Transurethral injection of autologous muscle precursor cells for treatment of female stress urinary incontinence: a prospective phase I clinical trial.

INTRODUCTION AND HYPOTHESIS: The purpose was to investigate the safety and feasibility of transurethral injections of autologous muscle precursor cells (MPCs) into the external urinary sphincter (EUS) to treat stress urinary incontinence (SUI) in female patients. METHODS: Prospective and randomised phase I clinical trial. Standardised 1-h pad test, International Consultation on Incontinence Questionnaire-Urinary Incontinence Short Form (ICIQ-UI-SF), urodynamic study, and MRI of the pelvis were performed at baseline and 6 months after treatment. MPCs gained through open muscle biopsy were transported to a GMP facility for processing and cell expansion. The final product was injected into the EUS via a transurethral ultrasound-guided route. Primary outcomes were defined as any adverse events (AEs) during follow-up. Secondary outcomes were functional, questionnaire, and radiological results. RESULTS: Ten female patients with SUI grades I-II were included in the study and 9 received treatment. Out of 8 AEs, 3 (37.5%) were potentially related to treatment and treated conservatively: 1 urinary tract infection healed with antibiotics treatment, 1 dysuria and 1 discomfort at biopsy site. Functional urethral length under stress was 25 mm at baseline compared with 30 mm at 6 months' follow-up (p=0.009). ICIQ-UI-SF scores improved from 7 points at baseline to 4 points at follow-up (p=0.035). MRI of the pelvis revealed no evidence of tumour or necrosis, whereas the diameter of the EUS muscle increased from 1.8 mm at baseline to 1.9 mm at follow-up (p=0.009). CONCLUSION: Transurethral injections of autologous MPCs into the EUS for treatment of SUI in female patients can be regarded as safe and feasible. Only a minimal number of expected and easily treatable AEs were documented.

Quality Requirements for Medicinal Cannabis and Respective Products in the European Union - Status Quo.

Medicinal cannabis and respective products have been available in EU member states as single-patient prescriptions without regular marketing authorizations for a couple of years. The Netherlands was the first member state to realize this; in the meantime other member states have followed. Today, aside from the Netherlands, Germany is the most important market for such products. The regulatory framework for the approval of medicinal cannabis and its distribution to patients in the EU member states is, however, not harmonized at all, and there are distinct national regulations. Regarding the quality of such products, the general requirements for herbal medicinal products as defined in the European Pharmacopoeia, national pharmacopoeias, and the EMA guidance documents in place beside GMP requirements in the EU are applicable. However, for a couple of aspects, every EU member state follows its own interpretation of these requirements. To facilitate free distribution of such products between EU member states in future and to harmonize requirements for quality and GMP, an EU-wide approach is needed. As a first step, this should be realized by implementing monographs for cannabis medicinal products in the European Pharmacopoeia.

Urine biomarkers can predict prostate cancer and PI-RADS score prior to biopsy.

Prostate-Specific Antigen (PSA) based screening of prostate cancer (PCa) needs refinement. The aim of this study was the identification of urinary biomarkers to predict the Prostate Imaging-Reporting and Data System (PI-RADS) score and the presence of PCa prior to prostate biopsy. Urine samples from patients with elevated PSA were collected prior to prostate biopsy (cohort = 99). The re-analysis of mass spectrometry data from 45 samples was performed to identify urinary biomarkers to predict the PI-RADS score and the presence of PCa. The most promising candidates, i.e. SPARC-like protein 1 (SPARCL1), Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), Alpha-1-microglobulin/bikunin precursor (AMBP), keratin 13 (KRT13), cluster of differentiation 99 (CD99) and hornerin (HRNR), were quantified by ELISA and validated in an independent cohort of 54 samples. Various biomarker combinations showed the ability to predict the PI-RADS score (AUC = 0.79). In combination with the PI-RADS score, the biomarkers improve the detection of prostate carcinoma-free men (AUC = 0.89) and of those with clinically significant PCa (AUC = 0.93). We have uncovered the potential of urinary biomarkers for a test that allows a more stringent prioritization of mpMRI use and improves the decision criteria for prostate biopsy, minimizing patient burden by decreasing the number of unnecessary prostate biopsies.

