onset of the cardiotoxicity of doxorubicin resulting from pretreatment with the bisdioxopiperazine ICRF-187. Although prior and ongoing clinical trials clearly indicate that ICRF-187 protects patients well against doxorubicin-induced heart damage, further investigations are required beforehigh doses of ICRF-187 can be used as a means of increasing the protective activity of this drug against doxorubicin-induced cardiotoxicity.This work was supported by the Cancer Research Campaign     

Pegylated liposome-encapsulated doxorubicin and cisplatin in the treatment of head and neck xenograft tumours

Purpose: To evaluate the in vitro and in vivo activity of unencapsulated doxorubicin (DOX) and cisplatin (CDDP) and their pegylated liposome encapsulated counterparts (PLED and PLEC) in a subcutaneous model of human squamous cell cancer of the head and neck. Methods: In vitro cytotoxicity was determined by means of the sulphorhodamine B assay and in vivo activity was assessed in terms of tumour growth delay following single intravenous doses of the various agents. Treatment-related toxicity was evaluated by means of serial weight measurement. Results: The IC₅₀ values for DOX (12.1-fold) and CDDP (21.5-fold) were lower than for their liposome-encapsulated counterparts. When the two unencapsulated agents were compared, the IC₅₀ value for DOX was 16-fold lower than that for CDDP. In the in vivo studies, liposomes containing DTPA (PLEDTPA) exerted no effect on KB xenograft tumours when compared to untreated controls (P > 0.1). PLED was significantly more effective than DOX at doses of 2 mg/kg, 4 mg/kg and 8 mg/kg (P < 0.001 for all comparisons). At the 8 mg/kg dose, 7/13 animals treated with PLED were free of disease at 60 days, compared to 0/12 treated with DOX. PLEC displayed superior activity in comparison to CDDP at the 4 mg/kg dose level (P < 0.001), although at doses of 2 mg/kg and 10 mg/kg this comparison only reached borderline statistical significance (0.1 > P > 0.05). The highest dose level of 20 mg/kg was fatal to all animals in the CDDP group but well-tolerated by the animals in the PLEC group. On the basis of serial weight measurements, both PLED and PLEC were shown to be tolerated better than DOX and CDDP. Conclusion: Both PLED and PLEC were shown to exert significant activity against head and neck xenograft tumours, with PLED showing particular efficacy. Received: 2 November 1999 / Accepted: 14 February 2000     

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 16 mg/kg, 0.8 mg/kg and 2.4 mg/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 (P < 0.01) 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 (P < 0.05) 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 (P < 0.01) when added to either DOX (MTS 2.0) or to DOX+PTX (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. Received: 7 October 1998 / Accepted: 3 December 1998     

Design of a multivalent galactoside ligand for selective targeting of HPMA copolymer-doxorubicin conjugates to human colon cancer cells

N-(2-hydroxypropyl)methacrylamide (HPMA)-based copolymers have been shown to be efficient carriers for anticancer drugs because of their versatile chemistry and good biocompatibility. As demonstrated with hepatocytes, targeting efficacy of anticancer drugs could be further improved when the drug (doxorubicin) was conjugated to HPMA copolymers with biorecognisable groups, such as simple carbohydrates. The present study was devised to learn whether the cluster (multivalent) construction of carbohydrate residues could improve the targeting capability of HPMA copolymer-doxorubicin (DOX) conjugates towards human colon adenocarcinoma cells. DOX was linked via a lysosomally degradable tetrapeptide sequence to HPMA copolymers bearing galactosamine (GalN), lactose (Lac), or multivalent galactose residues (TriGal) to produce targetable polymeric drug carriers. The effect of the type of sugar moiety and its three-dimensional cluster arrangement on biorecognition by three human colon-adenocarcinoma cell lines was studied. The role of galectin-3 in the biorecognition of HPMA copolymer conjugates was explored. Biorecognition of the targetable (glycoside-bearing) conjugates decreased their IC(50) doses in comparison to the non-targetable (non-glycosylated) conjugates. The biorecognition of the TriGal-containing HPMA copolymer-doxorubicin conjugate by the cells was superior with concomitant decrease of its IC(50) doses. It is suggested that the increased cytotoxicity of the glycosylated HPMA-copolymer-DOX conjugates toward human colon-adenocarcinoma cells was caused by their biorecognition and effective internalisation via receptor-mediated endocytosis. All three human colon adenocarcinoma cell lines tested, Colo-205, SW-480 and SW-620, expressed the galectin-3 protein and the galectin-3-specific RNA. However, contrary to expectation, Colo-205 cells did not express a detectable amount of galectin-3 on the cell surface. This suggests that the binding of the glycoside-bearing HPMA copolymer-DOX conjugates to the cells was mediated not only by galectin-3. We conclude that targeting of the anticancer agent, doxorubicin, using HPMA copolymer conjugates bearing multivalent galactoside residues can improve their cytotoxicity.     

