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Ian S Haworth - One of the best experts on this subject based on the ideXlab platform.
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electrophoretic mobility of duplex dna cross linked by Mechlorethamine at a cytosine cytosine mismatch pair
Electrophoresis, 2013Co-Authors: Rebecca M Romero, Pornchai Rojsittisak, Ian S HaworthAbstract:: The common nitrogen mustard, Mechlorethamine, can form a covalent cross-link between the two bases of a cytosine-cytosine mismatch pair within a DNA duplex. The cross-linked species can be readily separated from DNA monoadducts and unreacted strands using denaturing polyacrylamide gel electrophoresis. Here, using DNA 19 mer duplexes that are Mechlorethamine cross-linked at a C(4)-C(35), C(7)-C(32), C(10)-C(29), or C(13)-C(26) mismatch pair, we show that the denaturing polyacrylamide gel electrophoresis mobility of the cross-linked species is particularly sensitive to the proximity of the C-C cross-link to the duplex end. Species that are cross-linked at a C(4)-C(35) mismatch have greater mobilities than those cross-linked at C(7)-C(32) or C(13)-C(26), and the species with a central C(10)-C(29) cross-link have the lowest mobility. The mobility is also dependent on the proximity of the cross-link to a 5'-(32)P-phosphate or a 5'-fluorescein label. We interpret these results in terms of the conformational properties of the cross-linked species in the denaturing gel. The results are consistent with the retention of partial duplex character at the end proximal to the cross-link, with an influence on the mobility of the GC/AT ratio proximal to the cross-link and at the duplex end, and a small but discernible effect of the label.
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hplc uv maldi tof ms and esi ms ms analysis of the Mechlorethamine dna crosslink at a cytosine cytosine mismatch pair
PLOS ONE, 2011Co-Authors: Pornchai Rojsitthisak, Rebecca M Romero, Nutthapon Jongaroonngamsang, Ian S HaworthAbstract:Background Mechlorethamine [ClCH2CH2N(CH3)CH2CH2Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C) mismatch pair using gel electrophoresis. However, the atomic connectivity of this unusual crosslink is unknown.
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extrahelical cytosine bases in dna duplexes containing d gcc n d gcc n repeats detection by a Mechlorethamine crosslinking reaction
Nucleic Acids Research, 2001Co-Authors: Pornchai Rojsitthisak, Rebecca M Romero, Ian S HaworthAbstract:The cytosine–cytosine (C–C) pair is one of the least stable DNA mismatch pairs. The bases of the C–C mismatch are only weakly hydrogen bonded, and previous work has shown that, in certain sequence contexts, they can become unstacked from the core helix, and adopt an ‘extrahelical’ location. Here, using DNA duplexes with d[GCC]n·d[GCC]n fragments containing C–C mismatches in a 1,4 bp relationship, we show that cytosine bases of different formal mismatch pairs can be crosslinked by Mechlorethamine. For example, in the duplex d[CTCTCGCCGCCGCCGTATC]·d[GATACGCCGCCGCCGAGAG], where underlined cytosine bases are present as the formal C–C mismatch pairs C7–C32, C10–C29 and C13–C26, we show that two Mechlorethamine crosslinks form between C13 and C29 and between C10 and C32, in addition to crosslinks at C7–C32, C10–C29 and C13–C26 (we have reported previously the crosslinking of formal C–C pairs by Mechlorethamine). We interpret the formation of the C13–C29 and C10–C32 crosslinks as evidence of an extrahelical location of the crosslinkable cytosines. Such extrahelical cytosine bases have been observed previously for a single C–C mismatch pair (in the so-called E-motif conformation). In the E-motif, the extrahelical cytosines are folded back towards the 5′-end of the duplex, consistent with our crosslinking data, and also consistent with the absence of C7–C29 and C10–C26 crosslinks in the current work. Hence, our data provide evidence for an extended E-motif DNA (eE-DNA) conformation in short d[GCC]n·d[GCC]n repeat fragments, and raise the possibility that such structures might occur in much longer d[GCC]n·d[GCC]n repeat tracts.
