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Background: VERVE-101 is an investigational in vivo CRISPR base-editing medicine designed to alter a single DNA base in the PCSK9 gene, permanently turn off hepatic protein production, and thereby durably lower low-density lipoprotein cholesterol. We test the efficacy, durability, tolerability, and potential for germline editing of VERVE-101 in studies of nonhuman primates and a murine F1 progeny study. Methods: Cynomolgus monkeys were given a single intravenous infusion of a vehicle control (n=10) or VERVE-101 at a dose of 0.75 mg/kg (n=4) or 1.5 mg/kg (n=22) with subsequent follow-up up to 476 days. Two studies assessed the potential for germline editing, including sequencing sperm samples from sexually mature male nonhuman primates treated with VERVE-101 and genotyping offspring from female mice treated with the murine surrogate of VERVE-101 (VERVE-101mu). Results: Liver biopsies 14 days after dosing noted mean PCSK9 editing of 46% and 70% in monkeys treated with VERVE-101 at 0.75 and 1.5 mg/kg, respectively. This translated into mean reductions in blood PCSK9 (proprotein convertase subtilisin/kexin type 9) of 67% and 83% and reductions of low-density lipoprotein cholesterol of 49% and 69% at the 0.75 and 1.5 mg/kg doses, respectively, assessed as time-weighted average change from baseline between day 28 and up to 476 days after dosing. Liver safety monitoring noted a transient rise in alanine aminotransferase and aspartate aminotransferase concentrations after infusion that fully resolved by day 14 with no accompanying change in total bilirubin. In a subset of monkeys necropsied 1 year after dosing, no findings related to VERVE-101 were identified on macroscopic and histopathologic assessment of the liver and other organs. In the study to assess potential germline editing of male nonhuman primates, sperm samples collected after VERVE-101 dosing showed no evidence of PCSK9 editing. Among 436 offspring of female mice treated with a saturating dose of VERVE-101mu, the PCSK9 edit was transmitted in 0 of 436 animals. Conclusions: VERVE-101 was well tolerated in nonhuman primates and led to 83% lower blood PCSK9 protein and 69% lower low-density lipoprotein cholesterol with durable effects up to 476 days after dosing. These results have supported the initiation of a first-in-human clinical trial in patients with heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease.

VEGF pathway–targeting antiangiogenic drugs, such as bevacizumab, when combined with chemotherapy have changed clinical practice for the treatment of a broad spectrum of human cancers. However, adaptive resistance often develops, and one major mechanism is elevated tumor hypoxia and upregulated hypoxia-inducible factor-1α (HIF1α) caused by antiangiogenic treatment. Reduced tumor vessel numbers and function following antiangiogenic therapy may also affect intratumoral delivery of concurrently administered chemotherapy. Nonetheless, combining chemotherapy and bevacizumab can lead to improved response rates, progression-free survival, and sometimes, overall survival, the extent of which can partly depend on the chemotherapy backbone. A rational, complementing chemotherapy partner for combination with bevacizumab would not only reduce HIF1α to overcome hypoxia-induced resistance, but also improve tumor perfusion to maintain intratumoral drug delivery. Here, we evaluated bevacizumab and CRLX101, an investigational nanoparticle–drug conjugate containing camptothecin, in preclinical mouse models of orthotopic primary triple-negative breast tumor xenografts, including a patient-derived xenograft. We also evaluated long-term efficacy of CRLX101 and bevacizumab to treat postsurgical, advanced metastatic breast cancer in mice. CRLX101 alone and combined with bevacizumab was highly efficacious, leading to complete tumor regressions, reduced metastasis, and greatly extended survival of mice with metastatic disease. Moreover, CRLX101 led to improved tumor perfusion and reduced hypoxia, as measured by contrast-enhanced ultrasound and photoacoustic imaging. CRLX101 durably suppressed HIF1α, thus potentially counteracting undesirable effects of elevated tumor hypoxia caused by bevacizumab. Our preclinical results show pairing a potent cytotoxic nanoparticle chemotherapeutic that complements and improves concurrent antiangiogenic therapy may be a promising treatment strategy for metastatic breast cancer. Cancer Res; 76(15); 4493–503. ©2016 AACR.

Description of additional methods and procedures used in the study.

Bioluminescence imaging of mice bearing primary LM2-4 tumors (S1); Assessment of MVD, HIF-1α, and tumoral drug accumulation in PDX primary tumors (S2); Immunohistochemistry staining for CD31, CAIX, caspase 3, Ki67, and HIF-1α, as well as in vitro VEGF quantification (S3); CEUS imaging assessment of HCI-002 primary tumors (S4); Assessment of vessels with open lumen and tumor cell density in PDX tumors (S5); Time-intensity curves (TICs) of CEUS in primary MDA-MB-231/LM2-4 tumors (S6).

