Evaluation of alternative diluents for clinical use of collagenase clostridium histolyticum (CCH-aaes).

BACKGROUND: Collagenase clostridium histolyticum (CCH-aesthetic formulation [CCH-aaes]; QWO™ [Endo Aesthetics, Malvern PA, USA] is approved as a subcutaneous injection for treatment of cellulite. In the aesthetic practice, dilution of marketed products is commonly employed to tailor treatments to individual patients or off-label locations. Dilution beyond the 0.23 mg/ml achievable with the proprietary diluent supplied with the CCH-aaes lyophilized powder requires diluents readily available in clinic. AIM: To characterize the functionality and stability of CCH-aaes when reconstituted and/or diluted with alternative diluents, including normal saline, bacteriostatic saline, and/or proprietary diluent. PATIENTS/METHODS: Each dilution was assessed for purity using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), activity using collagenase (AUX-I) and gelatinase (AUX-II) assays, and aggregation using size-exclusion chromatography. RESULTS: When reconstituted with either saline or proprietary diluent, and diluted with proprietary diluent or saline, purity, activity, and stability of CCH-aaes is maintained for up to 24 h at 5°C or 25°C. In contrast, use of bacteriostatic saline to reconstitute and/or dilute CCH-aaes results in up to a 40% decrease in activity and aggregation of 5.3% of CCH-aaes protein. Importantly, inclusion of 2% lidocaine and 1:200 000 epinephrine does not negatively impact CCH-aaes purity, concentration, or activity for up to 24 h at 5°C or 25°C. CONCLUSIONS: From an efficacy and safety perspective, CCH-aaes must not be/should not be reconstituted and/or diluted with bacteriostatic saline to avoid injection of protein aggregates. Ideally, CCH-aaes should be reconstituted in proprietary diluent: further dilution with normal saline and addition of lidocaine and epinephrine is acceptable.

Long-term Curvature Deformity Characterization in Men Previously Treated With Collagenase Clostridium Histolyticum for Peyronie's Disease, Subgrouped by Penile Plaque Calcification.

OBJECTIVE: To examine the long-term (5-year) efficacy and safety of collagenase clostridium histolyticum (CCH) therapy in men with Peyronie's disease and varying degrees of plaque calcification. MATERIALS AND METHODS: CCH-treated adult men from the 12-month Investigation for Maximal Peyronie's Reduction Efficacy and Safety Studies I/II or 9-month open-label studies were eligible. Degree of plaque calcification (no calcification, noncontiguous stippled calcification, or calcification that did not interfere with CCH injection) was determined by penile x-ray or ultrasound. Penile curvature deformity and Peyronie's Disease Questionnaire responses were assessed annually for up to 5 years, with ≥6 months between consecutive visits. RESULTS: For no calcification group, from baseline to last (Reference) visit during the prior studies (n = 160), mean penile curvature improved by 20.9° ± 16.3° (39.3%) with CCH. Similar improvements with CCH from baseline to Reference were observed in stippled calcification (n = 27; improvement of 24.1° ± 20.2° [42.7%]) and calcification (n = 27; improvement of 21.7° ± 14.8° [43.3%]) subgroups. At Year 5 follow-up in no calcification group (n = 119), an additional 10.0% improvement in mean penile curvature vs Reference (4.3°) occurred. Penile curvature improvements seen at Reference in stippled calcification and calcification groups were maintained through Year 5. Additional numeric improvements in 3 Peyronie's Disease Questionnaire domains were observed at Year 5 visit vs baseline scores. No long-term safety issues were identified. CONCLUSION: This first report of long-term (5-year) CCH clinical trial outcomes in a population with penile plaque calcification demonstrates that nonsurgical intralesional CCH therapy is an appropriate Peyronie's disease treatment in men with penile plaque calcification that is stippled or does not impede CCH injection.

A comparative molecular field analysis (COMFA) of the structural determinants of heat-stable enterotoxins mediating activation of guanylyl cyclase C.

