Publications by authors named "Saliha Harrach"

13 Publications

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Animal Safety, Toxicology, and Pharmacokinetic Studies According to the ICH S9 Guideline for a Novel Fusion Protein tTF-NGR Targeting Procoagulatory Activity into Tumor Vasculature: Are Results Predictive for Humans?

Cancers (Basel) 2020 Nov 26;12(12). Epub 2020 Nov 26.

Department of Medicine A Hematology and Oncology, University Hospital Muenster, D-48149 Muenster, Germany.

Background: CD-13 targeted tissue factor tTF-NGR is a fusion protein selectively inducing occlusion of tumor vasculature with resulting tumor infarction. Mechanistic and pharmacodynamic studies have shown broad anti-tumor therapeutic effects in xenograft models.

Methods: After successful Good Manufacturing Practice (GMP) production and before translation into clinical phase I, ICH S9 (S6) guideline-conforming animal safety, toxicology, and pharmacokinetic (PK) studies were requested by the federal drug authority in accordance with European and US regulations.

Results: These studies were performed in mice, rats, guinea pigs, and beagle dogs. Results of the recently completed clinical phase I trial in end-stage cancer patients showed only limited predictive value of these non-clinical studies for patient tolerability and safety in phase I.

Conclusions: Although this experience cannot be generalized, alternative pathways with seamless clinical phase 0 microdosing-phase I dose escalation studies are endorsed for anticancer drug development and translation into the clinic.
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http://dx.doi.org/10.3390/cancers12123536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759859PMC
November 2020

First-In-Class CD13-Targeted Tissue Factor tTF-NGR in Patients with Recurrent or Refractory Malignant Tumors: Results of a Phase I Dose-Escalation Study.

Cancers (Basel) 2020 Jun 7;12(6). Epub 2020 Jun 7.

Department of Medicine A, Hematology, Oncology, University Hospital Muenster, D-48149 Muenster, Germany.

Background: Aminopeptidase N (CD13) is present on tumor vasculature cells and some tumor cells. Truncated tissue factor (tTF) with a C-terminal NGR-peptide (tTF-NGR) binds to CD13 and causes tumor vascular thrombosis with infarction.

Methods: We treated 17 patients with advanced cancer beyond standard therapies in a phase I study with tTF-NGR (1-h infusion, central venous access, 5 consecutive days, and rest periods of 2 weeks). The study allowed intraindividual dose escalations between cycles and established Maximum Tolerated Dose (MTD) and Dose-Limiting Toxicity (DLT) by verification cohorts.

Results: MTD was 3 mg/m tTF-NGR/day × 5, q day 22. DLT was an isolated and reversible elevation of high sensitivity (hs) Troponin T hs without clinical sequelae. Three thromboembolic events (grade 2), tTF-NGR-related besides other relevant risk factors, were reversible upon anticoagulation. Imaging by contrast-enhanced ultrasound (CEUS) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) showed major tumor-specific reduction of blood flow in all measurable lesions as proof of principle for the mode of action of tTF-NGR. There were no responses as defined by Response Evaluation Criteria in Solid Tumors (RECIST), although some lesions showed intratumoral hemorrhage and necrosis after tTF-NGR application. Pharmacokinetic analysis showed a t of 8 to 9 h without accumulation in daily administrations.

Conclusion: tTF-NGR is safely applicable with this regimen. Imaging showed selective reduction of tumor blood flow and intratumoral hemorrhage and necrosis.
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http://dx.doi.org/10.3390/cancers12061488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352358PMC
June 2020

Radiation synergizes with antitumor activity of CD13-targeted tissue factor in a HT1080 xenograft model of human soft tissue sarcoma.

PLoS One 2020 21;15(2):e0229271. Epub 2020 Feb 21.

Department of Medicine A, Hematology, Oncology, University Hospital Muenster, Muenster, Germany.

Background: Truncated tissue factor (tTF) retargeted by NGR-peptides to aminopeptidase N (CD13) in tumor vasculature is effective in experimental tumor therapy. tTF-NGR induces tumor growth inhibition in a variety of human tumor xenografts of different histology. To improve on the therapeutic efficacy we have combined tTF-NGR with radiotherapy.

