Development of an efficient stability-indicating LC-MS/MS method for the analysis of selexipag and characterization of its degradation products.

A new RP-HPLC method with a quick, sensitive and stable indication for the quantitative measurement of selexipag and its associated substances was developed and validated in the present study. In this new method, using the impurity-spiked solution, the chromatographic approach was optimized. Similarly, using the X-bridge phenyl column with isocratic elution of mobile phase containing acetonitrile and formic acid, selexipag and its impurities were separated. Recovery experiments obtained were satisfactory, and also the calibration graphs plotted for selexipag and its five impurities were found to be linear. The system validation parameters such as specificity, linearity, precision, accuracy and robustness were determined successfully. The obtained results indicated that the developed method was found to be useful for analyzing selexipag from its impurities. Further, using stress tests against acid, alkali, peroxide, reduction, thermal, hydrolysis and UV conditions, the present established method of HPLC was assessed and validated as per ICH Q2(R1) guidelines.

Oxidative degradation profile studies of tavaborole by a validated stability indicating RP-UPLC method: Isolation and characterization of novel degradant using 2D-NMR and HRMS.

The current research work reports a study on the degradation profile of tavaborole, which is an oxaborole antifungal drug used to treat infections in the toenails. This work also reports the chemical stability of tavaborole in different stress conditions along with the isolation and characterization of degradation products by high-resolution mass spectrometry and two-dimensional nuclear magnetic resonance techniques. A sensitive and reproducible stability-indicating ultra-performance liquid chromatography method was developed and validated for quantification of tavaborole bulk drug in the presence of degradation products. Significant degradation was observed during oxidative stress conditions using H2 O2 . It was observed that the drug was highly unstable under oxidation stress conditions and thus degradation profiles with various oxidizing reagents were studied. One unknown impurity (DP-1) was formed during peroxide degradation, which was isolated by reverse-phase preparative chromatography. The structure of this degradant was characterized by high-resolution mass spectrometry and multidimensional nuclear magnetic resonance techniques. The structure of this novel impurity DP-1 was identified as [4-fluoro-2-(hydroxymethyl)phenol], which was not reported as a degradant in the literature. An Acquity BEH C18 , 100 × 2.1 mm, 1.7 µm column was used to achieve the desired separation within a shorter runtime of 4.0 min. The method was validated for specificity, precision, linearity and accuracy over the concentration range of 5.0-400 µg ml-1 (r2 -0.9999) and limit of quantitation 5.0 µg ml-1 . This method is compatible with LCMS analysis which enables to identify the unknown impurities formed in the process.

Ensuring the safety of vaccine cell substrates by massively parallel sequencing of the transcriptome.

Massively parallel, deep, sequencing of the transcriptome coupled with algorithmic analysis to identify adventitious agents (MP-Seq™) is an important adjunct in ensuring the safety of cells used in vaccine production. Such cells may harbour novel viruses whose sequences are unknown or latent viruses that are only expressed following stress to the cells. MP-Seq is an unbiased and comprehensive method to identify such viruses and other adventitious agents without prior knowledge of the nature of those agents. Here we demonstrate its utility as part of an integrated approach to identify and characterise potential contaminants within commonly used virus and vaccine production cell lines. Through this analysis, in combination with more traditional approaches, we have excluded the presence of porcine circoviruses in the ATCC Vero cell bank (CCL-81), however, we found that a full length betaretrovirus related to SRV can be expressed in these cells, a factor that may be of importance in the production of certain vaccines. Similarly, insect cells are proving to be valuable for the production of virus like particles and sub-unit vaccines, but they can harbour a range of latent viruses. We show that following MP-Seq of the Trichoplusia ni (High Five cell line) transcriptome we were able to detect a contaminating, latent nodavirus and identify an expressed errantivirus genome. Collectively, these studies have reinforced the role of MP-Seq as an integral tool for the identification of contaminating agents in vaccine cell substrates.