The heat-stable enterotoxin binds to and activates guanylyl cyclase C (GC-C), regulating fluid and electrolyte secretion in intestinal epithelial cells. A COMFA model was developed to predict the primary interactions between GC-C agonists and their receptor. This model predicts that the amide backbone of Cys(5)-Cys(6)-Glu(7)-Leu(8), the beta carbon atoms of Cys(5)-Cys(6), and the side chains of Pro(12), Ala(13), and Ala(15) comprise the primary interactions of GC-C agonists with the receptor surface.

In vivo imaging of human colon cancer xenografts in immunodeficient mice using a guanylyl cyclase C--specific ligand.

UNLABELLED: Guanylyl cyclase C (GC-C) is a transmembrane receptor expressed by human intestinal cells and primary and metastatic colorectal adenocarcinomas but not by extraintestinal tissues or tumors. The Escherichia coli heat-stable enterotoxin analog, STa (5--18), is a 14--amino acid peptide that selectively binds to the extracellular domain of GC-C with subnanomolar affinity. This study examined the utility of a radiolabeled conjugate of STa (5--18) to selectively target and image extraintestinal human colon cancer xenografts in vivo in nude mice. METHODS: The STa conjugate, ethoxyethyl-mercaptoacetamidoadipoylglycylglycine-STa (5--18) (NC100586), was synthesized and labeled with (99m)Tc to produce (99m)Tc-NC100586. This compound was intravenously administered to nude mice bearing human colon cancer xenografts, and specific targeting was evaluated by biodistribution and gamma camera imaging. RESULTS: In CD-1 nude mice, biodistribution and scintigraphic imaging analyses showed selective uptake of (99m)Tc-NC100586 into human colon cancer xenografts that express GC-C but not into normal tissues that do not express GC-C. Similarly, (99m)Tc-NC100586 injected intravenously into CD-1 nude mice with human colon cancer hepatic metastases selectively accumulated in those metastases, and about 5-mm foci of tumor cells were visualized after ex vivo imaging of excised livers. Accumulation of (99m)Tc-NC100586 in human colon cancer xenografts reflected binding to GC-C because (99m)Tc-NC100588, an inactive analog that does not bind to GC-C, did not selectively accumulate in cancer xenografts compared with normal tissues. Also, coadministration of excess unlabeled STa (5--18) prevented accumulation of (99m)Tc-NC100586 in human colon cancer xenografts. Furthermore, (99m)Tc-NC100586 did not selectively accumulate in Lewis lung tumor xenografts, which do not express GC-C. CONCLUSION: This study showed that intravenously administered STa (5--18) selectively recognizes and binds to GC-C expressed by human colon cancer cells in vivo. Also shown was the ability to exploit this selective interaction to target imaging agents to extraintestinal human colon tumors in nude mice. These results suggest the utility of STa and GC-C for the development of novel targeted imaging and therapeutic agents with high specificity for metastatic colorectal tumors in humans.

Mechanism of action of colchicine in the treatment of gout.

PURPOSE: The aims of this article were to systematically review the literature about the mechanism of action of colchicine in the multimodal pathology of acute inflammation associated with gout and to consider the clinical utility of colchicine in other chronic inflammatory diseases. METHODS: The English-language literature on PubMed was searched for articles published between 1990 and October 2013, with a cross-reference to citations across all years. Relevant articles pertaining to the mechanism of action of colchicine and the clinical applications of colchicine in gout and other inflammatory conditions were identified and reviewed. FINDINGS: The molecular pathology of acute inflammation associated with gouty arthritis involves several concurrent pathways triggered by a variety of interactions between monosodium urate crystals and the surface of cells. Colchicine modulates multiple pro- and antiinflammatory pathways associated with gouty arthritis. Colchicine prevents microtubule assembly and thereby disrupts inflammasome activation, microtubule-based inflammatory cell chemotaxis, generation of leukotrienes and cytokines, and phagocytosis. Many of these cellular processes can be found in other diseases involving chronic inflammation. The multimodal mechanism of action of colchicine suggests potential efficacy of colchicine in other comorbid conditions associated with gout, such as osteoarthritis and cardiovascular disease. IMPLICATIONS: Colchicine has multiple mechanisms of action that affect inflammatory processes and result in its utility for treating and preventing acute gout flare. Other chronic inflammatory diseases that invoke these molecular pathways may represent new therapeutic applications for colchicine.

