Preparation, characterization, therapeutic efficacy and toxicity of liposomes, containing the antitumour drug cis-dichlorodiamineplatinum(II)

Cis-dichlorodiamineplatinum(II) (cis-DDP) was encapsulated in reverse phase evaporation vesicles (REV, 0.6 mg cis-DDP/ml lipid solution) and multilayered liposomes (MLV, 0.3 mg cis-DDP/ml lipid solution) with different cholesterol content. The identity of cis-DDP in free and encapsulated form was checked by various techniques. Particle size, homogeneity of liposomes and distribution of cis-DDP in REVs were shown by electron microscopy. The examination of entrapped cis-DDP in REVs relating to buffer and serum stability, in vitro and in vivo antitumour activity and nephrotoxicity proved that all points are strongly influenced by the cholesterol (CH) content. Enclosed cis-DDP in phosphatidyl (PC)-REV has the same, and in PC: CH-REV, a lower effect in vitro and in vivo compared to treatment with the free drug. Irrespective of the application of tumour cells and substance (i.v., i.p.) in optimal therapeutic doses, an equal increase in life-span (ILS) was registered with the free drug and with PC-cis-DDP-REV, while cis-DDP in PC: CH-REV had a significantly reduced effectiveness. Liposomal encapsulation of cis-DDP also influenced body weight change and leucocyte counts. The blood urea nitrogen (BUN) level, as an indicator of renal toxicity, was only moderately increased after very high doses of cis-DDP (24 mg/kg) in PC: CH-REV.     

In vivo gene transfer: focus on the kidney

Renal gene transfer techniques are being developed as a novelexperimental approach to understand the pathogenesis of renaldisease and to potentially develop new therapeutic tools. Wereview the currently available technology to introduce foreigngenetic material into renal tissue, i.e., retroviral, adenoviral,and liposomal transfer systems with their respective advantagesand caveats. Today, the transfer efficiency of these methodsappears to be sufficiently high to study the effects of transducedgenes on renal function and morphology in rat kidney. This willallow (i) the elucidation of the function of genes on the courseof renal disease in experimental animal models and (ii) themodulation of local expression of endogenous genes which presumptivelycontribute to renal pathology in these models. One strategyto accomplish this aim is the use of recombinant DNA technologyto design antisense DNA constructs or oligonucleotides, whichinterfere with the renal expression of target genes. We willalso discuss some of the shortcomings of the currently usedtechniques with respect to potential therapeutic use of genetransfer systems and gene modulation.     

Pharmaceutical Evaluation of Gas-Filled Microparticles as Gene Delivery System

Purpose. To produce and characterize a nonviral ultrasound-controlled release system of plasmid DNA (pDNA) encapsulated in gas-filled poly(D,L-lactide-co-glycolide) microparticles (PLGA-MPs). Methods. Different cationic polymers were used to form pDNA/polymer complexes to enhance the stability of pDNA during microparticle preparation. The physico-acoustical properties of the microparticles, particle size, pDNA integrity, encapsulation efficiency and pDNA release behavior were studied in vitro. Results. The microparticles had an average particle size of around 5 m. More than 50% of all microparticles contained a gas core, and when exposed to pulsed ultrasound as used for color Doppler imaging create a signal that yields typical color patterns (stimulated acoustic emission) as a result of the ultrasound-induced destruction of the microparticles. Thirty percent of the pDNA used was successfully encapsulated and approximately 10% of the encapsulated pDNA was released by ultrasound within 10 min. Conclusions. Plasmid DNA can be encapsulated in biodegradable gas-filled PLGA-MPs without hints for a structural disintegration. A pDNA release by ultrasound-induced microparticle-destruction could be shown in vitro.     

