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News Article | March 7, 2017
Site: www.realwire.com

Brussels/Munich, 7 March 2017 – UK businesses filed 5 142 patent applications with the European Patent Office (EPO) in 2016, an increase of +1.8% and the highest number since 2010. The UK thus bucked the trend of slightly decreasing patent applications from EU countries (Fig.: Growth of UK patent applications at the EPO). Last year, British companies and inventors were granted 2 931 patents[1] by the EPO (2015: 2 094) – the highest increase in ten years and a new record number of granted European patents. (Fig.: Growth of patents granted by the EPO to UK applicants) Overall, the European Patent Office received nearly 160 000 European patent applications in 2016, on par with the peak reached the year before (Fig.: Growth of European patent applications). There was strong growth again in applications from China (+24.8%) and from South Korea (+6.5%), a decrease in applications from the US (-5.9%) and Japan (-1.9%), whilst the volume of patent applications originating from the 38 member states of the European Patent Organisation remained almost stable last year (-0.2%). The top five countries of origin of applications were the US, Germany, Japan, France and Switzerland. The UK ranked 9th. (Fig.: Origin of applications) “The 2016 results confirm Europe’s attractiveness as a leading global marketplace for innovation,” said EPO President Benoît Battistelli. “In a rapidly changing political and economic landscape, companies from around the world have kept up their demand for patent protection in Europe. While we see impressive growth in applications from Asia, European companies maintain their role as drivers of innovation and economic growth in their home market, and are proving their resilience also in the face of unsettled economic conditions.” Rolls Royce No.1 UK patent applicant for the second consecutive year With 372 applications, Rolls Royce was the UK’s most active patent applicant at the EPO, followed by Unilever (237 applications), British Telecom (177), and BAE Systems (149). Oxford University was the highest ranked academic institution, climbing from 15th place in 2015 to 12th with 57 patent applications. (Fig. Top UK applicants at the EPO in 2016) Transport and Medical technology most important UK technology fields Transport, where many patent applications from the automotive, aeronautics and rail sectors are filed, was the UK’s main technology field with the highest number of patent applications for the second consecutive year, with a share of 7% of total UK applications at the EPO. The second most important field was Medical Technology, which increased its share of all UK applications from 5% in the previous year to 7% in 2016, followed by Measurement and Computer Technology, both fields with a 6% share. Within the ten most important technology fields, applications from the UK grew strongest in Basic Materials Chemistry (+24.5%), Medical technology (+22.4%), Biotechnology (20.9%) and Computer technology (+17%). East of England and South East England with strongest regional growth Greater London leads the regional ranking with a 30.2% share of all UK patent applications at the EPO (down from 31.8% in the previous year). (Fig: Leading UK regions for European patent applications) Greater London was also 9th European region for European patent applications. (Fig: Leading European regions for patent applications at the EPO). It was followed by East of England (11.7% share in 2016, up from 10.7% in the previous year), and South East England (11.2% share, up from 10.4%). These two regions also recorded the strongest growth among all UK regions (East of England +11.2%, and South East England +10.8%). Varied activity across Europe Among European countries there were marked differences in patenting activity at the EPO in 2016. Among the larger economies with higher patent application volumes, Belgium led the board, posting growth of 7% over 2015. Besides the UK, Italy (+4.5%), Austria (+2.6%), Spain (+2.6%), Switzerland (+2.5%) and Germany (+1.1%) also showed increases. Patent applications from France (-2.5%) and the Netherlands (-3.6%) dropped. The EPO also received fewer patent applications from some of the Nordic states, namely Finland (-8.8%), Sweden (-7.4%), and Denmark (-2.8%). (Fig.: Top 50 countries for applications) Most patents filed in medical technology Medical technology remained the field with the highest number of patent applications at the EPO (despite a slight drop of -2.1% in 2016), again followed by Digital communication and Computer technology. (Fig.: Technical fields with most applications). The strongest growth (of the top ten fields) was in Electrical machinery/apparatus/energy (+5.1%), followed by Transport (+3.6%) and Computer technology (+2.9%). Philips heads EPO company ranking Philips filed the most patent applications at the EPO in 2016, the second year running. Huawei was the new No. 2 (moving up from 11th in just three years), followed by Samsung, LG and United Technologies. The top 10 was made up of four companies from Europe, three from the US, two from Korea and one from China. (Fig.: Top 10 applicants in 2016) For detailed statistics, and a report on activities in 2016, see the EPO’s annual report at: www.epo.org/annual-report2016 [1] Patents are granted by the EPO after a detailed examination process of the patent application ensuring it fulfils all the requirements of patent protection. About the EPO With more than 7 000 staff, the European Patent Office (EPO) is one of the largest public service institutions in Europe. Its headquarters are in Munich and it also has offices in Berlin, Brussels, The Hague and Vienna. The EPO was founded with the aim of strengthening co-operation on patents in Europe. Through the EPO's centralised patent granting procedure, inventors are able to obtain high-quality patent protection in the 38 member states of the European Patent Organisation. The EPO is also the world's leading authority in patent information and patent searching.


