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Background: The Androgen Receptor (AR) remains the principal driver of castration-resistant prostate cancer during the transition from a localized to metastatic disease. Most ...patients initially respond to inhibitors of the AR pathway, but the response is often short-lived. The majority of patients progressing on enzalutamide or abiraterone exhibit genetic alterations in the AR locus, either in the form of amplifications or point mutations in the AR gene. Given these mechanisms of resistance, our goal is to eliminate the AR protein using the PROteolysis TArgeting Chimera (PROTAC) technology. Further, we sought to make an orally bioavailable AR PROTAC. Methods: Medicinal chemistry efforts yielded a small molecule AR PROTAC that simultaneously binds E3-ubiquitin ligase and AR, thus leading to ubiquitination and degradation of AR. This molecule has been characterized in in vitro and in vivo preclinical studies. Results: Our lead oral AR PROTAC degrades 92-98% of total AR in all cell lines tested, with 50% degradation concentration (DC
50
) < 1 nM. AR degradation suppresses the expression of AR-target gene PSA, inhibits cell proliferation, and induces potent apoptosis in VCaP cells. No activity is observed in AR-null cell lines, such as PC-3. While enzalutamide loses its activity in the presence of elevated androgens, AR PROTAC maintains its antiproliferative activity. Further, AR PROTAC is able to degrade all clinically relevant mutant AR proteins. A robust oral bioavailability is observed across multiple species and overall ADME properties are encouraging. Approximately 95% AR degradation is observed in AR-amplified VCaP xenografts at doses as low as 10 mg/kg. Congruent with AR degradation, a dose responsive tumor growth inhibition is observed in AR-dependent xenograft studies. Conclusions: In summary, we report the first orally bioavailable AR PROTAC that robustly degrades AR in vitro and in vivo.
The L1‐cell adhesion molecule (L1‐CAM) is highly expressed in various cancer types including ovarian carcinoma but is absent from most normal tissue. A chimeric monoclonal antibody, chCE7, ...specifically binds to human L1‐CAM and exhibits anti‐proliferative effects on L1‐CAM‐expressing tumor cells. The goal of this study was to evaluate the efficacy of a novel 177Lu‐chCE7 radioimmunotherapeutic agent and to compare it to a treatment protocol with unlabeled, growth‐inhibiting chCE7 in a mouse xenograft model of disseminated ovarian cancer. chCE7agl, an aglycosylated IgG1 variant with improved pharmacokinetics, was conjugated with 1,4,7,10‐tetraazacyclododecane‐N‐N′‐N′‐N‴‐tetraacetic acid (DOTA) and labeled with the low‐energy β‐emitter 177Lu. Tumor growth and survival were assessed after a single i.v. dose of 8 MBq (60 μg) radioimmunoconjugate in nude mice bearing either subcutaneous or intraperitoneal SKOV3.ip1 human ovarian cancer tumors. Therapeutic efficacy was compared with three times weekly i.p. administration of 10 mg/kg unconjugated chCE7. In vivo analysis of 177Lu‐chCE7agl biodistribution demonstrated high and specific accumulation of radioactivity at the tumor site with maximal tumor uptake of up to 48.0 ± 8.1% ID/g at 168 h postinjection. A single treatment with 177Lu‐DOTA‐chCE7agl caused significant retardation of tumor growth and prolonged median survival from 33 to 71 days, while administration of a nontargeted 177Lu‐immunoconjugate had no beneficial effect. Three times weekly i.p. application of unlabeled chCE7 10 mg/kg similarly increased survival from 44 to 72 days. We conclude that a single dose of 177Lu‐DOTA‐chCE7agl is as effective as repeated administration of nonradioactive chCE7 for treatment of small intraperitoneal tumors expressing L1‐CAM.
The insulin-like growth factor I receptor (IGF-IR) is a transmembrane tyrosine kinase that is essential to growth and development and also thought to provide a survival signal for the maintenance of ...the transformed phenotype. There has been increasing interest in further understanding the role of IGF-I signaling in cancer and in developing receptor antagonists for therapeutic application. We describe herein a novel animal model that involves transgenic expression of a fusion receptor that is constitutively activated by homodimerization. Transgenic mice that expressed the activated receptor showed aberrant development of the mammary glands and developed salivary and mammary adenocarcinomas as early as 8 weeks of age. Xenograft tumors and a cell line were derived from the transgenic animals and are sensitive to inhibition by a novel small-molecule inhibitor of the IGF-IR kinase. This new model should provide new opportunities for further understanding how aberrant IGF-IR signaling leads to tumorigenesis and for optimizing novel antagonists of the receptor kinase.
The discovery, synthesis, and characterization of 9H-carbazole-1-carboxamides as potent and selective ATP-competitive inhibitors of Janus kinase 2 (JAK2) are discussed. Display omitted
The discovery, ...synthesis, and characterization of 9H-carbazole-1-carboxamides as potent and selective ATP-competitive inhibitors of Janus kinase 2 (JAK2) are discussed. Optimization for JAK family selectivity led to compounds 14 and 21, with greater than 45-fold selectivity for JAK2 over all other members of the JAK kinase family.
Compound 3 (BMS-536924), a novel small-molecule inhibitor of the insulin-like growth factor receptor kinase with equal potency against the insulin receptor is described. The in vitro and in vivo ...biological activity of this interesting compound is also reported.