A Possibility to overcome P-glycoprotein (PGP)-mediated multidrug resistance by antibody-targeted drugs conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carrier

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing doxorubicin (DOX) and different targeting moieties were developed with the aim of specific chemotherapy. Two of them, HPMA-conjugated DOX and galactosamine-targeted DOX, are in phase II clinical trials in the U.K. We studied the effect of conjugates with different targeting moieties (anti-CD71, antithymocyte globulin, anti-CD4, transferrin) on human or mouse multidrug resistance (MDR) cell lines (CEM/VLB, P388-MDR). It was shown that targeting decreases the level of MDR for DOX and the level of MDR depends on the targeting moiety used. The combination of these conjugates with chemosensitisers (cyclosporin A, D, G) restored almost completely the sensitivity of MDR cell lines to that of parental sublines. These results suggest that different intracellular trafficking of these conjugates (in membrane-limited organelles) in contrast to free diffusion for low molecular weight compounds might partially overcome P-glycoprotein (Pgp)-mediated MDR. We also report here the development of biodegradable HPMA hydrogels suitable for prolonged release of the cytostatic drug and chemosensitiser as a potential approach to overcome MDR mediated by Pgp.     

Glutamine regulation of doxorubicin accumulation in hearts versus tumors in experimental rats

Purpose

Doxorubicin (DOX), an effective antineoplastic agent is known for its cardiotoxicity attributed mainly to free radical formation. Preliminary data indicated that oral glutamine (GLN) reduced cardiac oxidative damages in experimental rats treated with DOX. This study investigated the effect of GLN on DOX accumulation in tumors and normal tissues, troponin plasma concentration and functional alternations associated with DOX-induced myocardial damage.

Methods

Female Fisher344 rats (n = 40) with implanted MatBIII mammary tumors were randomized to receive oral GLN (1 g/kg/day) (n = 20) or to serve as controls (n = 20) and were treated with a single i.p. injection of 12 mg/kg DOX. Ten normal rats (n = 10) without treatment served as naive controls. Cardiac physiologic alterations resulting from DOX treatment in GLN-supplemented and control rats were assessed by micro-ultrasound imaging at 3 and 10 days after DOX injection. Ten rats from each GLN-supplemented and control groups were killed at 3 and 10 days after DOX administration. At killing, hearts, livers, spleens, kidneys, tumors and sera were examined for DOX concentration by measuring DOX natural fluorescence. Hearts were examined for Von Willebrand factor (vWF) expression using immunohistochemistry.

Results

Glutamine supplementation resulted in a significant reduction of DOX concentration in the normal tissues, without a significant effect on tumor DOX concentration. GLN-supplemented rats had lower plasma cTnI levels and lower cardiac levels of vWF. DOX-induced alterations in the echocardiographic parameters were significantly reduced in the GLN-supplemented rats.

Conclusions

These data indicate that GLN supplement is able to reduce DOX-induced cardiac damage and thus to enhance DOX therapeutic effectiveness.     