VERVE-101 is an investigationalCynomolgus monkeys were dosed with a single intravenous infusion of a vehicle control (N=10) or VERVE-101 at a dose of 0.75 mg/kg (N=4) or 1.5 mg/kg (N=22) with subsequent follow-up out to 476 days. Two studies assessed the potential for germline editing, including sequencing sperm samples from sexually mature male non-human primates treated with VERVE-101 and genotyping offspring from female mice treated with the murine surrogate of VERVE-101 (VERVE-101mu).Liver biopsies 14 days after dosing noted meanVERVE-101 was well-tolerated in in non-human primates and led to 83% lower blood PCSK9 protein and 69% lower LDL-C with durable effects up to 476 days following dosing. These results have supported initiation of a first-in-human clinical trial in patients with heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease.

Gene-editing technologies, which include the CRISPR–Cas nucleases1–3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a ‘once-and-done’ approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs. In a cynomolgus macaque model, CRISPR base editors delivered in lipid nanoparticles are shown to efficiently and stably knock down PCSK9 in the liver to reduce levels of PCSK9 and low-density lipoprotein cholesterol in the blood.

Standard lipid nanoparticles (LNPs) deliver gene editing cargoes to hepatocytes through receptor-mediated uptake via the low-density lipoprotein receptor (LDLR). Homozygous familial hypercholesterolemia (HoFH) is a morbid genetic disease characterized by complete or near-complete LDLR deficiency, markedly elevated blood low-density lipoprotein cholesterol (LDL-C) levels, and premature atherosclerotic cardiovascular disease. In order to enable in vivo liver gene editing in HoFH patients, we developed a novel LNP delivery technology that incorporates a targeting ligand—N-acetylgalactosamine (GalNAc)—which binds to the asialoglycoprotein receptor (ASGPR). In a cynomolgus monkey (Macaca fascicularis) non-human primate (NHP) model of HoFH created by somatic knockout of the LDLR gene via CRISPR-Cas9, treatment with GalNAc-LNPs formulated with an adenine base editor mRNA and a guide RNA (gRNA) targeting the ANGPTL3 gene yielded ~60% whole-liver editing and ~94% reduction of blood ANGPTL3 protein levels, whereas standard LNPs yielded minimal editing. Moreover, in wild-type NHPs, the editing achieved by GalNAc-LNPs compared favorably to that achieved by standard LNPs, suggesting that GalNAc-LNP delivery technology may prove useful across a range of in vivo therapeutic applications targeting the liver.

Novel nanoparticle-drug conjugates (NDCs) containing diverse, clinically relevant anticancer drug payloads (docetaxel, cabazitaxel, and gemcitabine) were successfully generated and tested in drug discovery studies. The NDCs utilized structurally varied linkers that attached the drug payloads to a β-cyclodextrin-PEG copolymer to form self-assembled nanoparticles. In vitro release studies revealed a diversity of release rates driven by linker structure-activity relationships (SARs). Improved in vivo pharmacokinetics (PK) for the cabazitaxel (CBTX) NDCs with glycinate-containing (1c) and hexanoate-containing linkers (2c) were demonstrated, along with high and sustained tumor levels (>168 h of released drug in tumor tissues). This led to potent efficacy and survival in both taxane- and docetaxel-resistant in vivo anticancer mouse efficacy models. Overall, the CBTX-hexanoate NDC 2c (CRLX522), demonstrated optimal and improved in vivo PK (plasma and tumor) and efficacy profile versus those of the parent drug, and the results support the potential therapeutic use of CRLX522 as a new anticancer agent.

Objective: The convective flow of CSF plays a crucial role for CNS to clear endogenous and xenobiotic substances. The objective of this study is to investigate the impact of modifying CSF flow with acetazolamide and arginine vasopressin (AVP) on the CNS clearance of ibuprofen and acetaminophen. Results: Microdialysis studies indicated that acetaminophen AUC ratio (Kp,uu) between brain ISF and unbound plasma increased from 0.40 ± 0.14 in the vehicle control group to 0.60 ± 0.27 in the acetazolamide treated group (P < 0.05). Conversely, acetaminophen’s steady-state ISFC to unbound_plasmaC ratio (Kp,uu) decreased from 0.44 ± 0.08 to 0.36 ± 0.07 upon IV infusion of 0.3 µg/hr AVP (P < 0.05). Using CSF concentration as a surrogate of unbound brain drug concentration, AVP treatment reduced the CSFAUC0-5hr/unbound_plasmaAUC0-5hr ratio from 1.63 to 0.85 for acetaminophen, and the CSFC4hr / unbound_plasmaC4hr ratio decreased from 0.91 ± 0.27 to 0.54 ± 0.12 for ibuprofen (P < 0.05). Conclusion: We have demonstrated that acetazolamide decreases the CNS clearance of acetaminophen, while AVP increases the CNS clearance of both acetaminophen and ibuprofen. Such changes are caused by altering the CSF production rates.

