Christian Che

Identification and Design of Peptides as a New Drug Delivery System for the Brain

By controlling access to the brain, the blood-brain barrier (BBB) restricts the entry of proteins and potential drugs to cerebral tissues. We demonstrate here the transcytosis ability of aprotinin and peptides derived from Kunitz domains using an in vitro model of the BBB and in situ brain perfusion. Aprotinin transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 10-fold greater than that of holo-transferrin. Sucrose permeability was unaffected by high concentrations of aprotinin, indicating that transcytosis of aprotinin was unrelated to changes in the BBCEC monolayer integrity. Alignment of the amino acid sequence of aprotinin with the Kunitz domains of human proteins allowed the identification and design of a family of peptides, named Angiopeps. These peptides, and in particular Angiopep-2, exhibit higher transcytosis capacity and parenchyma accumulation than aprotinin. Overall, these results suggest that these Kunitz-derived peptides could be advantageously used as a new brain delivery system for pharmacological agents that do not readily enter the brain.

Involvement of the low‐density lipoprotein receptor‐related protein in the transcytosis of the brain delivery vector Angiopep‐2

The blood–brain barrier (BBB) restricts the entry of proteins as well as potential drugs to cerebral tissues. We previously reported that a family of Kunitz domain‐derived peptides called Angiopeps can be used as a drug delivery system for the brain. Here, we further characterize the transcytosis ability of these peptides using an in vitro model of the BBB and in situ brain perfusion. These peptides, and in particular Angiopep‐2, exhibited higher transcytosis capacity and parenchymal accumulation than do transferrin, lactoferrin, and avidin. Angiopep‐2 transport and accumulation in brain endothelial cells were unaffected by the P‐glycoprotein inhibitor, cyclosporin A, indicating that this peptide is not a substrate for the efflux pump P‐glycoprotein. However, competition studies show that activated α2‐macroglobulin, a specific ligand for the low‐density lipoprotein receptor‐related protein‐1 (LRP1) and Angiopep‐2 can share the same receptor. In addition, LRP1 was detected in glioblastomas and brain metastases from lung and skin cancers. Fluorescent microscopy also revealed that Alexa488‐Angiopep‐2 co‐localized with LRP1 in brain endothelial cell monolayers. Overall, these results suggest that Angiopep‐2 transport across the BBB is, in part, mediated by LRP1.

New Angiopep-Modified Doxorubicin (ANG1007) and Etoposide (ANG1009) Chemotherapeutics With Increased Brain Penetration

This report describes the synthesis and preliminary biological characterization of 2 (ANG1007) and 3 (ANG1009), two new chemical entities under development for the treatment of primary and secondary brain cancers. 2 consists of three doxorubicin molecules conjugated to Angiopep-2, a 19-mer peptide that crosses the blood-brain barrier (BBB) by an LRP-1 receptor-mediated transcytosis mechanism. 3 has a similar structure, with the exception that three etoposide moieties are conjugated to Angiopep-2. Both agents killed cancer cell lines in vitro with similar IC(50) values and with apparently similar cytotoxic mechanisms as unconjugated doxorubicin and etoposide. 2 and 3 exhibited dramatically higher BBB influx rate constants than unconjugated doxorubicin and etoposide and pooled within brain parenchymal tissue. Passage through the BBB was similar in Mdr1a (-/-) and wild type mice. These results provide further evidence of the potential of this drug development platform in the isolation of novel therapeutics with increased brain penetration.