Alternative splicing to exon 11 of human prolactin receptor gene results in multiple isoforms including a secreted prolactin-binding protein.

Endocrine and autocrine prolactin (PRL) exerts effects on normal breast and breast cancer cells, and high serum PRL is a poor prognostic factor for colorectal cancer. Here we tested the hypothesis that short isoforms of the PRL receptor (PRLR) in human tissue regulate the actions of PRL in cancer. Using 3' RACE we isolated five splice variants of the human PRLR (hPRLR), three of which encode the complete extracellular binding domain. Two of these isoforms, short form 1a (SF1a) and short form 1b (SF1b), possess unique intracellular domains encoded by splicing to exon 11 from exons 10 and 9 respectively. A third novel isoform (delta7/11) reflects alternative splicing from exon 7 to exon 11 and encodes a secreted soluble PRL-binding protein. Additional splice variants of SF1b and delta7/11 that lacked exon 4 (delta4-SF1b and delta4-delta7/11) were also identified. Functional analyses indicated that hPRLR-SF1b is a strong dominant-negative to the differentiative function of the PRLR long form while hPRLR-SF1a is a weaker dominant-negative. Differential abundance of SF1a, SF1b and delta7/11 expression was detected in normal breast, colon, placenta, kidney, liver, ovary and pancreas, and breast and colon tumors. Taken together, these data indicate the presence of multiple isoforms of the hPRLR that may function to modulate the endocrine and autocrine effects of PRL in normal human tissue and cancer.

The safety of reusing injectable collagen: a multicenter microbiological study.

We have previously reported pilot data regarding the safety of saving partially used syringes of a glutaraldehyde cross-linked collagen for use in subsequent treatment sessions with the same individual. That single institution study involved 56 partially used syringes cultured for aerobic bacteria. Only one weakly positive culture was detected among these 56 samples, which prompted us to carry out this expanded study involving multiple centers and different injection techniques. Samples were collected from four centers. Following periurethral injection in an office setting, 166 partially used syringes of glutaraldehyde cross-linked collagen were refrigerated for between 1 and 104 weeks (average 58). Material from all 166 syringes was then cultured qualitatively and quantitatively for both aerobic and anaerobic organisms. Collagen from one syringe grew >100,000 colonies of Escherichia coli. All other cultures were negative. In the pilot study, one culture of 56 syringes was weakly positive for coagulase-negative staphylococcus. When the results from both studies were considered together, only two of 222 partially used syringes (0.9%) were contaminated. The background risk of local infection associated with periurethral collagen injection is approximately 0.29%. Using the statistical equation 'number needed to harm', we found that a clinician would have to reuse 111 syringes at a saving of $34,965 before he or she would cause a single local injection by so doing. Therefore, we feel that it may be cost-effective and safe to reinject material from a partially used syringe of glutaraldehyde cross-linked collagen during a subsequent treatment session on an individual.

Protective effect of suburethral slings on postoperative cystocele recurrence after reconstructive pelvic operation.

OBJECTIVE: The purpose of this study was to evaluate the independent effect of suburethral sling placement on the risk of cystocele recurrence after pelvic reconstructive operation. STUDY DESIGN: One hundred forty-eight women with cystoceles to or beyond the hymenal ring underwent pelvic reconstructive operation, with or without incontinence procedures, and were evaluated at 12 and 52 weeks after operation with a standardized pelvic examination. Rates of recurrent prolapse, at all sites, were statistically compared between subjects with and without suburethral slings. A multiple regression analysis was used to determine the independent effect of sling placement on the risk of recurrent cystoceles. RESULTS: Suburethral sling placement was associated with a 54.8% reduction in the mean rate of postoperative cystocele recurrence (P =.004). This protective effect was observed as early as 12 weeks and remained significant at 1-year follow up (42% vs 19%). A markedly reduced risk of cystocele recurrence was observed when women with sling procedures were compared with all other women, with those women who underwent other incontinence operations, and even with those women who had undergone prolapse repair with no incontinence procedure. The protective effect of the sling procedure remained highly significant (odds ratio, 0.29; P =.0003), even after controlling for potentially confounding variables in a multiple logistic regression model. CONCLUSION: Suburethral sling procedures appear to significantly reduce the risk of cystocele recurrence after pelvic reconstructive operation, in contrast with the effect of retropubic urethropexy and needle suspensions. These findings should be considered when the surgical treatment of stress incontinence that accompanies pelvic organ prolapse is being planned.

