Influence of Wound Fluid on the Transdifferentiation of Human Mesenchymal Bone Marrow Stem Cells into Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAF) in the tumor microenvironment have a decisive influence on tumor growth and metastatic behavior. The cellular origins as well as the stimuli leading to CAF formation are heterogenous, impeding a precise characterization. Aim of this study was to analyze the influence of cytokines secreted in the process of wound healing, tumor cell-associated paracrine-secreted factors, and direct cell-cell contact on the expression of the CAF-associated markers fibroblast activation protein (FAP), α-smooth muscle actin (α-SMA), thrombospondin-1 (THBS1), and tenascin-c (TNC) by RT-PCR in mesenchymal stem cells (MSC). Cells developed different morphological characteristics after incubation with wound fluid (WF). Moreover, expression of FAP and α-SMA in MSC was significantly reduced after WF compared to tumor-conditioned medium and in co-culture with tumor cells; THBS1 and TNC were not significantly altered after any of the different incubation methods. There were no alterations of expression patterns of FAP and α-SMA in the immunohistochemical analysis. Differ-ences in the cytokine composition of the media were found in the dot blot. The heterogeneity of the results emphasizes the complexity of the interactions of tumor cells and cells of the microenvironment, particularly through the addition of human-derived WF.

Evaluation of the drug-drug interaction potential of treosulfan using a physiologically-based pharmacokinetic modelling approach.

AIMS: The aim of this work is the development of a mechanistic physiologically-based pharmacokinetic (PBPK) model using in vitro to in vivo extrapolation to conduct a drug-drug interaction (DDI) assessment of treosulfan against two cytochrome p450 (CYP) isoenzymes and P-glycoprotein (P-gp) substrates. METHODS: A PBPK model for treosulfan was developed de novo based on literature and unpublished clinical data. The PBPK DDI analysis was conducted using the U.S. Food and Drug Administration (FDA) DDI index drugs (probe substrates) midazolam, omeprazole and digoxin for CYP3A4, CYP2C19 and P-gp, respectively. Qualified and documented PBPK models of the probe substrates have been adopted from an open-source online model database. RESULTS: The PBPK model for treosulfan, based on both in vitro and in vivo data, was able to predict the plasma concentration-time profiles and exposure levels of treosulfan applied for a standard conditioning treatment. Medium and low potentials for DDI on CYP3A4 (maximum area under the concentration-time curve ratio (AUCRmax = 2.23) and CYP2C19 (AUCRmax = 1.6) were predicted, respectively, using probe substrates midazolam and omeprazole. Treosulfan was not predicted to cause a DDI on P-gp. CONCLUSION: Medicinal products with a narrow therapeutic index (eg, digoxin) that are substrates for CYP3A4, CYP2C19 or P-gp should not be given during treatment with treosulfan. However, considering the comprehensive treosulfan-based conditioning treatment schedule and the respective pharmacokinetic properties of the concomitantly used drugs (eg, half-life), the potential for interaction on all evaluated mechanisms would be low (AUCR < 1.25), if concomitantly administered drugs are dosed either 2 hours before or 8 hours after the 2-hour intravenous infusion of treosulfan.

Non-clinical assessment of safety, biodistribution and tumorigenicity of human mesenchymal stromal cells.

Guidelines regulating the development of advanced therapy medicinal products (ATMPs) request nonclinical data for toxicity, biodistribution and tumorigenicity before mesenchymal stromal cell (MSC) products can be administered in large clinical trials. We assessed the biodistribution/persistence, safety and tumorigenicity of MC0518, a human allogeneic MSC product from pooled bone marrow mononuclear cells of eight healthy, adult, unrelated donors, which is currently investigated for the treatment of steroid-refractory acute Graft-versus-Host Disease (aGvHD) after hematopoietic stem cell transplantation. In our GLP studies, immuno-deficient mice were administered repeat doses of MC0518 (once weekly for 6 weeks, i.v.) at doses exceeding the proposed human clinical dose 20-60-fold. No signs of toxicity were observed in the combined biodistribution/toxicity study. Human MSCs in mouse tissues were detected by quantitative PCR (qPCR) and in situ hybridization (ISH). MC0518 showed initial trapping in the lung, occasional distribution into other organs and low tissue persistence beyond 24 h after application. No MSC-induced tumors of human origin were identified after a follow-up of six months. Additionally, we found that the combination of different detection methods (qPCR and ISH) is crucial for a reliable interpretation of biodistribution results. Our data suggest that MC0518 is safe for use in human.

