Reduced cardiotoxicity of doxorubicin given in the form ofN-(2-hydroxypropyl) methacrylamide conjugates: an experimental study in the rat

Summary A rat model was used to evaluate the general acute toxicity and the late cardiotoxicity of 4 mg/kg doxorubicin (DOX) given either as free drug or in the form of threeN-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates. In these HPMA copolymers, DOX was covalently bound via peptide linkages that were either non-biodegradable (Gly-Gly) or degradable by lysosomal proteinases (Gly-Phe-Leu-Gly). In addition, one biodegradable conjugate containing galactosamine was used; this residue was targeted to the liver. Over the first 3 weeks after the i.v. administration of free and polymer-bound DOX, all animals showed a transient reduction in body weight. However, the maximal reduction in body weight seen in animals that received polymer-bound DOX (4 mg/kg) was significantly lower than that observed in those that received free DOX (4 mg/kg) or a mixture of the unmodified parent HPMA copolymer and free DOX (4 mg/kg;P<0.01). Throughout the study (20 weeks), deaths related to cardiotoxicity were observed only in animals that received either free DOX or the mixture of HPMA copolymer and free DOX; in these cases, histological investigations revealed marked changes in the heart that were consistent with DOX-induced cardiotoxicity. Sequential measurements of cardiac output in surviving animals that received either free DOX or the mixture of HPMA copolymer and free DOX showed a reduction of 30% in function beginning at the 4th week after drug administration. The heart rate in these animals was 12% lower than that measured in age-matched control rats (P<0.05). Animals that were given the HPMA copolymer conjugates containing DOX exhibited no significant change in cardiac output throughout the study (P<0.05). In addition, no significant histological change was observed in the hearts of animals that received DOX in the form of HPMA copolymer conjugates and were killed at the end of the study. However, these animals had shown a significant increase in heart rate beginning at 8 weeks after drug administration (P<0.01). This study demonstrates that covalent binding of DOX to HPMA copolymer conjugates via both stable and biodegradable peptidyl linkages considerably reduces both the general acute toxicity and the late cardiotoxicity of DOX in the rat and could offer the potential for improving the therapeutic index in the clinical application of DOX.     

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     

In vivo CT measurement of blood-brain transfer constant of iopamidol in human brain tumors

Summary We have developed an in vivo method of measuring the blood-brain transfer constant (K) of iopamidol and the cerebral plasma volume (V_p) in brain tumors using a clinical X-ray CT scanner. In patient studies, Isovue 300 (iopamidol) was injected at a dosage of 1 ml/kg patient body weight. Serial CT scans of the tumor site and arterial blood samples from a radial artery were taken up to 48 min after injection. The leakage of iopamidol into the brain through the blood-brain barrier was modelled as an exchange process between two compartments, the intravascular plasma space and the tissue interstitial space. Using this model and the concentration measurements in blood plasma and tissue, quantitative estimates of K and V_p in brain tumors were obtained. In addition, distribution of the estimated values of K and V_p in tumors were displayed as false colour functional images overlaid on the conventional CT scan.In a study of twelve patients with anaplastic astrocytoma (n = 3), glioblastoma multiforme (n = 4) or metastases (n = 5) the mean K and VP values in tumor were found to be 0.0273 ± 0.0060 ml/min/g and 0.068 ± 0.11 ml/g respectively. These values were significantly higher than those in grey or white matter in the contralateral normal hemisphere (p < 0.05). The functional images showed variations in K and V_p within the tumor which were difficult to perceive in the original contrast enhanced CT scans.     

Maturation of epididymal spermatozoa in the nondomesticated guinea pigs Cavia aperea and Galea musteloides

The physiological changes occurring in spermatozoa in the male reproductive tract of 2 nondomesticated species of South American guinea pigs with different mating systems were studied. Cavia aperea, the wild ancestor of the domesticated guinea pig, has a polygynous mating system, whereas Galea musteloides exhibits promiscuous mating behavior. The epididymis of both species resembled that of the domesticated guinea pig, with a swathe of tubule convolutions (linking the 2 major parts of the organ) that was of smaller size in Cavia but not Galea. Higher relative epididymal weight was demonstrated in the promiscuous species. During their journey through the epididymis, spermatozoa from Galea developed their potential for motility expression more proximally than did those of Cavia, but motility developed into forward progression in the same region in both species. The maximal velocities exhibited by mature Cavia sperm in vitro were greater than those of Galea. Spermatozoa from Cavia were twice the length of those from Galea, they had larger heads, and the acrosomes of single sperm were more sensitive to disruption during morphological preparation. Only in Cavia did agglutination of sperm into rouleaux occur, after the potential for motility had been developed. Migration of the cytoplasmic droplet along the midpiece occurred in the same regions in both species and before agglutination in Cavia. It is suggested that the male's reproductive strategy (polygyny vs promiscuity) dictates the size of the testis and epididymis, whereas the female's reproductive physiology (induced ovulation vs cyclicity) influences the posttesticular development of sperm morphology and motility in the epididymis.     

