Jens Köhler

Afatinib, Erlotinib and Gefitinib in the First-Line Therapy of EGFR Mutation-Positive Lung Adenocarcinoma: A Review

Non-small cell lung cancer (NSCLC) consists of several histomorphologically defined phenotypes that display an enormous genetic variability. In recent years, epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinoma has emerged as a unique subset of NSCLC in terms of etiopathogenesis and tumor biology. Since the introduction of the reversible EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib, patients with metastatic EGFR mutation-positive lung cancer can be offered a therapeutic alternative that has proven its superiority over standard platinum-based chemotherapy. However, primary or acquired resistance limits the therapeutic success of these targeted agents. Irreversible inhibitors targeting all ErbB family receptor tyrosine kinases, such as afatinib and dacomitinib, have been developed to confer sustained disease control in ErbB-dependent cancers. The large LUX-Lung 3 phase III trial recently reported afatinib to be clearly superior over the most effective platinum doublet in patients with EGFR mutation-positive lung cancer. To fully exploit the clinical activity of afatinib, proactive management of its gastrointestinal and dermatologic toxicities is advised.

Synthesis and SAR studies of chiral non-racemic dexoxadrol analogues as uncompetitive NMDA receptor antagonists.

A series of chiral non-racemic dexoxadrol analogues with various substituents in position 4 of the piperidine ring was synthesized and pharmacologically evaluated. Only the enantiomers having (S)-configuration at the 2-position of the piperidine ring and 4-position of the dioxolane ring were considered. Key steps in the synthesis were an imino-Diels-Alder reaction of enantiomerically pure imine (S)-13, which had been obtained from d-mannitol, with Danishefskys Diene 14 and the replacement of the p-methoxybenzyl protective group with a Cbz-group. It was shown that (S,S)-configuration of the ring junction (position 2 of the piperidine ring and position 4 of the dioxolane ring) and axial orientation of the C-4-substituent ((4S)-configuration) are crucial for high NMDA receptor affinity. 2-(2,2-Diphenyl-1,3-dioxolan-4-yl)piperidines with a hydroxy moiety ((S,S,S)-5, K(i)=28nM), a fluorine atom ((S,S,S)-6, WMS-2539, K(i)=7nM) and two fluorine atoms ((S,S)-7, K(i)=48nM) in position 4 represent the most potent NMDA antagonists with high selectivity against σ(1) and σ(2) receptors and the polyamine binding site of the NMDA receptor. The NMDA receptor affinities of the new ligands were correlated with their electrostatic potentials, calculated gas phase proton affinities (negative enthalpies of deprotonation) and dipole moments. According to these calculations decreasing proton affinity and increasing dipole moment are correlated with decreasing NMDA receptor affinity.

Enantiomerically Pure 1,3-Dioxanes as Highly Selective NMDA and σ1 Receptor Ligands

We synthesized and investigated the NMDA and σ₁ receptor affinity of enantiomerically pure 2-(2-phenyl-1,3-dioxan-4-yl)ethanamines 17-26. The primary amines (R,R)-18-20 with an axially oriented phenyl moiety in position 2 interacted with high enantioselectivity (eudismic ratios 70-130) and high affinity (K(i)((R,R)-19) = 13 nM) with the PCP binding site of the NMDA receptor. Introduction of an N-benzyl moiety led to potent σ₁ ligands including compound (S,R)-22 (K(i) = 6 nM) with an equatorially oriented phenyl moiety in position 2.

Development of a highly sensitive and specific method for detection of circulating tumor cells harboring somatic mutations in non-small-cell lung cancer patients.

Background Oncogenic mutations are powerful predictive biomarkers for molecularly targeted cancer therapies. For mutation detection patients have to undergo invasive tumor biopsies. Alternatively, archival samples are used which may no longer reflect the actual tumor status. Circulating tumor cells (CTC) could serve as an alternative platform to detect somatic mutations in cancer patients. We sought to develop a sensitive and specific assay to detect mutations in the EGFR gene in CTC from lung cancer patients. Methods We developed a novel assay based on real-time polymerase chain reaction (PCR) and melting curve analysis to detect activating EGFR mutations in blood cell fractions enriched in CTC. Non-small-cell lung cancer (NSCLC) was chosen as disease model with reportedly very low CTC counts. The assay was prospectively validated in samples from patients with EGFR-mutant and EGFR-wild type NSCLC treated within a randomized clinical trial. Sequential analyses were conducted to monitor CTC signals during therapy and correlate mutation detection in CTC with treatment outcome. Results Assay sensitivity was optimized to enable detection of a single EGFR-mutant CTC/mL peripheral blood. CTC were detected in pretreatment blood samples from all 8 EGFR-mutant lung cancer patients studied. Loss of EGFR-mutant CTC signals correlated with treatment response, and its reoccurrence preceded relapse. Conclusions Despite low abundance of CTC in NSCLC oncogenic mutations can be reproducibly detected by applying an unbiased CTC enrichment strategy and highly sensitive PCR and melting curve analysis. This strategy may enable non-invasive, specific biomarker diagnostics and monitoring in patients undergoing targeted cancer therapies.

