scholarly journals Covalent 18F-Radiotracers for SNAPTag: A New Toolbox for Reporter Gene Imaging

2021 ◽  
Vol 14 (9) ◽  
pp. 897
Author(s):  
Sophie Stotz ◽  
Gregory D. Bowden ◽  
Jonathan M. Cotton ◽  
Bernd J. Pichler ◽  
Andreas Maurer
Keyword(s):  
Reporter Gene ◽  
Xenograft Model ◽  
Nuclear Imaging ◽  
Hek293 Cells ◽  
In Vivo Studies ◽  
Reporter Gene Imaging ◽  
Immunodeficient Mice ◽  
Biodistribution Studies ◽  
Sds Page

There is a need for versatile in vivo nuclear imaging reporter systems to foster preclinical and clinical research. We explore the applicability of the SNAPTag and novel radiolabeled small-molecule ligands as a versatile reporter gene system for in vivo nuclear imaging. SNAPTag is a high-affinity protein tag used in a variety of biochemical research areas and based on the suicide DNA repair enzyme O6-methylguanine methyl transferase (MGMT). Its ligands are well suited for reporter gene imaging as the benzyl guanine core scaffold can be derivatized with fluorescent or radiolabeled moieties for various applications. Three guanine-based SNAPTag ligands ([18F]FBBG, [18F]pFBG and [18F]mFBG) were synthesized in high yields and were (radio)chemically characterized. HEK293 cells were engineered to express the SNAPTag on the cell surface and served as cell model to assess target affinity by radiotracer uptake assays, Western blotting and SDS-PAGE autoradiography. A subcutaneous HEK293-SNAPTag xenograft model in immunodeficient mice was used for in vivo evaluation of [18F]FBBG and [18F]pFBG while the biodistribution of [18F]mFBG was characterized in naïve animals. The results were validated by ex vivo biodistribution studies and immunofluorescence staining of the xenografts. All three radiotracers were produced in high radiochemical purity, molar activity and good yields. Western blot analysis revealed successful SNAPTag expression by the transfected HEK293 cells. In vitro testing revealed high target affinity of all three tracers with an up to 191-fold higher signal in the HEK293-SNAPTag cells compared to untransfected cells. This was further supported by a prominent radioactive protein band at the expected size in the SDS-PAGE autoradiograph of cells incubated with [18F]FBBG or [18F]pFBG. The in vivo studies demonstrated high uptake in HEK293-SNAP xenografts compared to HEK293 xenografts with excellent tumor-to-muscle ratios (7.5 ± 4.2 for [18F]FBBG and 10.6 ± 6.2 for [18F]pFBG). In contrast to [18F]pFBG and its chemical analogue [18F]mFBG, [18F]FBBG showed no signs of unspecific bone uptake and defluorination in vivo. Radiolabeled SNAPTag ligands bear great potential for clinical applications such as in vivo tracking of cell populations, antibody fragments and targeted radiotherapy. With excellent target affinity, good stability, and low non-specific binding, [18F]FBBG is a highly promising candidate for further preclinical evaluation.

scholarly journals Covalent 18F-Radiotracers for SNAPTag: a New Toolbox for Reporter Gene Imaging

2021 ◽  
Author(s):  
Sophie Stotz ◽  
Gregory D. Bowden ◽  
Jonathan M. Cotton ◽  
Bernd J. Pichler ◽  
Andreas Maurer
Keyword(s):  
Reporter Gene ◽  
Xenograft Model ◽  
Nuclear Imaging ◽  
Hek293 Cells ◽  
In Vivo Studies ◽  
Reporter Gene Imaging ◽  
Immunodeficient Mice ◽  
Biodistribution Studies ◽  
Sds Page