Summary: The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform. : Finn et al. describe the development of a transient, biodegradable LNP-based CRISPR/Cas9 delivery system that achieves >97% knockdown of serum TTR levels following a single administration. Editing levels were stable for 12 months, despite the transient nature of the delivery system and the editing components. Keywords: CRISPR, Cas9, genome editing, LNP, lipid nanoparticle, TTR, CRISPR/Cas9, liver delivery, gene therapy, sgRNA

VEGF pathway–targeting antiangiogenic drugs, such as bevacizumab, when combined with chemotherapy have changed clinical practice for the treatment of a broad spectrum of human cancers. However, adaptive resistance often develops, and one major mechanism is elevated tumor hypoxia and upregulated hypoxia-inducible factor-1α (HIF1α) caused by antiangiogenic treatment. Reduced tumor vessel numbers and function following antiangiogenic therapy may also affect intratumoral delivery of concurrently administered chemotherapy. Nonetheless, combining chemotherapy and bevacizumab can lead to improved response rates, progression-free survival, and sometimes, overall survival, the extent of which can partly depend on the chemotherapy backbone. A rational, complementing chemotherapy partner for combination with bevacizumab would not only reduce HIF1α to overcome hypoxia-induced resistance, but also improve tumor perfusion to maintain intratumoral drug delivery. Here, we evaluated bevacizumab and CRLX101, an investigational nanoparticle–drug conjugate containing camptothecin, in preclinical mouse models of orthotopic primary triple-negative breast tumor xenografts, including a patient-derived xenograft. We also evaluated long-term efficacy of CRLX101 and bevacizumab to treat postsurgical, advanced metastatic breast cancer in mice. CRLX101 alone and combined with bevacizumab was highly efficacious, leading to complete tumor regressions, reduced metastasis, and greatly extended survival of mice with metastatic disease. Moreover, CRLX101 led to improved tumor perfusion and reduced hypoxia, as measured by contrast-enhanced ultrasound and photoacoustic imaging. CRLX101 durably suppressed HIF1α, thus potentially counteracting undesirable effects of elevated tumor hypoxia caused by bevacizumab. Our preclinical results show pairing a potent cytotoxic nanoparticle chemotherapeutic that complements and improves concurrent antiangiogenic therapy may be a promising treatment strategy for metastatic breast cancer. Cancer Res; 76(15); 4493–503. ©2016 AACR.

Reducing the production of larger aggregation-prone amyloid β-peptides (Aβ) remains an untested therapeutic approach for reducing the appearance and growth of Aβ plaques in the brain, which are a hallmark pathological feature of Alzheimer's disease. γ-Secretase modulators (GSMs) are therapeutics that impact γ-secretase-dependent cleavage of amyloid precursor protein to promote the production of shorter Aβ peptides that are less prone to aggregation and plaque deposition. This is accomplished without inhibiting overall γ-secretase function and cleavage of other substrates, which is believed to be a source of deleterious side effects. Here, we report the pharmacokinetic and pharmacodynamic properties of BIIB042, a novel bioavailable and brain-penetrant GSM. In cell-based assays, BIIB042 reduced the levels of Aβ42, increased the levels of Aβ38 and had little effect on the levels of Aβ40, the most abundant Aβ species. Similar pharmacodynamic properties were confirmed in the central nervous system and in plasma of mice and rats, and also in plasma of cynomolgus monkeys after a single oral dose of BIIB042. BIIB042 reduced Aβ42 levels and Aβ plaque burden in Tg2576 mice, which overexpress human amyloid precursor protein and serve as a model system for Alzheimer's disease. BIIB042 did not inhibit cleavage of other γ-secretase substrates in cell-based and in vivo signaling and cleavage assays. The pharmacodynamic effects of lowering Aβ42 in the central nervous system coupled with demonstrated efficacy in reducing plaque pathology suggests modulation of γ-secretase, with molecules like BIIB042, is a compelling therapeutic approach for the treatment of Alzheimer's disease.

