Merck and Cie

Schaffhausen, Switzerland

Merck and Cie

Schaffhausen, Switzerland
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Oswald M.,Merck Serono | Platscher M.,Merck and Cie | Geissler S.,Merck Serono | Goepferich A.,University of Regensburg
International Journal of Pharmaceutics | Year: 2016

Functionalized phospholipids are indispensable materials for the design of targeted liposomes. Control over the quality and quantity of phospholipids is thereby key in the successful development and manufacture of such formulations. This was also the case for a complex liposomal preparation composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), Cholesterol (CHO), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000). To this end, an RP-HPLC method was developed. Detection was done via evaporative light scattering (ELS) for liposomal components. The method was validated for linearity, precision, accuracy, sensitivity and robustness. The liposomal compounds had a non-linear quadratic response in the concentration range of 0.012-0.42 mg/ml with a correlation coefficient greater than 0.99 with an accuracy of method confirmed 95-105% of the theoretical concentration. Furthermore, degradation products from the liposomal formulation could be identified. The presented method was successfully implemented as a control tool during the preparation of functionalized liposomes. It underlined the benefit of HPLC analysis of phospholipids during liposome preparation as an easy and rapid control method for the functionalized lipid at each preparation step as well as for the quantification of all components. © 2015 Elsevier B.V. All rights reserved.


Ross T.L.,ETH Zurich | Honer M.,ETH Zurich | Muller C.,ETH Zurich | Groehn V.,Merck and Cie | And 2 more authors.
Journal of Nuclear Medicine | Year: 2010

The folate receptor is a proven target for folate-based diagnosis and treatment of cancer. Several folic acid conjugates have been developed as radiopharmaceuticals, but a suitable 18F-labeled folic acid derivative for routine clinical use is still lacking. The purpose of this study was to investigate the potential of 2′-18F-fluorofolic acid as a PET agent for folate receptor-positive tumors. Methods: The binding affinity of the cold reference compound 2′-fluorofolic acid was determined by in vitro displacement assays using human folate receptor-positive KB cells and 3H-folic acid. 18F labeling of 2′-fluorofolic acid was accomplished via a direct nucleophilic aromatic substitution of N 2-(N,N-dimethylamino-methylene)-2′-nitrofolic acid di-tertbutylester followed by acidic cleavage of the amino and carboxylic protecting groups. The new radiofolate was evaluated in nude mice bearing KB tumor xenografts under control and blocking conditions. Animals were either scanned from 75 to 105 min after injection of the radiotracer or sacrificed 75 min after injection for ex vivo biodistribution studies. Results: 2′-fluorofolic acid showed a high binding affinity (inhibition constant, 1.8 ± 0.1 nM) for the folate receptor. Direct aromatic 18F labeling of 2′-fluorofolic acid was achieved within 80 min via a convenient 2-step procedure in satisfactory radiochemical yields. The new radiotracer exhibited excellent pharmacokinetics with fast renal clearance and only moderate hepatobiliary elimination. Uptake of 29-18F-fluorofolic acid in folate receptor-positive KB tumors was high and specific, allowing a clear-cut visualization by PET. Conclusion: 2′-18F-fluorofolic acid, obtained via an integrated approach, is a promising PET agent for folate receptor-positive tumors and outperforms previously reported 18F-labeled folates. Copyright © 2010 by the Society of Nuclear Medicine, Inc.


Vakalopoulos A.,Bayer AG | Schmeck C.,Bayer AG | Thutewohl M.,Merck and Cie | Li V.,Bayer AG | And 5 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2011

Based on our former development candidate BAY 38-1315, optimization efforts led to the discovery of a novel chemical class of orally active cholesteryl ester transfer protein (CETP) inhibitors. The chromanol derivative 19b is a highly potent CETP inhibitor with favorable pharmacokinetic properties suitable for clinical studies. Chemical process optimization furnished a robust synthesis for a kilogram-scale process. © 2010 Elsevier Ltd. All rights reserved.