There is a need for versatile in vivo nuclear imaging reporter systems to foster preclinical and clinical research. We explore the applicability of the SNAPTag and novel radiolabeled small-molecule ligands as a versatile reporter gene system for in vivo nuclear imaging. SNAPTag is a high-affinity protein tag used in a variety of biochemical research areas and based on the suicide DNA repair enzyme O6-methylguanine methyl transferase (MGMT). Its ligands are well suited for reporter gene imaging as the benzyl guanine core scaffold can be derivatized with fluorescent or radiolabeled moieties for various applications. Three guanine-based SNAPTag ligands ([18F]FBBG, [18F]pFBG and [18F]mFBG) were synthesized in high yields and were (radio)chemically characterized. HEK293 cells were engineered to express the SNAPTag on the cell surface and served as cell model to assess target affinity by radiotracer uptake assays, Western blotting and SDS-PAGE autoradiography. A subcutaneous HEK293-SNAPTag xenograft model in immunodeficient mice was used for in vivo evaluation of [18F]FBBG amd [18F]pFBG while the biodistribution of [18F]mFBG was characterized in naïve animals. The results were validated by ex vivo biodistribution studies and immunofluorescence staining of the xenografts. All three radiotracers were produced in high radiochemical purity, molar activity and good yields. Western blot analysis revealed successful SNAPTag expression by the transfected HEK293 cells. In vitro testing revealed high target affinity of all three tracers with an up to 191-fold higher signal in the HEK293-SNAPTag cells compared to untransfected cells. This was further supported by a prominent radioactive protein band at the expected size in the SDS-PAGE autoradiograph of cells incubated with [18F]FBBG or [18F]pFBG. The in vivo studies demonstrated high uptake in HEK293-SNAP xenografts compared to HEK293 xenografts with excellent tumor-to-muscle ratios (7.5 ± 4.2 for [18F]FBBG and 10.6 ± 6.2 for [18F]pFBG). In contrast to [18F]pFBG and its chemical analogue [18F]mFBG, [18F]FBBG showed no signs of unspecific bone uptake and defluorination in vivo. Radiolabeled SNAPTag ligands bear great potential for clinical applications such as in vivo tracking of cell populations, antibody fragments and targeted radiotherapy. With excellent target affinity, good stability, and low non-specific binding, [18F]FBBG is a highly promising candidate for further preclinical evaluation.

scholarly journals Radiolabeling and Quantitative In Vivo SPECT/CT Imaging Study of Liposomes Using the Novel Iminothiolane-99mTc-Tricarbonyl Complex

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Zoltán Varga ◽  
Imola Cs. Szigyártó ◽  
István Gyurkó ◽  
Rita Dóczi ◽  
Ildikó Horváth ◽  
...  
Keyword(s):  
Specific Activity ◽  
Nuclear Imaging ◽  
Imaging Study ◽  
Size Exclusion ◽  
In Vivo Studies ◽  
The Novel ◽  
Tricarbonyl Complex ◽  
Biodistribution Studies ◽  
Exclusion Chromatography

The in vivo biodistribution of liposomal formulations greatly influences the pharmacokinetics of these novel drugs; therefore the radioisotope labeling of liposomes and the use of nuclear imaging methods for in vivo studies are of great interest. In the present work, a new procedure for the surface labeling of liposomes is presented using the novel 99mTc-tricarbonyl complex. Liposomes mimicking the composition of two FDA approved liposomal drugs were used. In the first step of the labeling, thiol-groups were formed on the surface of the liposomes using Traut’s reagent, which were subsequently used to bind 99mTc-tricarbonyl complex to the liposomal surface. The labeling efficiency determined by size exclusion chromatography was 95%, and the stability of the labeled liposomes in bovine serum was found to be 94% over 2 hours. The obtained specific activity was 50 MBq per 1 μmol lipid which falls among the highest values reported for 99mTc labeling of liposomes. Quantitative in vivo SPECT/CT biodistribution studies revealed distinct differences between the labeled liposomes and the free 99mTc-tricarbonyl, which indicates the in vivo stability of the labeling. As the studied liposomes were non-PEGylated, fast clearance from the blood vessels and high uptake in the liver and spleen were observed.

scholarly journals Efficacy and Biomarker Analysis of Adavosertib in Differentiated Thyroid Cancer

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3487
Author(s):  
Yu-Ling Lu ◽  
Ming-Hsien Wu ◽  
Yi-Yin Lee ◽  
Ting-Chao Chou ◽  
Richard J. Wong ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Cell Lines ◽  
Differentiated Thyroid Cancer ◽  
Xenograft Model ◽  
In Vivo Studies ◽  
Follicular Thyroid Cancer ◽  
Biomarker Analysis ◽  
Cancer Tumor