COPENHAGEN, Denmark, April 18, 2017 (GLOBE NEWSWIRE) -- Forward Pharma A/S (NASDAQ:FWP) (“we” or “Forward” or the “Company”), today reported financial results for the fourth quarter and year ended December 31, 2016. Net income for the fourth quarter December 31, 2016 was $9.6 million while net loss for the year ended December 31, 2016 was $33.3 million, or $0.20 and $(0.71) per diluted share, respectively. On a non-GAAP basis, after removing the effect of non-cash income and expense items, our fourth quarter and year ended 2016 net loss would have been $11.8 million and $40.9 million, or $(0.25) and $(0.87) per basic share, respectively. As of December 31, 2016, the Company had $138.7 million in cash, cash equivalents and marketable securities, with no debt outstanding. “We made substantial progress in creating shareholder value from our patent portfolio, most importantly signified by the Settlement and License Agreement with Biogen. We also received a ruling in the patent interference case with Biogen and are working diligently to advance the appeal to the Federal Circuit and prepare for the opposition proceeding for our European ‘355 patent in November,” said Dr. Claus Bo Svendsen, Chief Executive Officer of Forward. “Having received the $1.25 billion non-refundable payment from Biogen, we remain in the strongest financial condition in the history of the Company, positioning us to return significant near-term value to shareholders.” Fourth Quarter and Year Ended December 31, 2016 Financial and Operational Results The GAAP net loss for years ended 2016 and 2015 was $33.3 million and $37.0 million, respectively.  The GAAP net income for the fourth quarter of 2016 was $9.6 million compared to a net loss of $9.6 million for the fourth quarter of 2015.  Net income for the fourth quarter of 2016 includes a deferred tax benefit of $21.3 million primarily related to net operating loss carryforwards that will be utilized in 2017 based on our estimate that the Company will have taxable profits in 2017. Research and development expenses for the year ended 2016 and 2015 were $41.1 million and $33.7 million, respectively.  Research and development expenses were $10.3 million for the quarter ended December 31, 2016 compared to $8.6 million for the fourth quarter of 2015. The increase in costs for the year ended 2016 compared to 2015 was primarily due to an increase in expenses to register and advance our intellectual property and higher share-based compensation.  The increase in research and development expenses in the fourth quarter of 2016 versus the same period in 2015 was due to an increase in patent related costs.  We estimate that research and development costs will decrease in 2017 compared to 2016 as our development efforts for FP187 will be limited to finishing the work that was in process prior to entering into the License Agreement with Biogen discussed below. General and administrative expenses for the year ended 2016 and 2015 were $14.4 million and $15.9 million, respectively.  General and administrative expenses were $5.1 million for the quarter ended December 31, 2016 compared to $3.6 million for the fourth quarter of 2015. The decrease in costs for the year ended 2016 compared to 2015 was principally due to a $1.2 million reduction in share based compensation.  The increase in general and administrative expenses in the fourth quarter of 2016 versus the same period in 2015 was primarily due to professional fees. We expect our general and administrative costs will remain at current levels; however, expenses associated with protecting, defending and enforcing our patent rights that occur in the courts could increase in future periods. Non-cash stock based compensation expense included in total operating expenses was $14.3 million for the year ended 2016 versus $13.5 million for the year ended 2015.  As of December 31, 2016, the Company had $138.7 million in cash, cash equivalents and marketable securities. On January 17, 2017, Forward announced that it entered into a binding agreement with two wholly owned subsidiaries of Biogen and certain additional parties to enter into a Settlement and License Agreement (the “License Agreement”).  In February 2017, Forward received a non-refundable cash fee of $1.25 billion from Biogen in connection with the License Agreement.  During February and March 2017, to reduce the Company’s exposure to changes in foreign exchange rates, the Company converted the $1.25 billion into 1.17 billion Euros. On March 31, the Patent Trial and Appeal Board (“PTAB”) of the United States Patent and Trademark Office (“USPTO”) issued a decision in favor of Biogen in Patent Interference No. 106,023 regarding claims of Forward Pharma’s patent application 11/576,871 (the “’871 application”) that cover a method of treating multiple sclerosis (“MS”) using a 480 mg per day dose of dimethyl fumarate (“DMF”), the approved dose of Tecfidera®. We intend to appeal this decision.  