Identification of Urine Biomarkers to Improve Eligibility for Prostate Biopsy and Detect High-Grade Prostate Cancer.

PCa screening is based on the measurements of the serum prostate specific antigen (PSA) to select men with higher risks for tumors and, thus, eligible for prostate biopsy. However, PSA testing has a low specificity, leading to unnecessary biopsies in 50-75% of cases. Therefore, more specific screening opportunities are needed to reduce the number of biopsies performed on healthy men and patients with indolent tumors. Urine samples from 45 patients with elevated PSA were collected prior to prostate biopsy, a mass spectrometry (MS) screening was performed to identify novel biomarkers and the best candidates were validated by ELISA. The urine quantification of PEDF, HPX, CD99, CANX, FCER2, HRNR, and KRT13 showed superior performance compared to PSA. Additionally, the combination of two biomarkers and patient age resulted in an AUC of 0.8196 (PSA = 0.6020) and 0.7801 (PSA = 0.5690) in detecting healthy men and high-grade PCa, respectively. In this study, we identified and validated novel urine biomarkers for the screening of PCa, showing that an upfront urine test, based on quantitative biomarkers and patient age, is a feasible method to reduce the number of unnecessary prostate biopsies and detect both healthy men and clinically significant PCa.

Systemic availability and pharmacokinetics of thymol in humans.

Essential oil compounds such as found in thyme extract are established for the therapy of chronic and acute bronchitis. Various pharmacodynamic activities for thyme extract and the essential thyme oil, respectively, have been demonstrated in vitro, but availability of these compounds in the respective target organs has not been proven. Thus, investigation of absorption, distribution, metabolism, and excretion are necessary to provide the link between in vitro effects and in vivo studies. To determine the systemic availability and the pharmacokinetics of thymol after oral application to humans, a clinical trial was carried out in 12 healthy volunteers. Each subject received a single dose of a Bronchipret TP tablet, which is equivalent to 1.08 mg thymol. No thymol could be detected in plasma or urine. However, the metabolites thymol sulfate and thymol glucuronide were found in urine and identified by LC-MS/MS. Plasma and urine samples were analyzed after enzymatic hydrolysis of the metabolites by headspace solid-phase microextraction prior to GC analysis and flame ionization detection. Thymol sulfate, but not thymol glucuronide, was detectable in plasma. Peak plasma concentrations were 93.1+/-24.5 ng ml(-1) and were reached after 2.0+/-0.8 hours. The mean terminal elimination half-life was 10.2 hours. Thymol sulfate was detectable up to 41 hours after administration. Urinary excretion could be followed over 24 hours. The amount of both thymol sulfate and glucuronide excreted in 24-hour urine was 16.2%+/-4.5% of the dose.

Urinary excretion and metabolism of arbutin after oral administration of Arctostaphylos uvae ursi extract as film-coated tablets and aqueous solution in healthy humans.

Bearberry leaves and preparations made from them are traditionally used for urinary tract infections. The urinary excretion of arbutin metabolites was examined in a randomized crossover design in 16 healthy volunteers after the application of a single oral dose of bearberry leaves dry extract (BLDE). There were two groups of application using either film-coated tablets (FCT) or aqueous solution (AS). The urine sample analysis was performed by a validated HPLC coolarray method (hydroquinone) and a validated capillary electrophoresis method (hydroquinone-glucuronide, hydroquinone-sulfate). The total amounts of hydroquinone equivalents excreted in the urine from BLDE were similar in both groups. With FCT, 64.8% of the arbutin dose administered was excreted; with AS, 66.7% was excreted (p = 0.61). The maximum mean urinary concentration of hydroquinone equivalents was a little higher and peaked earlier in the AS group versus the FCT group, although this did not reach statistical significance (Cur max = 1.6893 micromol/ml vs. 1.1250 micromol/ml, p = 0.13; tmax (t midpoint) = 3.60 h vs. 4.40 h, p = 0.38). The relative bioavailability of FCT compared to AS was 103.3% for total hydroquinone equivalents. There was substantial intersubject variability. No significant differences between the two groups were found in the metabolite patterns detected (hydroquinone, hydroquinone-glucuronide, and hydroquinone-sulfate).