Hybrid [18F]-FDG PET/MRI including non-Gaussian diffusion-weighted imaging (DWI): Preliminary results in non-small cell lung cancer (NSCLC)

PURPOSE To assess the feasibility of non-Gaussian DWI as part of a FDG-PET/MRI protocol in patients with histologically proven non-small cell lung cancer. MATERIAL AND METHODS 15 consecutive patients with histologically proven NSCLC (mean age 61 ± 11 years) were included in this study and underwent whole-body FDG-PET/MRI following whole-body FDG-PET/CT. As part of the whole-body FDG-PET/MRI protocol, an EPI-sequence with 5 b-values (0, 100, 500, 1000 and 2000 s/mm(2)) was acquired for DWI of the thorax during free-breathing. Volume of interest (VOI) measurements were performed to determine the maximum and mean standardized uptake value (SUV(max); SUV(mean)). A region of interest (ROI) was manually drawn around the tumor on b=0 images and then transferred to the corresponding parameter maps to assess ADC(mono), D(app) and K(app). To assess the goodness of the mathematical fit R(2) was calculated for monoexponential and non-Gaussian analysis. Spearmans correlation coefficients were calculated to compare SUV values and diffusion coefficients. A Students t-test was performed to compare the monoexponential and non-Gaussian diffusion fitting (R(2)). RESULTS T staging was equal between FDG-PET/CT and FDG-PET/MRI in 12 of 15 patients. For NSCLC, mean ADC(mono) was 2.11 ± 1.24 × 10(-3) mm(2)/s, Dapp was 2.46 ± 1.29 × 10(-3) mm(2)/s and mean Kapp was 0.70 ± 0.21. The non-Gaussian diffusion analysis (R(2)=0.98) provided a significantly better mathematical fitting to the DWI signal decay than the monoexponetial analysis (R(2)=0.96) (p<0.001). SUV(max) and SUV(mean) of NSCLC was 13.5 ± 7.6 and 7.9 ± 4.3 for FDG-PET/MRI. ADC(mono) as well as Dapp exhibited a significant inverse correlation with the SUV(max) (ADC(mono): R=-0.67; p<0.01; Dapp: R=-0.69; p<0.01) as well as with SUV(mean) assessed by FDG-PET/MRI (ADC(mono): R=-0.66; p<0.01; Dapp: R=-0.69; p<0.01). Furthermore, Kapp exhibited a significant correlation with SUV(max) (R=0.72; p<0.05) and SUV(mean) as assessed by FDG-PET/MRI (R=0.71; p<0.005). CONCLUSION Simultaneous PET and non-Gaussian diffusion acquisitions are feasible. Non-Gaussian diffusion parameters show a good correlation with SUV and might provide additional information beyond monoexponential ADC, especially as non-Gaussian diffusion exhibits better mathematical fitting to the decay of the diffusion signal than monoexponential DWI.

Lestaurtinib Inhibits Histone Phosphorylation and Androgen-Dependent Gene Expression in Prostate Cancer Cells

Background Epigenetics is defined as heritable changes in gene expression that are not based on changes in the DNA sequence. Posttranslational modification of histone proteins is a major mechanism of epigenetic regulation. The kinase PRK1 (protein kinase C related kinase 1, also known as PKN1) phosphorylates histone H3 at threonine 11 and is involved in the regulation of androgen receptor signalling. Thus, it has been identified as a novel drug target but little is known about PRK1 inhibitors and consequences of its inhibition. Methodology/Principal Finding Using a focused library screening approach, we identified the clinical candidate lestaurtinib (also known as CEP-701) as a new inhibitor of PRK1. Based on a generated 3D model of the PRK1 kinase using the homolog PKC-theta (protein kinase c theta) protein as a template, the key interaction of lestaurtinib with PRK1 was analyzed by means of molecular docking studies. Furthermore, the effects on histone H3 threonine phosphorylation and androgen-dependent gene expression was evaluated in prostate cancer cells. Conclusions/Significance Lestaurtinib inhibits PRK1 very potently in vitro and in vivo. Applied to cell culture it inhibits histone H3 threonine phosphorylation and androgen-dependent gene expression, a feature that has not been known yet. Thus our findings have implication both for understanding of the clinical activity of lestaurtinib as well as for future PRK1 inhibitors.

Correlation of the Apparent Diffusion Coefficient (ADC) with the Standardized Uptake Value (SUV) in Lymph Node Metastases of Non-Small Cell Lung Cancer (NSCLC) Patients Using Hybrid 18F-FDG PET/MRI