Methods: Serum-stimulated human umbilical vein endothelial cells (HUVEC) and human HT1080 sarcoma cells were irradiated in vitro, and upregulated early-apoptotic phosphatidylserine (PS) on the cell surface was measured by standard flow cytometry. Increase of cellular procoagulant function in relation to irradiation and PS cell surface concentration was measured in a tTF-NGR-dependent Factor X activation assay. In vivo experiments with CD-1 athymic mice bearing human HT1080 sarcoma xenotransplants were performed to test the systemic therapeutic effects of tTF-NGR on tumor growth alone or in combination with regional tumor ionizing radiotherapy.

Results: As shown by flow cytometry with HUVEC and HT1080 sarcoma cells in vitro, irradiation with 4 and 6 Gy in the process of apoptosis induced upregulation of PS presence on the outer surface of both cell types. Proapoptotic HUVEC and HT1080 cells both showed significantly higher procoagulant efficacy on the basis of equimolar concentrations of tTF-NGR as measured by FX activation. This effect can be reverted by masking of PS with Annexin V. HT1080 human sarcoma xenografted tumors showed shrinkage induced by combined regional radiotherapy and systemic tTF-NGR as compared to growth inhibition achieved by either of the treatment modalities alone.

Conclusions: Irradiation renders tumor and tumor vascular cells procoagulant by PS upregulation on their outer surface and radiotherapy can significantly improve the therapeutic antitumor efficacy of tTF-NGR in the xenograft model used. This synergistic effect will influence design of future clinical combination studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229271PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034830PMC
May 2020

Notch Signaling Activity Determines Uptake and Biological Effect of Imatinib in Systemic Sclerosis Dermal Fibroblasts.

J Invest Dermatol 2019 02 28;139(2):439-447. Epub 2018 Sep 28.

Department of Orthopaedic Surgery, Otto-von-Guericke University Magdeburg, Germany. Electronic address:

Tyrosine kinase inhibitors have emerged as a therapeutic option for rheumatic diseases such as systemic sclerosis (SSc). Because tyrosine kinases like c-Abl kinase are important for fibroblast activation and fibrosis development in SSc, the c-Abl inhibitor imatinib was proposed for SSc treatment. Transporters for organic cations have become increasingly recognized as an important determinant for uptake and efficacy of tyrosine kinase inhibitors. Therefore, we investigated the role of organic cation transporters in the uptake of imatinib. Moreover, the influence of important SSc pathogenetic factors, like PDGF and Notch pathway activation on these uptake processes, has been studied. We showed that organic cation transporters OCT1-3, novel organic cation transporters OCTN1/2, and the multidrug and toxin extrusion protein MATE1 are expressed in healthy dermal and SSc fibroblasts. Decreased expression levels of MATE1 and decreased imatinib uptake were measured in SSc fibroblasts. In small interfering RNA experiments, MATE1 was identified as key transporter for imatinib uptake and biological effect in dermal fibroblasts. Furthermore, PDGF reduced imatinib uptake by decreasing MATE1 expression in SSc fibroblasts, but not in healthy fibroblasts. Blocking the Notch pathway in SSc fibroblasts increased MATE1 transporter expression and imatinib uptake. In conclusion, MATE1-mediated transport governs therapeutic efficacy of imatinib in SSc.
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http://dx.doi.org/10.1016/j.jid.2018.08.021DOI Listing
February 2019

Aminopeptidase N (CD13): Expression, Prognostic Impact, and Use as Therapeutic Target for Tissue Factor Induced Tumor Vascular Infarction in Soft Tissue Sarcoma.

Transl Oncol 2018 Dec 17;11(6):1271-1282. Epub 2018 Aug 17.

Department of Medicine A, Hematology, Oncology, University Hospital Muenster, Muenster, Germany.