N-(2-hydroxypropyl)methacrylamide copolymers targeted to the hepatocyte galactose-receptor: pharmacokinetics in DBA2 mice.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing doxorubicin (DOX) and galactosamine can be targeted to the hepatocyte galactose receptor for organ-specific chemotherapy of primary and metastatic liver cancer. Here we report the dose-dependent pharmacokinetics of this macromolecular conjugate. Following intravenous administration to mice most efficient liver targeting was seen at low dose (0.05 mg DOX kg-1), with receptor saturation observed using higher bolus doses. Repeated low dose bolus injections did not cause down-regulation of the galactose receptor and targeted drug delivery rates of greater than or equal to 2 micrograms DOX g-1 liver h-1 were achieved. DOX is released from such conjugates intracellularly via action of lysosomal proteinases. It was shown that isolated rat liver lysosomal enzymes (Tritosomes) can release unmodified DOX from the peptidyl side chain Gly-Phe-Leu-Gly at a rate greater than or equal to 3 micrograms DOX g-1 liver h-1 i.e. the hydrolytic capacity is greater than the observed rate of drug delivery to the liver lysosomes in vivo. Although most conjugate would be captured by normal hepatocytes following intravenous administration, it was shown that the human hepatoma cell line HepG2 retains the galactose receptor, accumulating and processing the conjugate efficiently. Potential dose limiting toxicities of such drug conjugates could include cardio- or hepatotoxicity. Administration of conjugate reduced the 15 min heart level of DOX approximately 100-fold compared with that observed for an equivalent dose of free drug. Preliminary experiments showed that plasma levels of alkaline phosphatase, alanine transaminase and asparate transaminase did not change following administration of HPMA copolymer-daunorubicin (DNR) (10 mg DNR kg-1) indicating no significant heptatoxicity.     

Antitumor activity of N-(2-hydroxypropyl) methacrylamide copolymer-Mesochlorine e6 and adriamycin conjugates in combination treatments.

This study demonstrates the selective tumor targeting and the antitumor efficacy of the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound mesochlorin e6 monoethylenediamine (Mce6) and HPMA copolymer-bound Adriamycin (ADR) in combination photodynamic therapy (PDT) and chemotherapy against human ovarian OVCAR-3 carcinoma xenografted in female athynmic mice. The concentrations of Mce6 and ADR in blood and tissues, in free or HPMA copolymer-bound form, were determined by fluorescence and high-performance liquid chromatography fluorescence assays, respectively. Xenograft responses to single and combination therapies were recorded. The peak concentration of HPMA copolymer-Mce6 conjugate in tumor was achieved 18 h after administration. For HPMA copolymer-bound drugs, the concentration ratios of liver and spleen versus muscle were significantly higher than those of free drugs. The HPMA copolymer-bound drugs demonstrated selective targeting and accumulation in the tumor, probably attributed to the enhanced permeability and retention effect. In vivo studies revealed that all tumors in the treatment groups showed significant responses after receiving any of the various types of therapy as compared with controls (P < 0.001). PDT with HPMA copolymer-Mce6 conjugate (PDTMC) at a dose of 13.4 mg/kg (1.5 mg/kg of Mce6 equivalent) and light doses of 110 J/cm2 at 12 and 18 h, respectively, resulted in significant suppression of the growth of OVCAR-3 tumors. Three courses of chemotherapy using 35 mg/kg (2.2 mg/kg of ADR equivalent) of HPMA copolymer-ADR conjugate (CHEMO) were effective in suppressing the growth of tumors. Single PDTMC plus multiple CHEMO exhibited significantly greater therapeutic efficacy than multiple CHEMO. In the group of mice receiving multiple PDTMC, tumor recurrence became obvious after day 20. However, 10 of 12 tumors exhibited complete responses in the group of mice receiving multiple PDTMC plus multiple CHEMO. The least to most effective treatments were ranked as follows: multiple CHEMO < single PDTMC plus multiple CHEMO < multiple PDTMC < multiple PDTMC plus multiple CHEMO. The results clearly demonstrate that: (a) HPMA copolymer-bound drugs exhibited selective tumor accumulation contrary to free drugs; (b) PDT using HPMA copolymer-Mce6 conjugate with multiple light irradiations was a better therapy than that with single light irradiation; and (c) combination chemotherapy and photodynamic therapy with HPMA copolymer-ADR and HPMA copolymer-Mce6 conjugates was the most effective regimen.     