Anterior or posterior sacrospinous vaginal vault suspension: long-term anatomic and functional evaluation.

OBJECTIVE: To compare vaginal anatomy and sexual function after the conventional posterior and anterior sacrospinous vault suspension. METHODS: A retrospective repeated measures cohort study included all 168 consecutive sacrospinous vault suspension procedures between July 1990 and February 1997. The posterior suspension (n = 92) used a posterior vaginal incision and pararectal dissection. Anterior suspension (n = 76) involved an anterior rather than posterior vaginal incision, retropubic perforation, and dissection of a paravaginal-paravesical rather than pararectal space to accommodate the vaginal vault. Two polytetrafluoroethylene (00) sutures anchored the anterior vaginal cuff (for the anterior sacrospinous suspension) or the posterior vaginal cuff (for the posterior sacrospinous suspension) to the ligament. Postoperative evaluation included an examination using the pelvic organ prolapse quantitative system, assessment of vaginal width and axis, and symptom questionnaire. RESULTS: Total vaginal length and apical suspension were slightly greater after the anterior suspension, and recurrent anterior vaginal relaxation was less likely. No differences were found in maximal dilator size or apical narrowing between the two groups. New onset dyspareunia was reported by two subjects in the anterior vault suspension group, and two in the posterior vault suspension group. Three of these four cases of de novo dyspareunia were attributable to either severe atrophy or recurrent prolapse, and none to vaginal narrowing or shortening. CONCLUSION: After anterior sacrospinous vault suspension, vaginal length and apical suspension were slightly increased, and recurrent anterior vaginal prolapse decreased compared with the posterior sacrospinous suspension technique. Upper vaginal caliber and sexual function appear well preserved using either technique.

Comparison of microtransducer and fiberoptic catheters for urodynamic studies.

OBJECTIVE: To assess the validity and reproducibility of a fiberoptic transducer urodynamic catheter for urethral closure pressure profiles and leak point pressure determination, using a microtransducer catheter as the standard. METHODS: Ninety women without significant pelvic organ prolapse underwent urodynamic evaluations with both fiberoptic and microtransducer catheters. Maximal urethral closure pressures and "leak point pressures" were repeatedly measured by the two catheters and statistically compared. The order of catheter use was randomized. RESULTS: Significantly lower mean maximal urethral closure pressures were recorded by the fiberoptic system than by the microtransducer system (28.9 cmH(2)O +/- 17.3 versus 43.2 cmH(2)O +/- 24.9, P <.001). The fiberoptic catheter predicted microtransducer values for maximum urethral closure pressure only within a range of 27 cmH(2)O. Mean "leak point pressure" recorded by the fiberoptic catheters (66.9 cmH(2)O +/- 2.9) was not significantly different than that recorded by the microtransducer catheters (66.4 cmH(2)O +/- 2.9, P =.97). CONCLUSION: There is a significant difference between maximum urethral closure pressure values recorded by the microtransducer and fiberoptic catheter systems. No significant difference was found between the two systems in measurement of Valsalva "leak point pressure."

Prospective randomized trial of polyglactin 910 mesh to prevent recurrence of cystoceles and rectoceles.