Oxidation of anthracyclines by peroxidase metabolites of salicylic Acid

Oxidation of anthracyclines leads to their degradation and inactivation. This process is carried out by peroxidases in the presence of a catalytic cofactor, a good peroxidase substrate. Here, we investigated the effect of salicylic acid, a commonly used anti-inflammatory and analgesic agent, on the peroxidative metabolism of anthracyclines. We report that at pharmacologically relevant concentrations, salicylic acid stimulates oxidation of daunorubicin and doxorubicin by myeloperoxidase and lactoperoxidase systems and that efficacy of the process increases markedly on changing the pH from 7 to 5. This pH dependence is positively correlated with the ease with which salicylic acid itself undergoes metabolic oxidation and involves the neutral form of the acid (pKa = 2.98). When salicylic acid reacted with a peroxidase and H2O2 at acid pH (anthracyclines omitted), a new metabolite with absorption maximum at 412 nm was formed. This metabolite reacted with anthracyclines causing their oxidation. It was tentatively assigned to biphenyl quinone, formed by oxidation of biphenol produced by dimerization of salicylic acid-derived phenoxyl radicals. The formation of this product was inhibited in a concentration-dependent manner by the anthracyclines, suggesting their scavenging of the salicylate phenoxyl radicals. Altogether, this study demonstrates that oxidation of anthracyclines is mediated by peroxidase metabolites of salicylic acid, such as phenoxyl radicals and the biphenol quinone. Given that cancer patients undergoing anthracycline chemotherapy may be administered salicylic acid-based drugs to control pain and fever, our results suggest that liberated salicylic acid could interfere with anticancer and/or cardiotoxic actions of the anthracyclines.     

Mechanism of action of bisphosphonates

In recent years, substantial progress has been made in understanding the mechanism for bisphosphonate suppression of bone turnover. Bisphosphonates can now be distinguished based on their molecular and cellular mechanisms of action. Simple bisphosphonates such as clodronate and etidronate inhibit bone resorption through induction of osteoclast apoptosis. Clodronate, and perhaps etidronate, triggers apoptosis by generating a toxic analog of adenosine triphosphate, which then targets the mitochondria, the energy center within the cell. For nitrogen-containing bisphosphonates, the direct intracellular target is the enzyme farnesyl diphosphate synthase in the cholesterol biosynthetic pathway. Its inhibition suppresses a process called protein geranylgeranylation, which is essential for the basic cellular processes required for osteoclastic bone resorption. Although nitrogen-containing bisphosphonates can induce osteoclast apoptosis, this is not necessary for their inhibition of bone resorption.     

Imaging-guided convection-enhanced delivery and gene therapy of glioblastoma

In a prospective phase I/II clinical study, we treated eight patients suffering from recurrent glioblastoma multiform with stereotactically guided intratumoral convection-enhanced delivery of an HSV-1-tk gene-bearing liposomal vector and systemic ganciclovir. Noninvasive identification of target tissue together with assessment of vector-distribution volume and the effects of gene therapy were achieved using magnetic resonance imaging and positron emission tomography. The treatment was tolerated well without major side effects. In two of eight patients, we observed a greater than 50% reduction of tumor volume and in six of eight patients focal treatment effects. Intracerebral infusion of contrast medium before vector application displayed substantial inhomogeneity of tissue staining indicating the need of test infusions to monitor the mechanical distribution of vectors. Visualization of therapeutic effects on tumor metabolism and documentation of gene expression using positron emission tomography indicated that molecular imaging technology appears to be essential for the further development of biological treatment strategies.     

Significance of genetic polymorphisms in glutathione S-transferase multigene family and lung cancer risk

A vast number of studies are focused on investigating genetic polymorphism in order to estimate genetic contribution to the development of cancer. Possible cancer susceptibility genes have been sought among oncogenes, tumor suppressor genes, DNA repair genes and genes encoding phase I and phase II enzymes. Large individual differences in the biotransformation of xenobiotics have been explained on the basis of genetic polymorphisms in some detoxifying enzymes, regardless of environmental and occupational exposure. Among these enzymes, glutathione S-transferases (GST) constitute a large multigene family of phase II enzymes involved in detoxification of potentially genotoxic chemicals. Five genetic polymorphisms of GST have been well documented. Total or partial deletions and (or) single nucleotide polymorphisms in alleles encoding GSTM1, GSTM3, GSTPI, GSTT1, GSTZ1 are associated with reduction of enzymatic activity toward several substrates of different GST isoenzymes. In addition, molecular epidemiology studies indicate that a single genetic polymorphism of glutathione S-transferase appears to be a moderate lung cancer risk factor. However, the risk is higher when interactions with more GST polymorphisms and other risk factors (e.g. cigarette smoking) occur. Individuals with decreased rate of detoxification, with "high risk" glutathione S-transferase genotypes have a slightly higher level of carcinogen-DNA adducts and more cytogenetic damages.