Myocardial toxicity of high-dose cyclophosphamide in rabbits treated with daunorubicin

Summary The present study was performed to evaluate experimentally the possible cardiotoxicity of high doses of cyclophosphamide after pretreatment with anthracyclines, a regimen used prior to bone marrow transplantation. A total of 27 rabbits received daunorubicin at a dose of 2.25 mg/kg per week for 10 weeks. At 1 week after the last daunorubicin dose, 13 of these rabbits received cyclophosphamide at 100 mg/kg per day ×2 (total dose, 200 mg/kg). All animals were killed after 1 additional week. Seven rabbits received cyclophosphamide at 100 mg/kg per day ×2 and two animals were given 50 mg/kg per day ×2 without additional treatment. In all, 18 untreated rabbits served as controls. At 3 h before the animals were killed, they received [^99mTc]-pyrophosphate i.v. Myocardial isotope activity was determined using a detector, and cardiac specimens were examined with a gamma-camera. Cardiotoxic effects were evaluated by myocardial isotope accumulation and pathologic changes were determined by morphology and by light and electron microscopy. The pathologic evaluation showed more frequent and wide-spread acute myocyte necrosis in daunorubicin/cyclophosphamide-treated rabbits as compared with those treated with daunorubicin or cyclophosphamide only. Myocardial isotope accumulation in rabbits treated with daunorubicin/cyclophosphamide was significantly higher then that in animals treated with either drug alone (20.001). Rabbits receiving cyclophosphamide as a single agent showed minor myocyte lesions but did not differ from controls in terms of isotope accumulation. We conclude that high-dose cyclophosphamide treatment on a dose schedule similar to that used prior to bone marrow transplantation and given soon after long-term daunorubicin therapy is considerably cardiotoxic.This study was supported by grant B85-04X-07156-01A from the Swedish Research Council and by grants from the Swedish Medical Society and by the Karolinska Institute, Solna, Sweden     

Timing of treatment with ICRF-187 and its effect on chronic doxorubicin cardiotoxicity

Studies were conducted to evaluate whether the timing of administration of ICRF-187 [(+)-1,2-bis(3,5 dioxopiperazinyl-1-yl)propane] would influence the degree of cardioprotection provided by this agent against the development of doxorubicin-induced chronic cardiomyopathy. Beagle dogs (8.5–14 kg) received either doxorubicin alone (1.75 mg/kg, i. v.,n=8), doxorubicin (1.75 mg/kg) simultaneously with ICRF-187 (35 mg/kg, i. v.,n=8), or doxorubicin (1.75 mg/kg) followed 2 h later by ICRF-187 (35 mg/kg,n=8). Control animals received ICRF-187 (35 mg/kg,n=4) or saline (n=4). All animals received a course of seven treatments, each given 3 weeks apart, and were killed 3 weeks after the last treatment. Semiquantitative grading of histologic sections of myocardium showed that as compared with animals treated with doxorubicin alone, the incidence and the severity of the doxorubicin-induced myocardial lesions were reduced in the two groups of animals given doxorubicin plus ICRF-187. However, protection was significantly better in dogs receiving ICRF-187 and doxorubicin simultaneously than in those given ICRF-187 2 h after doxorubicin. These observations were interpreted as indicating that the timing of administration of ICRF-187 with respect to that of doxorubicin is an important factor in determining the degree of cardioprotection and that there is a time window in which ICRF-187 exerts optimal effects.     

Tumour tropism and anti-cancer efficacy of polymer-based doxorubicin prodrugs in the treatment of subcutaneous murine B16F10 melanoma.

Doxorubicin (5 mg kg-1) was administered intravenously to C57 mice bearing subcutaneous B16F10 melanomas, distributing into the tumour with an area under the concentration-time curve (0-48 h; AUC) of 8.7 micrograms h g-1. Injection of doxorubicin-N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugate, containing 5 mg of doxorubicin equivalent per kg, mediated an AUC for free doxorubicin (i.e. doxorubicin released from the conjugate) of 15.2 micrograms h g-1 and for total doxorubicin (i.e. free plus conjugated) of 149.1 micrograms h g-1. An increased dose of doxorubicin-HPMA copolymer conjugate (18 mg of doxorubicin equivalent per kg) produced AUC values of 40.1 micrograms h g-1 and 671.7 micrograms h g-1 for free and total doxorubicin respectively. Hence administration of doxorubicin-HPMA copolymer conjugate achieved rises of 1.7- to 4.6-fold in tumour AUC (free doxorubicin) and 17.19 to 77.0-fold in tumour AUC (total doxorubicin). HPMA copolymers bearing fluorescein isothiocyanate accumulated in vascularised stromal regions, particularly in new growth sites at the tumour periphery. Treatment of mice with doxorubicin-HPMA copolymer conjugate achieved treated/control lifespans up to 320% (three doses of 27 mg of doxorubicin equivalent per kg) compared with only 133% using aggressive regimens of free doxorubicin (3 x 5 mg kg-1).     