Protein interacting with C kinase (PICK1) is a scaffolding protein that is present in dendritic spines and interacts with a wide array of proteins through its PDZ domain. The best understood function of PICK1 is regulation of trafficking of AMPA receptors at neuronal synapses via its specific interaction with the AMPA GluA2 subunit. Disrupting the PICK1-GluA2 interaction has been shown to alter synaptic plasticity, a molecular mechanism of learning and memory. Lack of potent, selective inhibitors of the PICK1 PDZ domain has hindered efforts at exploring the PICK1-GluA2 interaction as a therapeutic target for neurological diseases. Here, we report the discovery of PICK1 small molecule inhibitors using a structure-based drug design strategy. The inhibitors stabilized surface GluA2, reduced Aβ-induced rise in intracellular calcium concentrations in cultured neurons, and blocked long term depression in brain slices. These findings demonstrate that it is possible to identify potent, selective PICK1-GluA2 inhibitors which may prove useful for treatment of neurodegenerative disorders.

Many studies have focused on the challenges of small molecule uptake across the blood–brain barrier, whereas few in-depth studies have assessed the challenges with the uptake of antibodies into the central nervous system (CNS). In drug development, cerebrospinal fluid (CSF) sampling is routinely used as a surrogate for assessing CNS drug exposure and biomarker levels. In this report, we have studied the kinetic correlation between CSF and serum drug concentration–time profiles for five humanized monoclonal antibodies in rats and cynomolgus monkeys and analyzed factors that affect their CSF exposure. Upon intravenous (IV) bolus injection, antibodies entered the CNS slowly and reached maximum CSF concentration ( CSF T max ) in one to several days in both rats and monkeys. Antibody serum and CSF concentration–time curves converged until they became parallel after CSF T max was reached. Antibody half-lives in CSF ( CSF t ½ ) approximated their serum half-lives ( serum t ½ ). Although the intended targets of these antibodies were different, the steady-state CSF to serum concentration ratios were similar at 0.1–0.2% in both species. Independent of antibody target and serum concentration, CSF-to-serum concentration ratios for individual monkeys ranged by up to tenfold from 0.03 to 0.3%. Upon systemic administration, average antibodies CSF-to-serum concentration ratios in rats and monkeys were 0.1–0.2%. The CSF t ½ of the antibodies was largely determined by their long systemic t ½ ( systemic t ½ ).

The objectives of the study were to characterize the selectivity of dantrolene to breast cancer resistance protein (Bcrp) and to evaluate whether cerebrospinal fluid (CSF) can be used as a surrogate to assess brain exposures of BCRP and P-glycoprotein (Pgp) substrates. The impact of Bcrp and Pgp on dantrolene exposures in brain and CSF was examined in Bcrp and Mdr1a/1b knockout mice and was further investigated in wild-type mice in the presence of the Bcrp inhibitor (3 S ,6 S ,12a S )-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4- b ]indole-3-propanoic acid 1,1-dimethylethyl ester (Ko143), the Pgp inhibitor 6-[(2 S ,4 R ,6 E )-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporine A (PSC833), and the dual inhibitor N -(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). The effect of Bcrp and Pgp on digoxin exposures in brain and CSF was investigated in wild-type mice in the presence of the inhibitors. In vivo studies showed dantrolene exposures in brain and CSF, but not the blood, increased in Bcrp(−/−) and Mdr1a/1b(−/−)/Bcrp(−/−) mice, or in the presence of the Bcrp inhibitors Ko143 or GF120918. Inhibition of Pgp by GF120918 and PSC833 significantly increased digoxin exposures in brain, CSF, and blood to a lesser extent. Results from the present study demonstrated that inhibition of Bcrp and Pgp increased not only the exposures of dantrolene and digoxin in brain, but also the exposures in CSF. In addition, the change of exposures in CSF reflected the changes in brain. The present study strongly suggests that the dantrolene and digoxin exposures in CSF are primarily determined by the rapid transport from brain to CSF, and inhibition of Bcrp and Pgp exhibits little impact on using CSF as surrogates to assess brain exposures of Bcrp and Pgp substrates.

Starting from literature examples of nonsteroidal anti-inflammatory drugs (NSAIDs)-type carboxylic acid γ-secretase modulators (GSMs) and using a scaffold design approach, we identified 4-aminomethylphenylacetic acid 4 with a desirable γ-secretase modulation profile. Scaffold optimization led to the discovery of a novel chemical series, represented by 6b, having improved brain penetration. Further SAR studies provided analog 6q that exhibited a good pharmacological profile. Oral administration of 6q significantly reduced brain Aβ42 levels in mice and rats.