Aminopeptidase N (CD13) is expressed on tumor vasculature and tumor cells. It represents a candidate for targeted therapy, e.g., by truncated tissue factor (tTF)-NGR, binding to CD13, and causing tumor vascular thrombosis. We analyzed CD13 expression by immunohistochemistry in 97 patients with STS who were treated by wide resection and uniform chemo-radio-chemotherapy. Using a semiquantitative score with four intensity levels, CD13 was expressed by tumor vasculature, or tumor cells, or both (composite value, intensity scores 1-3) in 93.9% of the STS. In 49.5% tumor cells, in 48.5% vascular/perivascular cells, and in 58.8%, composite value showed strong intensity score 3 staining. Leiomyosarcoma and synovial sarcoma showed low expression; fibrosarcoma and undifferentiated pleomorphic sarcoma showed high expression. We found a significant prognostic impact of CD13, as high expression in tumor cells or vascular/perivascular cells correlated with better relapse-free survival and overall survival. CD13 retained prognostic significance in multivariable analyses. Systemic tTF-NGR resulted in significant growth reduction of CD13-positive human HT1080 sarcoma cell line xenografts. Our results recommend further investigation of tTF-NGR in STS patients. CD13 might be a suitable predictive biomarker for patient selection.
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http://dx.doi.org/10.1016/j.tranon.2018.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113655PMC
December 2018

CD13 as target for tissue factor induced tumor vascular infarction in small cell lung cancer.

Lung Cancer 2017 11 22;113:121-127. Epub 2017 Sep 22.

Gerhard-Domagk-Institute of Pathology, University of Muenster, 48149 Muenster, Germany.

Objectives: Zinc-binding protease aminopeptidase N (CD13) is expressed on tumor vascular cells and tumor cells. It represents a potential candidate for molecular targeted therapy, e.g. employing truncated tissue factor (tTF)-NGR, which can bind CD13 and thereby induce tumor vascular infarction. We performed a comprehensive analysis of CD13 expression in a clinically well characterized cohort of patients with small cell lung cancer (SCLC) to evaluate its potential use for targeted therapies in this disease.

Material And Methods: CD13 expression was analyzed immunohistochemically in 27 SCLC patients and correlated with clinical course and outcome. In CD-1 nude mice bearing human HTB119 SCLC xenotransplants, the systemic effects of the CD13-targeting fusion protein tTF-NGR on tumor growth were tested.

Results And Conclusion: In 52% of the investigated SCLC tissue samples, CD13 was expressed in tumor stroma cells, while the tumor cells were negative for CD13. No prognostic effect was found in the investigated SCLC study collective with regard to overall survival (p>0.05). In CD-1 nude mice, xenografts of CD13 negative HTB119 SCLC cells showed CD13 expression in the intratumoral vascular and perivascular cells, and the systemic application of CD13-targeted tissue factor tTF-NGR led to a significant reduction of tumor growth. We here present first data on the expression of CD13 in SCLC tumor samples. Our results strongly recommend the further investigation of tTF-NGR and other molecules targeted by NGR-peptides in SCLC patients. Considering the differential expression of CD13 in SCLC samples pre-therapeutic CD13 analysis is proposed for testing as investigational predictive biomarker for patient selection.
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http://dx.doi.org/10.1016/j.lungcan.2017.09.013DOI Listing
November 2017

Potential therapeutic impact of CD13 expression in non-small cell lung cancer.

PLoS One 2017 12;12(6):e0177146. Epub 2017 Jun 12.

Gerhard-Domagk-Institute of Pathology, University of Muenster, Muenster, Germany.

Background: Aminopeptidase N (CD13) is a zinc-binding protease that has functional effects on both cancerogenesis and tumor angiogenesis. Since CD13 is an antigen suitable for molecular targeted therapies (e.g. tTF-NGR induced tumor vascular infarction), we evaluated its impact in NSCLC patients, and tested the effects of the CD13-targeted fusion protein tTF-NGR (truncated tissue factor (tTF) containing the NGR motif: asparagine-glycine-arginine) in vivo in nude mice.

Methods: Expression of both CD13 and CD31 was studied in 270 NSCLC patients by immunohistochemistry. Clinical correlations and prognostic effects of the expression profiles were analyzed using univariate and multivariate analyses. In addition, a microarray-based analysis on the basis of the KM plotter database was performed. The in vivo effects of the CD13-targeted fusion protein tTF-NGR on tumor growth were tested in CD1 nude mice carrying A549 lung carcinoma xenotransplants.