Penetration of Treosulfan and its Active Monoepoxide Transformation Product into Central Nervous System of Juvenile and Young Adult Rats.

Treosulfan (TREO) is currently investigated as an alternative treatment of busulfan in conditioning before hematopoietic stem cell transplantation. The knowledge of the blood-brain barrier penetration of the drug is still scarce. In this paper, penetration of TREO and its active monoepoxide (S,S-EBDM) and diepoxide (S,S-DEB) into the CNS was studied in juvenile (JR) and young adult rats (YAR) for the first time. CD rats of both sexes (n = 96) received an intravenous dose of TREO 500 mg/kg b.wt. Concentrations of TREO, S,S-EBDM, and S,S-DEB in rat plasma, brain, and cerebrospinal fluid (CSF, in YAR only) were determined by validated bioanalytical methods. Pharmacokinetic calculations were performed in WinNonlin using a noncompartmental analysis and statistical evaluation was done in Statistica software. In male JR, female JR, male YAR, and female YAR, the brain/plasma area under the curve (AUC) ratio for unbound TREO was 0.14, 0.17, 0.10, and 0.07 and for unbound S,S-EBDM, it was 0.52, 0.48, 0.28, and 0.22, respectively. The CSF/plasma AUC ratio in male and female YAR was 0.12 and 0.11 for TREO and 0.66 and 0.64 for S,S-EBDM, respectively. Elimination rate constants of TREO and S,S-EBDM in all the matrices were sex-independent with a tendency to be lower in the JR. No quantifiable levels of S,S-DEB were found in the studied samples. TREO and S,S-EBDM demonstrated poor and sex-independent penetration into CNS. However, the brain exposure was greater in juvenile rats, so very young children might potentially be more susceptible to high-dose TREO-related CNS exposure than young adults.

Dicofol degradation to p,p'-dichlorobenzophenone - a potential antiandrogen.

In the present investigation, the degradation of the acaricide dicofol (also known as kelthane) was investigated with special emphasis on generation of p,p'-dichlorobenzophenone (DCBP) under alkaline conditions as well as induced by UV-light. Dicofol was also incubated in the presence and absence of microsomal preparations to measure potential metabolic formation of DCBP. The results indicate that the degradation of dicofol to DCBP primarily proceeds as an abiotic process via hydroxide ion catalysed elimination of a trichloromethyl anion. The generated anion picks up a proton from the solvent to generate chloroform. Microsomal metabolism does not appear to play a major role in the degradation of dicofol. DCBP is structurally analogous to the antiandrogen p,p'-dichlorodiphenylethene (DDE). We therefore investigated whether DCBP displays antiandrogenic properties. In an in vitro transactivation system utilising transiently transfected African green monkey kidney (COS-7) cells, DCBP showed potent antiandrogenic efficacy. This finding was confirmed by further studies in T47D human mammary carcinoma cells by measuring mRNA and protein expression of androgen dependent genes i.e. TRMP-2 (testosterone-repressed prostate message-2) mRNA and PSA (prostate-specific antigen) protein.

Microsurgical access flap in conjunction with enamel matrix derivative for the treatment of intra-bony defects: a controlled clinical trial.

OBJECTIVES: The aims of this controlled study were to clinically and radiographically evaluate the effect of a microsurgical approach for the treatment of intra-bony defects with and without an enamel matrix derivative (EMD). Parts of this study population were already published by Wachtel and colleagues in 2003. MATERIAL AND METHODS: Seventy intra-bony defects were randomly assigned to a microsurgical access flap with application of EMD (test group) and on the contra-lateral side to a microsurgical access flap alone (control group). Clinical and radiographic parameters were assessed at baseline and after 6 and 12 months. RESULTS: Both test and control treatments resulted in a statistically significant mean clinical attachment level (CAL) gain, probing pocket depth (PPD) reduction and radiographic bone fill. The test group yielded statistically significantly more CAL gain, PPD reduction and radiographic bone fill than the control group. Gingival recession increase after 12 months averaged 0.5 and 0.7 mm for the test and control groups, and did not reach statistical significance. Two weeks after surgery, primary wound closure was maintained in 91% of the test sites and 97% of the control sites. CONCLUSION: The combination of a microsurgical access flap with EMD seems to be superior to open flap debridement in terms of PPD reduction, CAL gain and radiographic bone fill. In the test as well as the control group, primary wound closure was successfully achieved.