We have investigated a novel series of acid-derived γ-secretase modulators as a potential treatment of Alzheimer's disease. Optimization based on cellular potency and brain pharmacodynamics after oral dosing led to the discovery of 10a (BIIB042). Compound 10a is a potent γ-secretase modulator, which lowered Aβ42, increased Aβ38, but had little to no effect on Aβ40 levels both in vitro and in vivo. In addition, compound 10a did not affect Notch signaling in our in vitro assessment. Compound 10a demonstrated excellent pharmacokinetic parameters in multiple species. Oral administration of 10a significantly reduced brain Aβ42 levels in CF-1 mice and Fischer rats, as well as plasma Aβ42 levels in cynomolgus monkeys. Compound 10a was selected as a candidate for preclinical safety evaluation.

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (−)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl) propan-1-ol was identified as a good SphK2 substrate and potent S1P1 agonist with good oral bioavailability.

Our nanoparticle-drug conjugates (NDCs), created at Cerulean Pharma Inc. by conjugating drug payloads to our novel β-cyclodextrin-PEG (CDP) copolymer, are designed to significantly mitigate a payload’s limitations by providing sustained drug delivery to the tumor and superior therapeutic index through controlled release kinetics. Cerulean has two NDCs in the clinic, CRLX101 and CRLX301, evidencing the translatability of our technology. CRLX101 has been dosed in over 400 patients and CRLX301 is in ongoing Phase 2a development. A key and differentiating feature of our NDC Platform is our linker technology, which is tailored for an optimal fit with the conjugation functionality of the API drug payloads (e.g., alcohol, carboxylic acid, amine, amide and urea functionality) and customizable to achieve desired drug release profiles. As an illustration of Cerulean’s ability to expand its NDC platform, we will present the biological and pharmacokinetic (PK) data supporting our drug combination platform of antibody nanoparticle-drug conjugates (ANDCs) and multi-drug nanoparticle-drug conjugates (mNDCs). ANDCs combine potentially any NDC with any conjugatable biologic to generate an ANDC with ultra-high drug-antibody ratios (DARs). Using our established NDC linker technology, we generated Herceptin-camptothecin ANDCs with DARs as high as 500, orders of magnitude higher than ADCs, and no more than 5-fold loss in binding (greater than 90% binding up to 300 DAR) compared to native Herceptin in our solution-based HER2 antigen binding assay. In addition, we provide evidence experimentally that rhodamine-labeled ANDCs penetrate and internalize in HER2+ tumor cells. The ANDCs demonstrate tunable in vitro release kinetics and long in vivo half lives in mouse PK studies, including sustained levels of released drug in tumors (greater than 300h) using an ANDC (average DAR 201) delivering an 8 mg/kg camptothecin dose. The observed mouse MTD for the ANDC was at least 3x higher than that of the corresponding NDC. The mNDCs take advantage of our existing NDC linker platform technology to conjugate diverse drug combos including DNA damaging agents (DDA) + DNA damage repair agents (DDR). We generated our POC camptothecin-olaparib mNDCs spanning a range of drug combination ratios (from 1:1 to 1:20) and all demonstrated distinguishable and tunable in vitro release rates. PK study of a 1:1 camptothecin-olaparib mNDC (~8mg/kg each drug) demonstrated very low clearance and sustained levels of released drug in tumor (greater than 72h). Our drug combination platforms, ANDCs and mNDCs, allow delivery of therapeutic agents to their respective biological targets at clinically-relevant doses, greatly increasing the diversity of drug combination possibilities. Note: This abstract was not presented at the meeting. Citation Format: Chester A. Metcalf, Derek van der Poll, Liang Zhao, Roy I. Case, Doug Lazarus, Donna Brown, Tiffany Halo, Lata Jayaraman, Christian Peters, Ellen Rohde, Scott Eliasof. Sustained and controlled in vivo therapeutic levels of drug payloads in tumors using two separate drug combination platforms: Antibody nanoparticle-drug conjugates and multi-drug nanoparticle-drug conjugates, with the potential for improved drug combinability and anticancer effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5135. doi:10.1158/1538-7445.AM2017-5135