If Forward Pharma ultimately prevails in the interference after all  appeals to the U.S. Court of Appeals for the Federal Circuit (“Federal Circuit”), and the ’871 application issues with claims covering treatment for MS by orally administering 480 mg per day of DMF, we anticipate that Biogen would be obligated to pay future royalties on net sales in the U.S. of Biogen’s DMF-containing products, including Tecfidera®, indicated for treating MS that would, but for the rights granted under the License Agreement, infringe a Company patent, provided that other conditions of the License Agreement are satisfied. Among the conditions that needs to be satisfied for any royalty to be payable by Biogen to us on net sales in the U.S. is the absence of generic entry in the U.S. having a particular impact as defined by the License Agreement. If Forward Pharma succeeds in the interference as described above and Biogen obtains an exclusive license to all intellectual property in the United States that is owned by Forward Pharma (the “U.S. Licensed Intellectual Property”) under the License Agreement and other conditions are satisfied, a royalty of 10% would be payable by Biogen on net sales in the U.S. of applicable infringing products from January 1, 2021 to December 31, 2028 (increasing to 20% from January 1, 2029) until the earlier of the expiration or invalidation of the patents owned by Forward Pharma in the U.S.  Biogen’s existing perpetual, irrevocable, co-exclusive license to all of the U.S. Licensed Intellectual Property will be converted into an irrevocable exclusive license to all of the U.S. Licensed Intellectual Property if all of the terms and conditions of the License Agreement are met within the time period set forth in the License Agreement, including the absence of legal restraints and termination or expiration of any required waiting period under the Hart-Scott-Rodino Antitrust Improvements Act of 1976, as amended. If Biogen does not obtain an exclusive license, and provided that other conditions of the License Agreement are satisfied, Biogen will maintain a co-exclusive license with respect to the U.S. Licensed Intellectual Property, and the royalties payable by Biogen to Forward Pharma on its net sales in the U.S. of applicable infringing products would instead be 1% from January 1, 2023, and Forward Pharma would retain the right to commercialize in the U.S. or assign its U.S. co-exclusive rights, on one occasion only, to a single third party. If we prevail after all appeals to the Federal Circuit, we expect our ’871 application, if ultimately issued, would be entitled to patent term adjustment extending the patent term to compensate the Company for time lost during prosecution and the interference, which the Company estimates would result in patent expiration in 2029 or later. The hearing for the pending opposition proceeding against the Company’s European patent EP2801355 (“Opposition Proceeding”) is currently scheduled for November 6 and 7, 2017.  If the Company obtains, as a result of the Opposition Proceeding, and any appeals therefrom, a patent with a claim covering oral treatment of MS with 480 mg per day of DMF, it would be eligible beginning on January 1, 2021 to collect a 10% royalty (increasing to 20% from January 1, 2029) until the earlier of the expiration or invalidation of the patents defined in the License Agreement, on a country-by-country basis on Biogen’s net sales outside the U.S. of DMF-containing products, including Tecfidera®, indicated for treating MS that, but for the rights granted under the License Agreement, would infringe a Company patent, provided that other conditions of the License Agreement are satisfied. Among the conditions that needs to be satisfied for any royalty to be payable by Biogen to us in a particular country is the absence of generic entry in that country having a particular impact as defined in the License Agreement. On March 1, 2017, the Company announced plans to finish its remaining research and development efforts and to pursue an organizational realignment to reduce personnel and operating expenses. We are currently finishing the research and development work that was in process prior to the effective date of the License Agreement and thereafter plan to suspend further development of our clinical candidate, FP187, pending the outcome of the appeal to the Federal Circuit regarding the decision in the interference with Biogen, and whether or not Biogen obtains an exclusive license in the