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dna interstrand crosslink formation by Mechlorethamine at a cytosine cytosine mismatch pair kinetics and sequence dependence
Archives of Biochemistry and Biophysics, 2001Co-Authors: Rebecca M Romero, Pornchai Rojsitthisak, Ian S HaworthAbstract:Abstract Expansion of the triplet repeat DNA sequence d[CGG] n · d[CCG] n is a characteristic of Fragile X syndrome, a human neurodegenerative disease. Stable intrastrand conformations formed by both d[CGG] n and d[CCG] n , and involving G–G and C–C mismatch pairs, respectively, are believed to be of importance in the development of the disease. We have shown previously that C–C mismatch pairs can be crosslinked covalently by Mechlorethamine, a nitrogen mustard alkylating agent, and hence this reaction may be of value as a probe for conformers of d[CCG] n . To characterize the Mechlorethamine C–C crosslink reaction further, here we report the kinetics and sequence dependence of formation of the crosslink species, using a series of model duplexes. The rate of reaction depends on the base sequence proximal to the C–C mismatch pair. Hence, in 19mer duplexes containing a central d[M 4 M 3 M 2 M 1 Cn 1 n 2 n 3 n 4 ] · d[N 4 N 3 N 2 N 1 Cm 1 m 2 m 3 m 4 ] sequence, where M–m and N–n are complementary base pairs, the amount of crosslink increased with increasing G–C content of the eight base pairs neighboring the C–C mismatch and with the proximity of the G–C pairs to the C–C mismatch. Molecular dynamics simulations of the solvated duplexes provided an explanation of these data. Hence, for a C–C pair flanked by G–C base pairs the mismatched cytosine bases remain stacked within the duplex, but for a C–C pair flanked by A–T base pairs, the simulations suggested local opening of the duplex around the C–C pair, making it a less effective target for Mechlorethamine.
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anomalous cross linking by Mechlorethamine of dna duplexes containing c c mismatch pairs
Biochemistry, 1999Co-Authors: Rebecca M Romero, Michael Mitas, Ian S HaworthAbstract:: Nitrogen mustards such as Mechlorethamine have previously been shown to covalently cross-link DNA through the N7 position of the two guanine bases of a d[GXC].d[GYC] duplex sequence, a so-called 1,3 G-G-cross-link, when X-Y = C-G or T-A. Here, we report the formation of a new Mechlorethamine cross-link with the d[GXC].d[GYC] fragment when X-Y is a C-C mismatch pair. Mechlorethamine cross-links this fragment preferentially between the two mismatched cytosine bases, rather than between the guanine bases. The cross-link also forms when one or both of the guanine bases of the d[GCC].d[GCC] fragment are replaced by N7-deazaguanine, and, more generally, forms with any C-C mismatch, regardless of the flanking base pairs. Piperidine cleavage of the cross-link species containing the d[GCC].d[GCC] sequence gives DNA fragments consistent with alkylation at the mismatched cytosine bases. We also provide evidence that the cross-link reaction occurs between the N3 atoms of the two cytosine bases by showing that the formation of the C-C cross-link is pH dependent for both Mechlorethamine and chlorambucil. Dimethyl sulfate (DMS) probing of the cross-linked d[GCC].d[GCC] fragment showed that the major groove of the guanine adjacent to the C-C mismatch is still accessible to DMS. In contrast, the known minor groove binder Hoechst 33258 inhibits the cross-link formation with a C-C mismatch pair flanked by A-T base pairs. These results suggest that the C-C mismatch is cross-linked by Mechlorethamine in the minor groove. Since C-C pairs may be involved in unusual secondary structures formed by the trinucleotide repeat sequence d[CCG]n, and associated with triplet repeat expansion diseases, Mechlorethamine may serve as a useful probe for these structures.
M Delaunay - One of the best experts on this subject based on the ideXlab platform.
