Expression of DNA damage response proteins in cervical cancer patients treated with radical chemoradiotherapy.

OBJECTIVE: The management of locally advanced cervical cancer has improved significantly with the advent of cisplatin-based chemoradiotherapy (CRT) as the primary treatment regimen. Nevertheless, a significant proportion of patients fail to respond or relapse on this treatment and have a very poor prognosis. Our goal was to determine the prognostic value of a panel of proteins involved in detection and repair of DNA damage. METHODS: We performed fluorescence immunohistochemistry, and used software analysis to assess expression of DNA damage response proteins ATM, DNA-PKcs, PARP-1, Ku70 and Ku86 in 117 pre-treatment specimens from patients with locally advanced cervical cancer. We compared expression to clinicopathologic correlates to determine prognostic significance. RESULTS: Five-year progression-free survival was significantly lower in the low expressors than in high expressors of ATM (35% vs. 58%, p=0.044) and PARP-1 (24% vs. 61%, p=0.003), and showed a trend to significance for DNA-PKcs (30% vs. 60%, p=0.050). Low expression of the same proteins also correlated significantly with lower overall survival. In multivariable analysis, adjusted for FIGO stage and tumor size, low ATM and PARP-1 expression was significantly associated with both poorer progression-free and overall survival. Pairwise analyses indicated that expression levels of these proteins were correlated. CONCLUSIONS: Expression of DNA damage response proteins in cervical cancer is associated with outcome in patients treated with CRT. Immunohistochemical analysis of these proteins may be useful in guiding treatment decisions in such patients.

The prognostic impact of a combined carbonic anhydrase IX and Ki67 signature in oral squamous cell carcinoma.

BACKGROUND: Tumour hypoxia is associated with impaired apoptosis, resistance to therapy and poor prognosis. We previously reported that high stromal expression of the endogenous marker of hypoxia, carbonic anhydrase IX (CAIX), is associated with significantly reduced survival in oral squamous cell carcinoma (OSCC). In addition to hypoxia, CAIX expression is regulated by proliferation-associated signalling. We hypothesised that incorporating Ki67, a proliferation marker, into our existing CAIX-based stratification of OSCC would identify patients with the least favourable prognosis. METHODS: Surgically resected tumours from 60 OSCC patients were analysed for CAIX, Ki67 and BAX expression using fluorescence immunohistochemistry and automated quantitative analysis (AQUA). RESULTS: In patients expressing high stromal CAIX (sCAIX), stratification by tumour Ki67 expression revealed significantly distinct survival outcomes (P=0.005). In our OSCC cohort, below-median Ki67 and top-quartile sCAIX expression (Ki67(lo)sCAIX(hi)) were associated with significantly worse disease-specific survival in univariate (HR 7.2 (2.5-20.4), P=0.001) and multivariate (HR 4.2 (1.4-12.8), P=0.011) analyses. Hypoxia is associated with decreased BAX expression; the Ki67(lo)sCAIX(hi) group was more strongly associated with low BAX expression than high sCAIX alone. CONCLUSION: These data suggest that combined analysis of tumour Ki67 and sCAIX expression may provide a more clinically relevant assessment of tumour hypoxia in OSCC.

The penetration of topically applied ointment containing hyaluronic acid in rabbit tissues.

The properties of hyaluronic acid used for treatment of acute and chronic joint disease are known for many years and this compound is widely used both in humans and animals. To obtain a therapeutic effect of a certain drug, the appropriate concentration in the target organ or tissue is important. The application of labeled compounds is one of the frequently applied techniques to estimate drug penetration into the skin and other body tissues or organs. The aim of the study was to evaluate the penetration of hyaluronic acid labeled with I-131 through the skin and its distribution within the knee joint and other internal organs in rabbits after a topical application of an ointment containing hyaluronic acid. The experiment was performed on 22 albino rabbits divided into control and examined groups. Fifteen rabbits were exposed to the multicomponent ointment containing hyaluronic acid labeled with I-131. Time of exposure was 48 hours. Hyaluronate penetrated to a high degree into the examined tissues. No significant differences in terms of leg tissue activity were observed between a leg tissue exposed to labeled ointment and that unexposed, suggesting that after topical administration, the active component of the ointment is delivered to the joint via the blood stream. Hyaluronate applied topically penetrates through the skin into the rabbit tissues and organs and into the joint fluid of both legs (exposed and not exposed). This route of administration seems to be useful for this drug delivery and allows to avoid unnecessary side effects.

Evaluation of skin penetration of topically applied drugs in humans by cutaneous microdialysis: acyclovir vs. salicylic acid.