U.S. If Biogen maintains a co-exclusive license, we expect to either assign our co-exclusive license rights to a single third party on one occasion only or reinitiate development of FP187, or initiate development of another DMF-containing formulation, in anticipation of a regulatory submission to the FDA. However, if Biogen prevails in the interference and IPR brought by the Coalition for Affordable Drugs, after any appeals to the Federal Circuit, we may be prevented from commercializing our lead product candidate, FP187, for MS in the U.S. at a 480 mg per day dose. Although we have previously released financial results on a quarterly basis, consistent with our plan to reduce expenses, we intend to release future financial results semi-annually, with our next report following the end of our second quarter. USPTO interference with Biogen: The related documents are publicly available on the USPTO interference website at https://acts.uspto.gov/ifiling/PublicView.jsp, using interference number 106023. Forward Pharma U.S. and European patents and patent applications can be found by using the following links:  USPTO: http://www.uspto.gov/  USPTO Public PAIR: http://portal.uspto.gov/pair/PublicPair  EPO: https://register.epo.org/regviewer About Forward Pharma: Forward Pharma A/S is a Danish biopharmaceutical company that commenced development in 2005 of FP187, a proprietary formulation of DMF for the treatment of inflammatory and neurological indications. The Company owns a significant intellectual property (IP) portfolio related to DMF formulations. The Company granted to Biogen an irrevocable license to all of its IP through the recent License Agreement and received from Biogen a non-refundable cash fee of $1.25 billion in February 2017. The Company has the opportunity to receive royalties from Biogen on sales of Tecfidera® or other DMF products for MS, dependent on, among other things, the anticipated appeal of the U.S. interference and the EP2801355 opposition outcome in Europe. Our principal executive offices are located at Østergade 24A, 1st Floor, 1100 Copenhagen K, Denmark and our American Depositary Shares are publicly traded on NASDAQ Stock Market (FWP).  For more information about the Company, please visit our web site at http://www.forward-pharma.com. Forward Looking Statements: Certain statements in this press release may constitute “forward-looking statements” of Forward Pharma A/S (the “Company”) within the meaning of the Private Securities Litigation Reform Act of 1995.  Forward-looking statements include, but are not limited to, statements which contain language such as “believe,” “expect,” “anticipate,” “hope,” “would”, “may”, and “potential.”  Forward-looking statements are predictions only, which involve known and unknown risks, uncertainties and other factors that may cause actual results to be materially different from those expressed in such statements.  Many such risks, uncertainties and other factors are taken into account as part of our assumptions underlying these forward-looking statements and include, among others, risks related to the following: the satisfaction of certain conditions, and the accuracy of certain representations of the Company, in the License Agreement entered into with subsidiaries of Biogen Inc. and certain other parties thereto; our ability to obtain, maintain, enforce and defend issued patents with royalty-bearing claims; our ability to successfully appeal the interference decision to the Federal Circuit; our ability to prevail in or obtain a favorable decision in the Opposition Proceeding, after all appeals; the issuance and term of our patents; future sales of Tecfidera®, including impact on such sales from competition, generic challenges, regulatory involvement and pricing pressures; the scope, validity and enforceability of our intellectual property rights in general and the impact on us of patents and other intellectual property rights of third parties; the timing, amount (if any) and tax consequences of any distribution to shareholders; and our ability to generate revenue from product sales in the U.S. directly or through an assignee of our U.S. co-exclusive license rights in the event Biogen does not obtain an exclusive license from us in the U.S. Certain of these and other risk factors are identified and described in detail in certain of our filings with the United States Securities and Exchange Commission, including our Annual Report on Form 20-F for the year ended December 31, 2016. We are providing this information as of the date of this release and do not undertake any obligation to update any forward-looking statements contained in this press release as a result of new information, future events or otherwise.