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treatment of early stage mycosis fungoides with twice weekly applications of Mechlorethamine and topical corticosteroids a prospective study
Archives of Dermatology, 2005Co-Authors: Julie De Quatrebarbes, E Esteve, Martine Bagot, P Souteyrand, M Beylotbarry, L Vaillant, M Delaunay, Philippe Bernard, M Dincan, N CordelAbstract:Objective To determine if a therapeutic regimen of twice-weekly applications of Mechlorethamine hydrochloride and betamethasone dipropionate cream is effective in the treatment of early-stage mycosis fungoides while increasing cutaneous tolerance. Design Prospective nonrandomized study conducted from November 1999 to November 2002. Setting Eleven university or hospital dermatology departments in France. Patients Sixty-four consecutive patients with newly diagnosed early-stage mycosis fungoides (stage IA, n = 33; stage IB, n = 26; stage IIA, n = 5). Interventions Patients were treated with twice-weekly applications of a 0.02% aqueous solution of Mechlorethamine followed by an application of betamethasone cream during a 6-month period. Main Outcome Measures The primary end point was the rate of complete response during the treatment. Secondary end points were mean delay to achieve complete response, rate of severe cutaneous reactions of intolerance, and rate of relapse after achieving complete response. Results Thirty-seven patients (58%) had a complete response after a mean ± SD treatment duration of 3.6 ± 2.5 months: 20 (61%) of 33 patients with stage IA disease, 15 (58%) of 26 patients with stage IB disease, and 2 (40%) of 5 patients with stage IIA disease. Eighteen patients (28%) developed severe cutaneous reactions of intolerance that necessitated treatment discontinuation. Relapse was observed in 17 patients (46%) after a mean ± SD time of 7.7 ± 6.5 months. Conclusions A regimen of twice-weekly applications of Mechlorethamine and betamethasone cream is an effective treatment for early-stage mycosis fungoides. The decreased frequency of applications provides an advantage to the patient by being easy to use with limited adverse effects.
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evaluation of a 1 h exposure time to Mechlorethamine in patients undergoing topical treatment
British Journal of Dermatology, 2002Co-Authors: P Foulc, M Delaunay, V Evrard, S Dalac, B Guillot, Jl Verret, B DrenoAbstract:SummaryBackground Mechlorethamine is frequently used in the treatment of cutaneous lymphoma, but its application is limited in 30–80% of cases because of cutaneous intolerance. Reducing the concentration to avoid this side-effect has been only modestly successful. Objectives To investigate whether a shorter application period could reduce the frequency of intolerance. Methods In an open prospective study in 39 patients with cutaneous T-cell lymphoma or parapsoriasis, Mechlorethamine was applied according to the usual practices of the participating physicians (number of weekly applications, treatment confined to lesions or performed over the entire body) and then washed off after 1 h in all cases. Results Cutaneous intolerance was observed in 19 of 39 patients (49%). Six of these patients showed allergic contact dermatitis to Mechlorethamine after a mean period of 9·3 weeks, while the other 13 developed irritant contact dermatitis after a longer period. Cutaneous intolerance did not differ significantly according to the number of applications per week or the extent of body area treated. The therapeutic response rate was 69%, and no difference in therapeutic efficacy was noted between daily and intermittent applications. Conclusions Comparison with published studies showed no significant difference in the number of cases of cutaneous intolerance after short-term application, although their occurrence was delayed. Therapeutic response was decreased appreciably by short-term application as compared with results in the literature.