Validated method for the determination of six metabolites derived from artichoke leaf extract in human plasma by high-performance liquid chromatography-coulometric-array detection.

A validated method was developed for the simultaneous determination of the hydroxycinnamates caffeic (CA), dihydrocaffeic (DHCA), ferulic (FA), dihydroferulic (DHFA), and isoferulic acid (IFA) and the flavonoid luteolin (LUT) in human plasma as metabolites derived from artichoke leaf extract. The method involves sample preparation followed by separation using high-performance liquid chromatography on reversed-phase material with a polar endcapping (Aqua-C(18), 250 x 4.6 mm). Selectivity and sensitivity towards the target compounds were achieved by electrochemical array detection (CoulArray). Calibration curves were constructed in the ranges 2.1-51.7 ng x mL(-1) (CA), 2.0-76.7 ng mL(-1) (DHCA), 2.2-53.7 ng x mL(-1) (FA), 2.1-79.2 ng x mL(-1) (DHFA), 1.1-52.6 ng x mL(-1) (IFA) and 2.1-258.6 ng x mL(-1) (LUT). Linearity could be shown for all target compounds over the entire calibration range. Values for within-day and between-day precision and accuracy were in accordance with the international guidelines for validation of bioanalytical methods. It is concluded that this newly developed method is appropriate for analysing samples from bioavailability and pharmacokinetic studies after oral administration of artichoke leaf extract.

Determination of thymol in human plasma by automated headspace solid-phase microextraction-gas chromatographic analysis.

A reliable and sensitive method was developed for determination of thymol in human plasma by automated headspace solid-phase microextraction (SPME). After enzymatic cleavage of thymol sulfate thymol was extracted by a 65 microm polydimethylsiloxane-divinylbenzene crimped fiber (Supelco) after addition of sodium chloride and phosphoric acid (85%). Desorption of the fiber was performed in the injection port of a gas chromatograph at 220 degrees C (HP 5890; 50 m x 0.2 mm I.D., 0.2 microm HP Innowax capillary column; flame ionization detection). Fibers were used repeatedly up to 40 analysis. The recovery was 5% after 35 min of extraction. The calibration curve was linear in the range of 8.1-203.5 ng ml(-1) with a limit of quantitation (LOQ) of 8.1 ng ml(-1). The within-day and between-day precision and accuracy were < or = 20% at the LOQ and <15% at higher concentrations according to international guidelines for validation of bioanalytical methods. After administration of a thymol-containing herbal extract only thymol sulfate, no free thymol, could be detected in human plasma, thus analysis of thymol was after enzymatic cleavage of thymol sulfate. It is concluded that the newly developed automated method can be used in clinical trials on bioavailability and pharmacokinetics of thymol-containing herbal medicinal products.

Validation of standardized high-performance thin-layer chromatographic methods for quality control and stability testing of herbals.

In herbal medicinal products the entire herbal drug or an herbal drug preparation is regarded as the active pharmaceutical ingredient, regardless of whether constituents with defined therapeutic activity are known. In quality control and stability testing of herbal medicinal products, fingerprint chromatograms are used as powerful tools to evaluate and compare the composition of compounds in such products. To fulfill the International Conference on Harmonization and Good Manufacturing Practice-based regulatory requirements in pharmaceutical quality control, chromatographic fingerprint analysis needs to be validated. Based on a standardized methodology, this paper provides a comprehensive concept for evaluating validation parameters for planar chromatographic fingerprinting by considering the stationary phase, sample application, developing solvent, chromatogram development, plate labeling, derivatization, documentation, and chromatographic equipment. Validation parameters addressed include stability of the analyte, selectivity, robustness testing, and method reproducibility.

Determination of ginsenosides from Panax ginseng using micellar electrokinetic chromatography.