Fast and efficient analysis of proteins in physiological fluids is of great interest to researchers and clinicians alike. Capillary electrophoresis (CE) has proven to be a potentially valuable tool for the separation of proteins in specimens. However, a generally acknowledged drawback of this technique is the limited sample volumes which can be loaded onto the CE capillary which results in a poor concentration limit of detection. In addition, matrix components in samples may also interfere with separation and detection of analytes. Membrane preconcentration–CE (mPC–CE) has proved to be effective in overcoming these problems. In this report, we describe the systematic evaluation of parameters affecting on-line preconcentration/clean-up and separation of protein mixtures by mPC–CE. Method development was carried out with a standard mixture of proteins (lysozyme, myoglobin, carbonic anhydrase, and human serum albumin). First, using MALDI-TOF-MS, membrane materials with cation-exchange (R-SO 3 H) or hydrophobic (C 2 , C 8 , C 18 , SDB) characteristics were evaluated for their potential to retain proteins in mPC cartridges. Hydrophobic membranes were found most suitable for this application. Next, all mPC–CE analysis of protein samples were performed in polybrene coated capillaries and parameters affecting sample loading, washing and elution, such as the composition and volume of the elution solvent were investigated. Furthermore, to achieve optimal mPC–CE performance for the separation of protein mixtures parameters affecting postelution focusing and electrophoresis, including the composition of the background electrolyte and a trailing stacking buffer were varied. Optimal conditions for mPC–CE analysis of proteins using a C 2 impregnated membrane preconcentration (mPC) cartridge were achieved with a background electrolyte of 5% acetic acid and 2 m M ammonium acetate, 60 nl of 80% acetonitrile in H 2 O as an elution solvent, and 60 nl of 0.5% ammonium hydroxide as a trailing stacking buffer. The developed method was used successfully to separate proteins in aqueous humor, which contains numerous proteins in a complex matrix of salts.

Information on the identity of inks adds to the circumstantial evidence in legal cases involving fraudulent documents. In combination with optical methods, multiple thin-layer chromatography (TLC) is currently the analytical tool used by forensic chemists to separate, compare and distinguish inks based on their dye composition. In our studies, capillary electrophoresis (CE) was used for the analysis of water-soluble fountain pen inks. Inks are complex mixtures of synthetic organic and inorganic dyes, surfactants, resins and other components. The investigations included the development of an electrophoretic separation method, the optimization of an extraction procedure for inks from paper as well as the evaluation of ultraviolet/visible (UV/VIS) absorbance and laser-induced fluorescence (LIF) detection for the analysis of inks by CE. Good results for the separation of 17 blue and black inks of different manufacturers and countries of origin were obtained with 100 mM borate buffer, pH 8.0, containing 20% methanol. The electropherograms of the inks and their extracts from paper showed patterns that were in most cases distinctly different from each other. Ultraviolet/visible scans can be used to compare spectra of the separated main and trace components of inks. Fluorescence detection at different excitation and emission wavelengths was more sensitive, but added to the complexity of the electropherograms due to the excitation of coextracted fluorescing paper components. The resolution power of CE combined with the information content provided by the detection modes investigated prove CE to be a powerful tool for the identification of water-soluble inks used on paper documents.

Cerulean Pharma Inc.'s Dynamic Tumor Targeting™ Platform creates nanoparticle-drug conjugates (NDCs) designed to significantly mitigate a payload's limitations by providing sustained drug delivery to the tumor and superior therapeutic index through controlled release kinetics. By conjugating drug payloads to our novel β-cyclodextrin-PEG copolymer through linker strategies that allow modulation of release and pharmacokinetics (PK), we provide advantages over entrapped nanoparticle strategies, e.g., polymeric nanoparticles and liposomes. Cerulean has two NDCs in the clinic, CRLX101 and CRLX301, evidencing the translatability of our technology. CRLX101 has been dosed in over 350 patients and CRLX301 is in an ongoing Phase 1/2a trial. Our Dynamic Tumor Targeting Platform is applicable to a diverse range of drug payload possibilities, including small molecules with a host of physiochemical properties, i.e., hydrophobic and hydrophilic payloads, and functional groups with chemical handles for conjugation. To illustrate the capabilities of our platform we will present the biological impact of a series of cabazitaxel-containing NDCs with linkers encompassing a diversity of in vitro release rates. In vivo PK studies showed high and sustained levels of released drug in tumor tissues (>168 hrs), and in vivo mouse tumor model studies demonstrated vastly improved efficacy, i.e., tumor regression and significant tumor growth delay, and survival over separately dosed cabazitaxel including efficacy in a taxane-resistant tumor model. Cerulean continues to expand its platform through the development of new and emerging capabilities to treat patients living with cancer. In this regard, we also will address the future evolution of NDCs, including the conjugation of multiple payloads to a single NDC and the development of antibody-conjugated NDCs. This abstract is also being presented as Poster B43. Citation Format: Chester Metcalf, III, Derek van der Poll, Liang Zhao, Tiffany Halo, Doug Lazarus, Adam Stockmann, Christian Peters, Donna Brown, Roy Case, Ellen Rohde, Lata Jayaraman, Hongwei Wang, Tiffany Crowell, Adrian Senderowicz, Scott Eliasof. Significant improvements in therapeutic index for conjugated payloads using a nanoparticle-drug conjugate (NDC) platform to provide sustained drug release and potentially improved anticancer effects. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR10.