Results: CD13 expression in tumor endothelial and vessel associated stromal cells was found in 15% of the investigated samples, while expression in tumor cells was observed in 7%. Although no significant prognostic impact was observed in the full NSCLC study cohort, both univariate and multivariate models identified vascular CD13 protein expression to correlate with poor overall survival in stage III and pN2+ NSCLC patients. Microarray-based mRNA analysis for either adenocarcinomas or squamous cell carcinomas did not reveal any significant effect. However, the analysis of CD13 mRNA expression for all lung cancer histologies demonstrated a positive prognostic effect. In vivo, systemic application of CD13-targeted tissue factor tTF-NGR significantly reduced CD13+ A549 tumor growth in nude mice.

Conclusions: Our results contribute a data basis for prioritizing clinical testing of tTF-NGR and other antitumor molecules targeted by NGR-peptides in NSCLC. Because CD13 expression in NSCLC tissues was found only in a specific subset of NSCLC patients, rigorous pre-therapeutic testing will help to select patients for these studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177146PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467809PMC
September 2017

Importance of the novel organic cation transporter 1 for tyrosine kinase inhibition by saracatinib in rheumatoid arthritis synovial fibroblasts.

Sci Rep 2017 04 28;7(1):1258. Epub 2017 Apr 28.

Experimental Nephrology, Department of Internal Medicine D, University Hospital Muenster, Muenster, 48149, Germany.

Recent therapeutic approaches of rheumatoid arthritis (RA) address the use of small molecules such as tyrosine kinase inhibitors (TKIs). However, the TKIs developed to date have important side effects and/or scarce efficacy in inflammatory diseases such as RA. Since intracellular effective TKIs must enter the cell to reach their intracellular targets, here we investigated the interaction of the TKI saracatinib, a dual inhibitor of c-Src and c-Abl signaling, with transporters for organic cations as well as the role of these transporters for the biological effect of saracatinib in human RA-synovial fibroblasts (hRASF). Saracatinib significantly reduced proliferation of hRASF. The cellular saracatinib uptake was mainly dependent on the human novel organic cation transporter 1 (hOCTN1), which showed the highest apparent affinity for saracatinib among all other transporters for organic cations analyzed here. In hRASF, saracatinib biologic function was dependent on hOCTN1. Further analysis showed that disease specific factors (pH, inflammatory cytokines such as TNFα) regulated saracatinib uptake in hRASF. The knowledge of which transporters mediate the specific uptake of TKIs in target cells and of how the expression and function of such transporters are regulated in RA is of highest priority to develop effective drugs for successful therapy with minimal side-effects.
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http://dx.doi.org/10.1038/s41598-017-01438-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430895PMC
April 2017

Combinatorial effects of doxorubicin and retargeted tissue factor by intratumoral entrapment of doxorubicin and proapoptotic increase of tumor vascular infarction.

Oncotarget 2016 Dec;7(50):82458-82472

Department of Medicine A (Hematology, Hemostaseology, Oncology and Pneumology), University Hospital of Muenster, Muenster, Germany.

Truncated tissue factor (tTF), retargeted to tumor vasculature by GNGRAHA peptide (tTF-NGR), and doxorubicin have therapeutic activity against a variety of tumors. We report on combination experiments of both drugs using different schedules. We have tested fluorescence- and HPLC-based intratumoral pharmacokinetics of doxorubicin, flow cytometry for cellular phosphatidylserine (PS) expression, and tumor xenograft studies for showing in vivo apoptosis, proliferation decrease, and tumor shrinkage upon combination therapy with doxorubicin and induced tumor vascular infarction. tTF-NGR given before doxorubicin inhibits the uptake of the drug into human fibrosarcoma xenografts in vivo. Reverse sequence does not influence the uptake of doxorubicin into tumor, but significantly inhibits the late wash-out phase, thus entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with doxorubicin in vitro increases PS concentrations in the outer layer of the cell membrane as a sign of early apoptosis. Cells expressing increased PS concentrations show comparatively higher procoagulatory efficacy on the basis of equimolar tTF-NGR present in the Factor X assay. Experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in athymic nude mice indeed show a combinatorial tumor growth inhibition applying doxorubicin and tTF-NGR in sequence over single drug treatment. Combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by new mechanisms, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in tumor endothelial and tumor cells.
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http://dx.doi.org/10.18632/oncotarget.12559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347705PMC
December 2016

Role of transporters in the distribution of platinum-based drugs.