A method for determination of ginsenosides in herbal medicinal products (HMPs) was developed using micellar electrokinetic chromatography (MEKC). Within 22 minutes 7 major ginsenosides were well separated. In order to demonstrate the accuracy, precision and robustness for the main target analyte the method was exemplarily validated for the determination of Rb1 according to ICH guidelines. Compared to chromatographic analysis, several benefits of capillary electrophoresis (CE) could be demonstrated such as high separation efficiency in an aqueous buffer without any organic solvent and shorter run time per assay.

Quantification of procyanidins in oral herbal medicinal products containing extracts of Crataegus species.

According to the European Pharmacopeia a photometric assay is used for the estimation of procyanidins in Crataegi fructus. This assay is also most commonly used for procyanidin analysis in herbal medicinal products (HMPs) containing extracts of hawthorn (Crataegus species). In order to find an appropriate method for the determination of oligomeric and polymeric procyanidins by analysing various preparations containing extracts of Crataegus, the Ph. Eur.-method was compared to an HPLC-method with chemical reaction detection (HPLC-CRD-method) and another conventional photometric assay using 4-dimethylamino-cinnamic-aldehyde (DMACA). Total procyanidins estimates obtained with the pharmacopeial method were, depending on the reference standard used, at least more than 50% higher than those obtained with the DMACA-assay. The determination of individual procyanidins could only be achieved by HPLC-CRD. Monomeric, dimeric, and trimeric procyanidins could be separated and detected individually, whereas no HPLC separation was possible for higher polymeric compounds. However, these compounds could be analysed as co-eluting groups. Using the DMACA method for the estimation of total oligomeric procyanidins and the HPLC-CRD method for quantification of the mono- up to trimeric procyanidins, some market leading herbal medicinal products from Germany containing extracts Crataegus species (C. monogyna Jacq., C. laevigata D.C., C. pentagyna Waldst. et Kit., C. nigra Waldst. et Kit, C. azarolus L.) were analysed. Procyanidin B2 (epicatechin-(4 beta-->8)-epicatechin) was isolated from Aesculus hippocastanum fruit shells as reference standard for calibration purposes. The structure elucidation was carried out by by means of MS and 1H-NMR. Quantitative 1H-NMR spectroscopy (qNMR) was applied for purity assessment.

Evaluation of glucoiberin reference material from Iberis amara by spectroscopic fingerprinting.

Increasing worldwide regulations require increased efforts toward validation of analytical and pharmacological reference materials. A detailed survey of glucoiberin, a prototype lead constituent of therapeutic value, using 1D/2D NMR, MS, and X-ray spectroscopy provided precise phytochemical data for structure assignment. Quantitative reference validation was achieved by the recently proposed qNMR method.

Pharmacokinetics and bioavailability of herbal medicinal products.

The use of herbs for treating various ailments dates back several centuries. Usually, herbal medicine has relied on tradition that may or may not be supported by empirical data. The belief that natural medicines are much safer than synthetic drugs has gained popularity in recent years and led to tremendous growth of phytopharmaceutical usage. Market driven information on natural products is widespread and has further fostered their use in daily life. In most countries there is no universal regulatory system that insures the safety and activity of phytopharmaceuticals. Evidence-based verification of the efficacy of HMPs (herbal medicinal products, botanicals) is still frequently lacking. However, in recent years, data on evaluation of the therapeutic and toxic activity of herbal medicinal products became available. The advances in analytical technology have led to discovery of many new active constituents and an ever-increasing list of putatively active constituents. Establishing the pharmacological basis for efficacy of HMPs is a constant challenge. Of particular interest is the question of bioavailability to assess to what degree and how fast compounds are absorbed after administration of HMPs. Of further interest is the elucidation of metabolic pathways (yielding potentially new active compounds), and the assessment of elimination routes and their kinetics. These data become an important issue to link data from pharmacological assays and clinical effects. Of interest are currently also interactions of herbal medicinal products with synthetically derived drug products. A better understanding of the pharmacokinetics and bioavailability of phytopharmaceuticals can also help in designing rational dosage regimens. In this review, pharmacokinetic and bioavailability studies that have been conducted for some of the more important or widely used phytopharmaceuticals are critically evaluated. Furthermore, various drug interactions are discussed which show that caution should be exercised when combining phytopharmaceuticals with chemically derived active pharmaceutical ingredients.