Transport characteristics of a novel peptide platform for CNS therapeutics

New and effective therapeutics that cross the blood‐brain barrier (BBB) are critically needed for treatment of many brain diseases. We characterize here a novel drug development platform that is broadly applicable for the development of new therapeutics with increased brain penetration. The platform is based on the Angiopep‐2 peptide, a sequence derived from ligands that bind to low‐density lipoprotein receptor‐related protein‐1 (LRP‐1), a receptor expressed on the BBB. Fluorescent imaging studies of a Cy5.5Angiopep‐2 conjugate and immunohistochemical studies of injected Angiopep‐2 in mice demonstrated efficient transport across the BBB into brain parenchyma and subsequent co‐localization with the neuronal nuclei‐selective marker NeuN and the glial marker glial fibrillary acidic protein (GFAP). Uptake of [125I]‐Angiopep‐2 into brain endothelial cells occurred by a saturable mechanism involving LRP‐1. The primary sequence and charge of Angiopep‐2 were crucial for its passage across the BBB. Overall, the results demonstrate the significant potential of this platform for the development of novel neurotherapeutics.

Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

Neurotensin (NT) has emerged as an important modulator of nociceptive transmission and exerts its biological effects through interactions with 2 distinct GPCRs, NTS1 and NTS2. NT provides strong analgesia when administered directly into the brain; however, the blood-brain barrier (BBB) is a major obstacle for effective delivery of potential analgesics to the brain. To overcome this challenge, we synthesized chemical conjugates that are transported across the BBB via receptor-mediated transcytosis using the brain-penetrant peptide Angiopep-2 (An2), which targets LDL receptor-related protein-1 (LRP1). Using in situ brain perfusion in mice, we found that the compound ANG2002, a conjugate of An2 and NT, was transported at least 10 times more efficiently across the BBB than native NT. In vitro, ANG2002 bound NTS1 and NTS2 receptors and maintained NT-associated biological activity. In rats, i.v. ANG2002 induced a dose-dependent analgesia in the formalin model of persistent pain. At a dose of 0.05 mg/kg, ANG2002 effectively reversed pain behaviors induced by the development of neuropathic and bone cancer pain in animal models. The analgesic properties of ANG2002 demonstrated in this study suggest that this compound is effective for clinical management of persistent and chronic pain and establish the benefits of this technology for the development of neurotherapeutics.

Abstract 2465: Targeting HER2-positive brain metastases by incorporating the brain-penetrant Angiopep-2 peptide to an anti-HER2 antibody and anti-HER2 antibody drug conjugate

Monoclonal antibodies directed against receptor tyrosine kinases such as HER2 have been demonstrated to reduce tumor size and increase survival. However, these agents achieve little to no brain penetration, making them ineffective against metastatic brain tumors. The blood-brain barrier (BBB), efficient at restricting entry of proteins such as mAbs and anticancer drugs into the brain, is comprised of capillary endothelial cells with tight junctions and efflux pumps. We have created a family of peptides (Angiopeps) which use receptor-mediated transcytosis to enter the brain. Conjugation of the Angiopep-2 (An2) to confer brain permeability has been validated for small molecules (ANG 1005, Phase II), peptides and proteins. The brain-penetrant An2 has also been incorporated to a humanized anti-HER2 mAb. This Angiopep-Antibody Conjugate, ANG4043, displays HER2 binding affinity and in vitro cytotoxic potency similar to that of native anti-HER2. ANG4043 demonstrates a high rate of entry into the brain. ANG4043 reduces the tumor size of BT-474 human breast cancer cells when implanted in the brain, consistent with achieving therapeutic concentrations. Here we describe chemical conjugation between three molecules: the An2, a cytotoxic drug (docetaxel or maytansine), and a mAb directed against HER2. These new An2-antibody-drug-conjugates (An2-ADCs) show a higher in vitro anti-proliferative potency than unconjugated mAb against HER2+ BT-474 and HC-19554 cells that are sensitive and resistant to Herceptin, respectively. Furthermore, they demonstrate a high rate of entry into the brain when compared to controls, leading to a reduction in brain tumor size and to an increase in the survival of mice bearing intracranial BT-474 tumors. Furthermore, An2-anti-HER2 derivatives are also efficacious in peripheral tissues since they inhibited the growth of subcutaneous BT-474 luciferase tumors. Overall, these data demonstrate that the conjugation of an Angiopep to therapeutic mAbs or ADCs can increase their efficacy in the CNS without affecting their anticancer properties outside of the brain. These results extend the validation of Angiopep conjugation beyond small anticancer drugs to include larger molecules such as therapeutic mAbs and ADCs for development of new brain-penetrant therapeutics for brain malignancies. Citation Format: Michel Demeule, Sanjoy Das, Christian Che, Gaoqiang Yang, Jean-Christophe Currie, Simon Lord-Dufour, Sasmita Tripathy, Anthony Regina, Jean-Paul Castaigne, Jean E. Lachowicz. Targeting HER2-positive brain metastases by incorporating the brain-penetrant Angiopep-2 peptide to an anti-HER2 antibody and anti-HER2 antibody drug conjugate. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2465. doi:10.1158/1538-7445.AM2015-2465

