Hydrogels based on low-methoxyl amidated citrus pectin and flaxseed gum formulated with tripeptide glycyl-l-histidyl-l-lysine improve the healing of experimental cutting wounds in rats.

Hydrogels based on natural and modified polysaccharides represent growing group of suitable matrices for the construction of effective wound healing materials. Bioactive tripeptide glycyl-l-histidyl-l-lysine and amino acid α-l-arginine are known to accelerate wound healing and skin repair. In this study, hydrogels based on low-methoxyl amidated citrus pectin or flaxseed gum were prepared and used for the transport of these healing agents to the experimental cutting wounds affected by extensive skin damage. Fourier-transform infrared spectroscopy, rheology, differential scanning calorimetry, scanning electron microscopy, swelling and release tests confirmed that these hydrogels differed in structure and physical properties. The cationic tripeptide was found to bind to carboxylic groups in LMA pectin, and the C3OH hydroxyl and ring oxygen O5 are involved in this interaction. The pectin hydrogel showed high viscosity and strong elastic properties, while the flaxseed gum hydrogel was characterised as a viscoelastic system of much lower viscosity. The former hydrogel released the drugs very slowly, while the latter hydrogel demonstrated zero order releasing kinetics optimal for drug delivery. In the in vivo wound healing testing on rats, both polysaccharide hydrogels improved the healing process mediated by the mentioned biomolecules. The tripeptide applied in the hydrogels showed significantly higher healing degree and lower healing time than in the control animals without treatment and when it was applied in an aqueous solution. Despite the absence of a synergistic effect, the mixture of the tripeptide and α-l-arginine in the hydrogels was also quite effective in wound healing. According to histological analysis, complete healing was achieved only when using the tripeptide in the flaxseed gum hydrogel. These observations might have an important prospect in clinical application of polysaccharide hydrogels.

Poly(2-ethyl-2-oxazoline) conjugates with doxorubicin for cancer therapy: In vitro and in vivo evaluation and direct comparison to poly[N-(2-hydroxypropyl)methacrylamide] analogues.

We designed and synthesized a new delivery system for the anticancer drug doxorubicin based on a biocompatible hydrophilic poly(2-ethyl-2-oxazoline) (PEtOx) carrier with linear architecture and narrow molar mass distribution. The drug is connected to the polymer backbone via an acid-sensitive hydrazone linker, which allows its triggered release in the tumor. The in vitro studies demonstrate successful cellular uptake of conjugates followed by release of the cytostatic cargo. In vivo experiments in EL4 lymphoma bearing mice revealed prolonged blood circulation, increased tumor accumulation and enhanced antitumor efficacy of the PEtOx conjugate having higher molecular weight (40 kDa) compared to the lower molecular weight (20 kDa) polymer. Finally, the in vitro and in vivo anti-cancer properties of the prepared PEtOx conjugates were critically compared with those of the analogous system based on the well-established PHPMA carrier. Despite the relatively slower intracellular uptake of PEtOx conjugates, resulting also in their lower cytotoxicity, there are no substantial differences in in vivo biodistribution and anti-cancer efficacy of both classes of polymer-Dox conjugates. Considering the synthetic advantages of poly(2-alkyl-2-oxazoline)s, the presented study demonstrates their potential as a versatile alternative to well-known PEO- or PHPMA-based materials for construction of drug delivery systems.

Hyperbilirubinemia Protects against Aging-Associated Inflammation and Metabolic Deterioration.

Mild constitutive hyperbilirubinemia is associated with a reduced risk of cardiovascular diseases, diabetes, and cancer. Since these pathologies are associated with aging, inflammation, and oxidative stress, we investigated whether hyperbilirubinemia interferes with ROS homeostasis in cell cultures and with inflammation, senescence, and mitochondrial dysfunction in aged rats. Human embryonic kidney cells and rat primary fibroblasts showed a dose-dependent decrease in the ratio of oxidized/reduced glutathione, intracellular H2O2 levels, and mitochondrial ROS production, with increasing bilirubin concentrations in the culture media. Compared to their normobilirubinemic siblings, aged hyperbilirubinemic Gunn rats showed significantly smaller amounts of visceral fat, better glucose tolerance, and decreased serum levels of proinflammatory cytokines TNFα, IL-1ß, and IL-18. Simultaneously, livers from Gunn rats showed decreased expression of senescence markers and cell cycle inhibitors p21 and p16. Mitochondria from aged Gunn rats showed higher respiration and lower H2O2 production compared to controls. In conclusion, we demonstrated that mildly elevated serum bilirubin is generally associated with attenuation of oxidative stress and with better anthropometric parameters, decreased inflammatory status, increased glucose tolerance, fewer signs of cellular senescence, and enhanced mitochondrial function in aged rats.