Green T.J.,University of British Columbia | Liu Y.,University of British Columbia | Dadgar S.,University of British Columbia | Li W.,University of British Columbia | And 2 more authors.
Journal of Nutrition | Year: 2013

Mandatory folic acid fortification of grains such as wheat flour has been introduced in several countries to reduce the incidence of neural tube defects. There are concerns, however, that folic acid could mask the hematologic signs of vitamin B-12 deficiency and lead to other adverse health outcomes in the population. Calcium L-5-methyltetrahydrofolic acid (L-5-MTHF), a synthetic form of reduced folate, should not mask vitamin B-12 deficiency and may be safer than folic acid. Unfortunately, L-5-MTHF is not stable in most food matrices such as bread. Microencapsulation of L-5-MTHF with sodium ascorbate and a modified starch is effective at preventing loss of the vitamin during baking and storage. Our aim was to assess the efficacy of wheat rolls fortified with microencapsulated L-5-MTHF or equimolar folic acid compared with wheat rolls containing no added folate (placebo) at increasing blood folate concentrations during 16 wk. Healthy men and women aged 18-45 y (n = 45) were randomly assigned to consume wheat rolls that contained L-5-MTHF (452 μg/d), the molar equivalent of folic acid (400 μg/d), or placebo. At 16 wk, the mean (95% CI) erythrocyte folate was 0.48 (0.27, 0.71) and 0.37 (0.17, 0.57) μmol/L higher in the L-5-MTHF (P < 0.001) and folic acid wheat roll (P = 0.001) groups, respectively, than in the placebo group. Likewise, the mean plasma folate was 23 (12, 34) and 23 (12, 34) nmol/L higher in the L-5-MTHF (P < 0.001) and folic acid wheat roll (P < 0.001) groups, respectively, than in the placebo group. There were no significant differences in blood folate concentrations between the L-5-MTHF and folic acid wheat roll groups. Both microencapsulated L-5-MTHF and folic acid-fortified wheat rolls increased blood folate concentrations compared with placebo. © 2013 American Society for Nutrition.


Burlina F.,University Pierre and Marie Curie | Morris C.,UK National Institute for Medical Research | Behrendt R.,Merck and Cie | White P.,Merck And Co. | Offer J.,UK National Institute for Medical Research
Chemical Communications | Year: 2012

We report a simplified procedure for the chemical ligation of peptides by using the sulfamylbutyryl linker as a mildly activating group capable of participating in ligation. When the peptidyl N-methylsulfonamide is directly added with excess thiols to ligation reactions, the speed of reaction is comparable to native chemical ligation. This journal is © The Royal Society of Chemistry 2012.


Haller S.,Paul Scherrer Institute | Reber J.,Paul Scherrer Institute | Brandt S.,University of Zürich | Bernhardt P.,Gothenburg University | And 4 more authors.
Nuclear Medicine and Biology | Year: 2015

Introduction: Application of therapeutic folate radioconjugates is a promising option for the treatment of folate receptor (FR)-positive tumors, although high uptake of radiofolates in the kidneys remains a critical issue. Recently, it was shown that enhancing the blood circulation of radiofolates results in increased tumor uptake and reduced retention of radioactivity in the kidneys. In this study, we investigated and compared the anti-tumor effects and potential long-term damage to the kidneys after application of an albumin-binding (177Lu-cm09), and a conventional (177Lu-EC0800) folate radioconjugate. Methods: In vivo studies were performed with KB tumor-bearing nude mice. 177Lu-EC0800 and 177Lu-cm09 were applied at variable quantities (10-30MBq/mouse), and the tumor growth was monitored over time. Mice without tumors were injected with the same radiofolates and investigated over eight months by determination of creatinine and blood urea nitrogen plasma levels and by measuring renal uptake of 99mTc-DMSA using SPECT. At the study end, the morphological changes were examined on renal tissue sections using variable staining methods. Results: Compared to untreated controls, dose-dependent tumor growth inhibition and prolonged survival was observed in all treated mice. In line with the resulting absorbed dose, the treatment was more effective with 177Lu-cm09 than with 177Lu-EC0800, enabling complete tumor remission after application of ≥20MBq (≥28Gy). Application of radiofolates with an absorbed renal dose ≥23Gy showed increased levels of renal plasma parameters and reduced renal uptake of 99mTc-DSMA. Morphological changes observed on tissue sections confirmed radionephropathy of variable stages. Conclusions: 177Lu-cm09 showed more favorable anti-tumor effects and significantly less damage to the kidneys compared to 177Lu-EC0800 as was expected based on improved tumor-to-kidney ratios. It was demonstrated that enhancing the blood circulation time of radiofolates was favorable regarding the risk-benefit profile of a therapeutic application. These results hold promise for future translation of the albumin-binder concept to the clinics, potentially enabling FR-targeted radionuclide therapy in patients. © 2015 Elsevier Inc.