BACKGROUND AND OBJECTIVE: Cutaneous drug application is used for both local drug therapy and systemic treatment. For both types of treatment, the drug concentration profile in, and transport across, the skin is important. To evaluate skin penetration of topically-applied drugs we recently used cutaneous microdialysis. The aim of this study was the use of this method for studying acyclovir and salicylic acid. METHOD: Five per cent acyclovir cream was applied on intact and tape-stripped skin of healthy volunteers and 5% salicylic acid ointment-onto intact skin of other volunteers. Microdialysis probes with 2 kDa molecular weight cut-off were inserted intradermally and were perfused with Ringer solution. Drug concentrations were measured by high-performance liquid chromatography. RESULTS: Following topical application of 5% acyclovir cream onto intact skin of eight healthy volunteers, no drug was determinable in the skin (cutaneous microdialysate) in any of the subjects studied. After partial removal of the stratum corneum the penetration of this drug into skin increased markedly. The mean maximum skin concentration was about 2 x 5 micromol/L after 2 x 4 +/- 0 x 7 h. Topically applied salicylic acid penetrated intact skin with a maximum concentration in the cutaneous microdialysate of 7 x 57 +/- 3 x 90 micromol/L after 5 x 3 +/- 0 x 4 h. CONCLUSION: Cutaneous microdialysis is a valuable method for estimating skin concentration of topically-applied drug. It allows evaluation after application to a small skin area, of about 2 cm(2), thereby reducing the risk of systemic toxicity. The method may be helpful for evaluating the influence of skin condition on the transport process.

Penetration of ciprofloxacin and its desethylenemetabolite into skin in humans after a single oral dose of the parent drug assessed by cutaneous microdialysis.

OBJECTIVE: To measure the concentration of ciprofloxacin and its desethylenemetabolite in plasma and cutaneous microdialysates and to compare ciprofloxacin penetration into cutaneous microdialysates against theoretically predicted penetration in a peripheral compartment. METHOD: A single oral dose of 0.5 g of the parent drug was administered to 10 healthy male volunteers. Microdialysis probes with 2 kDa molecular weight cut-off were inserted intradermally and were perfused with Ringer solution up to 8 h after drug ingestion. Drug and metabolite concentrations were measured by high performance liquid chromatography. RESULTS: Mean maximum concentrations of ciprofloxacin in plasma, cutaneous microdialysates and theoretical peripheral compartment were 7.01+/-1.69, 2.95+/-0.64 and 3.37+/-0.60 micromol/L, respectively, and were achieved after about 2.0+/-0.6, 2.4+/-0.9 and 4.8+/-0.9 h. The extent of penetration into cutaneous microdialysates and theoretical peripheral compartment relative to plasma were 0.550+/-0.150 and 0.788+/-0.131, respectively, and differed significantly. Similarly, time to maximum concentration as well as area under the concentration-time curve in these compartments also differed significantly unlike the maximum concentration. CONCLUSION: Microdialysis permits the evaluation of the penetration of drug and its metabolites into target tissues. Such evaluation is helpful to optimize treatment strategies. After a single 0.5 g oral dose, ciprofloxacin penetrated into skin and achieved concentrations above the minimum inhibitory concentrations for susceptible pathogens, recommended by the National Committee for Clinical Laboratory Standards (NCCLS).

Bullous systemic lupus erythematosus with antiphospholipid syndrome.

We report the case of a 44-year-old male with a 10-year history of manifestations of the rare form of bullous systemic lupus erythematosus (SLE) with coexisting antiphospholipid syndrome (APS) that remained undiagnosed until thrombotic-embolic episodes appeared and high titres of anticardiolipin (ACL) antibodies were detected. The patient fulfilled the criteria for SLE and the atypical cutaneous manifestations together with histopathological changes and a favourable response to sulphones were the grounds for the diagnosis of the bullous variety of SLE. Treatment with prednisolone, acenocoumarol and dapsone resulted in marked clinical improvement, reduction in antinuclear antibodies (ANAs) and normalization of ACL antibody titres.

Application of cutaneous microdialysis to evaluate metronidazole and its main metabolite concentrations in the skin after a single oral dose.

OBJECTIVE: To measure the concentration of metronidazole and its hydroxymetabolite in plasma and cutaneous microdialysates and to compare metronidazole penetration into cutaneous microdialysates against theoretical predicted penetration in a peripheral compartment. METHOD: A single oral dose of 2 g of the parent drug was administered to 10 healthy male volunteers. Microdialysis probes with 2 kDa molecular weight cut-off were inserted intradermally and were perfused with Ringer solution up to 8 h after drug ingestion. Drug and metabolite concentrations were measured by high performance liquid chromatography. RESULTS: Mean maximum concentration in plasma, cutaneous microdialysates and theoretical peripheral compartment were 214 +/- 49, 151 +/- 52 and 146 +/- 38 micromol/L, respectively, and were achieved after about 2.1 +/- 0.8, 2.8 +/- 1.0 and 6.0 +/- 2.9 h. The extent of penetration into cutaneous microdialysates and theoretical peripheral compartment relative to plasma were 0.672 +/- 0.196 and 0.675 +/-0.207, respectively, and did not differ significantly. Moreover, maximum concentration as well as area under concentration-time curve in these compartments also did not differ significantly. CONCLUSION: Use of cutaneous microdialysis technique permits the characterization of true systemic drug disposition, for optimizing drug treatment strategies.

