Histidine-Based Lipopeptides Enhance Cleavage of Nucleic Acids: Interactions with DNA and Hydrolytic Properties.

Interaction studies and cleavage activity experiments were carried out between plasmid DNA and a series of histidine-based lipopeptides. Specific fluorescent probes (ethidium bromide, Hoechst 33342, and pyrene) were used to monitor intercalation, minor groove binding, and self-assembly of lipopeptides, respectively. Association between DNA and lipopeptides was thus evidenced, highlighting the importance of both histidine and hydrophobic tail in the interaction process. DNA cleavage in the presence of lipopeptides was then detected by gel electrophoresis and quantified, showing the importance of histidine and the involvement of its side-chain imidazole in the hydrolysis mechanism. These systems could then be developed as synthetic nucleases while raising concern of introducing histidine in the design of lipopeptide-based transfection vectors.

Contact photoallergy to isothipendyl chlorhydrate.

Isothipendyl chlorhydrate is an azaphenothiazine, an active ingredient of an antipruriginous gel, Apaisyl gel® (Merck Médication Familiale, Dijon, France). Although Apaisyl gel is registered and used worldwide, we present the first case of contact photoallergy to isothipendyl chlorhydrate to our knowledge. The diagnosis suspected on the basis of a positive UVA photopatch test to chlorpromazine was confirmed by a strongly positive UVA Apaisyl gel photopatch test and our photophysical studies. This case confirms the need to keep the phenothiazines in the photopatch test standard series as a diagnostic marker of phenothiazine photoallergy.

Factors in the surgical management of primary eccrine porocarcinoma: prognostic histological factors can guide the surgical procedure.

BACKGROUND: Primary eccrine porocarcinoma is a rare cutaneous neoplasm and there is no consensus concerning its surgical management. OBJECTIVES: To conduct a retrospective analysis of all the cases of eccrine porocarcinoma seen in our hospital in order to identify prognostic factors. METHODS: Clinical data were retrieved from the patients' files. Each histological sample was reviewed by the pathologist and classified into three eccrine porocarcinoma subtypes: 'infiltrative', 'pushing' and 'pagetoid' eccrine porocarcinoma, according to Robson's criteria. Statistical methods were used to estimate the recurrence risk. RESULTS: Twenty-four patients were included. The surgical margins realized were ≥ 2 cm or < 2 cm in six and 13 patients, respectively, and slow Mohs procedure was performed in two patients. Ten, seven and two patients presented with 'infiltrative', 'pushing' or 'pagetoid' eccrine porocarcinoma, respectively. Eight patients (of 23) experienced recurrences (35%). The recurrence risk was linked to the histological subtype: two out of two pagetoid eccrine porocarcinoma, four out of 10 'infiltrative' and none of seven 'pushing' eccrine porocarcinoma recurred (P=0·026). The recurrence risk was not associated with epidermal growth factor receptor status, nor the margin realized. CONCLUSIONS: The recurrence risk of 'pushing' eccrine porocarcinoma is significantly lower than for 'infiltrative' eccrine porocarcinoma. Based on these considerations, a decisional algorithm might be proposed: first step, excisional biopsy of the primary eccrine porocarcinoma and identification of the histological subtype; second step, infiltrative or pagetoid porocarcinoma should be excised with an additional modified micrographic Mohs procedure, while no additional surgery is needed for 'pushing' eccrine porocarcinoma if the first excisional biopsy is complete. Although demonstrated in a limited case series, the histological subtype might guide the surgical management of primary eccrine porocarcinoma.

[Two cases of pulmonary melanoma metastasis treated with radiofrequency: An alternative to surgical excision]. / Deux cas de métastases pulmonaires de mélanome traitées par radiofréquence : une alternative à la chirurgie.

BACKGROUND: based on consensual recommendations, surgery remains the standard treatment for curable lung metastases. In this setting, radiofrequency ablation of limited metastases has been described in numerous studies in recent years. We report herein two patients presenting with low-burden lung metastases from malignant melanoma treated by radiofrequency. PATIENTS AND METHODS: two patients presented with one to two limited burden lung metastases from malignant melanoma, respectively. Both patients received neoadjuvant chemotherapy leading to disease stabilization, after which the lung metastases were treated by radiofrequency. Both patients had complete remission following radiofrequency, with 12 and 21 months follow-up respectively. DISCUSSION: surgical treatment of solitary or scant pulmonary metastases from melanoma has proved its efficacy with a gain in overall and disease-free survival. Nevertheless, this treatment cannot be proposed in patients with contraindications for anaesthesia or compromised pulmonary function. In this population, radiofrequency ablation appears to offer a potentially valuable alternative to surgery. Additionally, the related morbidity and duration of hospitalisation associated with radiofrequency seemed to be improved. As seen in these two cases, the efficacy and increased survival achieved with radiofrequency appears comparable to those obtained through surgery.