OBJECTIVE: Our aim was to evaluate the efficacy of polyglactin 910 mesh in preventing recurrent cystoceles and rectoceles. STUDY DESIGN: In a prospective, randomized, controlled trial, patients undergoing vaginal reconstructive surgery with cystoceles to the hymenal ring and beyond were randomly selected to undergo anterior and posterior colporrhaphy with or without polyglactin 910 mesh reinforcement. Results were evaluated preoperatively and at 2, 6, 12, and 52 weeks postoperatively. RESULTS: A total of 161 women were randomly selected for this study. One woman was excluded at the time of surgery, and 17 women were lost to follow-up. Eighty women received mesh, and 80 did not. Both groups were found to be equivalent with respect to age, parity, concomitant surgery, and menopausal and hormone replacement status. Preoperatively 49 women had a central cystocele to the hymenal ring and 111 women had cystoceles beyond the introitus; 91 women had a rectocele to the mid-vaginal plane, 31 to the hymenal ring, and 22 beyond the introitus. After 1 year, 30 (43%) of 70 subjects without mesh and 18 (25%) of 73 subjects with mesh had recurrent cystoceles beyond the mid-vaginal plane (P =.02). Eight women without mesh and 2 women with mesh had recurrent cystoceles to the hymenal ring (P =.04). No recurrent cystoceles beyond the hymenal ring occurred in either group. Multivariate logistic regression analysis showed concurrent slings to be associated with significantly fewer recurrent cystoceles (odds ratio, 0.32; P =.005), whereas the presence of mesh remained significantly predictive of fewer cystocele recurrences in this analysis. Thirteen recurrent rectoceles were noted 1 year postoperatively, with no differences between groups. CONCLUSION: Polyglactin 910 mesh was found to be useful in the prevention of recurrent cystoceles.

Quantitation of alternatively spliced estrogen receptor alpha mRNAs as separate gene populations.

Estrogen receptor (ER) mRNA undergoes alternative splicing generating transcripts that have deletions in various combination of exons. Although several reports have shown that the spliced variant mRNAs are expressed in both normal and malignant tissues, the exact functional role(s) of these molecules have not been established in estrogen induced signal transduction processes mainly due to practical limitations involved in their detection and quantitation. We have recently described a 'Splice Targeted Primer Approach' that can specifically detect splice variants without amplifying the wild type ERs [12]. In the current report, we describe strategies to quantify individual splice variant mRNAs as separate gene populations independent of wild type or other variants using ER exon 7Delta and exon 2Delta as models. We describe the methods of quantifying the exon 7Delta and exon 2Delta transcripts in two breast cancer cell lines, MCF-7 and LCC2, and a breast tumor using the splice-targeted primers in combination with template competition RT PCR. The exon 2Delta splice specific sense primer along with an anti-sense primer in exon 4 amplified a 412 bp product in both cell lines and the tumor that could be quantitated. The exon 7Delta splice targeted anti-sense primer along with a partner primer in exon 2 amplified four transcripts that have deletions in exon 7, exons 7 and 4, exons 7 and 3-4, and exons 7 and 3-5. These four transcripts could be simultaneously quantified by the template competition method described here. Our results also show that the estrogen-independent LCC2 cells express significantly higher levels of the above 7Delta transcripts compared to the estrogen-dependent MCF-7 cells.

Transvaginal bladder neck suspension to Cooper's ligament.

Numerous surgeries have been proposed for the treatment of genuine stress incontinence, with the goals to improve functional outcome and decrease complications and their associated morbidity. Two new, minimally invasive procedures, transvaginal retropubic urethropexy and transvaginal Cooper's ligament sling, are reviewed in this article. These procedures provide a completely transvaginal approach, without the use of abdominal incisions or bone anchors. The anterior point of suspension is Cooper's ligament. The transvaginal retropubic urethropexy is used for the treatment of genuine stress incontinence with urethral hypermobility, and the transvaginal sling also may be used in the presence of intrinsic sphincteric deficiency. The procedures are described and the recent outcomes discussed.

Transvaginal therapy of genuine stress incontinence.