Objective To compare the apparent diffusion coefficient (ADC) in lymph node metastases of non-small cell lung cancer (NSCLC) patients with standardized uptake values (SUV) derived from combined 18F-fluoro-deoxy-glucose-positron emission tomography/magnetic resonance imaging (18F-FDG PET/MRI). Material and Methods 38 patients with histopathologically proven NSCLC (mean age 60.1 ± 9.5y) received whole-body PET/CT (Siemens mCT™) 60min after injection of a mean dose of 280 ± 50 MBq 18F-FDG and subsequent PET/MRI (mean time after tracer injection: 139 ± 26 min, Siemens Biograph mMR). During PET acquisition, simultaneous diffusion-weighted imaging (DWI, b values: 0, 500, 1000 s/mm²) was performed. A maximum of 10 lymph nodes per patient suspicious for malignancy were analyzed. Regions of interest (ROI) were drawn covering the entire lymph node on the attenuation-corrected PET-image and the monoexponential ADC-map. According to histopathology or radiological follow-up, lymph nodes were classified as benign or malignant. Pearson’s correlation coefficients were calculated for all lymph node metastases correlating SUVmax and SUVmean with ADCmean. Results A total of 146 suspicious lymph nodes were found in 25 patients. One hundred lymph nodes were eligible for final analysis. Ninety-one lymph nodes were classified as malignant and 9 as benign according to the reference standard. In malignant lesions, mean SUVmax was 9.1 ± 3.8 and mean SUVmean was 6.0 ± 2.5 while mean ADCmean was 877.0 ± 128.6 x10-5 mm²/s in PET/MRI. For all malignant lymph nodes, a weak, inverse correlation between SUVmax and ADCmean as well as SUVmean and ADCmean (r = -0.30, p<0.05 and r = -0.36, p<0.05) existed. Conclusion The present data show a weak inverse correlation between increased glucose-metabolism and cellularity in lymph node metastases of NSCLC patients. 18F-FDG-PET and DWI thus may offer complementary information for the evaluation of treatment response in lymph node metastases of NSCLC.

Synthesis, Characterization, and Metabolism Studies of Fluspidine Enantiomers

The enantiomers of the potent σ1 ligand fluspidine (1) were prepared by using chiral preparative HPLC. Synthesis of racemic tosylate 2 and subsequent separation of enantiomers yielded (R)‐2 and (S)‐2 in excellent enantiomeric purities. The fluspidine enantiomers (R)‐1 and (S)‐1 were synthesized from (R)‐2 and (S)‐2 by nucleophilic substitution with tetra‐n‐butylammonium fluoride, affording (R)‐1 with 99.6 % ee and (S)‐1 with 96.4 % ee. Tosylates (R)‐2 and (S)‐2 can also serve as precursors for the radiosynthesis of enantiomerically pure radiotracers [18F](R)‐1 and [18F](S)‐1. The absolute configuration of the pure enantiomers was elucidated by comparison of their CD spectra with a calculated CD spectrum of a simplified model compound. In receptor binding studies, both enantiomers displayed very high σ1 receptor affinity and selectivity against the σ2 receptor. (R)‐Fluspidine ((R)‐1) is the eutomer, with a Ki value of 0.57 nM and a eudysmic ratio of 4. Incubation of (R)‐1 and (S)‐1 with rat liver microsomes led to the identification of seven and eight metabolites, respectively. Although the S‐configured enantiomer formed additional metabolite (S)‐1‐3, it is metabolically more stable than (R)‐1.

Synthesis of 4-(aminoalkyl) substituted 1,3-dioxanes as potent NMDA and σ receptor antagonists.

Elongation of the distance between the oxygen heterocycle and the basic amino moiety or ring expansion of the oxygen heterocycle of the NMDA receptor antagonists dexoxadrol and etoxadrol led to compounds with promising NMDA receptor affinity. Herein the combination of both structural features, i.e. elongation of the O-heterocycle--amine distance with a 1,3-dioxane ring is envisaged. The synthesis of aminoethyl-1,3-dioxanes 13, 22, 23 and 29 was performed by transacetalization of various acetals with pentane-1,3,5-triol, activation of the remaining free OH moiety with tosyl chloride and subsequent nucleophilic substitution. The corresponding 3-aminopropyl derivatives 33-35 were prepared by substitution of the tosylates with KCN and LiAlH4 reduction. The highest NMDA receptor affinity was found for 1,3-dioxanes with a phenyl and an ethyl residue at the acetalic position (23) followed by diphenyl (22) and monophenyl derivatives (13). Generally the NMDA affinity of primary amines is higher than the NMDA affinity of secondary and tertiary amines. Altogether the primary amine 23a (Ki=24 nM) represents the most promising NMDA receptor antagonist of this series exceeding the NMDA affinity of the mono-homologues (2-aminoethyl)-1,3-dioxolanes (3,4) and (aminomethyl)-1,3-dioxanes (5,6). Whereas the primary amine 23a turned out to be selective against σ1 and σ2 receptors the benzylamine 13d was identified as potent (Ki=19 nM) and selective σ1 antagonist, which showed extraordinarily high antiallodynic activity in the capsaicin assay.

The allosteric modulation of lipases and its possible biological relevance

BackgroundDuring the development of an enantioselective synthesis using the lipase from Mucor miehei an unusual reaction course was observed, which was analyzed precisely. For the first time an allosteric modulation of a lipase changing its selectivity was shown.TheoryConsidering the biological relevance of the discovered regulation mechanism we developed a theory that describes the regulation of energy homeostasis and fat metabolism.ConclusionThis theory represents a new approach to explain the cause of the metabolic syndrome and provides an innovative basis for further research activity.