Neoadjuvant chemotherapy: a new criterion for selection of candidate patients for surgery of low tumour burden metastases from malignant melanoma?

BACKGROUND: Surgery of limited metastatic lesions from malignant melanoma can achieve long-term remission and better survival than chemotherapy. Existing criteria for selection of candidate patients for this surgery do not seem sufficient to avoid useless excisions. OBJECTIVES: To test use of neoadjuvant chemotherapy as a new criterion in this setting. METHODS: All patients who underwent thoracic surgery for one or two lung metastases from melanoma during 1999-2007 were included in the study. Demographic and medical data were collected and analysed. Several possible prognostic factors were evaluated based on the overall survival curves. RESULTS: Thirteen patients were included in this retrospective study. All but two patients had no evidence of disease after surgery. Ten patients received neoadjuvant chemotherapy. Six responded (absence of progression) and four had progressive disease. Response to chemotherapy and no evidence of disease after surgery were predictive of long-term survival. CONCLUSIONS: Neoadjuvant chemotherapy can be considered as a new criterion for better selection of candidate patients for lung metastasis surgical resection. This would also avoid useless surgical procedures in rapidly progressive disease and give information on the chemosensibility of the metastatic disease. This study needs further confirmation, particularly with chemotherapy regimens that have demonstrated better objective responses.

Photochemical and photophysical properties of indoprofen.

The photophysical properties and photochemistry of indoprofen (INP) have been investigated. Absorption and emission spectroscopies in phosphate buffer, ethanol and ether show that INP photophysics is dominated by a singlet-singlet transition of pipi* character. INP fluoresces at room temperature, with a quantum yield approximately 0.04. Flash photolysis experiments together with the lack of phosphorescence at room temperature point to a very weak intersystem crossing. The photoreactivity of INP is centered on the propionic acid chain and gives rise to photoproducts similar to those obtained with other arylpropionic acids (ethyl, hydroxyethyl and acetyl derivatives). Thus, irradiation of INP in aqueous buffer results in photodecarboxylation and leads mainly to oxidative compounds whose proportions increase with increasing oxygen concentration. These data suggest a photoreactivity occurring from the excited singlet state.

Comparison of DNA damage photoinduced by ketoprofen, fenofibric acid and benzophenone via electron and energy transfer.

Ketoprofen (KP) and fenofibrate, respectively, anti-inflammatory and hypolipidemiant agents, promote anormal photosensitivity in patients and may induce photoallergic cross-reactions correlated to their benzophenone-like structure. Here, their ability to photosensitize the degradation of biological targets was particularly investigated in DNA. The photosensitization of DNA damage by KP and fenofibric acid (FB), the main metabolite of fenofibrate, and their parent compound, benzophenone (BZ), was examined on a 32P-end-labeled synthetic oligonucleotide in phosphate-buffered solution using gel sequencing experiments. Upon irradiation at lambda > 320 nm, piperidine-sensitive lesions were induced in single-stranded oligonucleotides by KP, FB and BZ at all G sites to the same extent. This pattern of damage, enhanced in D2O is characteristic of a Type-II mechanism. Spin trapping experiments using 2,2,6,6-tetramethyl-4-piperidone have confirmed the production of singlet oxygen during drug photolysis. On double-stranded oligonucleotides, highly specific DNA break occurred selectively at 5'-G of a 5'-GG-3' sequence, after alkali treatment. Prolonged irradiation led to the degradation of all G residues, with efficiency decreasing in the order 5'-GG > 5'-GA > 5'-GC > 5'-GT, in good agreement with the calculated lowest ionization potentials of stacked nucleobase models supporting the assumption of a Type-I mechanism involving electron transfer, also observed to a lesser extent with adenine. Cytosine sites were also affected but the action of mannitol which selectively inhibited cytosine lesions suggests, in this case, the involvement of hydroxyl radical, also detected by electronic paramagnetic resonance using 5,5-dimethyl-1-pyrrolidine-1-oxide as spin trap. On a double-stranded 32P-end-labeled 25-mer oligonucleotide containing TT and TTT sequences, the three compounds were found to photosensitize by triplet-triplet energy transfer the formation of cyclobutane thymine dimers detected using T4 endonuclease V.