Two minimally invasive techniques for treatment of genuine stress incontinence, a transvaginal retropubic urethropexy and a transvaginal sling, using Cooper's ligament as the anchoring structure, are reported along with the early results. These surgeries can be done easily in conjunction with vaginal reconstructive procedures. Twenty-seven women were operated on between October 1998 and September 1999. Seventeen women underwent the transvaginal retropubic urethropexy for genuine stress incontinence, whereas 10 women underwent the transvaginal sling for genuine stress incontinence with maximal urethral closure pressures less than 20 cm H(2)O. Postoperative urodynamics were done routinely at 4 months. Subjective follow-up was by routine postoperative visits or telephone survey. The mean follow-up of the retropubic urethropexy group was 6.5 months (range 1 to 12). Of these 17 women, 16 (94%) had no stress incontinence. Seven of 14 patients (50%) resolved their urge incontinence also. The mean follow-up of the transvaginal sling group was 2.8 months (range 1 to 4.5). Of the 10 patients in this group, 7 (70%) were cured of their stress incontinence. One patient in each group underwent only the anti-incontinence procedure without concomitant vaginal reconstructive procedures; both of these women were dry. Additional follow-up is required to see if these minimally invasive techniques produce successful results in the long term.

Recent developments in pelvic organ prolapse.

Pelvic organ prolapse is a common worldwide problem. Recent advances in our understanding of its pathophysiology, along with progress made in the evaluation and treatment of pelvic support defects, are discussed. Although the pathophysiology of this condition is still not completely understood, genetic factors and environmental factors are involved. Understanding these factors better will help us to approach treatment of pelvic organ prolapse in a more logical manner. Multiple surgical techniques are available for pelvic relaxation, with a wide range of success rates ranging from 77 to 97% for various procedures. New techniques need to be studied further before being incorporated into routine practice. Better standardization of evaluation methods can help in such clinical studies.

Is it safe to reuse a syringe of glutaraldehyde cross-linked collagen? A microbiological study.

PURPOSE: We evaluated the safety of saving partially used syringes of glutaraldehyde cross-linked collagen for subsequent treatment sessions in an individual. MATERIALS AND METHODS: After periurethral injection in an office setting 56 partially used syringes of glutaraldehyde cross-linked collagen were stored in a refrigerator for 1 to 61 weeks (mean 15). Collagen from all 56 syringes was then cultured qualitatively using a broth medium at 35C and semiquantitatively using a chocolate agar plate at 22 to 30C for 5 days each. RESULTS: A qualitative broth culture was positive for coagulase negative staphylococcus but the results of semiquantitative chocolate agar culture of material from the same syringe were negative. All cultures of the other 55 syringes were negative. CONCLUSIONS: The positive culture most likely resulted from contamination during periurethral injection or the culturing process. Minimal contamination from and the great potential cost savings of reusing glutaraldehyde cross-linked collagen for subsequent treatments in an individual indicate the need for an expanded study involving multiple centers.

Identification of twenty alternatively spliced estrogen receptor alpha mRNAs in breast cancer cell lines and tumors using splice targeted primer approach.