Multistage-targeted pH-responsive polymer conjugate of Auger electron emitter: optimized design and in vivo activity.

Auger electrons-emitting radioisotopes (such as iodine-125) are a potentially effective cancer treatment. They are extremely biologically effective, but only within a short range (nanometers). Their use as an effective cancer therapy requires that they will be transported within close proximity of DNA by an intercalator, where they induce double-strand breaks leading to cell death. This type of therapy may be even more beneficial when associated with drug delivery systems. In this report, we describe an optimized triple-targeted polymer delivery system for the intercalator ellipticine, which contains radioisotope iodine-125 with high specific radioactivity (63.2 GBq/mg). This compound is linked to an N-(2-hydroxypropyl)methacrylamide copolymer via an optimized acid-sensitive hydrazone linker. The system is stable at pH 7.4 (representing the pH of blood plasma), and the radioiodine-containing biologically active intercalator is released upon a decrease in pH (44% of the intercalator is released after 24h of incubation in pH 5.0 buffer, which mimics the pH in late endosomes). The active compound is a potent intercalator, as shown with direct titration with a DNA solution, and readily penetrates into cell nuclei, as observed by confocal microscopy. Its polymer conjugate is internalized into endosomes and releases the radioactive intercalator, which accumulates in the cell nuclei. In vivo experiments on mice with 4T1 murine breast cancer resulted in a statistically significant increase in the survival of mice treated with the polymer radioconjugate. The free radiolabeled intercalator was also shown to be effective, but it was less potent than the polymer conjugate.

Dual role of host Par2 in a murine model of spontaneous metastatic B16 melanoma.

AIM: We investigated differences of metastatic spread of normal proteinase-activated receptor-2 (Par2+/+) melanoma B16 in Par2-/- (knock-out) animals compared to C57Bl6 mice. MATERIALS AND METHODS: Nine knock-out mice B6.Cg-F2rl1tm1Mslb/J (Par2-/-) and nine C57Bl6/J controls were subcutaneously inoculated with B16 melanoma tissue cells. Twelve days after inoculation, all primary tumors were removed. Survival and metastatic spread was followed for up to 100 days after primary tumor extirpation. RESULTS: Excised primary tumors were on average larger in Par2-/- mice (360 mm3 vs. 221 mm3 in C57Bl6/J). Distant spontaneous metastases developed in only 3 of 9 of Par2-/- mice in comparison to 6 of 9 controls. The average survival time was 84 days in Par2-/- animals compared to 63 days in C57Bl6/J mice. CONCLUSION: Host Par2 melanoma model contributes to the limitation of local cancer progression in one area, while on the other hand is important for enhancing distant metastatic spread.

Anti-cancer effects of blue-green alga Spirulina platensis, a natural source of bilirubin-like tetrapyrrolic compounds.

Spirulina platensis is a blue-green alga used as a dietary supplement because of its hypocholesterolemic properties. Among other bioactive substances, it is also rich in tetrapyrrolic compounds closely related to bilirubin molecule, a potent antioxidant and anti-proliferative agent. The aim of our study was to evaluate possible anticancer effects of S. platensis and S. platensis-derived tetrapyrroles using an experimental model of pancreatic cancer. The anti-proliferative effects of S. platensis and its tetrapyrrolic components [phycocyanobilin (PCB) and chlorophyllin, a surrogate molecule for chlorophyll A] were tested on several human pancreatic cancer cell lines and xenotransplanted nude mice. The effects of experimental therapeutics on mitochondrial reactive oxygen species (ROS) production and glutathione redox status were also evaluated. Compared to untreated cells, experimental therapeutics significantly decreased proliferation of human pancreatic cancer cell lines in vitro in a dose-dependent manner (from 0.16 g•L-1 [S. platensis], 60 µM [PCB], and 125 µM [chlorophyllin], p<0.05). The anti-proliferative effects of S. platensis were also shown in vivo, where inhibition of pancreatic cancer growth was evidenced since the third day of treatment (p < 0.05). All tested compounds decreased generation of mitochondrial ROS and glutathione redox status (p = 0.0006; 0.016; and 0.006 for S. platensis, PCB, and chlorophyllin, respectively). In conclusion, S. platensis and its tetrapyrrolic components substantially decreased the proliferation of experimental pancreatic cancer. These data support a chemopreventive role of this edible alga. Furthermore, it seems that dietary supplementation with this alga might enhance systemic pool of tetrapyrroles, known to be higher in subjects with Gilbert syndrome.