Antibody recognition of a conformational epitope in a peptide antigen: Fv-peptide complex of an antibody fragment specific for the mutant EGF receptor, EGFRvIII.

Epitope mapping studies and the determination of the structure to 1.8 A resolution have been carried out for the antigen-binding fragment MR1 in complex with peptide antigen. MR1 is specific for the novel fusion junction of the mutant epidermal growth factor receptor EGFRvIII and has been reported to have a high degree of specificity for the mutant EGFRvIII over the wild-type EGF receptor. The structure of the complex shows that the peptide antigen residue side-chains found by epitope mapping studies to be critical for recognition are accommodated in pockets on the surface of the Fv. However, the most distinctive portion of the peptide antigen, the novel fusion glycine residue, makes no contact to the Fv and does not contribute directly to the epitope. The specificity of MR1 lies in the ability of this glycine residue to assume the restricted conformation needed to form a type II' beta-hairpin turn more easily, and demonstrates that a peptide antigen can be used to generate a conformational epitope.

Plasma and skin blister fluid concentrations of metronidazole and its hydroxy metabolite after oral administration.

Plasma and cantharidin-induced skin blister fluid concentrations of metronidazole and its main metabolite-hydroxymetronidazole were determined after a single and multiple oral doses. Metronidazole is nitroimidazole compound applied for the treatment of Protozoa infections. It is also active against anaerobic bacteria. The maximum concentrations of unchanged drug and its metabolite following a single oral dose of 2 g were observed after 1 +/- 1 and 11 +/- 2 h in plasma, whereas in blister fluid after 6 +/- 2 and 16 +/- 5 h, respectively. The average ratio of area under concentration time curve (AUC) in blister fluid to that of plasma was 1.02 +/- 0.12 for parent drug and 1.02 +/- 0.02 for the metabolite. After multiple doses of metronidazole (0.25 g every 8 h) the concentrations of unchanged drug in plasma and blister fluid, collected before the morning dose and 2 h after its administration, exceeded the minimal inhibitory concentrations for majority of susceptible pathogens. Hydroxymetronidazole concentrations in body fluids at steady-state amounted to about 30-50% of the parent drug and they could contribute to the overall activity against susceptible microorganisms since antibacterial activity of the metabolite is about 30-65% that of the metronidazole.

Penetration of tinidazole into skin blister fluid following its oral administration.

Plasma and skin blister fluid concentrations of tinidazole following a single oral dose of 2 g drug, and after multiple doses of 0.25 g every 12 h, were determined. Skin blisters were produced by direct application of 0.25% cantharidin ointment to the skin. The maximum concentration in plasma of about 36 mg.l-1 was observed after about 2 h, whereas in skin blister fluid the peak occurred after about 6 h and was 30 mg.l-1. The half-life in plasma was slightly shorter than in blister fluid at 17 and 19 h, respectively, but the difference was not significant. The penetration of tinidazole into cantharidin-induced skin blister fluid, defined according to Wise as the ratio of the AUCs in blister fluid and plasma was 1.00. During routine treatment with tinidazole (0.25 g every 12 h), the concentrations in plasma and blister fluid collected before and 3 h after the morning dose exceeded the minimal inhibitory concentrations for susceptible pathogens. The results provide a pharmacokinetic basis for the proven efficacy of tinidazole in the treatment of protozoal and anaerobic infections.

[Pharmacokinetics of sulphonamides administered in combination with trimethoprim]. / Farmakokinetyka sulfonamidów stosowanych lacznie z trimetoprimem.

Pharmacokinetic properties of sulfametrole, sulfamoxole, sulphamerazine, sulphadiazine, sulphamethoxazole and sulfamethopyrazine, i.e. the sulphonamides administered in combination with trimethoprim have been compared. From the pharmacokinetic point of view sulphadiazine seems to be the most optimal sulphonamide to apply jointly with trimethoprim. Some of long-acting sulphonamides like sulfamethopyrazine and sulfadimethoxine, used as components of above mentioned combinations, possess comparable clinical efficacy in spite of differences of their and trimethoprim half-lives.

O-dealkylation and acetylation of sulphamethomidine by the turtle Pseudemys scripta elegans.