Differentiated thyroid cancer (DTC) patients are usually known for their excellent prognoses. However, some patients with DTC develop refractory disease and require novel therapies with different therapeutic mechanisms. Targeting Wee1 with adavosertib has emerged as a novel strategy for cancer therapy. We determined the effects of adavosertib in four DTC cell lines. Adavosertib induces cell growth inhibition in a dose-dependent fashion. Cell cycle analyses revealed that cells were accumulated in the G2/M phase and apoptosis was induced by adavosertib in the four DTC tumor cell lines. The sensitivity of adavosertib correlated with baseline Wee1 expression. In vivo studies showed that adavosertib significantly inhibited the xenograft growth of papillary and follicular thyroid cancer tumor models. Adavosertib therapy, combined with dabrafenib and trametinib, had strong synergism in vitro, and revealed robust tumor growth suppression in vivo in a xenograft model of papillary thyroid cancer harboring mutant BRAFV600E, without appreciable toxicity. Furthermore, combination of adavosertib with lenvatinib was more effective than either agent alone in a xenograft model of follicular thyroid cancer. These results show that adavosertib has the potential in treating DTC.

scholarly journals Harnessing PET to track micro- and nanoplastics in vivo

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Outi Keinänen ◽  
Eric J. Dayts ◽  
Cindy Rodriguez ◽  
Samantha M. Sarrett ◽  
James M. Brennan ◽  
...  
Keyword(s):  
Sea Water ◽  
Accurate Determination ◽  
Nuclear Imaging ◽  
Imaging Data ◽  
Biodistribution Studies ◽  
Positron Emission ◽  
20 Nm ◽  
Pharmacokinetic Behavior

AbstractThe proliferation of plastics in the environment continues at an alarming rate. Plastic particles have been found to be persistent and ubiquitous pollutants in a variety of environments, including sea water, fresh water, soil, and air. In light of this phenomenon, the scientific and medical communities have become increasingly wary of the dangers posed to human health by chronic exposure to microplastics (< 5 mm diameter) and nanoplastics (< 100 nm diameter). A critical component of the study of the health effects of these pollutants is the accurate determination of their pharmacokinetic behavior in vivo. Herein, we report the first use of molecular imaging to track polystyrene (PS) micro- and nanoplastic particles in mammals. To this end, we have modified PS particles of several sizes—diameters of 20 nm, 220 nm, 1 µm, and 6 µm—with the chelator desferrioxamine (DFO) and radiolabeled these DFO-bearing particles with the positron-emitting radiometal zirconium-89 (89Zr; t1/2 ~ 3.3 d). Subsequently, positron emission tomography (PET) was used to visualize the biodistribution of these radioplastics in C57BL/6J mice at 6, 12, 24, and 48 h after ingestion. The imaging data reveal that the majority of the radioplastics remain in the gastrointestinal tract and are eliminated through the feces by 48 h post-ingestion, a result reinforced by acute biodistribution studies. Ultimately, this work suggests that nuclear imaging—and PET in particular—can be a sensitive and effective tool in the urgent and rapidly growing effort to study the in vivo behavior and potential toxicity of micro- and nanoplastics.

scholarly journals Visualisation of interstitial lung disease by molecular imaging of integrin αvβ3 and somatostatin receptor 2

2018 ◽  
Vol 78 (2) ◽  
pp. 218-227 ◽  
Author(s):  
Janine Schniering ◽  
Martina Benešová ◽  
Matthias Brunner ◽  
Stephanie Haller ◽  
Susan Cohrs ◽  
...  
Keyword(s):  
Interstitial Lung Disease ◽  
Lung Disease ◽  
Single Photon ◽  
Ex Vivo ◽  
Somatostatin Receptor ◽  
Photon Emission ◽  
Nuclear Imaging ◽  
Integrin Αvβ3 ◽  
Biodistribution Studies