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a prospective study of cutaneous intolerance to topical Mechlorethamine therapy in patients with cutaneous t cell lymphomas
Archives of Dermatology, 1999Co-Authors: E Esteve, Martine Bagot, Pascal Joly, P Souteyrand, M Beylotbarry, L Vaillant, M Delaunay, M F Avril, L Laroche, Florent GrangeAbstract:Objective To study the exact frequency and the histological features of cutaneous intolerance to Mechlorethamine (CIM) hydrochloride therapy in patients with cutaneous T-cell lymphomas, including Langerhans cell histiocytosis. Design A multicenter prospective study was conducted from January 1, 1994, to May 31, 1996, in 12 different hospitals in France. Patients Of the 52 patients with cutaneous T-cell lymphomas or Langerhans cell histiocytosis, 35 were men and 17 were women, aged 18 to 87 years. Of the 52 patients, 35 had mycosis fungoides, 8 had nonepidermotropic cutaneous lymphoma, 7 had lymphomatoid papulosis, 1 had Sezary syndrome, and 1 had Langerhans cell histiocytosis. Methods Patients were treated with topical applications of a 0.02% aqueous solution of Mechlorethamine. The diagnosis of CIM was determined by the presence of erythema and pruritus. Patients who developed CIM underwent closed patch testing with three 10-fold dilutions of 0.02% Mechlorethamine solution. A positive patch test result was the presence of erythema and pruritus, a weak result was the presence of simple erythema without pruritus, and a negative result was the absence of erythema and pruritus. Skin biopsy specimens from patients with positive patch test results were obtained in patients who developed CIM. The biopsy specimens were reviewed, and the results determined by 2 pathologists (E.T. and J.W.). The histopathological findings were classified in 3 categories: (1) spongiotic dermatitis, (2) irritant dermatitis, and (3) insignificant or normal. In September 1998, the referring physicians were contacted if Mechlorethamine therapy had been continued in patients with CIM. Results Of the 52 patients, 43 were evaluated for tolerance to Mechlorethamine therapy. Of the 43 patients, CIM developed in 23, from 4 days to 9 months after the initiation of Mechlorethamine therapy. Of those 23 patients, CIM developed within 3 months in 21 and within 1 month in 13. Closed patch tests were performed in 21 of the 23 patients who developed CIM. The results of the patch test were positive in 12, weak in 4, and negative in 5. Of these 21 patients, 14 skin biopsy specimens were obtained in 14 different patients who had positive or weak patch test results. The specimens showed histological features that were consistent with spongiotic dermatitis in 9 patients, irritant dermatitis in 2, and insignificant or normal in 3. All 9 patients with histological features of spongiotic dermatitis discontinued Mechlorethamine therapy. All 5 patients without histological features of spongiotic dermatitis were able to resume Mechlorethamine therapy. These results do not correlate with those of previous study results. Conclusions Mechlorethamine therapy is a cost-effective and easily administered treatment for cutaneous T-cell lymphomas. Our study shows that allergic dermatitis caused by Mechlorethamine therapy is an early and frequent adverse reaction in patients with cutaneous T-cell lymphomas. The most common histological feature of patients with CIM is spongiotic dermatitis.
Michael E Colvin - One of the best experts on this subject based on the ideXlab platform.
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nmr studies of the conjugation of Mechlorethamine with glutathione
Journal of Medicinal Chemistry, 1990Co-Authors: Michael P Gamcsik, Terence G Hamill, Michael E ColvinAbstract:: Many cancer cells are resistant to chemotherapeutic treatment with Mechlorethamine and other alkylating agents. These drug-resistant cells often show an increase in the intracellular concentration of glutathione and an increase in the activity of glutathione-S-transferase when compared to the sensitive cells. Both of these components are thought to be involved with inactivation of the drug either through conjugation with glutathione or by hydrolysis. NMR spectroscopy was used to monitor the nonenzymatic conjugation of Mechlorethamine with glutathione. Several intermediates along the pathway to the doubly glutathione substituted mustard, including both mustard-aziridinium adducts, can be observed. The assignment of the 1H NMR spectrum of these adducts are presented. At 30 degrees C, pH 7.0, no hydrolyzed mustard was detectable. With the use of 13C-labeled mustard, the conjugation reaction can be shown to proceed through an aziridinium intermediate rather than by direct nucleophilic substitution.
E Esteve - One of the best experts on this subject based on the ideXlab platform.