Antiproliferative effects of carbon monoxide on pancreatic cancer.

BACKGROUND: Carbon monoxide, the gaseous product of heme oxygenase, is a signalling molecule with a broad spectrum of biological activities. The aim of this study was to investigate the effects of carbon monoxide on proliferation of human pancreatic cancer. METHODS: In vitro studies were performed on human pancreatic cancer cells (CAPAN-2, BxPc3, and PaTu-8902) treated with a carbon monoxide-releasing molecule or its inactive counterpart, or exposed to carbon monoxide gas (500 ppm/24h). For in vivo studies, pancreatic cancer cells (CAPAN-2/PaTu-8902) were xenotransplanted subcutaneously into athymic mice, subsequently treated with carbon monoxide-releasing molecule (35 mg/kg b.w. i.p./day), or exposed to safe doses of carbon monoxide (500 ppm 1h/day; n = 6 in each group). RESULTS: Both carbon monoxide-releasing molecule and carbon monoxide exposure significantly inhibited proliferation of human pancreatic cancer cells (p<0.05). A substantial decrease in Akt phosphorylation was observed in carbon monoxide-releasing molecule compared with inactive carbon monoxide-releasing molecule treated cancer cells (by 30-50%, p<0.05). Simultaneously, carbon monoxide-releasing molecule and carbon monoxide exposure inhibited tumour proliferation and microvascular density of xenotransplanted tumours (p<0.01), and doubled the survival rates (p<0.005). Exposure of mice to carbon monoxide led to an almost 3-fold increase in carbon monoxide content in tumour tissues (p=0.006). CONCLUSION: These data suggest a new biological function for carbon monoxide in carcinogenesis, and point to the potential chemotherapeutic/chemoadjuvant use of carbon monoxide in pancreatic cancer.

Novel topical photodynamic therapy of prostate carcinoma using hydroxy-aluminum phthalocyanine entrapped in liposomes.

BACKGROUND: Clinically-approved anticancer photodynamic therapy (PDT) is now extensively studied for various cancer diagnoses. We focused on the treatment efficacy of topical administration of hydroxy-aluminum phthalocyanine (AlOH-PC) entrapped in liposomes against in vivo models of prostate carcinomas. MATERIALS AND METHODS: LNCaP and PC3 cells were subcutaneously injected into the right flank of athymic nude mice. Mice with grown tumours were used for in vivo efficacy studies. Firstly, we applied different doses of AlOH-PC to less aggressive LNCaP tumours to determine the effective dose. In later studies, we focused on more aggressive prostate tumours (PC3) using doses of liposomal-AlOH-PC gel formulation. Topical application of photosensitizers was followed by PDT irradiation (600-700 nm, 635 nm peak). Tumour growth was measured three times-a-week. RESULTS: Comparison of PDT of aggressive PC3 and less aggressive LNCaP prostate carcinomas showed that both tumour types are sensitive and treatable by liposomal formulation of AlOH-PC. For LNCaP tumours the efficient dose (100% experimental animals cured, n=8/8) was 4.5 mg/ml of AlOH-PC in the gel. Whereas, in the case of PC3 carcinomas, a dose of 4 mg/ml significantly postponed tumour growth, but no animals were cured (n=0/8); a sufficient curative dose (100% mice cured, n=8/8) was 6 mg/ml of AlOH-PC in the gel. CONCLUSION: Liposomal AlOH-PC gel has potential for effective PDT of prostate carcinomas.