ObjectiveTo evaluate integrin αvβ3 (alpha-v-beta-3)-targeted and somatostatin receptor 2 (SSTR2)-targeted nuclear imaging for the visualisation of interstitial lung disease (ILD).MethodsThe pulmonary expression of integrin αvβ3 and SSTR2 was analysed in patients with different forms of ILD as well as in bleomycin (BLM)-treated mice and respective controls using immunohistochemistry. Single photon emission CT/CT (SPECT/CT) was performed on days 3, 7 and 14 after BLM instillation using the integrin αvβ3-targeting 177Lu-DOTA-RGD and the SSTR2-targeting 177Lu-DOTA-NOC radiotracer. The specific pulmonary accumulation of the radiotracers over time was assessed by in vivo and ex vivo SPECT/CT scans and by biodistribution studies.ResultsExpression of integrin αvβ3 and SSTR2 was substantially increased in human ILD regardless of the subtype. Similarly, in lungs of BLM-challenged mice, but not of controls, both imaging targets were stage-specifically overexpressed. While integrin αvβ3 was most abundantly upregulated on day 7, the inflammatory stage of BLM-induced lung fibrosis, SSTR2 expression peaked on day 14, the established fibrotic stage. In agreement with the findings on tissue level, targeted nuclear imaging using SPECT/CT specifically detected both imaging targets ex vivo and in vivo, and thus visualised different stages of experimental ILD.ConclusionOur preclinical proof-of-concept study suggests that specific visualisation of molecular processes in ILD by targeted nuclear imaging is feasible. If transferred into clinics, where imaging is considered an integral part of patients’ management, the additional information derived from specific imaging tools could represent a first step towards precision medicine in ILD.

scholarly journals In vivo Monitoring of microRNA Biogenesis Using Reporter Gene Imaging

Theranostics ◽  
2013 ◽  
Vol 3 (12) ◽  
pp. 1004-1011 ◽  
Author(s):  
So Won Oh ◽  
Do Won Hwang ◽  
Dong Soo Lee
Keyword(s):  
Reporter Gene ◽  
Microrna Biogenesis ◽  
Reporter Gene Imaging ◽  
In Vivo Monitoring

scholarly journals Humanization, Radiolabeling and Biodistribution Studies of an IgG1-Type Antibody Targeting Uncomplexed PSA for Theranostic Applications

2021 ◽  
Vol 14 (12) ◽  
pp. 1251
Author(s):  
Joanna Strand ◽  
Kjell Sjöström ◽  
Urpo J. Lamminmaki ◽  
Oskar Vilhelmsson Timmermand ◽  
Sven-Erik Strand ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Specific Binding ◽  
Hek293 Cells ◽  
Castration Resistant Prostate Cancer ◽  
Castration Resistant ◽  
Biodistribution Studies ◽  
Indium 111 ◽  
Hormone Refractory ◽  
Theranostic Applications

Metastatic castration-resistant prostate cancer is today incurable. Conventional imaging methods have limited detection, affecting their ability to give an accurate outcome prognosis, and current therapies for metastatic prostate cancer are insufficient. This inevitably leads to patients relapsing with castration-resistant prostate cancer. Targeting prostate-specific antigens whose expression is closely linked to the activity in the androgen receptor pathway, and thus the pathogenesis of prostate cancer, is a possible way to increase specificity and reduce off-target effects. We have humanized and evaluated radioimmunoconjugates of a previously murine antibody, m5A10, targeting PSA intended for theranostics of hormone-refractory prostate cancer. The humanized antibody h5A10 was expressed in mammalian HEK293 cells transfected with the nucleotide sequences for the heavy and light chains of the antibody. Cell culture medium was filtered and purified by Protein G chromatography, and the buffer was changed to PBS pH 7.4 by dialysis. Murine and humanized 5A10 were conjugated with p-SCN-Bn-CHX-A”-DTPA. Surface plasmon resonance was used to characterize the binding to PSA of the immunoconjugates. Immunoconjugates were labeled with either indium-111 or lutetium-177. Biodistribution studies of murine and humanized 5A10 were performed in mice with LNCaP xenografts. 5A10 was successfully humanized, and in vivo targeting showed specific binding in xenografts. The results thus give an excellent platform for further theranostic development of humanized 5A10 for clinical applications.