CRLX101, an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin, is currently being clinically evaluated in multiple treatment-refractory solid tumors. In preclinical models, CRLX101 is believed to release camptothecin in the tumor in a slow and prolonged manner due to its long circulation half-life. CRLX101 has been shown preclinically to be a dual inhibitor of topoisomerase 1 and hypoxia-inducible factor 1α. It has demonstrated striking anti-tumor activity in several different tumor models. Camptothecin itself was identified as an active anti-tumor agent preclinically but was not developed clinically due to its poor tolerability in patients. The development of CRLX101, which has not shown significant toxicity in over 300 patients to date, offers a unique opportunity to improve cancer treatment in a meaningful way. We hypothesized that CRLX101 utilizes the enhanced permeability and retention (EPR) effect to accumulate selectively in tumors. In this study, we sought to mechanistically dissect the process of CRLX101 entry and accumulation into tumor cells using multiple methods, both in vitro and in xenograft tumors in vivo. Using confocal microscopy, we detected camptothecin fluorescence in CRLX101-treated tumor cells in culture as well as in tumor tissue from mice treated with CRLX101. We can co-localize this camptothecin with intact nanoparticles using an anti-PEG antibody that specifically detects the PEG loops in the NDCs. More recently, we have shown that camptothecin and anti-PEG co-localize specifically in tumors of patients treated with CRLX101 but not in adjoining normal tissue. We can also demonstrate that macropinocytosis and activation of actin polymerization play a role in the process by which tumor cells take up CRLX101. Using an anti-CD31 antibody, we can visualize the distance traversed by CRLX101 from the tumor vasculature over time. We have developed novel analytical methods to precisely quantify both released and CRLX101-conjugated camptothecin over time in CRLX101 treated tumor cells in vitro, as well as in tumor tissue from mice treated with CRLX101 in vivo. Using cell viability assays, we can correlate the kinetics of camptothecin released inside tumor cells to the degree of tumor cell kill. We believe that these data are an important step forward in understanding the precise mechanism(s) underlying selective delivery of CRLX101 into tumor tissue. Citation Format: Christian G. Peters, Douglas Lazarus, Donna Brown, Ningning Zhang, Adam P. Stockmann, Roy Case, Ellen Rohde, Scott Eliasof, Lata Jayaraman. Tumor selective localization of CRLX101, an investigational nanoparticle-drug conjugate of camptothecin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1345.

Background: In recent years, there has been a growing interest in automating small-molecule bioanalytical sample preparations specifically using the Hamilton MicroLab® STAR liquid-handling platform. In the most extensive work reported thus far, multiple small-molecule sample preparation assay types (protein precipitation extraction, SPE and liquid–liquid extraction) have been integrated into a suite that is composed of graphical user interfaces and Hamilton scripts. Using that suite, bioanalytical scientists have been able to automate various sample preparation methods to a great extent. However, there are still areas that could benefit from further automation, specifically, the full integration of analytical standard and QC sample preparation with study sample extraction in one continuous run, real-time 2D barcode scanning on the Hamilton deck and direct Laboratory Information Management System database connectivity. Results: We developed a new small-molecule sample-preparation automation system that improves in all of the aforementioned areas. Conclusion: The improved system presented herein further streamlines the bioanalytical workflow, simplifies batch run design, reduces analyst intervention and eliminates sample-handling error.

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.

The preparation of a reagentless glucose oxidase-based biosensor for application in a flow system is described. The enzyme was first codeposited with the redox polymer [Os(bpy)2(PVP)10Cl]Cl (bpy = bipyridyl, PVP = poly-4-vinylpyridine) and glutaradehyde on the surface of a platinum electrode, and subsequently covered with an electropolymerized layer of pyrrole, also containing oxidase. The electron transfer from the reduced FADH2 group in the core of the enzyme to the electrode surface is facilitated via the redox polymer/polypyrrole system. Due to the high permeability of both layers the amperometric detection of β- d -glucose can be carried out directly. Qualitative and quantitative investigations to optimize the composition of the solution for adsorption and electropolymerisation were performed. The characterisation of the sensor in terms of linearity, reproducibility, pH dependence, stability and the response to interfering substances was also carried out.