News Article | November 29, 2016
Site: globenewswire.com

COPENHAGEN, Denmark, Nov. 29, 2016 (GLOBE NEWSWIRE) -- Forward Pharma A/S (NASDAQ:FWP) (“we” or “Forward” or the “Company”), today reported financial results for the third quarter ended September 30, 2016.  Net loss for the third quarter ended September 30, 2016 was $11.0 million, or $0.23 per basic share versus a net loss of $15.8 million, or $0.34 per basic share for the third quarter of 2015.  On a non-GAAP basis, after removing the effect of non-cash income and expense items, our third quarter net loss for 2016 would have been $7.3 million, or $0.15 per basic share. As of September 30, 2016, the Company had $150.2 million in cash, cash equivalents and marketable securities, with no debt outstanding.  “We continue to advance our clinical programs and our intellectual property portfolio and look forward to the oral argument on our interference case to be held tomorrow at the USPTO,” said Peder Andersen, Chief Executive Officer of Forward. Third Quarter ended September 30, 2016 Financial and Operational Results The net loss for the third quarter of 2016 was $11.0 million compared to a net loss of $15.8 million for the comparable period in 2015. Research and development costs for the three month periods ended September 30, 2016 and 2015 were $7.7 million and $10.8 million, respectively. This decrease in the third quarter 2016 versus the prior year was related to a reduction in the use of contract manufacturers and clinical research organizations during our evaluation of options for an alternative Phase 3 clinical plan for FP187 in relapsing-remitting multiple sclerosis (RRMS). Research and development costs incurred in the third quarter of 2016 to conduct the ongoing interference case at the U.S. Patent and Trademark Office (USPTO) and opposition proceedings with the European Patent Office (EPO) were flat with the comparable period in 2015. Share-based compensation for the three month period ended September 30, 2016 was $1.6 million. We expect our use of contract manufacturers and clinical research organizations will likely return to historical levels in our further preparation for the start of our Phase 3 trial.  We expect our overall rate of research and development spending will increase in future quarters as our clinical and pharmaceutical development programs and patent office matters, including our pending interference action, advance. General and administrative costs for the three month periods ended September 30, 2016 and 2015 were $3.2 million and $4.6 million, respectively.  The decrease in expenses in the third quarter 2016 versus the prior year was principally due to a reduction in share-based compensation as well as lower legal, accounting and consulting fees.  In the third quarter 2016, share-based compensation decreased by $0.4 million versus the comparable period in 2015 to $1.7 million in connection with the vesting of equity awards issued during the years ended December 31, 2015 and 2014. We expect our rate of general and administrative spending could increase in the future in support of our expected increase in research and development related activities as well as the protection of our intellectual property portfolio including expenditures in connection with the lawsuits against Biogen in Europe. Non-cash stock-based compensation expense included in total operating expenses was $3.3 million for the third quarter of 2016 versus $3.8 million for the third quarter of 2015. We continue to advance our intellectual property portfolio.  As previously reported, on August 19, 2015, the USPTO re-declared the interference between Forward Pharma and a subsidiary of Biogen, Inc. regarding claims to the treatment of multiple sclerosis (MS), with a 480 mg daily dose of dimethyl fumarate (DMF), the active ingredient in Tecfidera®. The USPTO confirmed Forward Pharma as the senior party based on having an earlier benefit date of our U.S. Patent Application No. 11/576,871.  Biogen was deemed the junior party with respect to its U.S. Patent No. 8,399,514.  In August 2015, the parties filed priority statements and motions related to validity and benefit. The parties filed oppositions to motions on June 1, 2016.  Both parties filed replies to the oppositions on August 8, 2016.  