Construction and Characterization of a Novel Ribonuclease Immunotoxin Consisting of Two Ranpirnase (Rap) Molecules Fused to an Internalizing Anti-CD74 Humanized IgG4 Antibody.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3289-3289
Author(s):  
Sailaja S. Vanama ◽  
Puja Sapra ◽  
Hans J. Hansen ◽  
Ivan D. Horak ◽  
David M. Goldenberg ◽  
...  
Keyword(s):  
Hodgkin’S Lymphoma ◽  
Xenograft Model ◽  
Hodgkin's Lymphoma ◽  
Light Chains ◽  
Non Hodgkin’S Lymphoma ◽  
Non Hodgkin's Lymphoma ◽  
Sds Page

Abstract Ranpirnase (Rap), isolated from frog (Rana pipiens) oocytes, is a monomeric ribonuclease (MW 11800) that kills cells by degrading t-RNA upon internalization. Previous studies indicated that the cytotoxicity of Rap could be enhanced more than 10,000-fold when the enzyme is chemically conjugated to an internalizing antibody. Here we describe the construction and characterization of 2L-Rap-hLL1-γ4P, composed of two Rap molecules fused to hLL1, an internalizing anti-CD74 humanized monoclonal antibody. To reduce unwanted cytotoxicity, the IgG1 constant region of hLL1 was replaced with an IgG4 that contains a proline mutation in the hinge region. The Rap gene was inserted at the N-terminus of the light chain in the expression vector of hLL1 and expressed in NS0 mouse myeloma cells. The fusion protein was characterized by a variety of techniques, including SE-HPLC, SDS-PAGE, in vitro transcription translation (IVTT) assay using luciferase reporter system, and competition ELISA to measure the binding affinity for CD74. The in vitro potency was determined in non-Hodgkin’s lymphoma (Daudi) and multiple myeloma (MC/CAR) cell lines by MTS tetrazolium dye reduction assay. In vivo pharmacokinetics and biodistribution of radiolabeled 2L-Rap-hLL1- γ4P was compared to radiolabeled hLL1 mAb in naïve mice and in vivo therapeutic efficacy of 2L-Rap-hLL1- γ4P was determined in a xenograft model of Burkitt’s non-Hodgkin’s lymphoma (Daudi). Purified 2L-Rap-hLL1- γ4P was shown to be a single peak by SE-HPLC and its MW determined by MALDI-TOF to be 177,150, which is in agreement with the MW of one IgG (150,000) plus two Rap molecules (24,000). Reducing-SDS-PAGE of 2L-Rap-hLL1- γ4P revealed the presence of 3 bands, one corresponding to the heavy chain and the other two appearing to be derived from the Rap-fused light chains (38,526 and 36,700 by MS). Occurrence of the 2 light chains was shown to be due to glycosylation of Rap at the N69 residue. The binding affinity of 2L-Rap-hLL1- γ4P for CD74 was indistinguishable from that of hLL1. Both 2L-Rap-hLL1- γ4P and hLL1 bound to CD74 with subnanomolar affinity. The EC50 of RNase activity, as measured by the IVTT assay, was 300 pM for 2L-Rap-hLL1- γ4P and 30 pM for recombinant Rap (expressed in E. coil). In in vitro cytotoxicity assays, 2L-Rap-hLL1- γ4P was significantly cytotoxic against Daudi (EC50 280 pM) and the myeloma cell line, MC/CAR (EC50 50 nM). In contrast, free Rap or naked hLL1 did not demonstrate significant cytotoxicity at the concentrations tested. In vivo, the pharmacokinetic profile of 2L-Rap-hLL1- γ4P was almost identical to that of naked hLL1. Both 2L-Rap-hLL1- γ4P and hLL1 showed biphasic clearance from the circulation; the α and β half-life (t1/2) of 2L-Rap-hLL1- γ4P were 5 h and 119 h, respectively, and those of hLL1 were 4 h and 125 h, respectively. In tissue biodistribution studies, no significant difference was observed between 2L-Rap-hLL1- γ4P and hLL1 with regards to normal tissue uptake. Early efficacy results in the Daudi Burkitt’s non-Hodgkin’s lymphoma xenograft model demonstrate that treatment with a single dose of 2L-Rap-hLL1- γ4P as low as 1 μg/mouse significantly improves survival in comparison to untreated control mice (P<0.0001).