Behrendt R.,Merck and Cie | Huber S.,Merck and Cie | White P.,Merck And Co.
Journal of Peptide Science | Year: 2016

In our efforts to develop a universal solution to the problem of aspartimide formation in Fmoc SPPS, we investigated the application of our new β-trialkylmethyl protected aspartic acid building blocks to the synthesis of peptides containing the Asp-Gly motif. The Nα-Fmoc aspartic acid β-tri-(ethyl/propyl/butyl)methyl esters were used in the synthesis of the classic model peptide scorpion toxin II (VKDGYI), and their effectiveness in minimising aspartimide formation during extended piperidine treatments was evaluated. Furthermore, we compared their efficacy against that of the commonly used approach of adding acids to the Fmoc deprotection solution. Finally, we applied our aspartic acid building blocks to the stepwise Fmoc SPPS of teduglutide, a human GLP-2 analogue, whose synthesis is made challenging by extensive aspartimide formation. In all experiments, our approach led to almost complete reduction of aspartimide formation with accompanied suppression of aspartic acid epimerisation. © 2016 European Peptide Society and John Wiley & Sons, Ltd.


Betzel T.,ETH Zurich | Muller C.,Paul Scherrer Institute | Groehn V.,Merck and Cie | Muller A.,ETH Zurich | And 6 more authors.
Bioconjugate Chemistry | Year: 2013

The folate receptor (FR) has been identified as a valuable target for the imaging of cancer and activated macrophages, involved in inflammatory and autoimmune diseases via positron emission tomography (PET). Therefore, conjugates of folic acid have been synthesized by coupling of a radiolabeled prosthetic group to the glutamate part of folic acid (pendent approach). In this work, we present a novel class of folates, where the phenyl ring of folic acid was isosterically replaced by a pyridine moiety for direct labeling with [ 18F]fluoride (integrated approach). 3′-Azafolic acid and its 2′-halogenated derivatives (2′-chloro and 2′-fluoro) were evaluated in vitro to determine their binding affinity. 3′-Aza-2′- [18F]fluorofolic acid ([18F]6) was obtained, starting from N2-acetyl-3′-aza-2′-chlorofolic acid di-tert-butylester (2), in a maximum decay corrected radiochemical yield of about 9% in ≥98% radiochemical purity and high specific activities of 35-127 GBq/μmol. Binding affinity to the FR was high (IC50 = 0.8 ± 0.2 nM), and the radiotracer was stable in human plasma over 4 h at 37 C. No degradation or defluorination was detected after incubation of the radiotracer for 1 h at 37 C with human and murine liver microsomes and human S9-fraction. In vivo PET imaging and biodistribution studies with mice demonstrated a high and specific uptake in FR-positive KB tumor xenografts (12.59 ± 1.77% ID/g, 90 min p.i.). A high and specific accumulation of radioactivity was observed in the kidneys (57.33 ± 8.40% ID/g, 90 min p.i.) and salivary glands (14.09 ± 0.93% ID/g, 90 min p.i.), which are known to express the FR and nonspecific uptake found in the liver (10.31 ± 2.37% ID/g, 90 min p.i.). Preinjection of folic acid resulted in a >85% reduced uptake of [ 18F]6 in FR-positive tissues (xenografts, kidneys, and salivary glands). Furthermore, no radioactive metabolites were detected in the blood, urine, or tumor tissue, 30 min p.i. These characteristics indicate that this new 18F-labeled 3′-azafolate is an appropriate tool for imaging FR-positive (malignant) tissue. © 2012 American Chemical Society.