Photosensitivity to ketoprofen: mechanisms and pharmacoepidemiological data.

The topical use of nonsteroidal anti-inflammatory drugs (NSAIDs), widely used for moderate acute and chronic painful conditions, is one of several strategies used to improve the tolerability profile of NSAIDs, particularly with regard to gastric and renal adverse effects. However, topical NSAIDs can induce photosensitivity. Among the different NSAIDs used topically, ketoprofen has often been implicated in photosensitivity reactions. Photosensitivity includes both phototoxic and photoallergic reactions. Phototoxicity can be studied in the cell system and on biological targets such as cellular membranes or DNA. In hepatocyte cultures, data suggest that radical intermediates play a role in ketoprofen-photosensitised damage by cell membrane lysis. Photosensitised lysis of red blood cells has been employed as an indicator of membrane damage. Ketoprofen irradiation promotes the photolysis of erythrocyte suspensions. The drug is able to induce photoperoxidation of linoleic acid in the photo-induced lipid peroxidation process. The results obtained from the addition of radical scavengers suggest the involvement of free radicals in these processes. Ketoprofen may induce DNA damage in vitro upon irradiation. DNA, in the presence of ketoprofen, undergoes single strand breaks involving hydroxyl radicals as evidenced by the use of scavengers. Simultaneously with single strand breaks, pyrimidine dimers are formed by an energy transfer mechanism. The oxygen-dependence of both processes suggest competition between a radical process leading to DNA cleavage and a poorly efficient energy transfer between ketoprofen and pyrimidines at the origin of the dimerisation process. Photoallergy is due to a cell-mediated hypersensitivity response involving immunological reactions. Therefore, it only occurs in previously sensitised individuals and requires a latency period of sensitisation. Among NSAIDs, ketoprofen is the main drug involved in this photoallergic contact dermatitis. Cross-sensitivity reactions with other arylpropionic acid derivatives, such tiaprofenic acid, fenofibrate or oxybenzone-harbouring benzoyl ketone or benzophenone may also occur. Finally, the higher frequency of such adverse reactions with ketoprofen could be accounted for by its chemical structure and the variety of chemical reactions that give rise to phototoxic effects. The widespread and repeated use of these agents may lead to sensitisation, incurring a greater risk of systemic allergic reactions with oral NSAIDs or other drugs recognised to induce cross-reactions. Physicians and pharmacists should advise patients and inform them of the risks of topical NSAIDs which are often dispensed as over the counter drugs.

Comparison of the DNA damage photoinduced by fenofibrate and ketoprofen, two phototoxic drugs of parent structure.

Fenofibrate and ketoprofen (KP) are two drugs of similar structure derived from that of benzophenone. Both are photoallergic and promote cross reactions in patients. However, the cutaneous photosensitizing properties of KP also include phototoxic effects and are more frequently mentioned. To account for this difference in their in vivo properties, their in vitro photosensitizing properties on DNA were compared. First, it was shown that under irradiation at 313 nm, fenofibric acid (FB), the main metabolite of fenofibrate, photosensitized DNA cleavage by a radical mechanism similar to that proposed for KP but with a 50 times lower efficiency. Furthermore, FB did not photosensitize the formation of pyrimidine dimers into DNA in contrast to KP, which did promote this type of DNA damage. Their difference in efficiency as DNA breakers was compared to their relative photochemical reactivity and the quantum yield of FB photolysis was found to be eightfold lower than that of KP. The reactivity of these drugs cannot explain alone the difference in their photosensitizing properties. Other factors such as the magnitude of the ionic character of the photodecarboxylation pathway of these benzophenone-like drugs are considered in the discussion.

Nonsteroidal antiinflammatory drug-photosensitized formation of pyrimidine dimer in DNA.