Evaluation of topical photodynamic therapy of mammary carcinoma with an experimental gel containing liposomal hydroxyl-aluminium phthalocyanine.

BACKGROUND: Photodynamic therapy (PDT) is a clinically-accepted approach for the therapy of many types of cancer. This study focused on the treatment of mammarian carcinoma by topical administration of hydroxyl-aluminium phthalocyanine (AlOH-PC), compared to a clinically-approved photosensitizer (Metvix, Galderma & PhotoCure ASA, Inc., Oslo, Norway). MATERIALS AND METHODS: MDA-MB 231 cells were subcutaneously injected into the right flank of athymic nude mice. Mice with grown tumours were used for in vivo efficacy studies. Different doses of liposomal AlOH-PC were applied to determine the most effective dose. In later studies, Metvix or our liposomal-AlOH-PC gel formula were used. Topical application of photosensitizers was followed by the PDT irradiation at 600-700 nm (635 nm peak). Tumour growth was measured three times weekly. RESULTS: Therapeutic studies revealed that AlOH-PC treatment led to complete tumour remission in 90% (9/10) of experimental animals, whereas usage of the commercially available Metvix only postponed the tumour growth. Moreover, usage of liposomal AlOH-PC shortened the time allowed between the application of the photosensitizer and light exposure: for Metvix, hours are usually needed, while the tested liposomal AlOH-PC showed remarkable outcomes after only 10 min. CONCLUSION: Liposomal AlOH-PC gel appears to be potentially suitable for PDT of mammarian carcinoma.

Thermoresponsive polymeric radionuclide delivery system--an injectable brachytherapy.

Brachytherapy is of increasing popularity in clinical oncology for the local therapy of solid tumors due to high radiation doses delivered to malignant tissue while keeping the whole-body radiation burden low. Pronounced dose-dependent tumor growth reduction was achieved by single dose of injectable intratumoral brachytherapy with iodine-131-labeled thermoresponsive polymer [poly(N-isopropyl acrylamide)] in murine xenograft model (PC3 human prostate adenocarcinoma). The two doses of radionuclide were used, 2 MBq/mouse and 25 MBq/mouse. The higher dose caused gradual tumor volume reduction and 2 of 6 mice from this group were cured. The lower dose caused tumor growth retardation only. In both cases there were no signs of inflammation. The effects of both doses were statistically significant compared to untreated controls. Such injectable system should keep advantages of brachytherapy while making system administration easier and less invasive (injection instead of implantation), patient-tailored (splitting of doses into several depoes) and bioerodable.

Mapping the ribonucleolytic active site of bovine seminal ribonuclease. The binding of pyrimidinyl phosphonucleotide inhibitors.

Bovine seminal ribonuclease (BS-RNase) is a 27kDa homodimeric enzyme and a member of the pancreatic RNase A superfamily. It is the only RNase with a quaternary structure and it is a mixture of two dimeric forms. In the most abundant form the active site is formed by the swapping of the N-terminal segments. BS-RNase is a potent antitumor agent with severe side effects such as aspermatogenicity, and immunosuppression. As a first step towards the design of potent inhibitors of this enzyme we mapped its active site through the study of the binding of uridine 2'-phosphate (U2'p), uridine 3'-phosphate (U3'p), uridine 5'-diphosphate (UDP), cytidine 3'-phosphate (C3'p), and cytidine 5-phosphate (C5'p), by kinetics, and X-ray crystallography. These phosphonucleotides are potent inhibitors with C3'p being the most potent with a K(i) value of 22 microM. Absorption, distribution, metabolism, and excretion pharmacokinetic property predictions reveal U2'p, U3'p, and C5'p as the most promising with respect to oral bioavailability. In vivo studies on the aspermatogenic effect have shown that C3'p and C5'p inhibit significantly this biological action of BS-RNase.

Highly sensitive method for quantitative determination of bilirubin in biological fluids and tissues.