Dietary conjugated linoleic acid mixture affects the activity of intestinal acyl coenzyme A: cholesterol acyltransferase in hamsters

The present study was designed to study the mechanisms by which dietary conjugated linoleic acids (CLA) decrease serum cholesterol. Hamsters were fed a semi-synthetic diet containing 1 g cholesterol/kg diet with or without supplementation with 20 g linoleic acid (LA) and 20 g CLA/kg diet. After 8 weeks, serum fasting total cholesterol (TC) and triacylglycerol (TG) were significantly lower in the LA-supplemented and CLA-supplemented groups compared with those of the control (CTL) hamsters. In contrast to LA, CLA significantly lowered hepatic cholesterol but it increased the level of adipose tissue cholesterol, suggesting that the hypocholesterolaemic mechanism of CLA is different from that of LA. CLA decreased the activity of intestinal acyl CoA:cholesterol acyltransferase (ACAT) whereas LA had no effect on this enzyme. Consequently, CLA supplementation increased the faecal excretion of total neutral sterols, but it had no or little effect on the faecal acidic sterols. If the ACAT is associated with cholesterol absorption, the part of mechanisms by which CLA decreases serum cholesterol may involve down-regulation of intestinal ACAT activity.     

Complexed prostate specific antigen provides significant enhancement of specificity compared with total prostate specific antigen for detecting prostate cancer

PURPOSE: Determining serum total prostate specific antigen (PSA) has proved to be a valuable diagnostic aid for detecting prostatic carcinoma, although the lack of specificity has limited its usefulness. Studies indicate that the use of percent free PSA would improve specificity while maintaining sensitivity. Since complexed PSA represents the major proportion of measurable PSA in serum, we determined whether it represents a single test alternative to the use of percent free PSA for the early detection of prostate cancer. MATERIALS AND METHODS: Archival serum was obtained from 385 men with no evidence of malignancy on biopsy and 272 with biopsy confirmed prostate cancer. We determined the concentration and proportion of total, complexed and free PSA. RESULTS: Receiver operating characteristics analysis using total PSA results from all samples (range 0.32 to 117 ng./ml.) indicated that the areas under the curve for complexed PSA alone as well as the free-to-total and complexed-to-total PSA ratios were similar and significantly greater than those for total PSA alone. Within the range of 85% to 95% sensitivity receiver operating characteristics analysis revealed that the specificity of complexed PSA was higher than that of total PSA and equivalent to that of the free-to-total PSA ratio. We noted a similar improvement in specificity in the 4 to 10 ng./ml. total PSA range. Using published cutoff values for complexed, total and percent free PSA when total PSA was in the 4 to 10 ng./ml. range the sensitivity and specificity of complexed and percent free PSA were similar. Within the 4 to 10 ng./ml. total PSA range the population of patients with no evidence of malignancy and complexed PSA below the upper limit was different with respect to total PSA from that with no evidence of malignancy and free PSA greater than 25%. CONCLUSIONS: The measurement of complexed PSA represents an alternative to the use of percent free PSA, although the patient populations identified by the 2 tests are different.     

Enhancement of manumycin A-induced apoptosis by methoxyamine in myeloid leukemia cells.

Farnesyltransferase inhibitors (FTIs) are currently under investigation for leukemia treatment. We evaluated the FTI manumycin A (manumycin) in two myeloid leukemia cell lines (U937 and HL-60). Manumycin induced nitric oxide production and apoptosis of the leukemia cells. Nitric oxide or other reactive oxygen species may induce oxidative DNA damage, and the number of apurinic sites increased after manumycin treatment, which was reversed by concurrent treatment with N-acetyl-L-cysteine. Since repair of DNA damage is important to cell survival, we hypothesized that methoxyamine, an inhibitor of base-excision repair, would enhance the antineoplastic effect of manumycin. The combination of manumycin and methoxyamine resulted in enhanced apoptosis by six criteria increased annexin V binding, release of mitochondrial cytochrome c into the cytosol, activation of caspase-9, activation of caspase-3, specific cleavage of poly-adenosyl ribose polymerase, and increase in the sub-G1 cell cycle fraction. The drug combination enhanced inhibition on the soft agar clonogenic assay and on the formazan dye cell viability assay. The effects of manumycin or manumycin plus methoxyamine on apoptosis were blocked by N-acetyl-L-cysteine, and partially by nitric oxide synthase inhibitors or scavenger of peroxide. We conclude that methoxyamine enhances manumycin-induced apoptosis in myeloid leukemia cells.