Abstract 2657: Utilization of the Angiopep platform to enable brain penetration of therapeutic mAbs or Antibody-Drug Conjugates for treatment of brain tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Monoclonal antibodies directed against receptor tyrosine kinases such as HER2 have been demonstrated to reduce tumor size and increase survival. However, these agents achieve little to no brain penetration, making them ineffective against metastatic brain tumors. The blood-brain barrier (BBB), efficient at restricting entry of proteins such as mAbs into the brain, is comprised of capillary endothelial cells with tight junctions and efflux pumps. Entry of nutrients, hormones, and other required molecules is accomplished by processes such as receptor-mediated transcytosis. As low-density lipoprotein receptor-related protein 1 (LRP1) is known to perform this function in BBB endothelial cells, we have created a family of peptides (Angiopeps) designed for LRP1 recognition. Conjugation of the Angiopep-2 (An2) to confer brain permeability has been validated for small molecules (ANG 1005, Phase II), peptides and proteins. For example, therapeutic concentrations of an anti-HER2 mAb have been achieved with An2 conjugation. This Angiopep-Antibody Conjugate, ANG4043, displays HER2 binding affinity and in vitro cytotoxic potency similar to that of native anti-HER2. ANG4043 demonstrates a high rate of entry into the brain, consistent with achieving therapeutic concentrations. Mice intracranially implanted with BT-474 human breast cancer cells showed reduced brain tumor size when dosed with ANG4043 compared to controls and increased mice survival. Here, we also describe the chemical conjugation between a brain penetrant Angiopep, a cytotoxic agent and a mAb directed against HER2. This new peptide-antibody-drug-conjugate shows a much higher in vitro anti-proliferative potency against HER2+ BT-474 cells than the native antibody. Furthermore, this new peptide-drug-antibody-conjugate demonstrates a high rate of entry into the brain when compared to the unconjugated antibody. Overall, these data demonstrate that the addition of an Angiopep to therapeutic mAbs or Antibody Drug Conjugates (ADCs) can improve their brain penetration. These results extend the validation of Angiopep conjugation beyond small molecules and peptides to include larger molecules such as therapeutic mAbs or ADCs for development of new brain-penetrant antitumor therapeutics. Citation Format: Michel Demeule, Jean E. Lachowicz, Sasmita Tripathy, Giogang Yang, Sanjoy Das, Christian Che, Jean-Christophe Currie, Simon Lord-Dufour, Anthony Regina, Jean-Paul Castaigne. Utilization of the Angiopep platform to enable brain penetration of therapeutic mAbs or Antibody-Drug Conjugates for treatment of brain tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2657. doi:10.1158/1538-7445.AM2014-2657

Abstract 864: ANG4043: A new brain-penetrant peptide-antibody conjugate that reduces tumor growth in a HER2-positive orthotopic tumor model.