Unconjugated bilirubin (UCB) exhibits potent antioxidant and cytoprotective properties, but causes apoptosis and cytotoxicity at pathologically elevated concentrations. Accurate measurement of UCB concentrations in cells, fluids and tissues is needed to evaluate its role in redox regulation, prevention of atherosclerotic and malignant diseases, and bilirubin encephalopathy. In the present study, we developed and validated a highly sensitive method for tissue UCB determinations. UCB was extracted from rat organs with chloroform/methanol/hexane at pH 6.2 and then partitioned into a minute volume of alkaline buffer that was subjected to HPLC using an octyl reverse phase (RP) column. Addition of mesobilirubin as an internal standard corrected for losses of UCB during extraction. Recoveries averaged 75+/-5%. The detection limit was 10pmol UCB/g wet tissue. Variance was +/-2.5%. When used to measure UCB concentrations in tissues of jaundiced Gunn rats, this procedure yielded UCB levels directly comparable to published methods, and accurately determined very low tissue bilirubin concentrations (

Differences in antitumor effects of various statins on human pancreatic cancer.

Statins are widely used for the treatment of hypercholesterolemia. However, their inhibitory action on HMG-CoA reductase also results in the depletion of intermediate biosynthetic products, which importantly contribute to cell proliferation. The aim of the present study was to compare the effects of the individual commercially available statins on experimental pancreatic cancer. The in vitro effects of individual statins (pravastatin, atorvastatin, simvastatin, lovastatin, cerivastatin, rosuvastatin and fluvastatin) on the viability of human pancreatic cancer were evaluated in CAPAN-2, BxPc-3 and MiaPaCa-2 cell lines. The in vivo experiments were performed on nude mice xenotransplanted with CAPAN-2 cells. The mice received oral treatments either with a placebo, or with the statins mentioned earlier in a daily dose corresponding to a hypocholesterolemic dose in humans. The effect of these statins on the intracellular Ras protein, trafficking in MiaPaCa-2 transfected cells, was also investigated. Substantial differences in the tumor-suppressive effects of all statins were detected in both in vitro and in vivo experiments. While simvastatin exerted the highest tumor-suppressive effects in vitro, rosuvastatin (p = 0.002), cerivastatin (p = 0.002) and fluvastatin (p = 0.009) were the most potent compounds in an animal model. All statins (except pravastatin) inhibited intracellular Ras protein translocation. In summary, substantial tumor-suppressive effects of various statins on the progression of experimental pancreatic adenocarcinoma were demonstrated, with marked differences among individual statins. These results support greatly the potential of statins for the chemoadjuvant treatment of pancreatic cancer.

Radiation-induced production of PAR-1 and TGF-beta 1 mRNA in lung of C57Bl6 and C3H murine strains and influence of pharmacoprophylaxis by ACE inhibitors.

Transforming growth factor beta 1 (TGF-beta1) plays an important role in the development of radiation- and drug-induced organ diseases. Proteinases-activated receptor 1 (PAR-1) is involved in many pathophysiologic processes after its activation by serine proteases. The aim of the present study was to determine messenger RNA (mRNA) production of TGF-beta1 and PAR-1 in the lungs after local irradiation. Mice of C57BL/6 and C3H/J strains with different susceptibility to fibrosis development were exposed to a of 15Gy. Non-irradiated mice of both strains were used as negative controls. Control (irradiated) and irradiated angiotensin-converting enzyme (ACE) inhibitor-treated animals were examined simultaneously. The ACE inhibitor group was given butylaminiperindopril for 9 days after irradiation (15Gy) at a daily dose of 0.1 or 0.2mg/kg per rectum. On day 9, all mice were sacrificed, and the production of mRNA TGF-beta1 and PAR-1 in lung tissue was determined semiquantitatively using reverse transcriptase polymerase chain reaction, and immunohistochemical analysis of PAR-1 expression in pulmonary tissue was performed. In the fibrosing murine strain C57Bl/6, there was an increase in the mRNA TGF-beta1 and PAR-1 levels in lungs 9 days after irradiation as compared with non-irradiated controls and non-fibrosing murine strain C3H/J. In butylaminiperindopril-treated mice, a decrease in transcript of TGF-beta1 and PAR-1 was observed. Thus, PAR-1 is involved in radiation-induced lung fibrosis in correlation with TGF-beta1 production. Administration of ACEI influences PAR-1 and TGF-beta1 expression.

Establishment, growth and in vivo differentiation of a new clonal human cell line, EM-G3, derived from breast cancer progenitors.