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treatment of early stage mycosis fungoides with twice weekly applications of Mechlorethamine and topical corticosteroids a prospective study
Archives of Dermatology, 2005Co-Authors: Julie De Quatrebarbes, E Esteve, Martine Bagot, P Souteyrand, M Beylotbarry, L Vaillant, M Delaunay, Philippe Bernard, M Dincan, N CordelAbstract:Objective To determine if a therapeutic regimen of twice-weekly applications of Mechlorethamine hydrochloride and betamethasone dipropionate cream is effective in the treatment of early-stage mycosis fungoides while increasing cutaneous tolerance. Design Prospective nonrandomized study conducted from November 1999 to November 2002. Setting Eleven university or hospital dermatology departments in France. Patients Sixty-four consecutive patients with newly diagnosed early-stage mycosis fungoides (stage IA, n = 33; stage IB, n = 26; stage IIA, n = 5). Interventions Patients were treated with twice-weekly applications of a 0.02% aqueous solution of Mechlorethamine followed by an application of betamethasone cream during a 6-month period. Main Outcome Measures The primary end point was the rate of complete response during the treatment. Secondary end points were mean delay to achieve complete response, rate of severe cutaneous reactions of intolerance, and rate of relapse after achieving complete response. Results Thirty-seven patients (58%) had a complete response after a mean ± SD treatment duration of 3.6 ± 2.5 months: 20 (61%) of 33 patients with stage IA disease, 15 (58%) of 26 patients with stage IB disease, and 2 (40%) of 5 patients with stage IIA disease. Eighteen patients (28%) developed severe cutaneous reactions of intolerance that necessitated treatment discontinuation. Relapse was observed in 17 patients (46%) after a mean ± SD time of 7.7 ± 6.5 months. Conclusions A regimen of twice-weekly applications of Mechlorethamine and betamethasone cream is an effective treatment for early-stage mycosis fungoides. The decreased frequency of applications provides an advantage to the patient by being easy to use with limited adverse effects.
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a prospective study of cutaneous intolerance to topical Mechlorethamine therapy in patients with cutaneous t cell lymphomas
Archives of Dermatology, 1999Co-Authors: E Esteve, Martine Bagot, Pascal Joly, P Souteyrand, M Beylotbarry, L Vaillant, M Delaunay, M F Avril, L Laroche, Florent GrangeAbstract:Objective To study the exact frequency and the histological features of cutaneous intolerance to Mechlorethamine (CIM) hydrochloride therapy in patients with cutaneous T-cell lymphomas, including Langerhans cell histiocytosis. Design A multicenter prospective study was conducted from January 1, 1994, to May 31, 1996, in 12 different hospitals in France. Patients Of the 52 patients with cutaneous T-cell lymphomas or Langerhans cell histiocytosis, 35 were men and 17 were women, aged 18 to 87 years. Of the 52 patients, 35 had mycosis fungoides, 8 had nonepidermotropic cutaneous lymphoma, 7 had lymphomatoid papulosis, 1 had Sezary syndrome, and 1 had Langerhans cell histiocytosis. Methods Patients were treated with topical applications of a 0.02% aqueous solution of Mechlorethamine. The diagnosis of CIM was determined by the presence of erythema and pruritus. Patients who developed CIM underwent closed patch testing with three 10-fold dilutions of 0.02% Mechlorethamine solution. A positive patch test result was the presence of erythema and pruritus, a weak result was the presence of simple erythema without pruritus, and a negative result was the absence of erythema and pruritus. Skin biopsy specimens from patients with positive patch test results were obtained in patients who developed CIM. The biopsy specimens were reviewed, and the results determined by 2 pathologists (E.T. and J.W.). The histopathological findings were classified in 3 categories: (1) spongiotic dermatitis, (2) irritant dermatitis, and (3) insignificant or normal. In September 1998, the referring physicians were contacted if Mechlorethamine therapy had been continued in patients with CIM. Results Of the 52 patients, 43 were evaluated for tolerance to Mechlorethamine therapy. Of the 43 patients, CIM developed in 23, from 4 days to 9 months after the initiation of Mechlorethamine therapy. Of those 23 patients, CIM developed within 3 months in 21 and within 1 month in 13. Closed patch tests were performed in 21 of the 23 patients who developed CIM. The results of the patch test were positive in 12, weak in 4, and negative in 5. Of these 21 patients, 14 skin biopsy specimens were obtained in 14 different patients who had positive or weak patch test results. The specimens showed histological features that were consistent with spongiotic dermatitis in 9 patients, irritant dermatitis in 2, and insignificant or normal in 3. All 9 patients with histological features of spongiotic dermatitis discontinued Mechlorethamine therapy. All 5 patients without histological features of spongiotic dermatitis were able to resume Mechlorethamine therapy. These results do not correlate with those of previous study results. Conclusions Mechlorethamine therapy is a cost-effective and easily administered treatment for cutaneous T-cell lymphomas. Our study shows that allergic dermatitis caused by Mechlorethamine therapy is an early and frequent adverse reaction in patients with cutaneous T-cell lymphomas. The most common histological feature of patients with CIM is spongiotic dermatitis.