CRLX101, an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin conjugated to a biocompatible copolymer of cyclodextrin and polyethylene glycol (PEG), is currently being evaluated clinically in multiple treatment-refractory solid tumors. CRLX101 is a dual inhibitor of topoisomerase 1 (TOPO 1) and hypoxia-inducible factor 1α (HIF-1α) and has been shown pre-clinically to be active in many different tumor types. It has a long circulation half-life and is believed to release camptothecin in a slow and prolonged fashion in the tumor. Camptothecin is an active anti-tumor agent preclinically but was not developed clinically due to its poor tolerability in patients. The development of CRLX101, which has not demonstrated significant toxicity in over 250 patients dosed to date, offers a unique opportunity to improve cancer treatment in a meaningful way. We have previously published that in gastric cancer patients treated with CRLX101, camptothecin fluorescence can be visualized in tumor tissue but not adjoining normal tissue. Based on these data we hypothesized that CRLX101 utilizes the enhanced permeability and retention (EPR) effect to accumulate selectively in tumors. In this study, we sought to mechanistically dissect the process of CRLX101 entry and accumulation into tumor cells using multiple methods, both in vitro and in xenograft tumors in vivo. Using confocal microscopy, we can detect camptothecin fluorescence in CRLX101- treated tumor cells in culture as well as in tumor tissue from mice treated with CRLX101. We can co-localize this camptothecin with intact nanoparticles using an anti-PEG antibody that specifically detects the PEG loops in the NDCs. We can also demonstrate that macropinocytosis plays a role in the manner by which tumor cells take up CRLX101. Using an anti-CD31 antibody, we can visualize the distance traversed by CRLX101 from the tumor vasculature over time. We have developed novel analytical methods to precisely quantify both released and CRLX101-conjugated camptothecin over time in CRLX101 treated tumor cells in vitro, as well as in tumor tissue from mice treated with CRLX101 in vivo. Using cell viability assays, we can correlate the kinetics of camptothecin released inside tumor cells to the degree of tumor cell kill. We believe that these data are an important step forward in understanding the precise mechanism(s) underlying selective delivery of CRLX101 into tumor tissue. Citation Format: Christian G. Peters, Douglas Lazarus, Donna Brown, Ningning Zhang, Roy Case, Ellen Rohde, Scott Eliasof, Lata Jayaraman. Selective tumor localization of CRLX101, a novel nanoparticle-drug conjugate. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B37.

To support in vivo screening efforts for estrogen receptor (ER) subtype selective therapeutic agents, we initiated work to discover surrogate markers (biomarkers) in blood plasma that would change in response to ER subtype-specific action. We used a proteomic approach employing strong anion exchange chromatography (SAX), PAGE, and MS to identify potential plasma markers for selective ER-alpha action. The methodology was used to compare blood from vehicle-treated rats to blood from rats treated with either 17beta-estradiol (an ER-alpha/ER-beta agonist) or compound 1 (17alpha-ethynyl-[3,2-c]pyrazolo-19-nor-4-androstene-17beta-ol, an ER-alpha-selective agonist). Blood samples were first fractionated by SAX to separate fractions containing dominant common plasma proteins from fractions enriched for less-abundant plasma proteins. 1-D PAGE analysis of fractions depleted of dominant plasma proteins revealed treatment-specific changes in protein profiles. Protein bands that changed reproducibly in response to ER-alpha action were excised from the gel, separated by capillary LC, and identified by microspray ESI-MS. Using this method, the plasma levels of two proteins, transthyretin and apolipoprotein E, were shown to decrease in response to ER-alpha agonism. The method lacked the sensitivity to identify the known, 1000-fold less-abundant, estrogenic marker prolactin (PRL). However, using a commercial RIA and immunoblots, we showed that PRL levels increase significantly in response to treatment with the ER-alpha selective agonist, compound 1.

In the last few years capillary electrophoresis (CE) was successfully applied to a variety of analytical problems in the field of chiral separations. This article is intended to demonstrate the potential of CE for the determination of enantiomers. The separation is based on the addition of a suitable chiral selector to the buffer. Cyclodextrins perform as excellent selectors forming diastereomeric complexes of different stability with the enantiomeric forms of the analyte.Barbiturates were chosen for chiral electrophoretic separation. They are found in a variety of pharmaceuticals such as sedatives, hypnotics and antiepileptics and are mostly processed as racemic mixtures. Cyclodextrins were applied as chiral selectors. The complex formation depends essentially on the structure of the selector and the analyte, this fact is discussed extensively in the paper. The influence of pH, the type and the concentration of the chiral selector on the separation were investigated.The described experiment offers studen...