A new clonal cell line, EM-G3, was derived from a primary lesion of human infiltrating ductal breast carcinoma. The line consisted of cuboidal cells with occasional appearance of more differentiated branched cells apparently involved in cell-to-cell communication. The EM-G3 cells, population doubling time 34 h, are dependent on the epidermal growth factor. Multicolor fluorescence in situ hybridization (mFISH) analysis demonstrated a stable diploid genome with several genetic changes. Immunocytochemical analysis of EM-G3 in vitro revealed positivity for keratins (K) K5, K14, K18, nuclear protein p63, epithelial membrane antigen (EMA) and other proteins indicative of a pattern of mammary epithelium bipotent progenitors. Detection of integrins alpha-6, beta-1, and protein CD44 by cDNA array also pointed to the character of basal/stem cells. In contrast, dominant cells in the human original tumor showed the luminal character (K18+, K19+, K5-, K14-, and p63-). However, cells with the immunocytochemical profile similar to that of cultured EM-G3 cells were found in minor clusters in the patient's tumor sections. The EM-G3 cells formed limited tumors in nu/nu mice. The cells in mouse tumors were organized in primitive ductal-like structures consisting of 1-3 large central luminal-like cells (EMA+) surrounded by peripheral myoepithelial-like cells (p63+/EMA-). The large central cells gradually disintegrated, forming a pseudolumen. Apparently, EM-G3 cells are able to partially differentiate in vivo as well as in vitro. Our results indicate that EM-G3 cells were derived from a premalignant population of common progenitors of luminal and myoepithelial cells that were immortalized in an early stage of tumorigenesis.

Comparative anti-tumor efficacy of two orally administered platinum(IV) drugs in nude mice bearing human tumor xenografts.

The oral anti-tumor activity of a novel platinum(IV) complex, coded as LA-12, with a bulky adamantylamine ligand was evaluated and compared with another platinum(IV) complex satraplatin. The human carcinoma xenografts of colon HCT116, prostate PC3, and ovarian A2780 and A2780/cisR (resistant to cisplatin) were used to evaluate the in-vivo anti-tumor activity. The daily x 5 repeated dose regimen in equimolar doses of LA-12 and satraplatin, administered in 2 cycles, was selected for this evaluation. All doses of LA-12 and satraplatin were significantly effective in comparison with the control. The activities of LA-12 in all doses and all used tumor xenografts were higher than equimolar doses of satraplatin. The highest effect was reached with LA-12 at a dose of 60 mg/kg. The shapes of growth curves of ovarian carcinoma A2780 and its subline resistant to cisplatin after therapy with LA-12 were very similar. This shows that LA-12 is able to overcome resistance to cisplatin.

Preclinical anti-tumor activity of a new oral platinum(IV) drug LA-12.

A novel anti-tumor platinum(IV) complex, coded as LA-12, with a bulky adamantylamine ligand displaying oral activity was prepared and its oral activity was evaluated. The murine ADJ/PC6 plasmacytoma and human A2780 ovarian carcinoma tumor model were used to evaluate the in vivo anti-tumor activity of a single dose and also of repeated doses with comparison to the activity of cisplatin and of the platinum(IV) complex satraplatin. The acute toxicity of LA-12 in mice is relatively low (maximum tolerated dose 1000 mg/kg), and the effective dose is comparable to that of cisplatin and higher than that of satraplatin. The therapeutic index derived from this is very high (250). In the human tumor model, two repeated dose schedule regimens were evaluated. LA-12 exerted a significantly higher anti-tumor activity than other substances, i.e. cisplatin and satraplatin, in repeated doses on the murine ADJ/PC6 plasmacytoma tumor model. The dailyx5 repeated dose regimen was selected for further evaluation.

The effect of zinc salts on serum bilirubin levels in hyperbilirubinemic rats.