YM155, a Novel Survivin Suppressant, Improves the Antitumor Effect of Rituximab and Rituximab-Containing Regimens in Diffuse Large B Cell Lymphoma (DLBCL) Xenograft Mouse Models.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2718-2718
Author(s):  
Mari Nakata ◽  
Takahito Nakahara ◽  
Aya Kita ◽  
Keisuke Mitsuoka ◽  
Kentaro Yamanaka ◽  
...  
Keyword(s):  
Antitumor Effect ◽  
Cell Lymphoma ◽  
Tumor Regression ◽  
Xenograft Model ◽  
Body Weight Loss ◽  
B Cell Lymphoma ◽  
In Vivo Studies ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 2718 Poster Board II-694 Introduction: Survivin is a member of the inhibitor of apoptosis (IAP) family of proteins, and is highly expressed in many tumor types. Given its preferential expression in tumor cells, and its ability to block apoptosis and regulate cancer cell proliferation, survivin appears to be an attractive novel target for cancer therapy. YM155 is a novel, small molecule survivin suppressant (Nakahara et al., Cancer Research. 2007;67:8014–21). In this study, we evaluated the antitumor activity of YM155 alone and in combination with rituximab, R-ICE (rituximab + ifosfamide + carboplatin + etoposide), or [rituximab + cytarabin + cisplatin] in DLBCL xenograft models. Methods: Antiproliferative effect of YM155 in a panel of human DLBCL cell lines (DB, Pfeiffer, SU-DHL5, SU-DHL8, WSU-DLCL-2, and RL) was evaluated by sulforhodamine B assay. In in vivo studies, WSU-DLCL-2 and DB were subcutaneously implanted into male BALB/c nu/nu mice. When tumors reached a volume of 300 to 600 mm3, YM155 was administered as a 7-day continuous sc infusion, and the other drugs were administered via iv bolus. Dose and schedule of each drug were adjusted to clinical equivalent dose. PET imaging studies were performed using a Inveon PET/CT system (Siemens Medical Solusion). WSU-DLCL-2 xenografted mice were intravenously injected with [18F] FLT, and five-minute static PET scans were accqiured at 1h after injection. For each small-animal PET scan, region of interest was drawn over each tumor and over normal tissue on decay-corrected whole-body sagittal imagies. Results: In in vitro proliferation assays, YM155 showed potent antiproliferative activity against all six DLBCL cell lines, with GI50 values of 0.35 to 3.9 nM. In in vivo studies using WSU-DLCL2 xenograft model, YM155 at 1 and 3 mg/kg induced tumor regression without body weight loss. In combination studies using WSU-DLCL2 xenograft model, YM155 2 mg/kg enhanced antitumor effects of rituximab, R-ICE and [rituximab + cytarabin + cisplatin] without enhancement of the body weight loss. Tumor regression in the combination groups was sustained longer than single treatment groups, and even complete regressions were achievable. Moreover, combination of YM155 1 mg/kg and rituximab induced strong tumor regression in the DB xenograft model, while single-agent treatments did not show significant antitumor effect compared to vehicle control. In [18F]FLT-PET imaging, a significant reduction of FLT uptake in tumor was observed in rituximab combination group, which was more sensitive than the reduction in tumor volume. Conclusions: YM155 improves the antitumor effect of rituximab and rituximab-containing regimens in diffuse large B cell lymphoma (DLBCL) xenograft mouse models. Disclosures: Nakata: Astellas Pharma Inc.: Employment. Nakahara:Astellas Pharma Inc.: Employment. Kita:Astellas Pharma Inc.: Employment. Mitsuoka:Astellas Pharma Inc.: Employment. Yamanaka:Astellas Pharma Inc.: Employment. Kaneko:Astellas Pharma Inc.: Employment. Miyoshi:Astellas Pharma Inc.: Employment. Mori:Astellas Pharma Inc.: Employment. Koutoku:Astellas Pharma Inc.: Employment. Sasamata:Astellas Pharma Inc.: Employment.