The oral argument remains scheduled for November 30, 2016. Our development plan for our lead drug, FP187, is focused primarily on the RRMS indication. In consultation with a clinical research organization, we are continuing to prepare for our Phase 3 trial in RRMS. In addition to the single beta interferon-controlled design, we are evaluating options for an alternative Phase 3 clinical strategy in RRMS, which could shorten our time to commercialization and/or reduce costs. We expect to complete these evaluations as we continue to upscale production of FP187 during 2016, in anticipation of beginning the Phase 3 trial in mid-2017. We are currently performing additional Phase 1 studies on FP187. Our nonclinical development plan for FP187 is designed to support regulatory submissions with a complete toxicology package. This press release uses a non-GAAP financial measure of net loss that is not calculated in accordance with International Financial Reporting Standards as issued by the International Accounting Standards Board ("IASB"). The Company believes that the presentation of non-GAAP net loss is useful to investors because it excludes certain non-cash items that do not affect the Company’s liquidity, and foreign exchange rate changes are not within the control of the Company.  Non-cash items include: (i) share-based compensation expense and (ii) non-cash foreign exchange gains or (losses). However, there are limitations in the use of non-GAAP financial measures as they exclude certain income and expenses. Furthermore, the Company's non-GAAP financial measure may not be comparable with non-GAAP information provided by other companies. Any non-GAAP financial measure presented herein should be considered supplemental to, and not a substitute for, measures of financial performance prepared in accordance with IFRS. USPTO Interference with Biogen: The related documents are publicly available on the USPTO interference website at https://acts.uspto.gov/ifiling/PublicView.jsp, using interference number 106023. Forward Pharma U.S. and European patents and patent applications can be found by using the following links:  USPTO: http://www.uspto.gov/ USPTO Public Pair: http://portal.uspto.gov/pair/PublicPair EPO: https://register.epo.org/regviewer About Forward Pharma: Forward Pharma A/S is a Danish biopharmaceutical company developing FP187, a proprietary formulation of DMF (dimethyl fumarate) for the treatment of inflammatory and neurological indications. Since our founding in 2005, we have worked to advance unique formulations of DMF, which is an immune modulator, as a therapeutic agent to improve the health and well-being of patients with immune disorders including multiple sclerosis. FP187, our clinical candidate, is a DMF formulation in a delayed and slow release oral dose. Our principal executive offices are located at Østergade 24A, 1st Floor, 1100 Copenhagen K, Denmark, and our American Depositary Shares are publicly traded on NASDAQ Stock Market (FWP). For more information about the Company’s products and developments, please visit our web site at http://www.forward-pharma.com. Forward Looking Statements: Certain statements in this press release may constitute “forward-looking statements” of the Company within the meaning of the Private Securities Litigation Reform Act of 1995. Forward-looking statements include, but are not limited to, statements which contain language such as “believe,” “expect,” “anticipate,” “hope,” “would” and “potential.” Forward-looking statements are predictions only which involve known and unknown risks, uncertainties and other factors that may cause actual results to be materially different from those expressed in such statements. Many such risks, uncertainties and other factors are taken into account as part of our assumptions underlying these forward-looking statements and include, among others, the following: the Company’s ability to obtain, maintain and defend issued patents with protective claims; the issuance and term of patents; the Company’s ability to prevail in or obtain a favorable decision in any patent interference or infringement action; the Company’s ability to recover damages in any patent infringement action; uncertainties relating to our development plans and activities, including the commencement of any clinical trial and the results, timing, cost and location thereof; risks and uncertainties related to the scope, validity and enforceability of our intellectual property rights in general and the impact on us of patents and other intellectual property rights of third parties; our ability to commercialize and generate revenue from our sole clinical candidate, FP187; clinical development, and clinical trials of FP187 may not be successful; completion of required clinical trials may take longer than we anticipate, which could result in increased costs, limit our access to funding and delay or limit our ability to obtain regulatory approval for FP187; and our evaluation of alternative Phase 3 clinical strategies in RRMS may not be successful or shorten our time to commercialization and/or reduce costs. These and other factors are identified and described in detail in certain of our filings with the United States Securities and Exchange Commission, including our Annual Report on Form 20-F for the year ended December 31, 2015.