Front Pharmacol 2015 24;6:85. Epub 2015 Apr 24.

Experimental Nephrology, Medical Clinic D, University of Münster, University Hospital Münster Münster, Germany ; Interdisciplinary Center for Clinical Research (IZKF), University of Münster, University Hospital Münster Münster, Germany.

Platinum derivatives used as chemotherapeutic drugs such as cisplatin and oxaliplatin have a potent antitumor activity. However, severe side effects such as nephro-, oto-, and neurotoxicity are associated with their use. Effects and side effects of platinum-based drugs are in part caused by their transporter-mediated uptake in target and non target cells. In this mini review, the transport systems involved in cellular handling of platinum derivatives are illustrated, focusing on transporters for cisplatin. The copper transporter 1 seems to be of particular importance for cisplatin uptake in tumor cells, while the organic cation transporter (OCT) 2, due to its specific organ distribution, may play a major role in the development of undesired cisplatin side effects. In polarized cells, e.g., in renal proximal tubule cells, apically expressed transporters, such as multidrug and toxin extrusion protein 1, mediate secretion of cisplatin and in this way contribute to the control of its toxic effects. Specific inhibition of cisplatin uptake transporters such as the OCTs may be an attractive therapeutic option to reduce its toxicity, without impairing its antitumor efficacy.
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http://dx.doi.org/10.3389/fphar.2015.00085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408848PMC
May 2015

The organic cation transporter 3 (OCT3) as molecular target of psychotropic drugs: transport characteristics and acute regulation of cloned murine OCT3.

Pflugers Arch 2014 Mar 28;466(3):517-27. Epub 2013 Aug 28.

Experimental Nephrology, Department of Internal Medicine D, University of Münster, Albert-Schweitzer-Campus 1 (A14), 48149, Münster, Germany.

The organic cation transporter 3 (OCT3) is a widely expressed transporter for endogenous and exogenous organic cations. Of particular interest is OCT3 expression and function in the brain, where it plays a role in serotonin clearance and influences mood and behavior. Protein kinase signaling mediates rapid modulation of cerebral processes, but little is known about acute regulation of OCT3 by protein kinases. Therefore, we cloned mouse OCT3 (mOCT3) and generated a human embryonic kidney cell line stably expressing the transporter to study transport characteristics, acute regulation by protein kinases, and interaction with psychotropic drugs. Uptake measurement was performed using the fluorescent cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+), 1 μM) as a substrate. The translational value of these findings was determined by comparing results obtained with cloned mouse and human OCT3. mOCT3-mediated transport is membrane potential dependent and pH independent. ASP(+) uptake by mOCT3 and human OCT3 (hOCT3) was efficiently inhibited by 1-methyl-4-phenylpyridinium, tetrapentylammonium (TPA(+)), corticosterone, serotonin, and histamine and by the drugs ketamine, fluoxetine, and diazepam. The half maximal inhibitory concentrations of mOCT3 and hOCT3 for TPA(+), serotonin, diazepam, and ketamine are significantly different. Diazepam is a non-transported inhibitor. Furthermore, the activities of mOCT3 and hOCT3 are acutely regulated by the p56 (lck) tyrosine kinase by decreasing their V max. Studies with freshly isolated renal proximal tubules from mOCT1/2(-/-) mice, in which mOCT3 is the only OCT present, confirmed this regulation pathway. Only the activity of hOCT3 is regulated by calmodulin. These findings suggest that even though many transport properties of mOCT3 and hOCT3 are similar, there are also species-specific aspects of OCT3 function.
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http://dx.doi.org/10.1007/s00424-013-1335-8DOI Listing
March 2014

Transport mechanisms and their pathology-induced regulation govern tyrosine kinase inhibitor delivery in rheumatoid arthritis.

PLoS One 2012 20;7(12):e52247. Epub 2012 Dec 20.

Experimental Nephrology, Department of Internal Medicine D, University Hospital Münster, Münster, Germany.

Background: Tyrosine kinase inhibitors (TKIs) are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA) the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions.

Methodology/principal Findings: The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF). Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1β and IL-6, which all decreased the expression of hMATE1-mRNA and protein.

Conclusion/significance: The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important for developing intracellular acting drugs used in inflammatory diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052247PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527388PMC
June 2013