Sensitive in vitro test systems to determine androgenic/antiandrogenic activity.

We report on the establishment of one transgenic and two endogenous reporter gene assays to determine androgenic/antiandrogenic activity. A transient transactivation assay was developed in COS-7 African green monkey kidney cells. Three plasmids were co-transfected by electroporation: the human androgen receptor expression vector pSG5AR, the reporter gene vector pMamneoLuc, expressing luciferase under the control of the mouse mammary tumor virus (MMTV) promotor containing 4 hormone responsive elements (HREs), and the control plasmid pSVbeta. Transcriptional activation was measured by luciferase-mediated chemoluminescence. In T47D human breast cancer cells two endogenous reporter gene systems were established: one based on the androgen-induced expression of prostate-specific antigen (PSA), the other based on the androgen-repressed expression of testosterone repressed message 2 (TRPM-2). PSA protein was measured by enzyme-linked immunosorbent assay (ELISA), TRPM-2 m-RNA by reverse transcriptase polymerase chain reaction (RT-PCR). All three test systems were validated using the physiological androgen dihydrotestosterone (DHT) as agonist and the established antiandrogens hydroxyflutamide and p,p'-dichlorodiphenylethene (p,p'-DDE) as antagonists. The PSA assay was the most sensitive test system with an EC50 of 0.7 nM for DHT-induced response. The transient transactivation assay in COS-7 cells was less sensitive with an EC50 of 9.7 nM DHT. In the PSA assay hydroxyflutamide was a more potent antagonist (IC30 = 0.02 microM) than p,p'-DDE (IC30 = 0.9 microM). In the transient transactivation assay in COS-7 cells, both compounds elicited about 30% of the agonistic response induced by 100 nM DHT, thus showing partial agonistic properties. In summary, three highly sensitive and complementary in vitro test systems, together achieving enhanced specificity for detection and assessment of androgenic/antiandrogenic activity have been established and validated.

Microsurgical access flap and enamel matrix derivative for the treatment of periodontal intrabony defects: a controlled clinical study.

OBJECTIVES: Application of the guided tissue regeneration (GTR) principle and utilization of enamel matrix derivative (EMD) have both been shown to result in periodontal regeneration. While clinical investigations have demonstrated that the use of a microsurgical concept in combination with the GTR technique positively affects the percentage of primary closure and the amount of tissue preservation, no such information is available for EMD-treated periodontal defects. It was the aim of the present investigation to assess the clinical effect of the microsurgical access flap and EMD treatment with an emphasis on the evaluation of early wound healing. MATERIAL AND METHODS: Eleven patients displaying at least one pair of intrabony periodontal defects with an intrabony component of > or =3 mm participated in the study. At baseline and at 6 and 12 months after surgery, the following clinical parameters were assessed by a blinded examiner: oral hygiene status (API), gingival inflammation (BOP), probing pocket depth (PPD), clinical attachment level (CAL) and gingival recession (GR). Defects were randomly assigned to test or control treatment, which both consisted of a microsurgical access flap procedure designed for maximum tissue preservation. The exposed root surfaces of the test sites were conditioned with a 24% EDTA gel followed by EMD (Emdogain(R)) application. Primary flap closure was achieved by a 2-layered suturing technique. Postoperative healing was evaluated by a newly introduced early wound-healing index (EHI) at 1 and 2 weeks after surgery. RESULTS: Both test and control treatment resulted in a statistically significant mean CAL gain of 2.8 and 2.0 mm at 6 months, and 3.6 and 1.7 mm at 12 months, respectively (p<0.05). Differences in CAL gain between the two treatment modalities were statistically significant at both time points (p<0.05). Additional GR values after 12 months averaged 0.3 and 0.4 mm for test and control sites, respectively, and did not reach statistical significance (p> or =0.05). Two weeks after surgery, primary closure was maintained in 89% of the test sites and in 96% of the control sites. CONCLUSION: Both treatment modalities using the microsurgical flap procedure resulted in a high percentage of primary flap closure and maximum tissue preservation. In terms of PPD reduction and CAL gain, the combination with EMD application appeared to be superior to the microsurgical access flap alone.