Stecklum M.,Max Delbrück Center for Molecular Medicine | Stecklum M.,EPO GmbH | Wulf-Goldenberg A.,EPO GmbH | Purfurst B.,Max Delbrück Center for Molecular Medicine | And 6 more authors.
In Vitro Cellular and Developmental Biology - Animal | Year: 2014

In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction. © 2014, The Society for In Vitro Biology.


Wulf-Goldenberg A.,Epo GmbH | Keil M.,Max Delbrück Center for Molecular Medicine | Keil M.,Charité - Medical University of Berlin | Fichtner I.,Max Delbrück Center for Molecular Medicine | Eckert K.,Max Delbrück Center for Molecular Medicine
Tissue and Cell | Year: 2012

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34 + cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34 + cord blood stem cell preparations. © 2011 Elsevier Ltd.


Harnack U.,Charité - Medical University of Berlin | Eckert K.,epo GmbH | Pecher G.,Charité - Medical University of Berlin
Anticancer Research | Year: 2011

Background: Beta-(1-3),(1-6)-D-glucans demonstrate antitumor effects in vivo due to the activation of innate immune cells. Cyclophosphamide (CY) enhances natural or therapeutically induced antitumor immune responses by reducing the number and activity of regulatory T (Treg) cells. Materials and Methods: We tested whether oral administration of soluble beta-glucan augmented the inhibitory effect of intraperitoneally injected low-dose CY (30 mg/kg) on subcutaneously growing A20-lymphoma in Balb/c-mice. Results: Administration of CY one week after tumor inoculation significantly diminished tumor growth (p=0.009) and the absolute number of Treg cells in the peripheral blood compared with phosphate buffered saline-treated mice (p=0.036). Treatment of CY pre-conditioned lymphoma-bearing mice with daily beta-glucan (400 μg/mouse) between day 9 and day 13 after tumor injection significantly delayed onset of tumor growth, compared to mice which received only CY (p=0.01). Conclusion: Beta-(1-3),(1-6)-D-glucan could be useful in the treatment of lymphoma after low-dose chemotherapy with CY.


Twardziok M.,Charité - Medical University of Berlin | Twardziok M.,Free University of Berlin | Kleinsimon S.,Charité - Medical University of Berlin | Rolff J.,EPO GmbH | And 4 more authors.
PLoS ONE | Year: 2016

Ewing sarcoma is the second most common bone cancer in children and adolescents, with poor prognosis and outcome in ~70% of initial diagnoses and 10-15% of relapses. Hydrophobic triterpene acids and hydrophilic lectins and viscotoxins from European mistletoe (Viscum album L.) demonstrate anticancer properties, but have not yet been investigated for Ewing sarcoma. Commercial Viscum album L. extracts are aqueous, excluding the insoluble triterpenes. We recreated a total mistletoe effect by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilized with cyclodextrins. Ewing sarcoma cells were treated with viscum, TT and viscumTT in vitro, ex vivo and in vivo. In vitro and ex vivo treatment of Ewing sarcoma cells with viscum inhibited proliferation and induced apoptosis in a dose-dependent fashion, while viscumTT combination treatment generated a synergistic effect. Apoptosis occurred via intrinsic and extrinsic apoptotic pathways, evidenced by activation of both CASP8 and CASP9. We show that viscumTT treatment shifts the balance of apoptotic regulatory proteins towards apoptosis, mainly via CLSPN, MCL1, BIRC5 and XIAP downregulation. Viscum TT also demonstrated strong antitumor activity in a cell lineand patient-derived mouse model, and may be considered an adjuvant therapy option for pediatric patients with Ewing sarcoma. © 2016 Twardziok et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Busia L.,Bayer AG | Busia L.,Free University of Berlin | Faus H.,Bayer AG | Hoffmann J.,Bayer AG | And 2 more authors.
Molecular and Cellular Endocrinology | Year: 2011