Carfilzomib Platform Study Using The LAGκ-1A Multiple Myeloma Xenograft Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4452-4452
Author(s):  
Eric Sanchez ◽  
Mingjie Li ◽  
Suzie Vardanyan ◽  
Jillian Gottlieb ◽  
Kevin Delijani ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Disease Progression ◽  
Tumor Size ◽  
Single Agent ◽  
Xenograft Model ◽  
In Vivo Studies ◽  
Human Igg ◽  
Similar Treatment ◽  
Agent Treatment

Introduction We previously demonstrated that severe combined immunodeficient (SCID) mice bearing the human multiple myeloma (MM) xenograft LAGκ-1A treated with single agent carfilzomib or the alkylating agent (AA) cyclophosphamide (CY) did not show a reduction in tumor growth compared to vehicle-treated mice. In contrast, carfilzomib with CY resulted in a significant decrease in tumor size and IgG levels when compared to mice treated with single agent carfilzomib or CY or vehicle alone. We have also shown that the combination of carfilzomib and another AA, bendamustine, decreased tumor size and IgG levels, when compared to mice treated with single agents or vehicle alone. However, no data is available regarding sequencing of the proteasome inhibitors (PI) carfilzomib or bortezomib with the AA melphalan (MEL). Thus, we used our SCID-hu MM models to evaluate the sequencing of these drugs with MEL. These studies are critical as both PIs are now being used to treat MM. Thus, we evaluated the response, toxicity and survival of animals treated sequentially with these drugs. Methods Each naïve SCID mouse was surgically implanted with a 20 – 40 mm3 MM tumor piece into the left hind limb superficial gluteal muscle. Seven days post–implantation mice were randomized into treatment groups based on human immunoglobulin (Ig) G levels. Carfilzomib stock solution (2 mg/ml) was diluted to 3 mg/kg using 5% dextrose and administered twice weekly on two consecutive days via intravenous (i.v.) injection. Bortezomib stock solution (1 mg/ml) was diluted to 0.25 mg/kg using NaCl and administered twice weekly (Thursdays and Saturdays) via i.v. injection. MEL stock solution (3 mg/ml) was diluted to 1 mg/kg using PBS and administered once weekly via intraperitoneal injection. Mice (n = 10/group) were initially treated with carfilzomib or MEL alone until tumor progression. Progression was defined as an increase in paraprotein equal to or above 25% confirmed on one consecutive assessment. Mice initially treated with carfilzomib were randomized to continue to receive single agent carfilzomib, add in MEL alone or combine it with ongoing carfilzomib, substitute single agent bortezomib, or discontinue treatment altogether. A similar treatment strategy was evaluated with mice treated initially with MEL. At progression, these animals were continued on single agent MEL, carfilzomib added alone or with continuation of MEL, or discontinued treatment. Tumor size was measured using standard calipers and human IgG levels with an ELISA (Bethyl Laboratories, Montgomery, TX). This study was conducted according to protocols approved by the Institutional Animal Care and Use Committee. Results When carfilzomib was administered first, followed by the addition of MEL, a modest nonsignificant reduction in tumor size was observed compared to either drug alone. In addition, substitution of single agent bortezomib for carfilzomib showed no effect on tumor size. However, when MEL was administered first and carfilzomib was added after disease progression, at days 35 and 42 (end of study) post tumor implantation, mice treated with the combination showed a reduction in tumor volume compared to mice that discontinued melphalan (P = 0.0378 and P = 0.0105, respectively) whereas mice treated with carfilzomib alone showed no reduction in tumor size following progression from MEL. Notably, throughout the study there was a trend toward smaller tumors in mice receiving this combination when compared to mice receiving single agent treatment with carfilzomib or MEL alone or vehicle. Similar effects were observed on human IgG levels. Overall, all mice survived combination or single agent treatment with these agents. Conclusions These in vivo studies using our human MM LAGκ–1A SCID–hu model show that animals progressing from initial MEL treatment show a reduction in MM tumor burden when carfilzomib is added to MEL at progression. In contrast, mice progressing from initial carfilzomib treatment did not benefit from the addition of MEL at disease progression. No drug-related deaths occurred in any treatment group. This study demonstrates that using a different MM model (LAGκ-1A), that the PI carfilzomib can produce anti-tumor effects among mice progressing from single-agent MEL treatment, providing further support for the use of this PI as an agent that can help overcome drug resistance in MM. Disclosures: Berenson: Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau.

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