Phototoxic nonsteroidal antiinflammatory drugs (NSAIDs) may induce DNA damage in vitro upon irradiation. In this study, we investigated the ability of ketoprofen (KP), tiaprofenic acid (Tia), naproxen (NP) and indomethacin (IND) to photosensitize the formation of pyrimidine dimers and single strand breaks. Both kinds of damage were sought by analyzing DNA-drug mixtures irradiated at 313 nm by agarose gel electrophoresis. The formation of pyrimidine dimers was evidenced by using endonuclease V from bacteriophage T4 and compared to that induced by acetophenone, a well-known photosensitizer of thymine dimerization. Upon irradiation of DNA alone, pyrimidine dimers were observed while single strand breaks were not detected under our conditions. DNA, in the presence of NSAIDs, undergoes single strand breaks, the quantum yield of the DNA cleavage so induced (phiC) varying from 5 x 10(-4) for KP to 10(-5) for IND. The formation of dimers was only increased in the presence of KP or Tia. The quantum yields of pyrimidine dimers formed by photosensitization (phiD) were 2 x 10(-4) for KP and 10(-5) for Tia, respectively. The oxygen and concentration dependence of both processes was analyzed in the case of KP. In aerated solution, KP-photoinduced cleavage of DNA was predominant on the photodimerization process of pyrimidines, whereas in deaerated solution the cleavage was decreased and the dimerization increased. These results reflect competition between a radical process leading to DNA cleavage and a poorly efficient energy transfer between the drug and the pyrimidines at the origin of the dimerization process.

Structural determination of a glucuronide conjugate of flucytosine in humans.

The structure of a glucuronide metabolite of flucytosine (FC; 5-fluorocytosine), found in the urine of all patients treated with this antifungal drug, was determined. This compound is the O2-beta-glucuronide of FC. Its structure was established after isolation from urine and by comparing its spectroscopic characteristics with those of three FC glucuronides previously synthesized. This study is the first report of the identification of a glucuronide of a fluoropyrimidine drug in humans.

A new approach to the study of ifosfamide metabolism by the analysis of human body fluids with 31P nuclear magnetic resonance spectroscopy.

[31P] nuclear magnetic resonance spectroscopy was used to analyze body fluids from patients treated with ifosfamide (IF). This technique, which requires no labeled drug, allows a direct study of the biological sample with no need for extraction or derivatization and a simultaneous detection and quantification of all the different phosphorated metabolites in a single analysis. In urine, isophosphoramide mustard was detected in addition to the already known human urinary compounds [i.e., unchanged IF, carboxyifosfamide, 2-dechloroethylifosfamide, 3-dechloroethylifosfamide, ketoifosfamide]. 2,3-Didechloroethylifosfamide itself was not found, but two of its degradation compounds were detected, thus showing a minor route of didechloroethylation of IF in humans. Several other signals corresponding to unknown metabolites or to degradation compounds of IF metabolites were observed. None of them corresponded to IF-activated metabolites (4-hydroxyifosfamide, aldoifosfamide) or to conjugates of IF or its metabolites with mesna. The urinary excretion of IF and metabolites over 24 h amounted to 39 to 50% of the injected dose. Unmetabolized IF was the major compound in 0- to 8-h and 8- to 16-h fractions. 2-Dechloroethylifosfamide and 3-dechloroethylifosfamide were the main metabolites detected in each 8-h fraction. The two unknown compounds at 19.16 ppm and 16.06 ppm represented a non-negligible fraction of the excretion, above that of carboxyifosfamide. Only unchanged IF could be detected in plasma samples. Unmetabolized IF and 3-dechloroethylifosfamide were found in a cerebrospinal fluid sample. Neither IF nor IF metabolites could be observed in the corresponding plasma sample. This indicates a long persistence of these compounds in cerebrospinal fluid.

Study of the metabolism of flucytosine in Aspergillus species by 19F nuclear magnetic resonance spectroscopy.

The metabolism of flucytosine (5FC) in two Aspergillus species (Aspergillus fumigatus and A. niger) was investigated by 19F nuclear magnetic resonance spectroscopy. In intact mycelia, 5FC was found to be deaminated to 5-fluorouracil and then transformed into fluoronucleotides; the catabolite alpha-fluoro-beta-alanine was also detected in A. fumigatus. Neither 5-fluoroorotic acid nor 5-fluoro-2'-deoxyuridine-5'-monophosphate was detected in perchloric acid extracts after any incubation with 5FC. 5FC, 5-fluorouracil, and the classical fluoronucleotides 5-fluorouridine-5'-mono-, di-, and triphosphates were identified in the acid-soluble pool. Two hydrolysis products of 5-fluorouracil incorporated into RNA, 5-fluorouridine-2'-monophosphate and 5-fluorouridine-3'-monophosphate, were found in the acid-insoluble pool. No significant differences in the metabolic transformation of 5FC were noted in the two species of Aspergillus. The main pathway of 5FC metabolism in the two species of Aspergillus studied is thus the biotransformation into ribofluoronucleotides and the subsequent incorporation of 5-fluorouridine-5'-triphosphate into RNA.