While monoclonal antibodies to receptor tyrosine kinases such as HER2 have been demonstrated to reduce tumor size and increase survival, these agents achieve little to no brain penetration, making them ineffective against metastatic brain tumors. The blood-brain barrier (BBB), which restricts entry of proteins such as mAbs into the brain, is comprised of capillary endothelial cells with tight junctions and efflux pumps. Brain entry of nutrients, hormones, and other required molecules is accomplished by processes such as receptor-mediated transcytosis. As low-density lipoprotein receptor-related protein 1 (LRP1) is known to perform this function in BBB endothelial cells, we have created a family of peptides (Angiopeps) designed for LRP1 recognition. These proprietary Angiopeps can be used to create novel Peptide-Drug Conjugates that successfully cross the BBB by LRP1-mediated transcytosis. Here we describe chemical conjugation of Angiopep-2 (An2) to a mAb against HER2. This Peptide-Antibody Conjugate, ANG 4043, displays HER2 binding affinity and in vitro cytotoxic potency similar to that of native anti-HER2. ANG4043 demonstrates a high rate of entry into the brain, consistent with achieving therapeutic concentrations. The plasma half-life of ANG4043 is similar to that of Anti-HER2 in mouse. Mice with intracranially implanted BT-474 cells showed reduced brain tumor size when dosed with ANG4043 compared to controls. Overall, these data demonstrate that a brain-penetrant Peptide-Antibody Conjugate is efficacious in a mouse HER2-positive tumor model. These results extend the validation of An2 conjugation beyond small molecules and peptides to include larger molecules such as therapeutic mAbs for development of new brain-penetrant antitumor therapeutics. Citation Format: Jean E. Lachowicz, Michel Demeule, Christian Che, Sasmita Tripathy, Jean-Christophe Currie, Anthony Regina, Tran Nguyen, Simon Lord-Dufour, Jean-Paul Castaigne. ANG4043: A new brain-penetrant peptide-antibody conjugate that reduces tumor growth in a HER2-positive orthotopic tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 864. doi:10.1158/1538-7445.AM2013-864

Abstract 3578: New Angiochem-modified doxorubicin with increased brain penetration and efficacy against brain tumors

The blood-brain barrier (BBB) is mainly formed by brain capillary endothelial cells which are closely sealed by tight junctions and express high levels of active efflux transport proteins, including P-glycoprotein (Pgp). As a result, the overwhelming majority of small molecules, proteins and peptides do not cross the BBB. Angiochem9s engineered peptide compounds (EPiC) provides a non-invasive and flexible platform for small and large molecules to treat brain diseases. Based on these properties, we have created a portfolio of new drug entities composed of siRNA, peptides and mAbs, the most advanced of which is ANG1005 formed by chemical conjugation of our peptide to three molecules of paclitaxel. ANG1005 demonstrated safety and efficacy in two phase ½ clinical trials for the treatment of primary and secondary brain tumors in humans. In the present study, we have investigated the brain uptake of a new chemical entity formed by conjugation of the peptide Angiopep-2 (An2) with 3 molecules of the anti-cancer drug doxorubicin. Despite the clinical effectiveness of doxorubicin in the treatment of many malignant tumors, clinical trials involving systemic administration have demonstrated very limited efficacy in the treatment of gliomas. This limited efficacy can be explained by the poor penetration of the drug thru the BBB and by the effect of the multidrug resistance pump, P-gp. Here, we show by mice in-situ brain perfusion that the ANG-doxorubicin (ANG1007) is transported very efficiently across the BBB. The transport rate is higher than that of unconjugated doxorubicin by at least 10-fold. In vitro, ANG1007 inhibits cancer cell proliferation, with highly cytotoxic activities against various tumor cell lines. In addition, brain perfusion studies performed with P-gp knock-out mice showed that ANG1007 bypasses the drug efflux pump P-gp at the BBB. Furthermore, ANG1007 inhibits growth of glioblastoma s.c. tumors and increases survival of mice implanted with brain tumors. In conclusion, these data confirm that conjugation of chemotherapeutic drugs such as doxorubicin to the peptide Angiopep-2 significantly enhances their entry into the brain and further validate the use of Angiochem9s technology for new brain tumor therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3578.