Estrogen receptor (ER) alpha splice variant transcript profiles were analyzed by RT PCR in six ER positive breast cancer cell lines, MCF-7, T47D, ZR-75, LCC1, LCC2 and LCC9, three ER negative cell lines, MDA-MB-435, MDA-MB-235 and LCC6, and three ER positive malignant breast tumors using targeted primers which specifically anneal to the splice junctions of exon 2Delta, exon 3Delta, exons 2-3Delta, exon 4Delta, exon 5Delta, exon 6Delta and exon 7Delta. The partner primers were chosen such that largest possible transcripts were amplified between exons 1 and 8. The results described here show that each splice specific primer amplified not only the single exon deleted transcript but also a number of related transcripts that have deletions in various combinations of exons. The exon 2Delta specific primer amplified five transcripts that have deletions in exon 2, exons 2 and 7, exons 2, 5, and 7, exons 2 and 4-5, and exons 2 and 4-6. The exon 3Delta specific primer amplified two transcripts that have deletions in exon 3, and exons 3 and 7. The exon 2-3Delta specific primer amplified three products that have deletions in exons 2-3, exons 2-3 and 7 and exons 2-3, 5 and 7. The exon 4Delta specific primer amplified two products that have deletions in exon 4, and exons 4 and 7. The exon 5Delta specific primer amplified three transcripts, that have deletions in exon 5, exons 5 and 2, and exons 5, and 2-3. The 6Delta specific primer amplified only one transcript that has a deletion in exon 6. The 7Delta specific primer amplified four transcripts, that have deletions in exon 7, exons 7 and 4, exons 7 and 3-4, and exons 7 and 3-5. None of the above splice specific primers amplified the wild type ER sequences. The six ER positive cell lines differed in the patterns of the variant transcripts and among the three ER negative cell lines analyzed, only MDA-MB-435 showed the presence of exon 2Delta and exon 4Delta transcripts. Analyses in the tumor samples indicated that the above transcripts are extensively modified.

Alterations in the estrogen receptor alpha mRNA in the breast tumors of African American women.

Several recent reports have shown that the mortality rate with breast cancer is about three times higher in African American women than in other populations. In addition, the available data also indicate that the tumors are very aggressive and poorly differentiated with a very low frequency of hormone receptors. To gain an insight into the factors that may be responsible for their aggressive tumors, we investigated the transcript profiles of the estrogen receptor (ER), the most important prognostic factor in breast cancer, in the tumors derived from African American women. We analyzed 24 immunohistochemically ER+ and 6 ER- malignant tumors for ER mRNA by reverse transcription polymerase chain reaction using a number of primer pairs. For comparative purposes, 20 ER- malignant tumor issues derived from Caucasian patients were also included. Our results showed that only 15 of the ER+ tumors from African American women patients had full-length wild-type receptor transcripts and the others exhibited alterations/truncations in exon 8. We also found that the majority of tumors that had alterations/truncations in exon 8 did not express the naturally occurring, more abundant exon 7 deletion transcript. Most of the tumors expressed exon 2, exons 2-3, and exon 5 deletion variant transcripts. Unexpectedly, 2 of the 6 immunohistochemically ER- tumors showed full-length wild-type receptor mRNA but none of the variant transcripts.

Quantitation of estrogen receptor mRNA copy numbers in breast cancer cell lines and tumors.

Several clinical studies have suggested that the content of estrogen receptor (ER) in breast tumors influences the survival, tumor recurrence, and response to antiestrogen therapies. Therefore, the ability to precisely quantitate the ER content in tumor tissues will be of significant benefit to women with breast cancer. Although immunohistochemical and polymerase chain reaction (PCR) methods have been described for the detection and semiquantitation of ER, none of them precisely quantitate ER copy numbers in tumor samples. In the present report we describe a molecular approach to accurately quantitate ER mRNA copy numbers using a reverse-transcription PCR (RT-PCR) template competition method. A competitor template was devised by inserting unrelated nucleic acid sequences into an ER cDNA clone. A template competitive RT-PCR analysis was then performed to determine the number of copies of ER mRNA. As a standard of reference for the ER mRNA copy numbers from various samples, the mRNA copy numbers of a constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were also quantitated. The ER quantitations were performed in three positive cell lines, MCF-7, T47D, and ZR-75, and two positive tumor tissues by this approach. Our results described here show that among the cell lines studied, T47D expresses the highest copy numbers of ER. We also present here that ER as low as 10(3) copies per 10(5) copies of GAPDH can be detected and quantitated in tumor samples by the template competition method. In addition, the molecular approach can simultaneously detect, distinguish, and quantitate exon deletion variant copy numbers of ER. The results described in this report indicate that the ratios of exon 7 deletion variant to wild type in the tumor tissues are significantly higher than in the cell lines studied.