Aqueous fish extract increases survival in the mouse model of cytostatic toxicity

Background

Treatment of cancer patients with anthracycline antibiotic doxorubicin (DOX) may be complicated by development of acute and chronic congestive heart failure (CHF), malignant arrhythmias and death. The aim of this study was to test whether an aqueous low molecular weight (LMW) extract from cod muscle decreases acute mortality in the mouse model of acute CHF caused by DOX.

Methods

A LMW fraction (<500 Da) of the aqueous phase of cod light muscle (AOX) was used for treatment of male BALB/c mice (~25 g, n = 70). The animals were divided into four groups, DOX + AOX (n = 20), DOX + saline (NaCl) (n = 30), NaCl + AOX (n = 10) and NaCl only (n = 10). Echocardiography was performed in the separate subgroups (DOX treated n = 6 and controls n = 6) to verify the presence and the grade of acute CHF. The cod extract was delivered by subcutaneously implanted osmotic minipumps over the period of 2 weeks. High-dose injection of DOX was administered to randomly selected animals. The animals received single intraperitoneal injection of DOX (25 mg/kg) and were followed over two weeks for mortality.

Results

Mortality rate was 68% lower (p < 0.05) in the mice treated with the extract. The analyses of cod extract have shown strong antioxidative effect in vitro.

Conclusion

The aqueous LMW cod muscles extract decreases mortality in the mouse model of DOX induced acute CHF. This effect may be mediated by cardioprotection through antioxidative mechanisms.     

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 1 mg/kg DOX i.v., either alone or with 20 mg/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 DZR + DOX-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 DZR + DOX 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 DZR + DOX. There was also a marked testicular atrophy in the mature rats given either DOX or DZR + DOX 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. Received: 3 March 1998 / Accepted: 13 May 1998     

Long-term effects of 7-monohydroxyethylrutoside (monoHER) on DOX-induced cardiotoxicity in mice

Doxorubicin (DOX) is a potent antitumor agent for different types of cancer, but the cumulative, dose-related cardiotoxicity limits its clinical use. The incidence of abnormal cardiac function after treatment with DOX appears to increase with time. Therefore, late cardiotoxicity is—especially in young surviving patients—a major concern. The aim of this study was to evaluate in mice whether the semisynthetic flavonoid 7-monohydroxyethylrutoside (monoHER) also protected against DOX-induced cardiotoxicity after a long period of follow-up. Four groups of 6 Balb/c mice were treated weekly during 6 weeks with saline, DOX alone (4 mg/kg i.v.), DOX preceded by monoHER (500 mg/kg i.p.), or DOX preceded by monoHER followed by long-term weekly monoHER injections during the observation period of 6 months. Half of the mice treated with DOX only developed DOX-induced heart failure and died within 6 months of observation. Two mice co-treated with monoHER showed weight loss and shortness of breath, whereas one mouse was found dead in its cage known with weight loss. The group receiving DOX plus long-term repeated doses of monoHER started to lose weight. Five out of six mice in this group developed shortness of breath and died before the end of the study with symptoms of cardiac failure induced by DOX. Statistical comparison of the histological heart damage between the different experimental groups was not possible, because the animals died at different time-points in the observation period and DOX-induced cardiotoxicity progressed with time. Nevertheless, it was clear that the initial cardioprotective effect of monoHER was not prolonged during the half-year observation period. It was even suggested that addition of repeated doses of monoHER tended to aggravate DOX-induced cardiotoxicity. It cannot be excluded that the dose and frequency of monoHER administration is crucial in obtaining an optimal antioxidant activity without a pro-oxidant activity of monoHER. This work was supported in part by grant VU-97-1525 from the Koningin Wilhelmina Foundation, Amsterdam, The Netherlands.