The probiotic product Symbioflor2 (DSM 17252) is a bacterial concentrate of six different Escherichia coli genotypes, whose complete genome sequences are compared here, between each other as well as ...to other E. coli genomes. The genome sequences of Symbioflor2 E. coli components contained a number of virulence-associated genes. Their presence seems to be in conflict with a recorded history of safe use, and with the observed low frequency of adverse effects over a period of more than 6 years. The genome sequences were used to identify unique sequences for each component, for which strain-specific hybridization probes were designed. A colonization study was conducted whereby five volunteers were exposed to an exceptionally high single dose. The results showed that the probiotic E. coli could be detected for 3 months or longer in their stools, and this was in particular the case for those components containing higher numbers of virulence-associated genes. Adverse effects from this long-term colonization were absent. Thus, the presence of the identified virulence genes does not result in a pathogenic phenotype in the genetic background of these probiotic E. coli.
Abstract
The L1‐cell adhesion molecule (L1‐CAM) is highly expressed in various cancer types including ovarian carcinoma but is absent from most normal tissue. A chimeric monoclonal antibody, chCE7, ...specifically binds to human L1‐CAM and exhibits anti‐proliferative effects on L1‐CAM‐expressing tumor cells. The goal of this study was to evaluate the efficacy of a novel
177
Lu‐chCE7 radioimmunotherapeutic agent and to compare it to a treatment protocol with unlabeled, growth‐inhibiting chCE7 in a mouse xenograft model of disseminated ovarian cancer. chCE7agl, an aglycosylated IgG1 variant with improved pharmacokinetics, was conjugated with 1,4,7,10‐tetraazacyclododecane‐
N
‐
N
′‐
N
′‐
N
‴‐tetraacetic acid (DOTA) and labeled with the low‐energy β‐emitter
177
Lu. Tumor growth and survival were assessed after a single i.v. dose of 8 MBq (60 μg) radioimmunoconjugate in nude mice bearing either subcutaneous or intraperitoneal SKOV3.ip1 human ovarian cancer tumors. Therapeutic efficacy was compared with three times weekly i.p. administration of 10 mg/kg unconjugated chCE7.
In vivo
analysis of
177
Lu‐chCE7agl biodistribution demonstrated high and specific accumulation of radioactivity at the tumor site with maximal tumor uptake of up to 48.0 ± 8.1% ID/g at 168 h postinjection. A single treatment with
177
Lu‐DOTA‐chCE7agl caused significant retardation of tumor growth and prolonged median survival from 33 to 71 days, while administration of a nontargeted
177
Lu‐immunoconjugate had no beneficial effect. Three times weekly i.p. application of unlabeled chCE7 10 mg/kg similarly increased survival from 44 to 72 days. We conclude that a single dose of
177
Lu‐DOTA‐chCE7agl is as effective as repeated administration of nonradioactive chCE7 for treatment of small intraperitoneal tumors expressing L1‐CAM.
The probiotic Symbioflor 1 is a historical concoction of 10 isolates of Enterococcus faecalis Pulsed-field gel electrophoresis revealed two groups: one comprising eight identical clones (DSM16430, ...DSM16432, DSM16433, DSM16435 to DSM16439) and a further two isolates (DSM16431, DSM16434) with marginally different profiles. Here, we report a comparative analysis of the draft genome sequences of representative isolates.
Interleukin-10 homologues encoded by Herpes viruses such as Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) hold interesting structural and biological characteristics compared to human ...interleukin-10 (hIL-10) that render these proteins promising candidates for therapeutic application in inflammatory bowel disease (IBD). Intestinal delivery of cytokines using bacterial carriers as chassis represents a novel approach for treatment of IBD patients. For proof of concept, a Sec-dependent transporter construct was designed for secretory expression of recombinant viral IL-10 proteins in the periplasm of Escherichia coli laboratory strain BL21 (DE3), which might serve as part of a prospective lysis based delivery and containment system. The signal peptide of E. coli outer membrane protein F fused to the mature form of the viral IL-10 proteins enabled successful transport into the periplasm, a compartment which seems crucial for proper assembly of the dimeric configuration of the cytokines. Cytokine concentrations in different bacterial compartments were determined by ELISA and achieved yields of 67.8 ng/ml + or - 24.9 ng/ml for HCMV IL-10 and 1.5 mug/ml + or - 841.4 ng/ml for EBV IL-10 in the periplasm. Immunoblot analysis was used to confirm the correct size of the E. coli-derived recombinant cytokines. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the signal transduction cascade after IL-10 receptor interaction, as well as suppression of tumor necrosis factor alpha (TNF-alpha) release of lipopolysaccharide-stimulated mouse macrophages were used as read-out assays for proving in vitro biological activity of the E. coli derived, recombinant viral IL-10 counterparts. In this study, proof of principle is provided that E. coli cells are a suitable chassis for secretory expression of viral IL-10 cytokines encoded by codon-optimized synthetic genes fused to the E. coli ompF signal sequence. In vitro biological activity evidenced by activation of transcription factor STAT3 and suppression of TNF-alpha in mammalian cell lines was shown to be strictly dependent on export of viral IL-10 proteins into the periplasmic compartment. E. coli might serve as carrier system for in situ delivery of therapeutic molecules in the gut, thus representing a further step in the development of novel approaches for treatment of IBD.
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