The turtle Pseudemys scripta elegans acetylates and O-dealkylates sulphamethomidine. The yield of acetylation (3.1%) is about 0.7 times greater than the yield of O-dealkylation (4.3%).

Two-stage penetration of a single oral dose of sulphadimethoxine into skin blister fluid.

The time-dependent concentration curves of sulphadimethoxine in plasma and cantharidin-induced skin blister fluid have been evaluated following a single oral dose of 1 g. In contrast to other drugs, sulphadimethoxine exhibited two-stage penetration into the blister fluid, the second peak concentration being higher than the first. The maximum plasma concentration of 94.1 mg.l-1 was observed after 4 h, and in skin blister fluid the first peak of 25.6 mg.l-1 was found after 7 h, and the second of 58.0 mg.l-1 occurred after 30 h. The penetration of sulphadimethoxine into skin blister fluid, defined as the ratio of the AUC there to that in plasma was 0.748. The results suggest that sulphadimethoxine penetrates into skin blister fluid to a great extent from plasma and achieves concentrations exceeding the MIC for susceptible pathogens, but it requires a relatively long time to do so.

The comparison of some pharmacokinetic parameters of sulphadimethoxine estimated by high performance liquid chromatography and three spectrophotometric methods.

The influence of the method used for determination of drugs in biological fluids on the pharmacokinetic parameters of sulphadimethoxine was investigated in healthy adult human subjects. Sulphonamide concentrations were determined by four chemical methods: high performance liquid chromatography (HPLC) and three spectrophotometric techniques, i.e. Bratton-Marshall original method as well as Rieder's modification and author's modification of the Morris technique. The compatibility of pharmacokinetic parameter values calculated from these results was good, the correlation coefficients between HPLC and all spectrophotometric methods were high. It has also been shown that the phenotype of acetylation as well as moderate cigarette smoking which can induce some enzymes responsible for the formation of glucuronide conjugates, i.e. main metabolic pattern for sulphadimethoxine, does not affect the half-time of this drug.

Comparison of four methods for the determination of sulphonamide concentrations in human plasma.

Analysis of sulphadimethoxine in plasma from patients treated with this drug was performed with four chemical methods: Rieder's modification of the Bratton-Marshall technique and author's modification of the Morris technique, which are supposed to measure "potentially active fraction of sulphonamides", i.e., mainly unchanged sulphonamide; the Bratton-Marshall method, which enables the determination of "free sulphonamide", i.e., the sum of unchanged sulphonamide and glucuronide conjugate; and high performance liquid chromatography which made possible the determination of unchanged sulphadimethoxine. The spectrophotometric methods based upon Bratton-Marshall reaction gave very few higher results as compared with liquid chromatography. The differences between spectrophotometric and chromatographic methods were small; therefore, all four methods can be used for determination of sulphonamide concentrations in plasma during monitored treatment with these drugs.

Plasma and skin blister fluid concentrations of trimethoprim following its oral administration.

Plasma and skin blister fluid concentration-time curves following a single oral dose of trimethoprim have been evaluated. Skin blisters were produced by the cantharides technique, using patches with cantharidin ointment. Trimethoprim concentrations in plasma following multiple doses of 200 mg were also determined. The maximal concentration in plasma after a single oral dose of 400 mg trimethoprim was 3.95 +/- 1.08 mg/l, and it was observed after 2 h, whereas in skin blister fluid the level was 2.21 +/- 0.62 mg/l, and it was delayed for up to 6 h. This means that a certain time is required for drug transfer from the capillaries via the basal membrane into blister fluid. Penetration of the drug into blister fluid, defined as the ratio of the areas under the trimethoprim level time curve in skin blister fluid to that of plasma, was 0.826 +/- 0.096. The steady-state concentration of trimethoprim in plasma during routine treatment with 200-mg doses ranged between 2 and 3.5 mg/l.

Distribution of trimethoprim and sulphamethoxazole in blood during treatment with co-trimoxazole.

Changes in the distribution of sulphamethoxazole and trimethoprim in whole blood, plasma and erythrocytes at steady-state in patients treated with cotrimoxazole have been studied. Unlike sulphamethoxazole, trimethoprim was weakly bound to erythrocytes and was partially liberated when the erythrocytes were rinsed with isotonic saline. The maximal steady-state concentration of trimethoprim in whole blood was 3 mg/l, but calculated on the basis of the concentration determined in erythrocytes it was 1.8 mg/l. Erythrocytes may be of great significance in trimethoprim distribution as carriers of a readily liberated reservoir of the drug. Acetylated sulphamethoxazole derivatives occurred in a higher percentage in erythrocytes at the maximal steady-state concentration (9.9%) than at the level (7.3%), which may help in interpreting the behaviour of this metabolite in other cells in the organism.