OBJECTIVES: Intestinal metabolism of bilirubin is implicated in the pathogenesis of neonatal jaundice and Crigler-Najjar syndrome. In the present study the authors investigated the effect of oral administration of zinc salts on serum bilirubin levels in hyperbilirubinemic rats. METHODS: Bilirubin-binding activities of zinc sulfate and water-insoluble zinc methacrylate were determined in vitro. Congenitally hyperbilirubinemic Gunn rats and artificially hyperbilirubinemic Wistar rats were used in in vivo studies. Animals were fed a normal diet for 1 week and then a treatment diet of either zinc sulfate or zinc methacrylate for additional 2 weeks. Serum and fecal bile pigments were determined at the end of each phase. Biliary bilirubin secretion rates were determined in hyperbilirubinemic Wistar rats fed zinc methacrylate. RESULTS: Substantial bilirubin-binding activities of zinc salts were demonstrated in in vitro experiments. Treatment with oral zinc salts significantly decreased serum bilirubin levels in Gunn rats (166 +/- 53 versus 123 +/- 38 and 206 +/- 34 versus 131 +/- 31 micromol/L, P < 0.05 for zinc methacrylate and zinc sulfate, respectively). A similar effect of zinc methacrylate was also observed in hyperbilirubinemic Wistar rats (102 +/- 10 versus 14 +/- 4 micromol/L, P < 0.0001). In accord, biliary bilirubin secretion decreased significantly in these animals (45 +/- 11 versus 28 +/- 4 nmol/h 100g body weight, P < 0.02). In contrast to zinc sulfate, treatment with zinc methacrylate did not lead to the elevation of serum zinc levels. CONCLUSIONS: Oral administration of zinc salts efficiently decreased serum bilirubin levels in hyperbilirubinemic rats, presumably as a result of inhibition of enterohepatic circulation of bilirubin. This approach might be useful in the treatment of severe unconjugated hyperbilirubinemias.

The impact of intestinal microflora on serum bilirubin levels.

BACKGROUND/AIMS: Intestinal microflora plays an important role in the pathogenesis of neonatal jaundice by inhibiting enterosystemic circulation of bilirubin. The present study aimed to investigate the influence of intestinal microflora on serum bilirubin levels in hyperbilirubinemic Gunn rats. METHODS: After a baseline phase Gunn rats received oral antibiotics (either clindamycin/neomycine or co-trimethoxazole for four days, phase II). Intestinal colonization was carried out either with a bilirubin-reducing strain of C. perfringens or C. pasteurianum incapable of reducing bilirubin (phase III). Serum bilirubin and fecal bile pigments were determined at the end of each phase. RESULTS: Oral administration of clindamycin/neomycine resulted in the disappearance of fecal urobilinoids. Simultaneously, serum bilirubin increased dramatically (186+/-31 vs. 289+/-35 micromol/l, P=0.004). Intestinal colonization with C. perfringens led to reappearance of fecal urobilinoid production accompanied with a partial decrease of serum bilirubin (289+/-35 vs. 239+/-17 micromol/l, P=0.013), whereas the effect of C. pasteurianum on bile pigment metabolism was negligible. Co-trimethoxazole therapy had no effect on serum and intestinal metabolism of bilirubin. CONCLUSIONS: Intestinal microflora greatly affects intravascular metabolism of bilirubin. Prolonged use of certain antibiotics in man may lead to an increase in serum bilirubin levels, while the enhancement of intestinal catabolism may have an opposite effect.

Effect of hyaluronidase and PEG chain conjugation on the biologic and antitumor activity of RNase A.

Subcutaneous application of bovine RNase A conjugated to HYase (bovine hyaluronidase), polyethylene glycol (PEG) and HYase+PEG resulted in a marked reduction of the width of the spermatogenic layers of the mouse testes. The number of sperms in caput epididymidis was significantly decreased in mice injected with conjugated RNase A. There was not any significant embryotoxic effect of free RNase A even conjugated with HYse, PEG and HYse+PEG. The immunogenicity, expressed in production of antibodies against free RNase A or conjugates with PEG, was very low. However, the immunogenic action of this enzyme conjugated only to HYase was much higher and produced the same immunogenicity as HYase itself. The immunogenic effect of RNase A+HYase conjugate decreased when PEG was joined to this conjugate. The inhibitory effect of RNase A conjugated to HYase, PEG and HYase+PEG on human ML-2 cells studied in vitro, was practically ineffective. On the other side, when RNase A conjugated to HYase or PEG was administered intraperitoneally into the mice bearing human melanoma, the antitumor effect was pronounced.