The ovarian steroid hormone progesterone is essential for normal mammary gland physiology but may also play a role in breast cancer. Highly potent and selective antiprogestins may therefore represent a new treatment option for this disease. Here we studied the effects of the new antiprogestin Lonaprisan on the T47D breast cancer cell line. Strong inhibition of cell proliferation and arrest in the G0/G1 phase were observed, as well as induction of a senescence-like phenotype. This was accompanied by p21 induction through direct binding of Lonaprisan-bound progesterone receptor (PR) to the promoter. Reduction of p21 levels blunted the antiproliferative effects of Lonaprisan. Mutation analysis showed that intact PR DNA-binding properties were needed for p21 induction. Phosphorylation of PR Ser345 was stimulated by Lonaprisan, but this post-translational modification was not required for p21 promoter activation, nor was the interaction with c-Src needed. These results support the rationale for using antiprogestins in breast cancer treatment and warrant further studies to better understand their mode of action. © 2010 Elsevier Ireland Ltd.


Schmieder R.,Bayer AG | Hoffmann J.,EPO GmbH | Becker M.,EPO GmbH | Bhargava A.,Shakti BioResearch | And 9 more authors.
International Journal of Cancer | Year: 2014

Regorafenib, a novel multikinase inhibitor, has recently demonstrated overall survival benefits in metastatic colorectal cancer (CRC) patients. Our study aimed to gain further insight into the molecular mechanisms of regorafenib and to assess its potential in combination therapy. Regorafenib was tested alone and in combination with irinotecan in patient-derived (PD) CRC models and a murine CRC liver metastasis model. Mechanism of action was investigated using in vitro functional assays, immunohistochemistry and correlation with CRC-related oncogenes. Regorafenib demonstrated significant inhibition of growth-factor-mediated vascular endothelial growth factor receptor (VEGFR) 2 and VEGFR3 autophosphorylation, and intracellular VEGFR3 signaling in human umbilical vascular endothelial cells (HuVECs) and lymphatic endothelial cells (LECs), and also blocked migration of LECs. Furthermore, regorafenib inhibited proliferation in 19 of 25 human CRC cell lines and markedly slowed tumor growth in five of seven PD xenograft models. Combination of regorafenib with irinotecan significantly delayed tumor growth after extended treatment in four xenograft models. Reduced CD31 staining indicates that the antiangiogenic effects of regorafenib contribute to its antitumor activity. Finally, regorafenib significantly delayed disease progression in a murine CRC liver metastasis model by inhibiting the growth of established liver metastases and preventing the formation of new metastases in other organs. In addition, our results suggest that regorafenib displays antimetastatic activity, which may contribute to its efficacy in patients with metastatic CRC. Combination of regorafenib and irinotecan demonstrated an increased antitumor effect and could provide a future treatment option for CRC patients. What's new? Regorafenib is a multikinase inhibitor with antiangiogenic activity recently approved in the US and in Europe for the treatment of metastatic colorectal cancer in patients who failed previous therapies. Here, a research team led by Bayer Pharma AG, the discoverer of the drug, confirms inhibition of key mediators of angiogenesis and lymphangiogenesis (VEGFR2 and VEGFR3) as the potential antiangiogenic mechanism of action of the drug. Regorafenib further inhibited growth of established and prevented formation of new liver metastases, and in combination with the chemotherapeutic drug irinotecan led to significant tumor growth delay in four patient-derived colorectal cancer xenograft models. The authors speculate that combination treatments including regorafenib may provide novel therapeutic opportunities for patients with therapy-resistant colorectal cancer. © 2013 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.


In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34(+) cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation. Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34(+) cord blood stem cell preparations.

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