John C Reepmeyer - One of the best experts on this subject based on the ideXlab platform.
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stability of the nitrogen mustard Mechlorethamine in novel formulations for dermatological use
International Journal of Pharmaceutics, 2008Co-Authors: W A Ritschel, Wei Ye, Lucinda F Buhse, John C ReepmeyerAbstract:Abstract Long term stability measurements were made for the nitrogen mustard Mechlorethamine HCl at a concentration of 0.02% in six topical formulations: Aquaphor ® ointment, Transcutol ® , Labrasol ® , 10% Transcutol ® in Aquaphor ® , 10% Transcutol ® in Labrasol ® , and Aquaphilic ® ointment. The drug decomposed gradually in Aquaphor ® ointment at room temperature, dropping to 95% in 4 weeks, 85% in 12 weeks, and 78% in 39 weeks. On the other hand, the drug decomposed rapidly in Aquaphilic ® ointment, giving an assay of less than 20% of its initial concentration after 24 h at room temperature. Generally, Mechlorethamine HCl was more stable in Aquaphor ® ointment than in formulations containing Transcutol ® or Labrasol ® . However, the addition of the free radical inhibitor, BHT, significantly enhanced the stability of Mechlorethamine in Transcutol ® and Labrasol ® formulations. Four BHT-stabilized Transcutol ® and Labrasol ® formulations gave assays in ranges of 92–99% at the end of 4 weeks, 77–98% at the end of 12 weeks, and 38–93% at the end of 41 weeks.
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modifications and insights into a method for the analysis of the nitrogen mustard Mechlorethamine by high performance liquid chromatography
Analytica Chimica Acta, 2008Co-Authors: John C Reepmeyer, Wei Ye, W A RitschelAbstract:Abstract Previously, a method was presented for the analysis of Mechlorethamine by derivatization of this unstable nitrogen mustard to bis(2-phenylthioethyl)methylamine (PTEMA), a stable compound suitable for analysis by HPLC with UV detection [J.C. Reepmeyer, J. Chromatogr. A, 1085 (2005) 262]. Mechlorethamine HCl served as a reference standard and it was derivatized in situ simultaneously with samples of Mechlorethamine HCl in ointment preparations. This paper presents the synthesis of PTEMA on a gram scale, synthesis of its picrate salt, bis(2-phenylthioethyl)methylamine picrate (PTEMAP), and isolation of the picrate as a crystalline solid. PTEMAP may serve as a reference standard replacing the toxic Mechlorethamine HCl. Insights into the handling, storage, drying, and hygroscopic properties of Mechlorethamine HCl and PTEMAP are discussed. In addition, one step following the derivatization procedure in the original method is recognized as a potential for error, and a procedure relating to the order of addition of reagents is presented to avoid this error. The method has been extended to the analysis of Mechlorethamine in aqueous solutions.
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analysis of the nitrogen mustard Mechlorethamine in topical pharmaceutical preparations by high performance liquid chromatography
Journal of Chromatography A, 2005Co-Authors: John C ReepmeyerAbstract:Abstract Mechlorethamine in topical pharmaceutical formulations was derivatized with benzenethiol to form the disubstitution product and analyzed by normal-phase HPLC on silica gel using dibutyl phthalate as an internal standard. The derivatization reaction, purification, and isolation were conveniently performed in a single test tube. Analyses were successfully performed on three types of ointment formulations: anhydrous hydrophobic petrolatum-based ointments, anhydrous hydrophilic ointments, and hydrous hydrophilic ointments. Precision for the analysis of Mechlorethamine standard or Mechlorethamine in ointments ranged from 0.08 to 0.52% RSD (n = 6). Recoveries from ointments spiked with 0.02% Mechlorethamine hydrochloride were 98.4–100.4%. The chromatograms were clean, showing minimal or no interference from ointment excipients or reagents.