Fischer C.R.,ETH Zurich | Groehn V.,Merck and Cie | Reber J.,Paul Scherrer Institute | Schibli R.,ETH Zurich | And 3 more authors.
Molecular Imaging and Biology | Year: 2013

Purpose: The folate receptor (FR) is a promising target for nuclear imaging due to its overexpression in many different cancer types. A drawback of using folate radioconjugates is the high accumulation of radioactivity in the kidneys. Therefore, the aim of this study was to develop a 18 F-labeled folate conjugate with an albumin-binding entity to enhance the blood circulation time and hence improve the tumor-to-kidney ratio. Procedures: The novel 18 F-folate was prepared by conjugation of a 18 F-labeled glucose azide to an alkyne-functionalized folate precursor containing an albumin-binding entity via Cu(I)-catalyzed 1,3-dipolar cycloaddition. The radioconjugate was tested in vitro on FR-positive KB tumor cells and by biodistribution and positron emission tomography (PET) imaging studies using KB tumor-bearing mice. Results: The radiosynthesis of the albumin-binding [ 18 F]fluorodeoxyglucose-folate ([18 F]3) resulted in a radiochemical yield of 1-2 % decay corrected (d.c.) and a radiochemical purity of ≥95 %. The specific activity of [18 F]3 ranged from 20 to 50 GBq/μmol. In vitro experiments revealed FR-specific binding of [18 F]3 to KB tumor cells. In vivo we found an increasing uptake of [18 F]3 into tumor xenografts over time reaching a value of ∼ 15 % injected dose (ID)/g at 4 h post-injection (p.i.). Uptake in the kidneys (∼ 13 % ID/g; 1 h p.i.) was approximately fourfold reduced compared to previously published 18 F-labeled folic acid derivatives. An excellent visualization of tumor xenografts with an unprecedentedly high tumor-to-kidney ratio (∼ 1) was obtained by PET imaging. Conclusions: [18 F]3 showed a favorable accumulation in tumor xenografts compared to the same folate conjugate without albumin-binding properties. Moreover, the increased tumor-to-kidney ratios improved the PET imaging quality significantly, in spite of a somewhat higher background radioactivity which was a consequence of the slower blood clearance of [18 F]3. © 2013 World Molecular Imaging Society.


PubMed | Merck and Cie, Laue Langevin Institute, Sahlgrenska University Hospital and Paul Scherrer Institute
Type: Journal Article | Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine | Year: 2014

In recent years, (47)Sc has attracted attention because of its favorable decay characteristics (half-life, 3.35 d; average energy, 162 keV; E, 159 keV) for therapeutic application and for SPECT imaging. The aim of the present study was to investigate the suitability of (47)Sc for radionuclide therapy in a preclinical setting. For this purpose a novel DOTA-folate conjugate (cm10) with an albumin-binding entity was used.(47)Sc was produced via the (46)Ca(n,)(47)Ca[Formula: see text](47)Sc nuclear reaction at the high-flux reactor at the Institut Laue-Langevin. Separation of the (47)Sc from the target material was performed by a semi-automated process using extraction chromatography and cation exchange chromatography. (47)Sc-labeled cm10 was tested on folate receptor-positive KB tumor cells in vitro. Biodistribution and SPECT imaging experiments were performed in KB tumor-bearing mice. Radionuclide therapy was conducted with two groups of mice, which received either (47)Sc-cm10 (10 MBq) or only saline. Tumor growth and survival time were compared between the two groups of mice.Irradiation of (46)Ca resulted in approximately 1.8 GBq of (47)Ca, which subsequently decayed to (47)Sc. Separation of (47)Sc from (47)Ca was obtained with 80% yield in only 10 min. The (47)Sc was then available in a small volume (500 L) of an ammonium acetate/HCl (pH 4.5) solution suitable for direct radiolabeling. (47)Sc-cm10 was prepared with a radiochemical yield of more than 96% at a specific activity of up to 13 MBq/nmol. In vitro (47)Sc-cm10 showed folate receptor-specific binding and uptake into KB tumor cells. In vivo SPECT/CT images allowed the visualization of accumulated radioactivity in KB tumors and in the kidneys. The therapy study showed a significantly delayed tumor growth in mice, which received (47)Sc-cm10 (10 MBq, 10 Gy) resulting in a more than 50% increase in survival time, compared with untreated control mice.With this study, we demonstrated the suitability of using (47)Sc for therapeutic purposes. On the basis of our recent results obtained with (44)Sc-folate, the present work confirms the applicability of (44)Sc/(47)Sc as an excellent matched pair of nuclides for PET imaging and radionuclide therapy.

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