MSI-1436 reduces acute food intake without affecting dopamine transporter activity.

Many therapies designed to reduce food intake and body weight act, in part, by blocking the dopamine transporter (DAT) - a protein responsible for clearing extracellular dopamine (DA) after release thereby terminating its action. Here, we found that a single injection of the drug trodusquemine (MSI-1436) decreased food intake in rats. To assess the effects of MSI-1436 on DAT function, fast-scan cyclic voltammetry was used to measure DA concentration changes in the ventral striatum. DA release was evoked by electrical stimulation of the ventral tegmental area every 5 min. After 3 baseline measurements, rats were injected with MSI-1436 (10 mg/kg), the known DAT blocker bupropion (80 mg/kg) or saline and evoked DA release and reuptake were monitored for an additional hour. Neither saline nor MSI-1436 caused a significant change in the magnitude of evoked release from baseline values whereas bupropion caused a significant increase. In addition, neither saline nor MSI-1436 significantly increased DA decay rates while such an increase was observed with bupropion. Thus, over a time course when MSI-1436 suppresses food intake it does not affect DAT function. The results support MSI-1436 as an anti-obesity treatment which spares DAT.

Inhibition of PTP1B by trodusquemine (MSI-1436) causes fat-specific weight loss in diet-induced obese mice.

Trodusquemine (MSI-1436) causes rapid and reversible weight loss in genetic models of obesity. To better predict the potential effects of trodusquemine in the clinic, we investigated the effects of trodusquemine treatment in a murine model of diet-induced obesity (DIO). Trodusquemine suppressed appetite, reduced body weight (BW) in a fat-specific manner, and improved plasma insulin and leptin levels in mice. Screening assays revealed that trodusquemine selectively inhibited protein-tyrosine phosphatase 1B (PTP1B), a key enzyme regulating insulin and leptin signaling. Trodusquemine significantly enhanced insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) beta and STAT3, direct targets of PTP1B, in HepG2 cells in vitro and/or hypothalamic tissue in vivo. These data establish trodusquemine as an effective central and peripheral PTP1B inhibitor with the potential to elicit noncachectic fat-specific weight loss and improve insulin and leptin levels.

STa peptide analogs for probing guanylyl cyclase C.

Guanylyl cyclase C (GC-C), universally overexpressed on primary and metastatic colorectal carcinoma cells, is activated by endogenous ligands, guanylin, and uroguanylin, and by exogenous 18-residue heat-stable enterotoxins (STa) produced by diarrheagenic bacteria. Two 12-residue STa analogs with alternate combinations of two interlocked disulfide bonds, peptides 3 and 6, were synthesized by orthogonal solid phase synthesis routes. Peptides 3 and 6 bound GC-C with a rank order potency of STa > peptide 3 > peptide 6. Peptides 3 and 6 behaved as agonists in stimulating cGMP production. The results reveal that the toxic domain of STa can be reduced to 12 amino acids.

Statistical algorithm for assuring similar efficiency in standards and samples for absolute quantification by real-time reverse transcription polymerase chain reaction.

Reverse transcription (RT) followed by the polymerase chain reaction (PCR) is the method of choice for quantifying rare transcripts in biological samples. A key assumption underlying the absolute quantification of transcripts is similar amplification efficiencies of all external standards and samples. However, efficiencies can vary between individual reactions, a problem that can be magnified when quantifying transcripts of low abundance. Here, an algorithm to assure that calibration standards and samples meet the assumption of similar amplification efficiencies underlying absolute quantification is presented. Individual reaction efficiency is estimated by fitting an exponential growth model to the fluorescence data in the exponential phase of the reaction. Next, reactions of standards with outlying estimates of amplification rates are eliminated using the boxplot outlier detection rule. Then, estimates of amplification rates of outlier-free standards are employed to define exact tolerance intervals, which are used to eliminate kinetic outliers from test samples. This algorithm was employed to eliminate kinetic outliers prior to defining the baseline expression of guanylyl cyclase C mRNA, a marker for colorectal cancer, in blood of healthy volunteers. These studies demonstrate that elimination of kinetic outliers from calibration standards and test samples improves the accuracy of absolute transcript quantification by RT-PCR.